SAMPLING DEVICE AND ANALYSIS OF METHYLATED DNA
20220071605 · 2022-03-10
Inventors
- Robert F. Eisele (Carlsbad, CA, US)
- Michael E. Williams (San Diego, CA, US)
- Prasad G. Iyer (Rochester, MN, US)
- Paul Marrione (Madison, WI, US)
- Hatim T. Allawi (Madison, WI)
- Graham P. Lidgard (Madison, WI, US)
- Maria Giakoumopoulos (Madison, WI, US)
- William R. Taylor (Rochester, MN, US)
- Tracy C. Yab (Rochester, MN, US)
- Douglas W. Mahoney (Rochester, MN, US)
- John B. Kisiel (Rochester, MN, US)
- David A. Ahlquist (Rochester, MN, US)
Cpc classification
A61B10/0038
HUMAN NECESSITIES
A61B2010/0061
HUMAN NECESSITIES
C12Q1/6883
CHEMISTRY; METALLURGY
International classification
A61B10/02
HUMAN NECESSITIES
A61B10/00
HUMAN NECESSITIES
Abstract
The invention relates to cell sampling devices. In particular, the present invention relates to ingestible cell sampling devices for sampling cells in a subject, and methods of use for detecting abnormalities in a subject using the same. Provided herein is technology for neoplasia screening, and particularly, but not exclusively, to methods, compositions, and related uses for detecting the presence of cancer.
Claims
1. An ingestible cell sampling device comprising: i) an abrasive sponge housed within a dissolvable capsule, the dissolvable capsule comprising an exterior surface exposed to an external environment, wherein the abrasive sponge is retained in a compressed state by the dissolvable capsule.; ii) a molded cap comprising a cap interior surface and a cap exterior surface; and iii) a string having a first end attached to the molded cap, wherein the string passes through a portion of the abrasive sponge.
2. The ingestible cell sampling device of claim 1, further comprising an unswallowable handle attached to the string.
3. The ingestible cell sampling device of claim 2, wherein the string has a second end, wherein the unswallowable handle is attached to the string at the second end.
4. The ingestible cell sampling device of claim 1, wherein the dissolvable capsule comprises one or more openings, wherein a portion of the abrasive sponge is exposed to the external environment at the one or more openings.
5. The ingestible cell sampling device of claim 1, wherein the dissolvable capsule comprises a first end and a second end, wherein: a) the first end is closed and the second end is closed; or b) the first end is closed and the second end is open.
6. The ingestible cell sampling device of claim 5, wherein the cap interior surface is in contact with the exterior surface of the capsule at the first closed end, and the cap exterior surface is in contact with the external environment.
7. The ingestible cell sampling device of claim1, wherein the cap interior surface is in contact with the abrasive sponge.
8. The ingestible cell sampling device of claim 7, wherein the cap exterior surface is in contact with an internal surface of the capsule at the first closed end and/or with the external environment.
9. The ingestible cell sampling device of claim 7, wherein the cap interior surface is attached to the abrasive sponge by an adhesive.
10. The ingestible cell sampling device of claim 1, wherein the cap interior surface is attached to the abrasive sponge.
11. The ingestible cell sampling device of claim 1, wherein the string is attached to the molded cap by a knot and/or an adhesive.
12. A system or kit for obtaining a cell sample from a subject, comprising an ingestible cell sampling device of claim 1; and further comprising one or more of: i) a container to receive an abrasive sponge comprising collected cells; ii) a cell preservative reagent, preferably a buffer reagent; iii) a microscope slide; iv) an assay plate; v) a local anesthetic treatment, preferably a local anaesthetic spray; vi) a component of a drinkable solution; preferably a pre-mixed drinkable solution; and vii) a lubricant, preferably a lubricant gel or liquid.
13. A method of obtaining a cell sample from a subject, comprising: a) orally administering an abrasive sponge housed within a dissolvable capsule of a ingestible cell sampling device of to the subject, wherein the ingestible cell sampling device comprises: i) an abrasive sponge housed within a dissolvable capsule, the dissolvable capsule comprising an exterior surface exposed to an external environment, wherein the abrasive sponge is retained in a compressed state by the dissolvable capsule.; ii) a molded cap comprising a cap interior surface and a cap exterior surface; and iii) a string having a first end attached to the molded cap, wherein the string passes through a portion of the abrasive sponge, and b) withdrawing from the subject the abrasive sponge, wherein the abrasive sponge collects a cell sample from the subject during the withdrawing.
14. A method of characterizing an esophageal cell sample from a subject, comprising: a) preparing DNA from the esophageal cell sample from the subject; b) treating the DNA with bisulfite to produce bisulfite-treated DNA; and c) amplifying the bisulfite-treated DNA to determine an amount of at least one methylated biomarker gene selected from the group of methylation biomarker genes consisting of ANKRD13B, BMP3, CD1D, CDKN2A , CHST2, CNNM1, DIO3, DOCK2, DTX1, ELMO1, FER1L4, FERMT3, FLI1, GRIN2D, HUNK, JAM3, LRRC4, NDRG4, OPLAH, PDGFD, PKIA, PPP2R5C, QKI, SEP9, SFMBT2, SLC12A8, TBX15, TSPYL5, VAV3, ZNF304, ZNF568, ZNF671, and ZNF682; d) assaying the bisulfite-treated DNA from the esophageal cell sample for an amount of a reference DNA; and e) comparing the amount of the at least one methylated biomarker gene to the amount of the reference DNA to determine a methylation state for the at least one methylation biomarker gene in the esophageal cell sample.
15. The method of claim 14, comprising obtaining the esophageal cell sample from the subject by a method comprising: a) orally administering an abrasive sponge housed within a dissolvable capsule of a ingestible cell sampling device of to the subject, wherein the ingestible cell sampling device comprises: i) an abrasive sponge housed within a dissolvable capsule, the dissolvable capsule comprising an exterior surface exposed to an external environment, wherein the abrasive sponge is retained in a compressed state by the dissolvable capsule.; ii) a molded cap comprising a cap interior surface and a cap exterior surface; and iii) a string having a first end attached to the molded cap, wherein the string passes through a portion of the abrasive sponge and b) withdrawing from the subject the abrasive sponge, wherein the abrasive sponge collects an esophageal cell sample from the subject during the withdrawing.
16. The method of claim 14, wherein determining the methylation state for the at least one methylation biomarker gene in the esophageal cell sample comprises determining the methylation state of no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 methylation biomarker genes selected from the group consisting of ANKRD13B, BMP3, CD1D, CDKN2A , CHST2, CNNM1, DIO3, DOCK2, DTX1, ELMO1, FER1L4, FERMT3, FLI1, GRIN2D, HUNK, JAM3, LRRC4, NDRG4, OPLAH, PDGFD, PKIA, PPP2R5C, QKI, SEP9, SFMBT2, SLC12A8, TBX15, TSPYL5, VAV3, ZNF304, ZNF568, ZNF671, and ZNF682.
17. The method of claim 16, wherein the at least one methylated biomarker gene is selected from the group of methylation biomarker genes consisting of: ANKRD13B, CHST2, CNNM1, DOCK2, DTX1, FER1L4, FERMT3, FLI1, GRIN2D, JAM3, LRRC4, OPLAH, PDGFD, PKIA, PPP2R5C, QKI, SEP9, SFMBT2, SLC12A8, TBX15, TSPYL5, VAV3, ZNF304, ZNF568, and ZNF671.
18. The method of claim 16, wherein the at least one methylated biomarker gene is selected from the group of methylation biomarker genes consisting of: BMP3, NDRG4, VAV3, SFMBT2, DIO3, HUNK, ELMO1, CD1D, CDKN2A, and OPLAH.
19. The method of claim 14, wherein the reference DNA is methylated.
20. The method of claim 14, wherein determining the amount of the at least one methylated biomarker gene comprises using one or more methods selected from the group consisting of methylation-specific PCR, quantitative methylation-specific PCR, methylation-sensitive DNA restriction enzyme analysis, quantitative bisulfite pyrosequencing, flap endonuclease assay, PCR-flap assay, and bisulfite genomic sequencing PCR.
Description
DESCRIPTION OF THE DRAWINGS
[0182] These and other features, aspects, and advantages of the present technology will become better understood with regard to the following drawings:
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DETAILED DESCRIPTION OF THE INVENTION
[0203] The technology relates to cell sampling devices. In particular, the present invention relates to ingestible cell sampling devices and their use in methods for detecting various abnormalities in a subject. Provided herein is technology relating to selection and use of nucleic acid markers for use in assays for detection and quantification of DNA, e.g., methylated DNA. In particular, the technology relates to use of methylation assays to detect cancer in the digestive system, e.g., in the esophagus or the colon.
[0204] In this detailed description of the various embodiments, for purposes of explanation, numerous specific details are set forth to provide a thorough understanding of the embodiments disclosed. One skilled in the art will appreciate, however, that these various embodiments may be practiced with or without these specific details. In other instances, structures and devices are shown in block diagram form. Furthermore, one skilled in the art can readily appreciate that the specific sequences in which methods are presented and performed are illustrative and it is contemplated that the sequences can be varied and still remain within the spirit and scope of the various embodiments disclosed herein.
[0205] The ingestible cell sampling devices described herein are advantageous in that the devices provide improved safety for use in a subject. For example, the ingestible cell sampling devices described herein are designed to prevent detachment of the string from the molded cap, thus preventing loss of the device within a subject. As another example, the ingestible cell sampling devices described herein are designed to minimize the risk of the sponge detaching from the string during retrieval of the device, thus also preventing loss of the device within the subject. Furthermore, the devices described herein use materials that prevent laceration to the esophagus upon withdrawal of the device. The devices are easily swallowed by the subject, with a rapid dissolution of the capsule and expansion of the sponge, thus minimizing the total time required to collect an esophageal sample from the subject. Furthermore, the sponge comprises multiple features that enable for maximal surface area to capture sufficient tissue from a subject. Accordingly, described herein are ingestible cell sampling devices with maximal sampling capabilities having enhanced safety and tolerability for use in a subject.
[0206] In some embodiments, provided herein are ingestible cell sampling devices. The devices comprise an abrasive sponge housed within a dissolvable capsule, a molded cap, and a string attached to the molded cap.
[0207] The abrasive sponge may comprise any suitable material. Preferably, the material is capable of being compressed and retained in a compressed state by the dissolvable capsule. For example, the abrasive sponge may comprise a reticulated material. In some embodiments, the reticulated material comprises 10-35 pores per inch of material. For example, the reticulated material may comprise about 10, about 15, about 20, about 25, about 30, or about 35 pores per inch of material. The material may be any suitable porous, low-density material capable of collecting esophageal cells from a subject. For example, the abrasive sponge may comprise reticulated polyurethane.
[0208] The porosity of the abrasive sponge may be at least 80%. For example, the porosity may be at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.
[0209] The abrasive sponge may be of any suitable size and shape. The size and shape of the sponge may depend on the size of the capsule. In some embodiments, the abrasive sponge is of a suitable size and shape to allow for compression into a capsule suitable for oral administration (e.g. ingestion), and subsequent facile removal from the esophagus and throat of a subject after dissolution of the capsule and restoration of the sponge to its uncompressed size. For example, the sponge may be spherical in shape with a diameter of about 20-40 mm in an uncompressed state. For example, the sponge may be spherical in shape with a diameter of about 20 mm, about 25 mm, about 30 mm, about 35 mm, or about 40 mm in an uncompressed state. For example, the sponge may be spherical in shape with a diameter of 20 mm, 21 mm, 22 mm, 23 mm, 24 mm, 25 mm, 26 mm, 27 mm, 28 mm, 29 mm, or 30 mm in an uncompressed state.
[0210] The abrasive sponge may be compressed to a suitable size and housed within the dissolvable capsule in a compressed state. For example, the compressed sponge may have a diameter of about 1 mm to about 15 mm. For example, the compressed sponge may have a diameter of 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 11 mm, 12 mm, 13 mm, 14 mm, or 15 mm in a compressed state.
[0211] In some embodiments, the dissolution of the dissolvable capsule releases the abrasive sponge from compression and allows the abrasive sponge to expand. In some embodiments, the abrasive sponge expands to its uncompressed size following dissolution of the dissolvable capsule. In some embodiments, the abrasive sponge expands to substantially the same size as its original, uncompressed size prior to packaging within the capsule. As a nonlimiting example, the abrasive sponge may expand to within 10% of the original, uncompressed size following dissolution of the dissolvable capsule. For example, the sponge may expand to within 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the original, uncompressed size following dissolution of the dissolvable capsule. For example, the original, uncompressed size may be 30 mm and the sponge may expand to 27-30mm following dissolution of the capsule.
[0212] In some embodiments, the uncompressed sponge is uniform in shape. For example, the uncompressed sponge may be uniformly spherical in shape. In some embodiments, the uncompressed sponge may be spherical in shape with a protrusion that extends a portion of the abrasive sponge material into the dissolvable capsule. This protrusion is exemplified in
[0213] In some embodiments the abrasive sponge is formed to have concavities or indentations, or other external or internal spaces devoid of sponge material (“void spaces”), as may be provided by removing at least one portion of the abrasive sponge. As used herein in reference to a shape of an abrasive sponge, a “removed” portion of a sponge refers a void space in the shaped abrasive sponge, e.g., a portion that would be removed from a simple solid form, e.g., a sphere or cylinder, to produce the final shape comprising one or more void spaces. It is understood that an abrasive sponge may be manufactured in a final form that comprises such a void space, such that no sponge material need be physically “removed” during manufacture. In some embodiments, at least one portion of material may be removed from the center of the abrasive sponge. For example, the sponge may be spherical in shape and a portion of the material may be removed from the center of the abrasive sponge. For example, such embodiments are exemplified in
[0214] Any suitable sized portion may be removed from the sponge. For example, 5%, 10%, 15%, 20%, 25%, 30%, 35% 40%, 45%, 50%, or more of the material may be removed from the sponge. In some embodiments, removal of a portion of the material facilitates compression of the sponge into a suitable sized capsule for ingestion by a subject. In some embodiments, removal of a portion of the material from the sponge facilitates rapid expansion of the sponge following dissolution of the dissolvable capsule. In some embodiments, removal of a portion of the material from the sponge increases the surface area of the sponge available for collecting esophageal cells in the subject.
[0215] The abrasive sponge is housed within a dissolvable capsule. Accordingly, the abrasive sponge may be compressed into a cylindrical shape to fit into the dissolvable capsule. The dissolvable capsule may comprise any suitable material. For example, the dissolvable capsule may comprise gelatin, starch, or a cellulosic material as known in the art. In some embodiments, the dissolvable may be a vegan or vegetarian capsule (e.g., exclusive of all animal products, or free of products from certain types of animals; e.g. gelatin-free capsule).
[0216] The dissolvable capsule may be made of a suitable material. In some embodiments, the dissolvable capsule comprises a suitable material that dissolves within 10 minutes of entering the stomach cavity of a subject. For example, the dissolvable capsule may dissolve approximately within 10 minutes, within 9 minutes, within 8 minutes, within 7 minutes, within 6 minutes, within 5 minutes, within 4 minutes, within 3 minutes, within 2 minutes, or within 1 minute of exposure to the stomach cavity of a subject. Preferably, the dissolvable capsule dissolves within 5 minutes of exposure to the stomach cavity of the subject.
[0217] In some embodiments, the dissolvable capsule comprises a first closed end and a second closed end. For example, a dissolvable capsule comprising a first closed end and a second open end is shown in
[0218] In some embodiments, the dissolvable capsule comprises one or more openings, such that a portion of the abrasive sponge is exposed to the external environment at the one or more openings. The presence of one or more openings may facilitate a faster dissolution time of the capsule upon ingestion by a subject. In some embodiments, the dissolvable capsule comprises one opening. In some embodiments, the dissolvable capsule comprises two or more openings. Representative images of capsules containing one or more openings are shown in
[0219] The one or more openings may be any suitable size and shape that allow for exposure of the abrasive sponge to the external environment without substantially diminishing the ability of the capsule to retain the sponge in a compressed state. The one or more openings may be in any suitable location on the dissolvable capsule. For example, the dissolvable capsule may comprise one or more openings on a closed end of the capsule. As another example, the dissolvable capsule may comprise one or more openings on a cylindrical edge of the capsule.
[0220] The ingestible cell sampling device further comprises a molded cap. In some embodiments, the molded cap is hemispherical in shape. In some embodiments, the molded cap is cylindrical in shape (e.g., a button). In some embodiments, the molded cap is connected to the capsule. For example, the molded cap may be connected to the capsule by an adhesive. In some embodiments, the molded cap is connected to the abrasive sponge. For example, the molded cap may be connected to the abrasive sponge by an adhesive. In some embodiments, the cell sampling device may comprise a capsule comprising a first closed end and a second closed end, and a hemispherical molded cap may cover one of the closed ends of the capsule. For example, the molded cap may comprise an internal surface in contact with an external surface of one end of the capsule and an exterior surface in contact with the external environment (as exemplified in
[0221] In some embodiments, the molded cap comprises an interior surface in contact with the abrasive sponge and an exterior surface in contact with an internal surface of one end of the capsule. For example, the capsule may comprise a first closed end and a second closed end, and a hemispherical molded cap may comprise an interior surface in contact with the abrasive sponge and an exterior surface in contact with the internal surface of one closed end of the capsule (e.g., the molded cap is fitted within the capsule). In some embodiments, the capsule may comprise a first closed end and a second closed end, and a cylindrically shaped molded cap (e.g., a button) may be fitted inside the capsule. In such embodiments, the button may be fitted inside the capsule such that the rim of the molded cap is in contact with the capsule, the bottom surface of the molded cap is in contact with the abrasive sponge, and the top surface of the molded cap is not in direct contact with the interior surface of the capsule (as exemplified in
[0222] In embodiments wherein the interior surface of the molded cap is in contact with the abrasive sponge, the interior surface of the molded cap may be attached to the abrasive sponge. For example, the interior surface of the molded cap may be attached to the abrasive sponge by an adhesive.
[0223] In embodiments where a surface of the molded cap is in contact with the capsule, the molded cap may be attached to the capsule (e.g. by an adhesive).
[0224] The ingestible cell sampling device further comprises a string attached to the molded cap. The string may be attached to the molded cap by any suitable means, including but not limited to crimping, over-molding, adhesive, melting, wrapping, or taping. In some embodiments, the string is attached to the molded cap by a knot. Any suitable type of knot may be used. For example, the knot may be a hitch knot. The term “hitch knot” refers to a type of knot used to tie a string to an object or to another string. The term encompasses many distinct types of hitch knots, including an alternate ring hitching, anchor bend variant, bale sling hitch, barrel hitch, becket hitch, blackwall hitch, blake's hitch, boom hitch, bottom loaded release hitch, buntline hitch, cat's paw, chain hitch, clinging clara, clove hitch, continuous ring hitching, cow hitch variant, cow hitch with toggle, cow hitch, double half hitches, Farrimond friction hitch, garda hitch, ground-line hitch, half hitch, halter hitch, highpoint hitch, highwayman's hitch, hitching tie, icicle hitch, killick hitch, knute hitch, lighterman's hitch, magnus hitch, marline hitching, marlinespike hitch, masthead knot, midshipman's hitch, munter hitch, munter friction hitch, ossel hitch, palomar knot, pile hitch, prusik knot, reverse half hitches, round hitch, round turn and two half hitches, sailor's gripping hitch, sailor's hitch, siberian hitch, single hitch, slippery hitch, snell knot, snuggle hitch, taut-line hitch, timber hitch, trilene knot, trucker's hitch, tugboat hitch, uni knot, or a wagoner's hitch knot. In some embodiments, the hitch knot is a double overhand knot.
[0225] In some embodiments, the knot may be a binding knot. The term “binding knot” refers to a type of knot used to keep an object or multiple objects together, using a string that passes at least once around them. Suitable binding knots include, for example, a boa knot, a bottle sling, a bowline knot, a constrictor knot, a corned beef knot, a granny knot, a ground-line hitch, a Miller's knot, a Packer's knot, a reef knot, a strangle knot, a surgeon's knot, a thief knot, a jamming knot, a sheet bend, or a common whipping knot. The type of knot may be selected to allow for ease of manufacturing while also providing a stable means of connecting the string to the molded cap.
[0226] The molded cap may comprise any suitable feature to enable attachment of the string to the cap. For example, the molded cap may comprise two holes through which the string can be threaded and tied into a suitable knot. The string can be threaded through the first hole, pass through the external environment, and re-enter the interior of the capsule by passing through the second hole, before a knot can be tied on the interior of the capsule. As another example, the molded cap may comprise a bar on which the string can be secured (as shown in
[0227] The string may comprise any suitable material. For example, the string may be a suture material (e.g. surgical suture material). The suture material may be made from a variety of materials, including biological materials or synthetic materials. For example, the suture material may comprise synthetic materials such as nylon, polyester, PVDF, polypropylene, or combinations thereof.
[0228] The string should be of a suitable thickness to allow for facile ingestion by the subject without causing lacerations to the throat. In some embodiments, the string has a thickness of 0.3 mm to 0.7 mm. For example, the string may have a thickness of 0.3 mm, 0.35 mm, 0.4 mm, 0.45 mm, 0.5 mm, 0.55 mm, 0.6 mm, 0.65 mm, or 0.7 mm.
[0229] The string should be of a suitable length to allow for retrieval of the device after dissolution of the dissolvable capsule in the subject. Accordingly, the string should be long enough to allow for the abrasive sponge contained within the dissolvable capsule to reach the stomach cavity of the subject, while retaining enough string for the subject or a physician to be able to grip the string to initiate retrieval of the device. For example, the string may be at least 60 cm long. In some embodiments, the string may be 60cm to 80cm long. For example, the string may be 60 cm, 61 cm, 62 cm, 63 cm, 64 cm, 65 cm, 66 cm, 67 cm, 68c cm, 69 cm, 70 cm, 71 cm, 72 cm, 73 cm, 74 cm, 75 cm, 76 cm, 77 cm, 78 cm, 79 cm, or 80 cm long.
[0230] In some embodiments, the string may comprise markings on the string to judge the amount of string that has been swallowed. Such markings would assist in determining that the dissolvable capsule has traveled to the desired area (e.g. the stomach cavity of the subject). The markings may be spaced any suitable distance apart. For example, the markings may be spaced 1-80 cm apart. For example, the markings may be placed about 1 cm, about 5 cm, about 10 cm, about 15 cm, about 20 cm, about 25 cm, about 30 cm, about 35 cm, or about 40 cm apart.
[0231] The string should have a suitable tensile strength to minimize the risk of the string breaking during ingestion and/or retrieval of the device. For example, the string should have a suitable tensile strength to allow for the string to be pulled to retrieve the device from the subject after dissolution of the dissolvable capsule. In some embodiments, the string may comprise a handle or a grip to facilitate retrieval of the device and/or prevent swallowing of the entire string. For example, the string may comprise a handle or a grip on the end of the string that does not contain the capsule. The handle or grip may be of any suitable size and shape to facilitate retrieval and prevent swallowing of the string. The handle or grip may be an open shape (e.g. bar shape, T-shape, X-shape, etc.) or a closed shape (e.g. circular or semi-circular shape, rectangular shape, triangular shape, etc.), and may be formed from the same material as the string (e.g., may be a loop or knot in the string) or may comprise different material (e.g., plastic, metal, etc.).
[0232] In some embodiments, the string passes through a portion of the abrasive sponge. Accordingly, passing the string through the sponge will help secure the sponge to the molded cap, such that the sponge is not lost within the subject after dissolution of the dissolvable capsule. In some embodiments, the string passes through at least one surface of the dissolvable capsule. For example, the string may pass through the first closed end of the dissolvable capsule, through the abrasive sponge, and then attach to the molded cap. In some embodiments, the string may pass through the first closed end of the dissolvable capsule, through the abrasive sponge, through the second closed end of the dissolvable capsule, and then attach to the molded cap.
[0233] Further described herein are methods for collecting cells from a subject. The methods comprise providing an ingestible cell sampling device described herein to the subject. Suitable methods for providing an ingestible cell sampling device to a subject are described in U.S. Pat. No. 4,735,214, U.S. Pat. No. 10,327,742, and U.S. Pat. No. 10,292,687, each of which are incorporated herein by reference in their entireties. For example, the subject may swallow the ingestible cell sampling device described herein and a suitable amount of time may pass prior to retrieving the device from the subject. For example, the subject may swallow the ingestible cell sampling device and 10 minutes or less may be allowed to pass prior to retrieval. For example, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, or 1 minute may pass prior to retrieval. Retrieval may comprise having the subject, physician, or otherwise suitable person grab and pull on the string at a suitable rate to allow for comfortable retrieval of the device from the subject. Esophageal cells may be harvested from the abrasive sponge by any suitable means and subsequently analyzed to determine whether one or more abnormalities are present in the subject. In some embodiments, the esophageal cells may be harvested and placed in a suitable stabilization buffer prior to analysis. For example, a stabilization buffer may comprise any suitable agent or combination of agents that prevent unwanted damage to the cells (e.g. cell lysis) or damage/degradation of the nucleic acids (e.g. DNA or RNA) contained within the cell sample.
[0234] In some embodiments, esophageal cells are harvested from the abrasive sponge and analyzed to determine whether an esophageal disorder is present in the subject. Analysis may be performed by any suitable method, including protein-based tests, tissue/cell examinations (e.g., microscopy or other visual inspections), and/or nucleic acid detection assays. For example, analysis may be performed by protein-based techniques to analyze one or more biomarkers of interest. Protein-based techniques include, for example, immunohistochemistry, ELISA, western blot, flow cytometry, fluorescent in-situ hybridization (FISH), fluorescence analysis of cell sorting (FACS), mass spectrometry, etc. For example, protein-based techniques may be performed using one or more antibodies against at least one biomarker protein of interest. The biomarker protein(s) may be detected using an antibody capable of reacting with the protein(s), and subsequent visualization of the antibody. The antibody may be a polyclonal antibody or a monoclonal antibody. The use of secondary, tertiary or further antibodies may advantageously employed in order to amplify the signal and facilitate detection.
[0235] In some embodiments, esophageal cells are harvested from the abrasive sponge and examined to determine whether an esophageal disorder is present in the subject. For example, cells may be harvested from the sponge, plated on an appropriate medium, and examined by microscopy or other visual examination to determine whether characteristics indicative of an esophageal disorder are present in the cells. In some embodiments, cells may be harvested from the sponge, plated, and inspected using a microscope to determine whether one or more cancer cells are present. In some embodiments, diagnosis of an esophageal disorder may be made by visualization of a specific cell type, such as a columnar cell, which may be indicative of gastroesophageal reflux disease or complications thereof, including Barrett's Esophagus or esophageal adenocarcinoma.
[0236] In some embodiments, esophageal cells are harvested from the abrasive sponge and one or more nucleic acid detection assays are performed to determine whether an esophageal disorder is present in the subject. For example, esophageal cells may be harvested from the abrasive sponge following use in a subject, and the cells may be analyzed by one or more nucleic acid detection assays to detect levels of one or more biomarkers of an esophageal disorder. Suitable methods (e.g. nucleic acid detection assays) and biomarkers for detecting esophageal disorders are described in U.S. patent application Ser. No. 15/881,409 of Allawi, et al., filed Jan. 26, 2018, (including, e.g., ANKRD13B, CHST2, CNNM1, DOCK2, DTX1, FER1L4, FERMT3, FLI1, GRIN2D, JAM3, LRRC4, OPLAH, PDGFD, PKIA, PPP2R5C, QKI, SEP9, SFMBT2, SLC12A8, TBX15, TSPYL5, VAV3, ZNF304, ZNF568, and ZNF671), and U.S. Pat. No. 10,435,755, (including, e.g., BMP3, NDRG4, VAV3, SFMBT2, D103, HUNK, ELMO1, CD1D, CDKN2A; and OPLAH), both of which are incorporated herein by reference in their entireties.
[0237] Biomarkers selected from this group may comprise 1 biomarker, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 biomarkers, alone or in any combination or subcombination, without limitation. For example, in certain embodiments, the biomarker or group of biomarkers is selected from the group consisting of ANKRD13B, CHST2, CNNM1, DOCK2, DTX1, FER1L4, FERMT3, FLI1, GRIN2D, JAM3, LRRC4, OPLAH, PDGFD, PKIA, PPP2R5C, QKI, SEP9, SFMBT2, SLC12A8, TBX15, TSPYL5, VAV3, ZNF304, ZNF568, and ZNF671, while in some embodiments, the biomarker or group of biomarkers is selected from the group consisting of BMP3, NDRG4, VAV3, SFMBT2, D103, HUNK, ELMO1, CD1D, CDKN2A, and OPLAH.
[0238] In some embodiments, analysis of target DNAs comprises analysis of multiple different DNAs in a single reaction. Typical instrumentation for real-time detection of amplification reactions allows for simultaneous detection and quantification of only 3-5 fluorescent dyes. This is mainly because spectral overlap between fluorophores makes it difficult to distinguish one dye from another when the many dyes with overlap excitation and/or emission spectra are used together. When detection of a specific disease from a biological specimen requires a panel comprising more than about 5 different markers, this presents a challenge, especially when the size of the sample is limited and the markers are present in low levels, a situation often requiring use of the entirety of a sample in a single amplification run.
[0239] In some embodiments, methods described herein allow for detection of multiple different markers in the same sample by having each sample produce a result from the same dye. In the embodiment described in detail herein, multiplexed flap cleavage assays (e.g., QuARTS flap endonuclease assays) for multiple different markers produce initial cleavage products that use the same FRET cassette to produce fluorescent signal.
[0240] In preferred embodiments, the combined assay comprises several different probe oligonucleotides that each have a portion that hybridizes to a different target nucleic acid, but that all have essentially the same 5′ arm sequence. Cleavage of the probes in the presence of their respective target nucleic acids all release the same 5′ arm, and all of the released arms then combine with FRET cassettes having the same flap-binding sequence and the same dye to produce fluorescence signal by endonuclease cleavage of the FRET cassette. In other embodiments, the probes for different targets may have different flap arms that report to different FRET cassettes, wherein the different FRET cassettes all use the same reporter fluorophore.
[0241] Combining assays in this manner has multiple advantages. For example, a sample can provide a result if any one of the target sequences associated with a condition (e.g., a disease state, such as colorectal cancer) is detected in the assay, without the need to divide the sample into multiple different assays, Further, if more than one of the target sequences provides such a result, aggregation of these signals into a single dye channel may provide a stronger signal over background, providing more certainty for the assay result. During development of the methods described herein, it was surprisingly found that combining a large number of primers and flap assay probes for detecting multiple different target sequences, along with a shared FRET cassette, in a single amplification plus flap cleavage assay reaction did not increase background signal in no-target controls or in negative samples.
[0242] In some embodiments, different target sequences reporting to a single FRET cassette and single dye channel may not be from different marker genes or regions, but may be from different regions within a single marker (e.g., a single methylation marker gene). As described in Example 4, configuring assays to detect multiple regions of a single marker gene in an assay where all the regions report to a single dye, e.g., via a single FRET cassette, boosts the level of detectable signal from the copies of the target gene present in the reaction.
[0243] In yet other embodiments, the different target sequences to be detected may be a mixture of multiple regions of one marker, along with one or more regions of a different marker or markers. The different target sequences may comprise any combination of methylation markers, mutation markers, deletions, insertions, or any other manner of nucleic acid variants detectable in an assay such as a QuARTS amplification/flap cleavage assay.
[0244] In some embodiments, a marker is a region of 100 or fewer bases, the marker is a region of 500 or fewer bases, the marker is a region of 1000 or fewer bases, the marker is a region of 5000 or fewer bases, or, in some embodiments, the marker is one base. In some embodiments the marker is in a high CpG density promoter.
[0245] The technology is not limited by sample type. For example, in some embodiments the sample is a stool sample, a tissue sample, sputum, a blood sample (e.g., plasma, serum, whole blood), an excretion, or a urine sample.
[0246] Furthermore, the technology is not limited in the method used to determine methylation state. In some embodiments the assaying comprises using methylation specific polymerase chain reaction, nucleic acid sequencing, mass spectrometry, chip or array hybridization, methylation specific nuclease, mass-based separation, or target capture. In some embodiments, the assaying comprises use of a methylation specific oligonucleotide. In some embodiments, the technology uses massively parallel sequencing (e.g., next-generation sequencing) to determine methylation state, e.g., sequencing-by-synthesis, real-time (e.g., single-molecule) sequencing, bead emulsion sequencing, nanopore sequencing, etc.
[0247] The technology provides reagents for detecting a differentially methylated region (DMR). In some embodiments are provided an oligonucleotide comprising a sequence complementary to a chromosomal region having Kit embodiments are provided, e.g., a kit comprising a bisulfite reagent; and a control nucleic acid comprising a chromosomal region having an annotation selected from VAV3; ZNF671; CHST2; FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; GRIN2D, and QKI and having a methylation state associated with a subject who does not have a cancer (e.g., colon cancer). In some embodiments, kits comprise a bisulfite reagent and an oligonucleotide as described herein. In some embodiments, kits comprise a bisulfite reagent; and a control nucleic acid comprising a sequence from such a chromosomal region and having a methylation state associated with a subject who has colon cancer.
[0248] The technology is related to embodiments of compositions (e.g., reaction mixtures). In some embodiments are provided a composition comprising a nucleic acid comprising a chromosomal region having an annotation selected from VAV3; ZNF671; CHST2; FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; GRIN2D, and QKJ and a bisulfite reagent. Some embodiments provide a composition comprising a nucleic acid comprising a chromosomal region having an annotation selected from VAV3; ZNF671; CHST2; FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; GRIN2D, and QKI and an oligonucleotide as described herein. Some embodiments provide a composition comprising a nucleic acid comprising a chromosomal region having an annotation selected from VAV3; ZNF671; CHST2; FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; GRIN2D, and QKJ and a methylation-sensitive restriction enzyme. Some embodiments provide a composition comprising a nucleic acid comprising a chromosomal region having an annotation selected from VAV3; ZNF671; CHST2; FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; GRIN2D, and QKJ and a polymerase.
[0249] Additional related method embodiments are provided for screening for a neoplasm (e.g., colon carcinoma) in a sample obtained from a subject, e.g., a method comprising determining a methylation state of a marker in the sample comprising a base in a chromosomal region having an annotation selected from VAV3; ZNF671; CHST2; FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; GRIN2D, and QKI; comparing the methylation state of the marker from the subject sample to a methylation state of the marker from a normal control sample from a subject who does not have colon cancer; and determining a confidence interval and/or a p value of the difference in the methylation state of the subject sample and the normal control sample. In some embodiments, the confidence interval is 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9% or 99.99% and the p value is 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, or 0.0001. Some embodiments of methods provide steps of reacting a nucleic acid comprising a chromosomal region having an annotation selected from VAV3; ZNF671; CHST2; FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; GRIN2D, and QKI with a bisulfite reagent to produce a bisulfite-reacted nucleic acid; sequencing the bisulfite-reacted nucleic acid to provide a nucleotide sequence of the bisulfite-reacted nucleic acid; comparing the nucleotide sequence of the bisulfite-reacted nucleic acid with a nucleotide sequence of a nucleic acid comprising the chromosomal region from a subject who does not have colon cancer to identify differences in the two sequences; and identifying the subject as having a neoplasm when a difference is present.
[0250] Systems for screening for colon cancer in a sample obtained from a subject are provided by the technology. Exemplary embodiments of systems include, e.g., a system for screening for colon cancer in a sample obtained from a subject, the system comprising an analysis component configured to determine the methylation state of a sample, a software component configured to compare the methylation state of the sample with a control sample or a reference sample methylation state recorded in a database, and an alert component configured to alert a user of a cancer-associated methylation state. An alert is determined in some embodiments by a software component that receives the results from multiple assays (e.g., determining the methylation states of multiple markers, e.g., a chromosomal region having an annotation selected from VAV3; ZNF671; CHST2; FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; GRIN2D, and QKI and calculating a value or result to report based on the multiple results. Some embodiments provide a database of weighted parameters associated with each chromosomal region having an annotation selected from VAV3; ZNF671; CHST2; FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; GRIN2D, and QKI provided herein for use in calculating a value or result and/or an alert to report to a user (e.g., such as a physician, nurse, clinician, etc.). In some embodiments all results from multiple assays are reported and in some embodiments one or more results are used to provide a score, value, or result based on a composite of one or more results from multiple assays that is indicative of a colon cancer risk in a subject.
[0251] In some embodiments of systems, a sample comprises a nucleic acid comprising a chromosomal region having an annotation selected from VAV3; ZNF671; CHST2; FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; GRIN2D, and QKJ. In some embodiments the system further comprises a component for isolating a nucleic acid, a component for collecting a sample such as a component for collecting a stool sample. In some embodiments, the system comprises nucleic acid sequences comprising a chromosomal region having an annotation selected from VAV3; ZNF671; CHST2; FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; GRIN2D, and QKL In some embodiments the database comprises nucleic acid sequences from subjects who do not have colon cancer. Also provided are nucleic acids, e.g., a set of nucleic acids, each nucleic acid having a sequence comprising a chromosomal region having an annotation selected from VAV3; ZNF671; CHST2; FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; GRIN2D, and QKJ.
[0252] Related system embodiments comprise a set of nucleic acids as described and a database of nucleic acid sequences associated with the set of nucleic acids. Some embodiments further comprise a bisulfite reagent and some embodiments further comprise a nucleic acid sequencer.
[0253] In certain embodiments, methods for characterizing a sample obtained from a human subject are provided, comprising a) obtaining a sample from a human subject; b) assaying a methylation state of one or more markers in the sample, wherein the marker comprises a base in a chromosomal region having an annotation selected from the following groups of markers: VAV3; ZNF671; CHST2; FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; GRIN2D, and QKJ; and c) comparing the methylation state of the assayed marker to the methylation state of the marker assayed in a subject that does not have a neoplasm.
[0254] In some embodiments, the technology is related to assessing the presence of and methylation state of one or more of the markers identified herein in a biological sample. These markers comprise one or more differentially methylated regions (DMR) as discussed herein. Methylation state is assessed in embodiments of the technology. As such, the technology provided herein is not restricted in the method by which a gene's methylation state is measured. For example, in some embodiments the methylation state is measured by a genome scanning method. For example, one method involves restriction landmark genomic scanning (Kawai et al. (1994) Mol. Cell. Biol. 14: 7421-7427) and another example involves methylation-sensitive arbitrarily primed PCR (Gonzalgo et al. (1997) Cancer Res. 57: 594-599). In some embodiments, changes in methylation patterns at specific CpG sites are monitored by digestion of genomic DNA with methylation-sensitive restriction enzymes followed by Southern analysis of the regions of interest (digestion-Southern method). In some embodiments, analyzing changes in methylation patterns involves a PCR-based process that involves digestion of genomic DNA with methylation-sensitive restriction enzymes prior to PCR amplification (Singer-Sam et al. (1990) Nucl. Acids Res. 18: 687). In addition, other techniques have been reported that utilize bisulfite treatment of DNA as a starting point for methylation analysis. These include methylation-specific PCR (MSP) (Herman et al. (1992) Proc. Natl. Acad. Sci. USA 93: 9821-9826) and restriction enzyme digestion of PCR products amplified from bisulfite-converted DNA (Sadri and Hornsby (1996) Nucl. Acids Res. 24: 5058-5059; and Xiong and Laird (1997) Nucl. Acids Res. 25: 2532-2534). PCR techniques have been developed for detection of gene mutations (Kuppuswamy et al. (1991) Proc. Natl. Acad. Sci. USA 88: 1143-1147) and quantification of allelic-specific expression (Szabo and Mann (1995) Genes Dev. 9: 3097-3108; and Singer-Sam et al. (1992) PCR Methods Appl. 1: 160-163). Such techniques use internal primers, which anneal to a PCR-generated template and terminate immediately 5′ of the single nucleotide to be assayed. Methods using a “quantitative Ms-SNuPE assay” as described in U.S. Pat. No. 7,037,650 are used in some embodiments.
[0255] Upon evaluating a methylation state, the methylation state is often expressed as the fraction or percentage of individual strands of DNA that is methylated at a particular site (e.g., at a single nucleotide, at a particular region or locus, at a longer sequence of interest, e.g., up to a ˜100-bp, 200-bp, 500-bp, 1000-bp subsequence of a DNA or longer) relative to the total population of DNA in the sample comprising that particular site. Traditionally, the amount of the unmethylated nucleic acid is determined by PCR using calibrators. Then, a known amount of DNA is bisulfite treated and the resulting methylation-specific sequence is determined using either a real-time PCR or other exponential amplification, e.g., a QuARTS assay (e.g., as provided by U.S. Pat. Nos. 8,361,720; 8,715,937; 8,916,344; and 9,212,392).
[0256] For example, in some embodiments methods comprise generating a standard curve for the unmethylated target by using external standards. The standard curve is constructed from at least two points and relates the real-time Ct value for unmethylated DNA to known quantitative standards. Then, a second standard curve for the methylated target is constructed from at least two points and external standards. This second standard curve relates the Ct for methylated DNA to known quantitative standards. Next, the test sample Ct values are determined for the methylated and unmethylated populations and the genomic equivalents of DNA are calculated from the standard curves produced by the first two steps. The percentage of methylation at the site of interest is calculated from the amount of methylated DNAs relative to the total amount of DNAs in the population, e.g., (number of methylated DNAs)/(the number of methylated DNAs+number of unmethylated DNAs)×100.
[0257] Also provided herein are compositions and kits for practicing the methods. For example, in some embodiments, reagents (e.g., primers, probes) specific for one or more markers are provided alone or in sets (e.g., sets of primer pairs for amplifying a plurality of markers). Additional reagents for conducting a detection assay may also be provided (e.g., enzymes, buffers, positive and negative controls for conducting QuARTS, PCR, sequencing, bisulfite, or other assays). In some embodiments, the kits containing one or more reagents necessary, sufficient, or useful for conducting a method are provided. Also provided are reactions mixtures containing the reagents. Further provided are master mix reagent sets containing a plurality of reagents that may be added to each other and/or to a test sample to complete a reaction mixture.
[0258] Methods for isolating DNA suitable for these assay technologies are known in the art. In particular, some embodiments comprise isolation of nucleic acids as described in U.S. Pat. 9,000,146, which is incorporated herein by reference in its entirety.
[0259] Genomic DNA may be isolated by any means, including the use of commercially available kits. Briefly, wherein the DNA of interest is encapsulated by a cellular membrane the biological sample must be disrupted and lysed by enzymatic, chemical, or mechanical means. The DNA solution may then be cleared of proteins and other contaminants, e.g., by digestion with proteinase K. The genomic DNA is then recovered from the solution. This may be carried out by means of a variety of methods including salting out, organic extraction, or binding of the DNA to a solid phase support. The choice of method will be affected by several factors including time, expense, and required quantity of DNA. All clinical sample types comprising neoplastic matter or pre-neoplastic matter are suitable for use in the present method, e.g., cell lines, histological slides, biopsies, paraffin-embedded tissue, body fluids, stool, colonic effluent, urine, blood plasma, blood serum, whole blood, isolated blood cells, cells isolated from the blood, and combinations thereof.
[0260] The technology is not limited in the methods used to prepare the samples and provide a nucleic acid for testing. For example, in some embodiments, a DNA is isolated from a stool sample or from blood or from a plasma sample using direct gene capture, e.g., as detailed in U.S. Pat. Nos. 8,808,990 or 9,000,146, or by a related method. In some embodiments, a DNA is isolated from cells collected using an ingestible cell sampling device.
I. Methylation Assays to Detect Colon Cancer
[0261] Candidate methylated DNA markers were identified by unbiased whole methylome sequencing of selected colon cancer case and colon control tissues. The top marker candidates were further evaluated in 89 cancer and 95 normal plasma samples. DNA extracted from patient tissue samples was bisulfite treated and then candidate markers and reference genes (e.g., (3-actin or B3GALT6) as a normalizing genes were assayed by Quantitative Allele-Specific Real-time Target and Signal amplification (QuARTS amplification). QuARTS assay chemistry yields high discrimination for methylated marker selection and screening.
[0262] On receiver operator characteristics analyses of individual marker candidates, areas under the curve (AUCs) ranged from 0.63 to 0.75. At 92.6% specificity, a combined panel of 12 methylation markers (VAV3; ZNF671; CHST2; FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; GRIN2D, and QKI) plus an assay for the CEA protein yielded a sensitivity of 67.4% across all stages of colon cancer.
II. Methylation Detection Assays and Kits
[0263] The markers described herein find use in a variety of methylation detection assays. The most frequently used method for analyzing a nucleic acid for the presence of 5-methylcytosine is based upon the bisulfite method described by Frommer, et al. for the detection of 5-methylcytosines in DNA (Frommer et al. (1992) Proc. Natl. Acad. Sci. USA 89: 1827-31 explicitly incorporated herein by reference in its entirety for all purposes) or variations thereof. The bisulfite method of mapping 5-methylcytosines is based on the observation that cytosine, but not 5-methylcytosine, reacts with hydrogen sulfite ion (also known as bisulfite). The reaction is usually performed according to the following steps: first, cytosine reacts with hydrogen sulfite to form a sulfonated cytosine. Next, spontaneous deamination of the sulfonated reaction intermediate results in a sulfonated uracil. Finally, the sulfonated uracil is desulfonated under alkaline conditions to form uracil. Detection is possible because uracil base pairs with adenine (thus behaving like thymine), whereas 5-methylcytosine base pairs with guanine (thus behaving like cytosine). This makes the discrimination of methylated cytosines from non-methylated cytosines possible by, e.g., bisulfite genomic sequencing (Grigg G, & Clark S, Bioessays (1994) 16: 431-36; Grigg G, DNA Seq. (1996) 6: 189-98),methylation-specific PCR (MSP) as is disclosed, e.g., in U.S. Pat. No. 5,786,146, or using an assay comprising sequence-specific probe cleavage, e.g., a QuARTS flap endonuclease assay (see, e.g., Zou et al. (2010) “Sensitive quantification of methylated markers with a novel methylation specific technology” Clin Chem 56: A199; and in U.S. Pat. Nos. 8,361,720; 8,715,937; 8,916,344; and 9,212,392.
[0264] Some conventional technologies are related to methods comprising enclosing the DNA to be analyzed in an agarose matrix, thereby preventing the diffusion and renaturation of the DNA (bisulfite only reacts with single-stranded DNA), and replacing precipitation and purification steps with a fast dialysis (Olek A, et al. (1996) “A modified and improved method for bisulfite-based cytosine methylation analysis” Nucleic Acids Res. 24: 5064-6). It is thus possible to analyze individual cells for methylation status, illustrating the utility and sensitivity of the method. An overview of conventional methods for detecting 5-methylcytosine is provided by Rein, T., et al. (1998) Nucleic Acids Res. 26: 2255.
[0265] The bisulfite technique typically involves amplifying short, specific fragments of a known nucleic acid subsequent to a bisulfite treatment, then either assaying the product by sequencing (Olek & Walter (1997) Nat. Genet. 17: 275-6) or a primer extension reaction (Gonzalgo & Jones (1997) Nucleic Acids Res. 25: 2529-31; WO 95/00669; U.S. Pat. No. 6,251,594) to analyze individual cytosine positions. Some methods use enzymatic digestion (Xiong & Laird (1997) Nucleic Acids Res. 25: 2532-4). Detection by hybridization has also been described in the art (Olek et al., WO 99/28498). Additionally, use of the bisulfite technique for methylation detection with respect to individual genes has been described (Grigg & Clark (1994) Bioessays 16: 431-6,; Zeschnigk et al. (1997) Hum Mol Genet. 6: 387-95; Feil et al. (1994) Nucleic Acids Res. 22: 695; Martin et al. (1995) Gene 157: 261-4; WO 9746705; WO 9515373).
[0266] Various methylation assay procedures can be used in conjunction with bisulfite treatment according to the present technology. These assays allow for determination of the methylation state of one or a plurality of CpG dinucleotides (e.g., CpG islands) within a nucleic acid sequence. Such assays involve, among other techniques, sequencing of bisulfite-treated nucleic acid, PCR (for sequence-specific amplification), Southern blot analysis, and use of methylation-sensitive restriction enzymes.
[0267] For example, genomic sequencing has been simplified for analysis of methylation patterns and 5-methylcytosine distributions by using bisulfite treatment (Frommer et al. (1992) Proc. Natl. Acad. Sci. USA 89: 1827-1831). Additionally, restriction enzyme digestion of PCR products amplified from bisulfite-converted DNA finds use in assessing methylation state, e.g., as described by Sadri & Hornsby (1997) Nucl. Acids Res. 24: 5058-5059 or as embodied in the method known as COBRA (Combined Bisulfite Restriction Analysis) (Xiong & Laird (1997) Nucleic Acids Res. 25: 2532-2534).
[0268] COBRA™ analysis is a quantitative methylation assay useful for determining DNA methylation levels at specific loci in small amounts of genomic DNA (Xiong & Laird, Nucleic
[0269] Acids Res. 25:2532-2534, 1997). Briefly, restriction enzyme digestion is used to reveal methylation-dependent sequence differences in PCR products of sodium bisulfite-treated DNA. Methylation-dependent sequence differences are first introduced into the genomic DNA by standard bisulfite treatment according to the procedure described by Frommer et al. (Proc. Natl. Acad. Sci. USA 89:1827-1831, 1992). PCR amplification of the bisulfite converted DNA is then performed using primers specific for the CpG islands of interest, followed by restriction endonuclease digestion, gel electrophoresis, and detection using specific, labeled hybridization probes. Methylation levels in the original DNA sample are represented by the relative amounts of digested and undigested PCR product in a linearly quantitative fashion across a wide spectrum of DNA methylation levels. In addition, this technique can be reliably applied to DNA obtained from microdissected paraffin-embedded tissue samples.
[0270] Typical reagents (e.g., as might be found in a typical COBRA™-based kit) for COBRA™ analysis may include, but are not limited to: PCR primers for specific loci (e.g., specific genes, markers, regions of genes, regions of markers, bisulfite treated DNA sequence, CpG island, etc.); restriction enzyme and appropriate buffer; gene-hybridization oligonucleotide; control hybridization oligonucleotide; kinase labeling kit for oligonucleotide probe; and labeled nucleotides. Additionally, bisulfite conversion reagents may include: DNA denaturation buffer; sulfonation buffer; DNA recovery reagents or kits (e.g., precipitation, ultrafiltration, affinity column); desulfonation buffer; and DNA recovery components.
[0271] Assays such as “MethyLight™” (a fluorescence-based real-time PCR technique) (Eads et al., Cancer Res. 59:2302-2306, 1999), Ms-SNuPE™ (Methylation-sensitive Single Nucleotide Primer Extension) reactions (Gonzalgo & Jones, Nucleic Acids Res. 25:2529-2531, 1997), methylation-specific PCR (“MSP”; Herman et al., Proc. Natl. Acad. Sci. USA 93:9821-9826, 1996; U.S. Pat. No. 5,786,146), and methylated CpG island amplification (“MCA”; Toyota et al., Cancer Res. 59:2307-12, 1999) are used alone or in combination with one or more of these methods.
[0272] The “HeavyMethyl™” assay, technique is a quantitative method for assessing methylation differences based on methylation-specific amplification of bisulfite-treated DNA. Methylation-specific blocking probes (“blockers”) covering CpG positions between, or covered by, the amplification primers enable methylation-specific selective amplification of a nucleic acid sample.
[0273] The term “HeavyMethyl™ MethyLight™” assay refers to a HeavyMethyl™ MethyLight™ assay, which is a variation of the MethyLight™ assay, wherein the MethyLight™ assay is combined with methylation specific blocking probes covering CpG positions between the amplification primers. The HeavyMethyl™ assay may also be used in combination with methylation specific amplification primers.
[0274] Typical reagents (e.g., as might be found in a typical MethyLight™-based kit) for HeavyMethyl™ analysis may include, but are not limited to: PCR primers for specific loci (e.g., specific genes, markers, regions of genes, regions of markers, bisulfite treated DNA sequence, CpG island, or bisulfite treated DNA sequence or CpG island, etc.); blocking oligonucleotides; optimized PCR buffers and deoxynucleotides; and Taq polymerase.
[0275] MSP (methylation-specific PCR) allows for assessing the methylation status of virtually any group of CpG sites within a CpG island, independent of the use of methylation-sensitive restriction enzymes (Herman et al. Proc. Natl. Acad. Sci. USA 93:9821-9826, 1996; U.S. Pat. No. 5,786,146). Briefly, DNA is modified by sodium bisulfite, which converts unmethylated, but not methylated cytosines, to uracil, and the products are subsequently amplified with primers specific for methylated versus unmethylated DNA. MSP requires only small quantities of DNA, is sensitive to 0.1% methylated alleles of a given CpG island locus, and can be performed on DNA extracted from paraffin-embedded samples. Typical reagents (e.g., as might be found in a typical MSP-based kit) for MSP analysis may include, but are not limited to: methylated and unmethylated PCR primers for specific loci (e.g., specific genes, markers, regions of genes, regions of markers, bisulfite treated DNA sequence, CpG island, etc.); optimized PCR buffers and deoxynucleotides, and specific probes.
[0276] The MethyLight™ assay is a high-throughput quantitative methylation assay that utilizes fluorescence-based real-time PCR (e.g., TaqMan®) that requires no further manipulations after the PCR step (Eads et al., Cancer Res. 59:2302-2306, 1999). Briefly, the MethyLight™ process begins with a mixed sample of genomic DNA that is converted, in a sodium bisulfite reaction, to a mixed pool of methylation-dependent sequence differences according to standard procedures (the bisulfite process converts unmethylated cytosine residues to uracil). Fluorescence-based PCR is then performed in a “biased” reaction, e.g., with PCR primers that overlap known CpG dinucleotides. Sequence discrimination occurs both at the level of the amplification process and at the level of the fluorescence detection process.
[0277] The MethyLight™ assay is used as a quantitative test for methylation patterns in a nucleic acid, e.g., a genomic DNA sample, wherein sequence discrimination occurs at the level of probe hybridization. In a quantitative version, the PCR reaction provides for a methylation specific amplification in the presence of a fluorescent probe that overlaps a particular putative methylation site. An unbiased control for the amount of input DNA is provided by a reaction in which neither the primers, nor the probe, overlie any CpG dinucleotides. Alternatively, a qualitative test for genomic methylation is achieved by probing the biased PCR pool with either control oligonucleotides that do not cover known methylation sites (e.g., a fluorescence-based version of the HeavyMethyl™ and MSP techniques) or with oligonucleotides covering potential methylation sites.
[0278] The MethyLight™ process is used with any suitable probe (e.g. a “TaqMan®” probe, a Lightcycler® probe, etc.) For example, in some applications double-stranded genomic DNA is treated with sodium bisulfite and subjected to one of two sets of PCR reactions using TaqMan® probes, e.g., with MSP primers and/or HeavyMethyl blocker oligonucleotides and a TaqMan® probe. The TaqMan® probe is dual-labeled with fluorescent “reporter” and “quencher” molecules and is designed to be specific for a relatively high GC content region so that it melts at about a 10° C. higher temperature in the PCR cycle than the forward or reverse primers. This allows the TaqMan® probe to remain fully hybridized during the PCR annealing/extension step.
[0279] As the Taq polymerase enzymatically synthesizes a new strand during PCR, it will eventually reach the annealed TaqMan® probe. The Taq polymerase 5′ to 3′ endonuclease activity will then displace the TaqMan® probe by digesting it to release the fluorescent reporter molecule for quantitative detection of its now unquenched signal using a real-time fluorescent detection system.
[0280] Typical reagents (e.g., as might be found in a typical MethyLight™-based kit) for MethyLight™ analysis may include, but are not limited to: PCR primers for specific loci (e.g., specific genes, markers, regions of genes, regions of markers, bisulfite treated DNA sequence, CpG island, etc.); TaqMan® or Lightcycler® probes; optimized PCR buffers and deoxynucleotides; and Taq polymerase.
[0281] The QM™ (quantitative methylation) assay is an alternative quantitative test for methylation patterns in genomic DNA samples, wherein sequence discrimination occurs at the level of probe hybridization. In this quantitative version, the PCR reaction provides for unbiased amplification in the presence of a fluorescent probe that overlaps a particular putative methylation site. An unbiased control for the amount of input DNA is provided by a reaction in which neither the primers, nor the probe, overlie any CpG dinucleotides. Alternatively, a qualitative test for genomic methylation is achieved by probing the biased PCR pool with either control oligonucleotides that do not cover known methylation sites (a fluorescence-based version of the HeavyMethyl™ and MSP techniques) or with oligonucleotides covering potential methylation sites.
[0282] The QM™ process can be used with any suitable probe, e.g., “TaqMan®” probes, Lightcycler® probes, in the amplification process. For example, double-stranded genomic DNA is treated with sodium bisulfite and subjected to unbiased primers and the TaqMan® probe. The TaqMan® probe is dual-labeled with fluorescent “reporter” and “quencher” molecules, and is designed to be specific for a relatively high GC content region so that it melts out at about a 10° C. higher temperature in the PCR cycle than the forward or reverse primers. This allows the TaqMan® probe to remain fully hybridized during the PCR annealing/extension step. As the Taq polymerase enzymatically synthesizes a new strand during PCR, it will eventually reach the annealed TaqMan® probe. The Taq polymerase 5′ to 3′ endonuclease activity will then displace the TaqMan® probe by digesting it to release the fluorescent reporter molecule for quantitative detection of its now unquenched signal using a real-time fluorescent detection system. Typical reagents (e.g., as might be found in a typical QM™-based kit) for QM™ analysis may include, but are not limited to: PCR primers for specific loci (e.g., specific genes, markers, regions of genes, regions of markers, bisulfite treated DNA sequence, CpG island, etc.); TaqMan® or Lightcycler® probes; optimized PCR buffers and deoxynucleotides; and Taq polymerase.
[0283] The Ms-SNuPE™ technique is a quantitative method for assessing methylation differences at specific CpG sites based on bisulfate treatment of DNA, followed by single-nucleotide primer extension (Gonzalgo & Jones, Nucleic Acids Res. 25:2529-2531, 1997). Briefly, genomic DNA is reacted with sodium bisulfite to convert unmethylated cytosine to uracil while leaving 5-methylcytosine unchanged. Amplification of the desired target sequence is then performed using PCR primers specific for bisulfite-converted DNA, and the resulting product is isolated and used as a template for methylation analysis at the CpG site of interest.
[0284] Small amounts of DNA can be analyzed (e.g., microdissected pathology sections) and it avoids utilization of restriction enzymes for determining the methylation status at CpG sites.
[0285] Typical reagents (e.g., as might be found in a typical Ms-SNuPE™-based kit) for Ms-SNuPE™ analysis may include, but are not limited to: PCR primers for specific loci (e.g., specific genes, markers, regions of genes, regions of markers, bisulfite treated DNA sequence, CpG island, etc.); optimized PCR buffers and deoxynucleotides; gel extraction kit; positive control primers; Ms-SNuPE™ primers for specific loci; reaction buffer (for the Ms-SNuPE reaction); and labeled nucleotides. Additionally, bisulfite conversion reagents may include: DNA denaturation buffer; sulfonation buffer; DNA recovery reagents or kit (e.g., precipitation, ultrafiltration, affinity column); desulfonation buffer; and DNA recovery components.
[0286] Reduced Representation Bisulfite Sequencing (RRBS) begins with bisulfite treatment of nucleic acid to convert all unmethylated cytosines to uracil, followed by restriction enzyme digestion (e.g., by an enzyme that recognizes a site including a CG sequence such as MspI) and complete sequencing of fragments after coupling to an adapter ligand. The choice of restriction enzyme enriches the fragments for CpG dense regions, reducing the number of redundant sequences that may map to multiple gene positions during analysis. As such, RRBS reduces the complexity of the nucleic acid sample by selecting a subset (e.g., by size selection using preparative gel electrophoresis) of restriction fragments for sequencing. As opposed to whole-genome bisulfite sequencing, every fragment produced by the restriction enzyme digestion contains DNA methylation information for at least one CpG dinucleotide. As such, RRBS enriches the sample for promoters, CpG islands, and other genomic features with a high frequency of restriction enzyme cut sites in these regions and thus provides an assay to assess the methylation state of one or more genomic loci.
[0287] A typical protocol for RRBS comprises the steps of digesting a nucleic acid sample with a restriction enzyme such as MspI, filling in overhangs and A-tailing, ligating adaptors, bisulfite conversion, and PCR. See, e.g., et al. (2005) “Genome-scale DNA methylation mapping of clinical samples at single-nucleotide resolution” Nat Methods 7: 133-6; Meissner et al. (2005) “Reduced representation bisulfite sequencing for comparative high-resolution DNA methylation analysis” Nucleic Acids Res. 33: 5868-77.
[0288] In some embodiments, a quantitative allele-specific real-time target and signal amplification (QuARTS) assay is used to evaluate methylation state. Three reactions sequentially occur in each QuARTS assay, including amplification (reaction 1) and target probe cleavage (reaction 2) in the primary reaction; and FRET cleavage and fluorescent signal generation (reaction 3) in the secondary reaction. When target nucleic acid is amplified with specific primers, a specific detection probe with a flap sequence loosely binds to the amplicon. The presence of the specific invasive oligonucleotide at the target binding site causes a 5′ nuclease, e.g., a FEN-1 endonuclease, to release the flap sequence by cutting between the detection probe and the flap sequence. The flap sequence is complementary to a non-hairpin portion of a corresponding FRET cassette. Accordingly, the flap sequence functions as an invasive oligonucleotide on the FRET cassette and effects a cleavage between the FRET cassette fluorophore and a quencher, which produces a fluorescent signal. The cleavage reaction can cut multiple probes per target and thus release multiple fluorophore per flap, providing exponential signal amplification. QuARTS can detect multiple targets in a single reaction well by using FRET cassettes with different dyes. See, e.g., in Zou et al. (2010) “Sensitive quantification of methylated markers with a novel methylation specific technology” Clin Chem 56: A199). In embodiments, described herein, the QuARTS assay can also be configured to detect multiple different targets in or different regions of the same target using the same FRET cassette, producing an additive fluorescence signal from a single dye.
[0289] In some embodiments, the bisulfite-treated DNA is purified prior to the quantification. This may be conducted by any means known in the art, such as but not limited to ultrafiltration, e.g., by means of Microcon™ columns (manufactured by Millipore™). The purification is carried out according to a modified manufacturer's protocol (see, e.g., PCT/EP2004/011715, which is incorporated by reference in its entirety). In some embodiments, the bisulfite treated DNA is bound to a solid support, e.g., a magnetic bead, and desulfonation and washing occurs while the DNA is bound to the support. Examples of such embodiments are provided, e.g., in WO 2013/116375. In certain preferred embodiments, support-bound DNA is ready for a methylation assay immediately after desulfonation and washing on the support. In some embodiments, the desulfonated DNA is eluted from the support prior to assay.
[0290] In some embodiments, fragments of the treated DNA are amplified using sets of primer oligonucleotides according to the present invention (e.g., see
[0291] Methods for isolating DNA suitable for these assay technologies are known in the art. In particular, some embodiments comprise isolation of nucleic acids as described in U.S. patent application Ser. No. 13/470,251 (“Isolation of Nucleic Acids”, published as US 2012/0288868), incorporated herein by reference in its entirety.
[0292] In some embodiments, the markers described herein find use in QUARTS assays performed on stool samples. In some embodiments, methods for producing DNA samples and, in particular, to methods for producing DNA samples that comprise highly purified, low-abundance nucleic acids in a small volume (e.g., less than 100, less than 60 microliters) and that are substantially and/or effectively free of substances that inhibit assays used to test the DNA samples (e.g., PCR, INVADER, QuARTS assays, etc.) are provided. Such DNA samples find use in diagnostic assays that qualitatively detect the presence of, or quantitatively measure the activity, expression, or amount of, a gene, a gene variant (e.g., an allele), or a gene modification (e.g., methylation) present in a sample taken from a patient. For example, some cancers are correlated with the presence of particular mutant alleles or particular methylation states, and thus detecting and/or quantifying such mutant alleles or methylation states has predictive value in the diagnosis and treatment of cancer.
[0293] Many valuable genetic markers are present in extremely low amounts in samples and many of the events that produce such markers are rare. Consequently, even sensitive detection methods such as PCR require a large amount of DNA to provide enough of a low-abundance target to meet or supersede the detection threshold of the assay. Moreover, the presence of even low amounts of inhibitory substances compromise the accuracy and precision of these assays directed to detecting such low amounts of a target. Accordingly, provided herein are methods providing the requisite management of volume and concentration to produce such DNA samples.
[0294] In some embodiments, the sample comprises blood, serum, plasma, or saliva. In some embodiments, the subject is human. Such samples can be obtained by any number of means known in the art, such as will be apparent to the skilled person. Cell free or substantially cell free samples can be obtained by subjecting the sample to various techniques known to those of skill in the art which include, but are not limited to, centrifugation and filtration. Although it is generally preferred that no invasive techniques are used to obtain the sample, it still may be preferable to obtain samples such as tissue homogenates, tissue sections, and biopsy specimens. The technology is not limited in the methods used to prepare the samples and provide a nucleic acid for testing. For example, in some embodiments, a DNA is isolated from a stool sample or from blood or from a plasma sample using direct gene capture, e.g., as detailed in U.S. Pat. Nos. 8,808,990 and 9,169,511, and in WO 2012/155072, or by a related method.
[0295] The analysis of markers can be carried out separately or simultaneously with additional markers within one test sample. For example, several markers can be combined into one test for efficient processing of multiple samples and for potentially providing greater diagnostic and/or prognostic accuracy. In addition, one skilled in the art would recognize the value of testing multiple samples (for example, at successive time points) from the same subject. Such testing of serial samples can allow the identification of changes in marker methylation states over time. Changes in methylation state, as well as the absence of change in methylation state, can provide useful information about the disease status that includes, but is not limited to, identifying the approximate time from onset of the event, the presence and amount of salvageable tissue, the appropriateness of drug therapies, the effectiveness of various therapies, and identification of the subject's outcome, including risk of future events.
[0296] The analysis of biomarkers can be carried out in a variety of physical formats. For example, the use of microtiter plates or automation can be used to facilitate the processing of large numbers of test samples. Alternatively, single sample formats could be developed to facilitate immediate treatment and diagnosis in a timely fashion, for example, in ambulatory transport or emergency room settings.
[0297] It is contemplated that embodiments of the technology are provided in the form of a kit. The kits comprise embodiments of the compositions, devices, apparatuses, etc. described herein, and instructions for use of the kit. Such instructions describe appropriate methods for preparing an analyte from a sample, e.g., for collecting a sample and preparing a nucleic acid from the sample. Individual components of the kit are packaged in appropriate containers and packaging (e.g., vials, boxes, blister packs, ampules, jars, bottles, tubes, and the like) and the components are packaged together in an appropriate container (e.g., a box or boxes) for convenient storage, shipping, and/or use by the user of the kit. It is understood that liquid components (e.g., a buffer) may be provided in a lyophilized form to be reconstituted by the user. Kits may include a control or reference for assessing, validating, and/or assuring the performance of the kit. For example, a kit for assaying the amount of a nucleic acid present in a sample may include a control comprising a known concentration of the same or another nucleic acid for comparison and, in some embodiments, a detection reagent (e.g., a primer) specific for the control nucleic acid. The kits are appropriate for use in a clinical setting and, in some embodiments, for use in a user's home. The components of a kit, in some embodiments, provide the functionalities of a system for preparing a nucleic acid solution from a sample. In some embodiments, certain components of the system are provided by the user.
[0298] In some embodiments the technology relates to the analysis of any sample associated with colon cancer. For example, in some embodiments the sample comprises a tissue and/or biological fluid obtained from a patient. In some embodiments, the sample comprises a secretion. In some embodiments, the sample comprises sputum, blood, serum, plasma, gastric secretions, colon tissue samples, colon cells or colon DNA recovered from stool. In some embodiments, the subject is human. Such samples can be obtained by any number of means known in the art, such as will be apparent to the skilled person.
[0299] In some embodiments, diagnostic assays identify the presence of a disease or condition in an individual. In some embodiments, the disease is cancer (e.g., colon cancer). In some embodiments, markers whose aberrant methylation is associated with a colon cancer (e.g., one or more markers selected from the markers listed in Table 1, or preferably one or more of VAV3; ZNF671; CHST2; FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; GRIN2D, QKI, FER1L4) are used. In some embodiments, an assay further comprises detection of a reference gene (e.g., (3-actin, ZDHHC1, B3GALT6).
[0300] In some embodiments, the technology finds application in treating a patient (e.g., a patient with colon cancer, with early-stage colon cancer, or who may develop colon cancer), the method comprising determining the methylation state of one or more markers as provided herein and administering a treatment to the patient based on the results of determining the methylation state. The treatment may be administration of a pharmaceutical compound, a vaccine, performing a surgery, imaging the patient, performing another test. Preferably, said use is in a method of clinical screening, a method of prognosis assessment, a method of monitoring the results of therapy, a method to identify patients most likely to respond to a particular therapeutic treatment, a method of imaging a patient or subject, and a method for drug screening and development.
[0301] In some embodiments, the technology finds application in methods for diagnosing colon cancer in a subject. The terms “diagnosing” and “diagnosis” as used herein refer to methods by which the skilled artisan can estimate and even determine whether or not a subject is suffering from a given disease or condition or may develop a given disease or condition in the future. The skilled artisan often makes a diagnosis on the basis of one or more diagnostic indicators, such as for example a biomarker, the methylation state of which is indicative of the presence, severity, or absence of the condition.
[0302] Along with diagnosis, clinical cancer prognosis relates to determining the aggressiveness of the cancer and the likelihood of tumor recurrence to plan the most effective therapy. If a more accurate prognosis can be made or even a potential risk for developing the cancer can be assessed, appropriate therapy, and in some instances less severe therapy for the patient can be chosen. Assessment (e.g., determining methylation state) of cancer biomarkers is useful to separate subjects with good prognosis and/or low risk of developing cancer who will need no therapy or limited therapy from those more likely to develop cancer or suffer a recurrence of cancer who might benefit from more intensive treatments.
[0303] As such, “making a diagnosis” or “diagnosing”, as used herein, is further inclusive of making a determination of a risk of developing cancer or determining a prognosis, which can provide for predicting a clinical outcome (with or without medical treatment), selecting an appropriate treatment (or whether treatment would be effective), or monitoring a current treatment and potentially changing the treatment, based on the measure of the diagnostic biomarkers disclosed herein.
[0304] Further, in some embodiments of the technology, multiple determinations of the biomarkers over time can be made to facilitate diagnosis and/or prognosis. A temporal change in the biomarker can be used to predict a clinical outcome, monitor the progression of colon cancer, and/or monitor the efficacy of appropriate therapies directed against the cancer. In such an embodiment for example, one might expect to see a change in the methylation state of one or more biomarkers disclosed herein (and potentially one or more additional biomarker(s), if monitored) in a biological sample over time during the course of an effective therapy.
[0305] The technology further finds application in methods for determining whether to initiate or continue prophylaxis or treatment of a cancer in a subject. In some embodiments, the method comprises providing a series of biological samples over a time period from the subject; analyzing the series of biological samples to determine a methylation state of at least one biomarker disclosed herein in each of the biological samples; and comparing any measurable change in the methylation states of one or more of the biomarkers in each of the biological samples. Any changes in the methylation states of biomarkers over the time period can be used to predict risk of developing cancer, predict clinical outcome, determine whether to initiate or continue the prophylaxis or therapy of the cancer, and whether a current therapy is effectively treating the cancer. For example, a first time point can be selected prior to initiation of a treatment and a second time point can be selected at some time after initiation of the treatment. Methylation states can be measured in each of the samples taken from different time points and qualitative and/or quantitative differences noted. A change in the methylation states of the biomarker levels from the different samples can be correlated with risk for developing colon, prognosis, determining treatment efficacy, and/or progression of the cancer in the subject.
[0306] In preferred embodiments, the methods and compositions of the invention are for treatment or diagnosis of disease at an early stage, for example, before symptoms of the disease appear. In some embodiments, the methods and compositions of the invention are for treatment or diagnosis of disease at a clinical stage.
[0307] As noted above, in some embodiments multiple determinations of one or more diagnostic or prognostic biomarkers can be made, and a temporal change in the marker can be used to determine a diagnosis or prognosis. For example, a diagnostic marker can be determined at an initial time, and again at a second time. In such embodiments, an increase in the marker from the initial time to the second time can be diagnostic of a particular type or severity of cancer, or a given prognosis. Likewise, a decrease in the marker from the initial time to the second time can be indicative of a particular type or severity of cancer, or a given prognosis. Furthermore, the degree of change of one or more markers can be related to the severity of the cancer and future adverse events. The skilled artisan will understand that, while in certain embodiments comparative measurements can be made of the same biomarker at multiple time points, one can also measure a given biomarker at one time point, and a second biomarker at a second time point, and a comparison of these markers can provide diagnostic information.
[0308] As used herein, the phrase “determining the prognosis” refers to methods by which the skilled artisan can predict the course or outcome of a condition in a subject. The term “prognosis” does not refer to the ability to predict the course or outcome of a condition with 100% accuracy, or even that a given course or outcome is predictably more or less likely to occur based on the methylation state of a biomarker. Instead, the skilled artisan will understand that the term “prognosis” refers to an increased probability that a certain course or outcome will occur; that is, that a course or outcome is more likely to occur in a subject exhibiting a given condition, when compared to those individuals not exhibiting the condition. For example, in individuals not exhibiting the condition, the chance of a given outcome (e.g., suffering from colon cancer) may be very low.
[0309] In some embodiments, a statistical analysis associates a prognostic indicator with a predisposition to an adverse outcome. For example, in some embodiments, a methylation state different from that in a normal control sample obtained from a patient who does not have a cancer can signal that a subject is more likely to suffer from a cancer than subjects with a level that is more similar to the methylation state in the control sample, as determined by a level of statistical significance. Additionally, a change in methylation state from a baseline (e.g., “normal”) level can be reflective of subject prognosis, and the degree of change in methylation state can be related to the severity of adverse events. Statistical significance is often determined by comparing two or more populations and determining a confidence interval and/or ap value. See, e.g., Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York, 1983, incorporated herein by reference in its entirety. Exemplary confidence intervals of the present subject matter are 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9% and 99.99%, while exemplary p values are 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, and 0.0001.
[0310] In other embodiments, a threshold degree of change in the methylation state of a prognostic or diagnostic biomarker disclosed herein can be established, and the degree of change in the methylation state of the biomarker in a biological sample is simply compared to the threshold degree of change in the methylation state. A preferred threshold change in the methylation state for biomarkers provided herein is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 50%, about 75%, about 100%, and about 150%. In yet other embodiments, a “nomogram” can be established, by which a methylation state of a prognostic or diagnostic indicator (biomarker or combination of biomarkers) is directly related to an associated disposition towards a given outcome. The skilled artisan is acquainted with the use of such nomograms to relate two numeric values with the understanding that the uncertainty in this measurement is the same as the uncertainty in the marker concentration because individual sample measurements are referenced, not population averages.
[0311] In some embodiments, a control sample is analyzed concurrently with the biological sample, such that the results obtained from the biological sample can be compared to the results obtained from the control sample. Additionally, it is contemplated that standard curves can be provided, with which assay results for the biological sample may be compared. Such standard curves present methylation states of a biomarker as a function of assay units, e.g., fluorescent signal intensity, if a fluorescent label is used. Using samples taken from multiple donors, standard curves can be provided for control methylation states of the one or more biomarkers in normal tissue, as well as for “at-risk” levels of the one or more biomarkers in tissue taken from donors with colon cancer.
[0312] The analysis of markers can be carried out separately or simultaneously with additional markers within one test sample. For example, several markers can be combined into one test for efficient processing of a multiple of samples and for potentially providing greater diagnostic and/or prognostic accuracy. In addition, one skilled in the art would recognize the value of testing multiple samples (for example, at successive time points) from the same subject. Such testing of serial samples can allow the identification of changes in marker methylation states over time. Changes in methylation state, as well as the absence of change in methylation state, can provide useful information about the disease status that includes, but is not limited to, identifying the approximate time from onset of the event, the presence and amount of salvageable tissue, the appropriateness of drug therapies, the effectiveness of various therapies, and identification of the subject's outcome, including risk of future events.
[0313] The analysis of biomarkers can be carried out in a variety of physical formats. For example, the use of microtiter plates or automation can be used to facilitate the processing of large numbers of test samples. Alternatively, single sample formats could be developed to facilitate immediate treatment and diagnosis in a timely fashion, for example, in ambulatory transport or emergency room settings.
[0314] In some embodiments, the subject is diagnosed as having colon cancer if, when compared to a control methylation state, there is a measurable difference in the methylation state of at least one biomarker in the sample. Conversely, when no change in methylation state is identified in the biological sample, the subject can be identified as not having colon cancer, not being at risk for the cancer, or as having a low risk of the cancer. In this regard, subjects having colon cancer or risk thereof can be differentiated from subjects having low to substantially no cancer or risk thereof. Those subjects having a risk of developing colon cancer can be placed on a more intensive and/or regular screening schedule. On the other hand, those subjects having low to substantially no risk may avoid being subjected to screening procedures, until such time as a future screening, for example, a screening conducted in accordance with the present technology, indicates that a risk of colon cancer has appeared in those subjects.
[0315] As mentioned above, depending on the embodiment of the method of the present technology, detecting a change in methylation state of the one or more biomarkers can be a qualitative determination or it can be a quantitative determination. As such, the step of diagnosing a subject as having, or at risk of developing, colon cancer indicates that certain threshold measurements are made, e.g., the methylation state of the one or more biomarkers in the biological sample varies from a predetermined control methylation state. In some embodiments of the method, the control methylation state is any detectable methylation state of the biomarker. In other embodiments of the method where a control sample is tested concurrently with the biological sample, the predetermined methylation state is the methylation state in the control sample. In other embodiments of the method, the predetermined methylation state is based upon and/or identified by a standard curve. In other embodiments of the method, the predetermined methylation state is a specifically state or range of state. As such, the predetermined methylation state can be chosen, within acceptable limits that will be apparent to those skilled in the art, based in part on the embodiment of the method being practiced and the desired specificity, etc.
[0316] Over recent years, it has become apparent that circulating epithelial cells, representing metastatic tumor cells, can be detected in the blood of many patients with cancer. Molecular profiling of rare cells is important in biological and clinical studies. Applications range from characterization of circulating epithelial cells (CEpCs) in the peripheral blood of cancer patients for disease prognosis and personalized treatment (See e.g., Cristofanilli M, et al. (2004) N Engl J Med 351:781-791; Hayes D F, et al. (2006) Clin Cancer Res 12:4218-4224; Budd G T, et al., (2006) Clin Cancer Res 12:6403-6409; Moreno J G, et al. (2005) Urology 65:713-718; Pantel et al., (2008) Nat Rev 8:329-340; and Cohen S J, et al. (2008) J Clin Oncol 26:3213-3221). Accordingly, embodiments of the present disclosure provide compositions and methods for detecting the presence of metastatic cancer in a subject by identifying the presence of methylated markers in plasma or whole blood.
Esophageal Cell Samples
[0317] Exemplary nucleic acid assay designs are shown in
[0318] In some embodiments, the biomarker may be ZNF682. Exemplary primers and probes for ZNF682 are shown in
[0319] In some embodiments, the biomarker may be VAV3. Exemplary primers and probes for VAV3 are shown in
[0320] In some embodiments, the biomarker may be NDRG4. Exemplary primers and probes for NDRG4 are shown in
[0321]
[0322]
[0323] In some embodiments, the one or more biomarkers are normalized against a reference marker. Suitable methods and reference markers are described in U.S Pat. No. 10,465,248 and U.S. patent application Ser. No. 16/318,580, the entire contents of each of which are incorporated herein by reference. In some embodiments, the reference marker is selected from β-actin, ZDHHC1, and B3GALT6.
[0324] In some embodiments, the reference marker may be ZDHHC1. Exemplary primers and probes for ZDHHC1 are shown in
[0325] In some embodiments, the reference marker may be B3GALT6. Exemplary primers and probes for B3GALT6 are shown in
[0326] In some embodiments, the reference marker may be β-actin. Exemplary primers and probes for β-actin are shown in
EXPERIMENTAL EXAMPLES
Example 1
Sample Preparation Methods
Methods for DNA Isolation and QUARTS Assay
[0327] The following provides exemplary method for DNA isolation prior to analysis, and an exemplary QuARTS assay, such as may be used in accordance with embodiments of the technology. Application of QuARTS technology to DNA from blood and various tissue samples is described in this example, but the technology is readily applied to other nucleic acid samples, as shown in other examples.
DNA Isolation From Cells and Plasma
[0328] For cell lines, genomic DNA may be isolated from cell conditioned media using, for example, the “Maxwell® RSC ccfDNA Plasma Kit (Promega Corp., Madison, Wis.). Following the kit protocol, 1 mL of cell conditioned media (CCM) is used in place of plasma, and processed according to the kit procedure. The elution volume is 100 μL, of which 70 μL are generally used for bisulfite conversion. See also U.S. Patent Appl. Ser. Nos. 62/249,097, filed Oct. 30, 2015; Ser. Nos. 15/335,111 and 15/335,096, both filed Oct. 26, 2016; and International Appl. Ser. No. PCT/US16/58875, filed Oct. 26, 2016, each of which is incorporated herein by reference in its entirety, for all purposes.
[0329] An example of a complete process for isolating DNA from a blood sample for use, e.g., in a detection assay, is provided in this example. Optional bisulfite conversion and detection methods are also described.
I. Blood Processing
[0330] Whole blood is collected in anticoagulant EDTA or Streck Cell-Free DNA BCT tubes. An exemplary procedure is as follows: [0331] 1. Draw 10 mL whole blood into vacutainers tube (anticoagulant EDTA or Streck BCT), collecting the full volume to ensure correct blood to anticoagulant ratio. [0332] 2. After collection, gently mix the blood by inverting the tube 8 to 10 times to mix blood and anticoagulant and keep at room temperature until centrifugation, which should happen within 4 hours of the time of blood collection. [0333] 3. Centrifuge blood samples in a horizontal rotor (swing-out head) for 10 minutes at 1500 g (±100 g) at room temperature. Do not use brake to stop centrifuge. [0334] 4. Carefully aspirate the supernatant (plasma) at room temperature and pool in a centrifuge tube. Make sure not to disrupt the cell layer or transfer any cells. [0335] 5. Carefully transfer 4mL aliquots of the supernatant into cryovial tubes. [0336] 6. Close the caps tightly and place on ice as soon as each aliquot is made. This process should be completed within 1 hour of centrifugation. [0337] 7. Ensure that the cryovials are adequately labeled with the relevant information, including details of additives present in the blood. [0338] 8. Specimens can be kept frozen at −20° C. for a maximum of 48 hours before transferring to a −80° C. freezer.
II. Preparation of a Synthetic Process Control DNA
[0339] Complementary strands of methylated zebrafish DNA are synthesized having the sequences as shown below using standard DNA synthesis methods such as phosphoramidite addition, incorporating 5-methyl C bases at the positions indicated. The synthetic strands are annealed to create a double-stranded DNA fragment for use as a process control.
TABLE-US-00001 SEQ ID Oligo Name NO: Oligo Sequence Zebrafish RASSF1 me 177 5-TCCAC/iMe- synthetic Target dC/GTGGTGCCCACTCTGGACAGGTGGAGCAGAGGGAAGGTGGT Sense Strand G/iMe-dC/GCATGGTGGG/iMe-dC/GAG/iMe-dC/G/iMe- dC/GTG/iMe-dC/GC CTGGAGGACCC/iMe-dC/GATTGGCTGA/iMe- dC/GTGTAAACCAGGA/iMe-dC/GA GGACATGACTTTCAGCCCTGCAGCCAGACACAGCTGAGCTGGTGT GACCTGTGTGGAGAGTTCATCTGG-3 Zebrafish RASSF1 me 178 5- synthetic Target CCAGATGAACTCTCCACACAGGTCACACCAGCTCAGCTGTGTCTGG Anti-Sense Strand CTGCAGGGCTGAAAGTCATGTCCT/iMe- dC/GTCCTGGTTTACA/iMe-dC/GTCAGCCAAT/iMe- dC/GGGGTCCTCCAGG/iMe-dC/GCA/iMe-dC/G/iMe- dC/GCT/iMe-dC/GC CCACCATG/iMe- dC/GCACCACCTTCCCTCTGCTCCACCTGTCCAGAGTGG GCACCA/iMe-dC/GGTGGA-3
A. Annealing and Preparation of Concentrated Zebrafish (ZF-RASS F1 180mer) Synthetic Process Control [0340] 1. Reconstitute the lyophilized, single stranded oligonucleotides in 10 mM Tris, pH 8.0, 0.1 mM EDTA, at a concentration of 1 μM. [0341] 2. Make 10X Annealing Buffer of 500mM NaCl, 200mM Tris-HCl pH 8.0, and 20mM MgCl.sub.2. [0342] 3. Anneal the synthetic strands:
[0343] In a total volume of 100 combine equimolar amounts of each of the single-stranded oligonucleotides in 1× annealing buffer, e.g., as shown in the table below:
TABLE-US-00002 Final Conc. Volume Stock (copies/μl in 1 ml added Component Conc. final volume) (μL) Zebrafish RASSF1 me 1 μM 1.0E+10 16.6 synthetic Target Sense Strand Zebrafish RASSF1 me 1 μM 1.0E+10 16.6 synthetic Target Anti-Sense Strand Annealing Buffer 10X NA 10.0 Water NA NA 56.8 total vol. 100.0 μL [0344] 4. Heat the annealing mixture to 98° C. for 11-15 minutes. [0345] 5. Remove the reaction tube from the heat and spin down briefly to collect condensation to bottom of tube. [0346] 6. Incubate the reaction tube at room temp for 10 to 25 minutes. [0347] 7. Add 0.9 mL fish DNA diluent (20 ng/mL bulk fish DNA in Te (10 mM Tris-HCl pH8.0, 0.1 mM EDTA)) to adjust to the concentration of zebrafish RASSF 1 DNA fragment to 1.0×10.sup.10 copies/μl of annealed, double-stranded synthetic zebrafish RASSF 1 DNA in a carrier of genomic fish DNA. [0348] 8. Dilute the process control to a desired concentration with 10 mM Tris, pH 8.0, 0.1 mM EDTA, e.g., as described in the table below, and store at either −20° C. or −80° C.
TABLE-US-00003 Target Total Initial Concentration Addition Te Volume Final Concentration 1.00E+10 copies/μL 10 μL 990 μL 1000 μL 1.00E+08 copies/μL 1.00E+08 copies/μL 10 μL 990 μL 1000 μL 1.00E+06 copies/μL
B. Preparation of 100× Stock Process Control (12,000 copies/μL Zebrafish RASSF 1 DNA in 200 ng/μL bulk Fish DNA) [0349] 1. Thaw reagents [0350] 2. Vortex and spin down thawed reagents [0351] 3. Add the following reagents into a 50 mL conical tube
TABLE-US-00004 Reagent Initial Concentration Final Concentration Volume to add (mL) Stock carrier fish DNA 10 μg/μL 200 ng/μL 0.40 Zebrafish (ZF-RASS F1 180mer) 1.00E+06 copies/μL 1.20E+04 copies/μL 0.24 10 mM Tris, pH 8.0, 0.1 mM EDTA NA NA 19.36 Total Volume 20.00 [0352] 4. Aliquot into labeled 0.5 mL tubes and store @−20° C.
C. Preparation of 1× Stock of Process Control (120 copies/μL Zebrafish RASSF 1 DNA in 2 ng/μL Fish DNA) [0353] 1. Thaw reagents [0354] 2. Vortex and spin down thawed reagents [0355] 3. Add the following reagents into a 50 mL conical tube:
TABLE-US-00005 Reagent 1 mL 5 mL 10 mL 100x Zebrafish Process Control 10 μL 50 μL 100 μL 10 mM Tris, pH 8.0, 0.1 mM EDTA 990 μL 4950 μL 9900 μL [0356] 4. Aliquot 0.3 mL into labeled 0.5 mL tubes and store @−20° C.
III. DNA extraction from plasma [0357] 1. Thaw plasma, prepare reagents, label tubes, and clean and setup biosafety cabinet for extraction [0358] 2. Add 300 μL Proteinase K (20 mg/mL) to one 50 mL conical tube for each sample. [0359] 3. Add 2 - 4 mL of plasma sample to each 50 mL conical tube (do not vortex). [0360] 4. Swirl or pipet to mix and let sit at room temp for 5 min. [0361] 5. Add 4 - 6 mL of lysis buffer 1 (LB1) solution to bring the volume up to approximately 8 mL.
[0362] LB1 Formulation: [0363] 0.1 mL of 120 copies/μL of zebrafish RASSF1 DNA process control, as described above; [0364] 0.9 -2.9 mL of 10 mM Tris, pH 8.0, 0.1 mM EDTA (e.g., use 2.9 mL for 2 mL plasma samples) [0365] 3 mL of 4.3 M guanidine thiocyanate with 10% IGEPAL (from a stock of 5.3 g of IGEPAL CA-630 combined with 45 mL of 4.8 M guanidine thiocyanate) [0366] 6. Invert tubes 3 times. [0367] 7. Place tubes on bench top shaker (room temperature) at 500 rpm for 30 minutes at room temperature. [0368] 8. Add 200 μL of silica binding beads (16 μg of particles/μL) and mix by swirling. [0369] 9. Add 7 mL of lysis buffer 2 (LB2) solution and mix by swirling.
[0370] LB2 Formulation: [0371] 4 mL 4.3 M guanidine thiocyanate mixed with 10% IGEPAL [0372] 3 mL 100% Isopropanol
(Lysis buffer 2 may be added before, after, or concurrently with the silica binding beads) [0373] 10. Invert tubes 3 times. [0374] 11. Place tubes on bench top shaker at 500 rpm for 30 minutes at room temperature. [0375] 12. Place tubes on capture aspirator and run program with magnetic collection of the beads for 10 minutes, then aspiration. This will collect the beads for 10 minutes then remove all liquid from the tubes. [0376] 13. Add 0.9 mL of Wash Solution 1 (3 M guanidine hydrochloride or guanidine thiocyanate, 56.8% EtOH) to resuspend binding beads and mix by swirling. [0377] 14. Place tubes on bench top shaker at 400 rpm for 2 minute at room temperature.
(All subsequent steps can be done on a STARlet automated platform.) [0378] 15. Mix by repeated pipetting then transfer containing beads to 96 deep well plate. [0379] 16. Place plate on magnetic rack for 10 min. [0380] 17. Aspirate supernatant to waste. [0381] 18. Add 1 mL of Wash Solution 2 (80% Ethanol, 10 mM Tris pH 8.0). [0382] 19. Mix for 3 minutes. [0383] 20. Place tubes on magnetic rack for 10 min. [0384] 21. Aspirate supernatant to waste. [0385] 22. Add 0.5 mL of Wash Solution 2. [0386] 23. Mix for 3 minutes. [0387] 24. Place tubes on magnetic rack for 5 min. [0388] 25. Aspirate supernatant to waste. [0389] 26. Add 0.25 mL of Wash Solution 2. [0390] 27. Mix for 3 minutes. [0391] 28. Place tubes on magnetic rack for 5 min. [0392] 29. Aspirate supernatant to waste. [0393] 30. Add 0.25 mL of Wash Solution 2. [0394] 31. Mix for 3 minutes. [0395] 32. Place tubes on magnetic rack for 5 min. [0396] 33. Aspirate supernatant to waste. [0397] 34. Place plate on heat block at 70° C., 15 minutes, with shaking. [0398] 35. Add 125 μL of elution buffer (10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA). [0399] 36. Incubate 65° C. for 25 minutes with shaking. [0400] 37. Place plate on magnet and let the beads collect and cool for 8 minutes. [0401] 38. Transfer eluate to 96-well plate and store at −80° C. The recoverable/transferrable volume is about 100 μL.
IV. Pre-Bisulfite DNA Quantification
[0402] To measure DNA in samples using ACTB gene and to assess zebrafish process control recovery, the DNA may be measured prior to further treatment. Setup a QuARTS PCR-flap assay using 10 μL of the extracted DNA using the following protocol: [0403] 1. Prepare 10× Oligo Mix containing forward and reverse primers each at 2 μM, the probe and FRET cassettes at 5 μM and deoxynucleoside triphosphates (dNTPs) at 250 μM each. (See below for primer, probe, and FRET sequences)
TABLE-US-00006 SEQ ID Concentration Oligo Sequence (5′-3′) NO: (μM) ZF RASSF1 UT CGCATGGTGGGCGAG 179 2 forward primer ZF RASSF1 UT ACACGTCAGCCAATCGGG 180 2 reverse primer ZF RASSF1 UT CCACGGACG GCGCGTGCGTTT/3C6/ 181 5 Probe (Arm 3) Arm 5 FAM /FAM/TCT/BHQ-1/ 182 5 FRET AGCCGGTTTTCCGGCTGAGACGTCCGTGG/3C6/ ACTB forward CCATGAGGCTGGTGTAAAG 164 2 primer 3 ACTB Reverse CTACTGTGCACCTACTTAATACAC 165 2 primer 3 ACTB probe CGCCGAGGGCGGCCTTGGAG/3C6/ 166 5 with Arm 1 Arm 1 /Q670/TCT/BHQ-2/ 174 5 QUASAR670 AGCCGGTTTTCCGGCTGAGACCTCGGCG/3C6/ FRET dNTP mix 2500 [0404] 2. Prepare a master mix as follows:
TABLE-US-00007 Component Volume per reaction (μL) Water 15.50 10X oligo Mix 3.00 20X Quarts Enzyme Mix* 1.50 total volume 20.0 *20X enzyme mix contains 1 unit/μL GoTaq Hot start polymerase (Promega), 292 ng/μL Cleavase 2.0 flap endonuclease(Hologic). [0405] 3. Pipette 10 μL of each sample into a well of a 96 well plate. [0406] 4. Add 20 μL of master mix to each well of the plate. [0407] 5. Seal plate and centrifuge for 1 minutes at 3000 rpm. [0408] 6. Run plates with following reaction conditions on an ABI7500 or Light Cycler 480 real time thermal cycler
TABLE-US-00008 QuARTS Assay Reaction Cycle: Signal Ramp Rate Number Acqui- Stage Temp/Time (° C. per second) of Cycles sition Pre-incubation 95° C./3 min 4.4 1 No Amplification 1 95° C./2 sec 4.4 5 No 63° C./30 sec 2.2 No 70° C./30 sec 4.4 No Amplification 2 95° C./20 sec 4.4 40 No 53° C./1 min 2.2 Yes 70° C./30 sec 4.4 No Cooling 40° C./30 sec 2.2 1 No
V. Bisulfite Conversion and Purification of DNA
[0409] 1. Thaw all extracted DNA samples from the DNA extraction from plasma step and spin down DNA. [0410] 2. Reagent Preparation:
TABLE-US-00009 Component Abbreviation Name Formulation BIS SLN Bisulfite Conversion 56.6% Ammonium Bisulfite Solution DES SLN Desulfonation 70% Isopropyl alcohol, 0.1N NaOH Solution BND BDS Binding Beads Maxwell RNA Beads (16 mg/mL), (Promega Corp.) BND SLN Binding Solution 7M Guanidine HCl CNV WSH Conversion Wash 10 mM Tris-HCl, 80% Ethanol ELU BUF Elution Buffer 10 mM Tris, 0.1 mM EDTA, pH 8.0 [0411] 3. Add 5 μL of 100 ng/μL BSA DNA Carrier Solution to each well in a deep well plate (DWP). [0412] 4. Add 80 μL of each sample into the DWP. [0413] 5. Add 5 μL of freshly prepared 1.6N NaOH to each well in the DWP(s). [0414] 6. Carefully mix by pipetting with pipette set to 30-40 μL to avoid bubbles. [0415] 7. Incubate at 42° C. for 20 minutes. [0416] 8. Add 120 μL of BIS SLN to each well. [0417] 9. Incubate at 66° C. for 75 minutes while mixing during the first 3 minutes. [0418] 10. Add 750 μL of BND SLN [0419] 11. Pre-mix of silica beads (BND BDS) and add of 50 μL of Silica beads (BND BDS) to the wells of DWP. [0420] 12. Mix at 30° C. on heater shaker at 1,200 rpm for 30 minutes. [0421] 13. Collect the beads on a plate magnet for 5 minutes followed by aspiration of solutions to waste. [0422] 14. Add 1 mL of wash buffer (CNV WSH) then move the plate to a heater shaker and mix at 1,200 rpm for 3 minutes. [0423] 15. Collect the beads on a plate magnet for 5 minutes followed by aspiration of solutions to waste. [0424] 16. Add 0.25 mL of wash buffer (CNV WSH) then move the plate to the heater shaker and mix at 1,200 rpm for 3 minutes. [0425] 17. Collect the beads on a plate magnet followed by aspiration of solutions to waste. [0426] 18. Add of 0.2 mL of desulfonation buffer (DES SLN) and mix at 1,200 rpm for 7 minutes at 30° C. [0427] 19. Collect the beads for 2 minutes on the magnet followed by aspiration of solutions to waste. [0428] 20. Add of 0.25 mL of wash buffer (CNV WSH) then move the plate to the heater shaker and mix at 1,200 rpm for 3 minutes. [0429] 21. Collect the beads for 2 minutes on the magnet followed by aspiration of solutions to waste. [0430] 22. Add of 0.25 mL of wash buffer (CNV WSH) then move the plate to the heater shaker and mix at 1,200 rpm for 3 minutes. [0431] 23. Collect the beads for 2 minutes on the magnet followed by aspiration of solutions to waste. [0432] 24. Allow the plate to dry by moving to heater shaker and incubating at 70° C. for 15 minutes while mixing at 1,200 rpm. [0433] 25. Add 80 μL of elution buffer (ELU BFR) across all samples in DWP. [0434] 26. Incubated at 65° C. for 25 minutes while mixing at 1,200 rpm. [0435] 27. Manually Transfer eluate to 96well plate and store at −80° C. [0436] 28. The recoverable/transferrable volume is about 65 μL.
VI. QuARTS-X Multiplex Flap Assay for Methylated DNA Detection and Quantification
[0437] A. Multiplex PCR (mPCR) Setup: [0438] 1. Prepare a 10× primer mix containing forward and reverse primers for each methylated marker of interest to a final concentration of 750 nM each. Use 10 mM Tris-HCl, pH 8, 0.1 mM EDTA as diluent, as described in the examples above. [0439] 2. Prepare 10X multiplex PCR buffer containing 100 mM MOPS, pH 7.5, 75 mM MgCl.sub.2, 0.08% Tween 20, 0.08% IGEPAL CA-630, 2.5 mM dNTPs. [0440] 3. Prepare multiplex PCR master mix as follows:
TABLE-US-00010 Component Volume per reaction (μL) Water 9.62 10X Primer Mix (0.75 μM each) 7.5 mPCR Buffer 7.5 Hot Start GoTaq (5 units/μl) 0.38 total volume 25.0 [0441] 4. Thaw DNA and spin plate down. [0442] 5. Add 25 μL of master mix to a 96 well plate. [0443] 6. Transfer 50 μL of each sample to each well. [0444] 7. Seal plate with aluminum foil seal (do not use strip caps) [0445] 8. Place in heated-lid thermal cycler and proceed to cycle using the following profile, for about 5 to 20 cycles, preferably about 10 to 13 cycles:
TABLE-US-00011 Stage Temp/Time Number of Cycles Pre-incubation 95° C./5 min 1 Amplification 1 95° C./30 sec 12 64° C./60 sec Cooling 4° C./hold 1 [0446] 9. After completion of the thermal cycling, perform a 1:10 dilution of amplicon as follows: [0447] a) Transfer 180 μL of 10 mM Tris-HCl, pH 8, 0.1 mM EDTA to each well of a deep well plate. [0448] b) Add 20 μL of amplified sample to each pre-filled well. [0449] c) Mix the diluted samples by repeated pipetting using fresh tips and a 200 μL pipettor (be careful not to generate aerosols). [0450] d) Seal the diluted plate with a plastic seal. [0451] e) Centrifuge the diluted plate at 1000 rpm for 1 min. [0452] f) Seal any remaining multiplex PCR product that has not been diluted with a new aluminum foil seal. Place at −80° C.
B. QuARTS Assay on Multiplex-Amplified DNA:
[0453] 1. Thaw fish DNA diluent (20 ng/μL) and use to dilute plasmid calibrators (see, e.g., U.S. patent application Ser. No. 15/033,803, which is incorporated herein by reference) needed in the assay. Use the following table as a dilution guide:
TABLE-US-00012 Initial Plasmid Final plasmid μL of μL of total Concentration, Concentration, plasmid diluent volume, copies per μL copies per μL to add to add μL 1.00E+05 1.00E+04 5 45 50 1.00E+04 1.00E+03 5 45 50 1.00E+03 1.00E+02 5 45 50 1.00E+02 1.00E+01 5 45 50 [0454] 2. Prepare 10× triplex QuARTS oligo mix using the following table for markers A, B, and C (e.g., markers of interest, plus run control and internal controls such as β-actin or B3GALT6 (see, e.g., U.S. Pat. Appln. Ser. No. 62/364,082, incorporated herein by reference).
TABLE-US-00013 Concen- SEQ ID tration Oligo Sequence (5′-3′) NO: (μM) Marker A Forward NA 2 primer Marker A Reverse NA 2 primer Marker A probe- NA 5 Arm 1 Marker B Forward NA 2 primer Marker B Reverse NA 2 primer Marker B probe- NA 5 Arm 5 Marker C Forward NA 2 primer Marker C Reverse NA 2 primer Marker C probe- NA 5 Arm 3 Arm 1 HEX FRET /HEX/TCT/BHQ-1/ 171 5 AGCCGGTTTTCCGGCTG AGACCTCGGCG/3C6/ Arm 5 FAM FRET /FAM/TCT/BHQ-1/ 172 5 AGCCGGTTTTCCGGCTG AGACGTCCGTGG/3C6/ Arm 3 QUASAR-670 /Q670/TCT/BHQ-2/ 173 5 FRET AGCCGGTTTTCCGGCTGA GACTCCGCGTC/3C6/ dNTP mix 250
[0455] For example, the following might be used to detect bisulfate-treated β-actin, B3GALT6, and zebrafish RASSF1 markers:
TABLE-US-00014 Concen- Oligo SEQ ID tration Description Sequence (5′-3′) NO: (μM) ZF RASSF1 BT TGCGTATGGTGGGCGAG 160 2 Forward primer ZF RASSF1 BT CCTAATTTACACGTCAACCAATCGAA 161 2 Reverse primer ZF RASSF1 BT CCACGGACGGCGCGTGCGTTT/3C6/ 162 5 probe-Arm 5 B3GALT6 Forward GGTTTATTTTGGTTTTTTGAGTTTTCGG 8 2 primer B3GALT6 Reverse TCCAACCTACTATATTTACGCGAA 9 2 primer B3GALT6 probe- CGCCGAGGGCGGATTTAGGG/3C6/ 10 5 Arm 1 BTACT Forward GTGTTTGTTTTTTTGATTAGGTGTTTAAGA 168 2 primer BTACT Reverse CTTTACACCAACCTCATAACCTTATC 169 2 primer BTACT probe- GACGCGGAGATAGTGTTGTGG/3C6/ 170 5 Arm 3 Arm 1 HEX FRET /HEX/TCT/BHQ-1/ 171 5 AGCCGGTTTTCCGGCTGAGACCTCGGCG/3C6/ Arm 5 FAM FRET /FAM/TCT/BHQ-1/ 172 5 AGCCGGTTTTCCGGCTGAGACGTCCGTGG/3C6/ Arm 3 QUASAR- /Q670/TCT/BHQ-2/ 173 5 670 FRET AGCCGGTTTTCCGGCTGAGACTCCGCGTC/3C6/ dNTP mix 2500 [0456] 3. Prepare a QuARTS flap assay master mix using the following table:
TABLE-US-00015 Component Volume per reaction (μL) Water 15.5 10X Triplex Oligo Mix 3.0 20X QuARTS Enzyme mix 1.5 total volume 20.0 *20X enzyme mix contains 1 unit/μL GoTaq Hot start polymerase (Promega), 292 ng/μL Cleavase 2.0 flap endonuclease(Hologic). [0457] 4. Using a 96 well ABI plates, pipette 20 μL of QuARTS master mix into each well. [0458] 5. Add 10 μL of appropriate calibrators or diluted mPCR samples. [0459] 6. Seal plate with ABI clear plastic seals. [0460] 7. Centrifuge the plate using 3000 rpm for 1 minute. [0461] 8. Place plate in ABI thermal cycler programmed to run the following thermal protocol then start the instrument.
TABLE-US-00016 QuARTS Reaction Cycle: Signal Ramp Rate Number of Acqui- Stage Temp/Time (° C. per second) Cycles sition Pre-incubation 95° C./3 min 4.4 1 none Amplification 1 95° C./2 sec 4.4 5 none 63° C./30 sec 2.2 none 70° C./30 sec 4.4 none Amplification 2 95° C./20 sec 4.4 40 none 53° C./1 min 2.2 Yes 70° C./30 sec 4.4 none Cooling 40° C./30 sec 2.2 1 none
[0462] Aliquots of the diluted pre-amplified DNA (e.g., 10 μL) are used in a QuARTS PCR-flap assay, e.g., as described above. See also U.S. Patent Appl. Ser. No. 62/249,097, filed Oct. 30, 2015; Ser. No. 15/335,096, filed Oct. 26, 2016, and PCT/US16/58875, filed Oct. 26, 2016, each of which is incorporated herein by reference in its entirety, for all purposes.
Example 2
Selection and Testing of Methylation Markers for Colorectal Cancer Detection in Plasma
[0463] Reduced Representation Bisulfite Sequencing (RRBS) data was obtained on tissues from 19 patients with colon cancer, 19 patients with polyps, 19 healthy patients, and 19 healthy patients buffy coat extracted DNA.
[0464] After alignment to an in silico bisulfite-converted version of the human genome sequence, average methylation at each CpG island was computed for each sample type (i.e., tissue or buffy coat) and marker regions were selected based on the following criteria: [0465] Regions were selected to be 50 base pairs or longer. [0466] For QuARTS flap assay designs, regions were selected to have a minimum of 1 methylated CpG under each of: a) the probe region, b) the forward primer binding region, and c) the reverse primer binding region. For the forward and reverse primers, it is preferred that the methylated CpGs are close to the 3′-ends of the primers, but not at the 3′terminal nucleotide. Exemplary flap endonuclease assay oligonucleotides are shown in
[0470] Based on the criteria above, the markers ANKRD13B; CHST2; CNNM1; DOCK2; DTX1; FERMT3; FLI1; GRIN2D; JAM3; LRRC4; OPLAH; PDGFD; PKIA; PPP2R5C; QKI; SEP9; SFMBT2; SLC12A8; TBX15; TSPYL5; VAV3; ZDHHC1; ZNF304; ZNF568;and ZNF671 were selected and QuARTS flap assays were designed for them, as shown in
[0471] The 25 markers selected from the tissue screening results were triplexed with the assay for bisulfite-converted β-actin and used for testing DNA isolated from plasma samples as described above. CEA protein in the plasma was measured using a Luminex Magplex assay, per manufacturer protocol (Luminex Corp.) DNA from 2 mL of plasma samples (89 cancer and 95 normal) was extracted and eluted in 125 μL. 10 μL aliquots of the extracted DNA were used in a QuARTS assay to detect (3-actin and zebrafish synthetic targets. 80 μL aliquots of the DNA were bisulfite-converted as described in Example 1, and eluted in 70 μL.
[0472] A multiplex PCR reaction was performed on 50 μL aliquots of the bisulfite-converted DNA samples, using the forward and reverse primers for the targets shown in
[0473] Based on individual marker sensitivities, the following 12 methylation markers were selected for further analysis: VAV3, ZNF671, CHST2, FLI1, JAM3, SFMBT2, PDGFD, DTX1, TSPYL5, ZNF568, GRIN2D, and QKI.
[0474] All 12 markers were pre-amplified together using primers as shown for these markers in
TABLE-US-00017 CHST2 FLI1 BTACT VAV3 ZNF671 BTACT TSPYL5 ZNF568 BTACT JAM3 SFMBT2 BTACT PDGFD DTX1 BTACT GRIN2D QKI BTACT ZFRASSF1 B3GALT6 BTACT
[0475] In addition to the above, the CEA protein was measured for the same samples, as described above. The data and results are shown in
TABLE-US-00018 Marker Sensitivity @ 90% specificity ZNF671 49% TSPYL5 46% QKI 41% JAM3 40% DTX1 40% GRIN2D 38% ZNF568 37% CEA protein 36% FLI1 36% SFMBT2 35% PDGFD 35% CHST2 33% VAV3 31%
[0476] At 95% individual cutoff of the individual markers, the following final sensitivity was obtained for using the combined data set.
TABLE-US-00019 Cancer Stage Negative Positive Total # of samples Sensitivity I 14 7 21 33% II 7 18 25 72% III 7 17 24 71% IV 1 18 19 95% Overall 60 89 67%
The combined specificity of the assay was (88/95=92.6%).
[0477] Thus, the combination of these 12 markers plus CEA protein resulted in 67% sensitivity (88 of 95 cancers) for all of the cancer tissues tested, with 92.6% specificity. This panel of methylated DNA markers assayed on tissue achieves extremely high discrimination for all types of colon cancer while remaining negative in normal colon tissue. Assays for this panel of markers can be also be applied to blood or bodily fluid-based testing, and finds applications in, e.g., colon cancer screening.
Multiple Target Sequences Reporting to One Dye
[0478] The following experiments related to amplification flap cleavage assays that are configured to have multiple target-specific primary cleavage reactions report to a single FRET cassette, thereby producing fluorescence signal in a single dye channel. Different targets to be detected may be, for example, different markers or genes, different mutations, or different regions of a single marker or gene. Example 3 relates to detecting methylation of multiple different markers associated with cancer, e.g., colorectal cancer, using a single FRET cassette and dye channel, and Example 4 relates to detecting multiple regions within a single marker using a single FRET cassette and dye channel.
[0479] Reagents used in the following experiments:
TABLE-US-00020 Reagents Sequence (5′-3′) VAV3_877 Forward Primer TCGGAGTCGAGTTTAGCGC (SEQ ID NO: 108) VAV3_877 Reverse Primer v2 CGAAATCGAAAAAACAAAAACCGC (SEQ ID NO: 109) VAV3_877 Probe (arm 5) CCACGGACGCGGCGTTCGCGA/3C6/ (SEQ ID NO: 146) VAV3_11878 forward primer GAGTCGAGTTTTAGGTTATTCGGT (SEQ ID NO: 150) VAV3_11878 reverse primer CGTCGAACATAAAACCGTAAAAACAA (SEQ ID NO: 151) VAV3_11878 probe (arm 5) CCACGGACGATACGCGCAATA/3C6/ (SEQ ID NO: 152) SFM8T2_897 Forward Primer v5 GTCGTCGTTCGAGAGGGTA (SEQ ID NO: 88) SFMBT2_897 Forward Primer v4 GAACAAAAACGAACGAACGAACA (SEQ ID NO: 89) SFMBT2_897 Probe (arm 5) v5 CCACGGACGATCGGTTTCGTT/3C6/ (SEQ ID NO: 90) SFMBT2_897 probe (arm 1) CGCCGAGGATCGGTTTCGTT/3C6/ (SEQ ID NO: 141) SFMBT2_895 forward primer GCGACGTAGTCGTCGTTGT (SEQ ID NO: 144) SFMBT2_895 reverse primer CCAACGCGAAAAAAACGCG (SEQ ID NO: 145) SFMBT2_895 probe (arm 1) CGCCGAGGGAAAACGCGAAA/3C6/ (SEQ ID NO: 146) CHST2_7890 Forward Primer GTATAGCGCGATTTCGTAGCG (SEQ ID NO: 13) CHST2_7890 Reverse Primer AATTACCTACGCTATCCGCCC (SEQ ID NO: 14) CHST2_7890 Probe (arm 5) CCACGGACGCGAACATCCTCC/3C6/ (SEQ ID NO: 15) CHST2_7890 probe (arm 1) CGCCGAGGCGAACATCCTCC/3C6/ (SEQ ID NO: 175) CHST2_7889 forward primer CGAGTTCGGTAGTTGTACGTAGA (SEQ ID NO: 138) CHST2_7889 reverse primer CGAAATACGAACGCGAAATCTAAAACT (SEQ ID NO: 139) CHST2_7889 probe (arm 5) CCACGGACGTCGTCGATACCG/3C6/ (SEQ ID NO: 140) CHST2_7889 probe (arm 1) CGCCGAGG-TCGTCGATACCG/3C6/ (SEQ ID NO: 176) BTACT_FP65 Forward Primer GTGTTTGTTTTTTTGATTAGGTGTTTAAGA SEQ ID NO: 139 BTACT_RP65 Reverse Primer CTTTACACCAACCTCATAACCTTATC SEQ ID NO: 140 BTACT Probe A3 GACGCGGAGATAGTGTTGTGG/3C6/ SEQ ID NO: 141 Arm 1 FRET cassette HEX SEQ ID NO: 170 Arm 5 FRET cassette FAM SEQ ID NO: 171 Arm 1 FRET cassette QUASAR-670 SEQ ID NO: 174 Arm 3 FRET cassette QUASAR-670 SEQ ID NO: 173 ECOR1 digested pUC57 plasmid (Genscript) containing SFMBT2_897 insert ECOR1 digested pUC57 plasmid (Genscript) containing CHST2_7890 insert ECOR1 digested pUC57 plasmid (Genscript) containing VAV3 insert ECOR1 digested pUC57 plasmid (Genscript) containing BTACT insert VAV3/BTACT Biplexed plasmids, serially diluted from 1e+04 copies/pi SFMBT2_897/BTACT Biplexed plasmids, serially diluted from 1e+04 copies/μL CHST2_7890/BTACT Biplexed plasmids, serially diluted from 1e+04 copies/μL SFMBT2_897/VAV3/BTACT Biplexed plasmids, 1e+04 copies/μL CHST2_7890/VAV3/BTACT Biplexed plasmids, 1e+04 copies/μL CHST2_7890/SFMBT2_897/BTACT Biplexed plasmids, 1e+04 copies/μL VAV3/CHST2_7890/SFMBT2_897/BTACT Triplexed plasmids, 1e+04 copies/μL CHST2_7889 + 7890 Calibration curve dilution set (1e4−1e0 cp/ul) SFMBT2_895 + 897 Calibration curve dilution set (1e4−1e0 cp/ul) VAV3_877 + 11878 Calibration curve dilution set (1e4−1e0 cp/ul) VAV3/BTACT 10X Oligo Mix SFMBT2_897/BTACT 10X Oligo Mix CHST2_7890/BTACT 10X Oligo Mix VAV3/SFMBT2_897/CHST2_7890/BTACT 10X Oligo Mix VAV3/SFMBT2_897 (100 nM F. Primer)/CHST2_7890/BTACT 10X Oligo Mix VAV3/SFMBT2_897 (50 nM F. Primer)/CHST2_7890/BTACT 10X Oligo Mix VAV3/SFMBT2_897 (200 nM Probe)/CHST2_7890/BTACT 10X Oligo Mix VAV3/SFMBT2_897 (250 nM Probe)/CHST2_7890/BTACT 10X Oligo Mix VAV3/SFMBT2_897 (100 nM Probe)/CHST2_7890/BTACT 10X Oligo Mix VAV3 (400 nM Primers)/SFM8T2_897 (200 nM Probe)/CHST2_7890/BTACT 10X Oligo Mix VAV3 (750 nM Probe)/SFM8T2_897 (200 nM Probe)/CHST2_7890/BTACT 10X Oligo Mix 20X Enzyme mix, 1 U/μL Go Taq Hot Start polymerase (Promega), 292 ng/μL Cleavase 2.0 (Hologic) fDNA Diluent, 20 ng/μL fish DNA in 10 mM Tris, 0.1 mM EDTA fDNA Diluent, 20 ng/μL fish DNA in 10 mM Tris, 0.1 mM EDTA Mol. Biol. Grade water dNTPs, 25 mM (each dNTP)
Example 3
Multiple Markers Reporting to One Dye
[0480] As discussed above, in some embodiments it is desirable to have a larger number of markers in a single reaction, using a single FRET cassette and single dye channel. In developing a test for detecting multiple markers reporting to a single FRET cassette and single dye, markers having similar reaction efficiencies (i.e. that produce the same amount of detectable signal per target copy) were selected for combining in a multiplexed reaction reporting to a single dye channel. An advantage of combining detection assays that have the same or similar reaction efficiencies is that any individual calibrator for one of the assays may be used as a calibration standard for any and all of the efficiency-matched detection assays.
[0481] Three markers were selected for testing in a multiple marker/one dye system (SFMBT2, VAV3, and CHST2). These target DNAs were mixed in an oligonucleotide mix in which the assay oligonucleotides for all three markers were configured to report to the same FRET cassette and therefore to the same dye (FAM). The three disease-associated markers reporting to the FAM dye were combined in the same reaction with reagents to detect bisulfite-converted β-actin DNA (using a QUASAR 670 FRET cassette) as a control.
[0482] When testing on plasmid calibrators was performed, the data showed that using the multiple markers reporting to a single dye is an efficient approach that overcomes the need to run markers in separate wells.
Example 3.1
[0483] For QuARTS flap endonuclease assays for multiple different markers to be run in a multiplex reaction reporting to a single FRET cassette, the reaction efficiency for each individual marker was first analyzed so that the reactions could be balanced when combined in a multiplex configuration. Assays were run to determine the assay performance of three selected markers (VAV3, SFMBT2_897 and CHST2_7890) reporting to one dye (FAM), biplexed with bisulfite-converted β-actin (BTACT), which was configured to produce signal reporting to the Quasar 670 channel.
[0484] The assays were also configured to determine whether each marker would exhibit similar QuARTS assay performance (slopes/intercepts/Cps) when the three markers are reporting to the same channel (FAM).
[0485] An oligonucleotide mix comprising reagents to detect all three methylation markers reporting to a FAM FRET cassette was prepared. The oligonucleotide mix comprised reagents for detecting BTACT reporting to Quasar 670 as a control. This oligonucleotide mix was tested against plasmid targets containing individual plasmids comprising the marker target DNAs and BTACT DNA. Calculations were done to see whether a calibrator curve for one marker could be used to quantitate the other markers accurately. All reactions were done in replicates of 4.
Protocol:
[0486] Stock Plasmid dilutions comprising one marker plasmid and one BTACT control plasmid each (see Reagent Table, above) were prepared as follows, in a diluent of 20 ng/μL of fish DNA in 10 mM Tris, 0.1 mM EDTA:
TABLE-US-00021 SFMBT2_897/BTACT Copies in stock Copies final plasmid mix solution, /μL mixture/μL μL to add SFMBT2_897 Plasmid 1.00E+05 1.00E+04 50 BTACT Plasmid 1.00E+05 1.00E+04 50 Fish DNA Diluent NA NA 400 total volume NA NA 500
TABLE-US-00022 CHST2_7890/BTACT plasmid mix Ci, cp/μL Cf, cp/μL μL to add CHST2_7890 Plasmid 1.00E+05 1.00E+04 50 BTACT Plasmid 1.00E+05 1.00E+04 50 fDNA Diluent NA NA 400 total volume NA NA 500
TABLE-US-00023 VAV3/BTACT plasmid mix Ci, cp/μL Cf, cp/μL μL to add VAV3 Plasmid 1.00E+05 1.00E+04 50 BTACT Plasmid 1.00E+05 1.00E+04 50 fDNA Diluent NA NA 400 total volume NA NA 500
From the 3 plasmid mixtures prepared above, the following dilutions were prepared:
TABLE-US-00024 Cf, cp/μL Ci, cp/μL df μL Ci to add μL diluent total volume 1.00E+05 1.00E+04 10 50 450 500 1.00E+04 1.00E+03 10 50 450 500 1.00E+03 1.00E+02 10 50 450 500 1.00E+02 1.00E+01 10 50 450 500 1.00E+01 1.00E+00 10 50 450 500
10× Oligonucleotide mixes comprising assay oligonucleotides (primers, probes, FRET cassettes) and dNTPs were made as follows:
TABLE-US-00025 Final Reaction 10X oligo Mix Marker Reagent Concentration (μM) Concentration (μM) VAV3 VAV3 Forward Primer 0.2 2 VAV3 VAV3 Reverse Primer v2 0.2 2 VAV3 VAV3 Probe A5 0.5 5 SFMBT2_897 SFMBT2_897 Forward Primer v5 0.2 2 SFMBT2_897 SFMBT2_897 Forward Primer v4 0.2 2 SFMBT2_897 SFMBT2_897 Probe A5 v5 0.5 5 CHST2_7890 CHST2_7890 Forward Primer 0.2 2 CHST2_7890 CHST2_7890 Reverse Primer 0.2 2 CHST2_7890 CHST2_7890 Probe A5 0.5 5 Arm 5 FAM FRET Cassette 0.5 5 BTACT ACTB_BT_FP65 Forward Primer 0.2 2 BTACT ACTB_BT_RP65 Reverse Primer 0.2 2 BTACT ACTB BT Probe A3 0.5 5 Arm 3 QUASAR FRET cassette 0.5 5 dNTPs (each dNTP) 250 2500
QuARTS Flap Endonuclease Assay Reaction Set-Up:
[0487] Master mixes for the QuARTS amplification reactions are prepared as follows:
TABLE-US-00026 Master Mix Formulation: 96 well plate - μL vol of stock to μL vol for38 Reagent add per reaction reactions ddH2O 15.50 589 10X oligo Mix 3.00 114 20X Enzyme Mix 1.50 57 total volume master mix 20.0 760 use 20 ul master mix per well and add 10 ul sample for 96 well plate = 30 ul final rxn vol Sample* 10 20.0
Reactions were set up as follows: [0488] Pipette 20 μl of master mix into a 96-well QuARTS plate, using a multichannel pipette
[0489] Add 10 μl of a sample
[0490] Seal plate and centrifuge for 1 min. at 3000 rpm.
[0491] Run the plates using the following conditions on the LightCycler480, detecting on FAM, HEX and Quasar 670 channels: 465-510, 533-580, and 618-660 nm
TABLE-US-00027 QuARTS Assay Reaction Cycle: Signal Ramp Rate Number Acqui- Stage Temp/Time (° C. per second) of Cycles sition Pre-incubation 95° C./3 min 4.4 1 No Amplification 1 95° C./20 sec 4.4 5 No 63° C./30 sec 2.2 No 70° C./30 sec 4.4 No Amplification 2 95° C./20 sec 4.4 40 No 53° C./1 min 2.2 Yes 70° C./30 sec 4.4 No Cooling 40° C./30 sec 2.2 1 No
Results:
[0492] Strand counts using VAV3/BTACT Plasmid Calibrator Standard Curve:
TABLE-US-00028 VAV3/BTACT Plasmid Calibrator Standard Curve Slope −3.147684 Intercept 32.08568 Efficiency 107.8%
TABLE-US-00029 VAV3/BTACT Plasmid Calibrator Calibrator Strands/Rxn Average Cp Calculated Average Strands 200000 15.36 205,254 20000 18.66 18,432 2000 21.58 2,178 200 24.88 194
TABLE-US-00030 SFMBT2_897/BTACT Plasmid Calibrator Calibrator Strands/Rxn Average Cp Calculated Average Strands 200000 13.87 612,036 20000 17.17 54,780 2000 19.64 9,021 200 22.12 1,470
TABLE-US-00031 CHST2_7890/BTACT Plasmid Calibrator Calibrator Strands/Rxn Average Cp Calculated Average Strands 200000 15.17 235,836 20000 18.05 28,813 2000 20.39 5,200 200 23.03 752
Strand counts using SFMBT2_897/BTACT Plasmid Calibrator Standard Curve:
TABLE-US-00032 SFMBT2_897/BTACT Plasmid Calibrator Standard Curve Slope −2.720157 Intercept 28.53753 Efficiency 133.1%
TABLE-US-00033 VAV3/BTACT Plasmid Calibrator Calibrator Strands/Rxn Average Cp Calculated Average Strands 200000 15.36 69,636 20000 18.66 4,282 2000 21.58 362 200 24.88 22
TABLE-US-00034 SFMBT2_897/BTACT Plasmid Calibrator Calibrator Strands/Rxn Average Cp Calculated Average Strands 200000 13.87 246,543 20000 17.17 15,101 2000 19.64 1,873 200 22.12 229
TABLE-US-00035 CHST2_7890/BTACT Plasmid Calibrator Calibrator Strands/Rxn Average Cp Calculated Average Strands 200000 15.17 81,777 20000 18.05 7,180 2000 20.39 990 200 23.03 106
Strand counts using CHST2_7890//BTACT Plasmid Calibrator Standard Curve:
TABLE-US-00036 CHST2_7890/BTACT Plasmid Calibrator Standard Curve Slope −2.59121 Intercept 29.01007 Efficiency 143.2%
TABLE-US-00037 VAV3/BTACT Plasmid Calibrator Calibrator Strands/Rxn Average Cp Calculated Average Strands 200000 15.36 184,582 20000 18.66 9,878 2000 21.58 738 200 24.88 39
TABLE-US-00038 SFMBT2_897/BTACT Plasmid Calibrator Calibrator Strands/Rxn Average Cp Calculated Average Strands 200000 13.87 695,942 20000 17.17 37,096 2000 19.64 4,147 200 22.12 458
TABLE-US-00039 CHST2_7890/BTACT Plasmid Calibrator Calibrator Strands/Rxn Average Cp Calculated Average Strands 200000 15.17 218,505 20000 18.05 16,997 2000 20.39 2,123 200 23.03 203
[0493] These data show: [0494] No cross reactivity or background signal was generated when markers and controls were amplified and detected together; [0495] Cp values were similar for CHST2_7890 and VAV3; [0496] Cp values for SFMBT2_897 come up at an earlier cycle than CHST2_7890 and VAV3, showing that this is a faster QuARTS assay reaction; [0497] SFMBT2_897 calibrator and oligonucleotide mix combination underestimates the count of strands present for VAV3 and CHST2_7890 because of the faster SFMBT2_897 reaction; [0498] The CHST2_7890 calibrator provides a VAV3 calculation indicating assay performance equal to the CHST2_7890 assay reaction, but overestimates the amount of SFMBT2_897; [0499] The VAV3 calibrator provides a CHST2_7890 calculation indicating assay performance equal to the VAV3 assay reaction, but produces an overestimate of the amount of SFMBT2_897; and [0500] To balance the reactions, the QuARTS assay performance in detecting SFMBT2_897 needs to be reduced to match that of SFMBT2_897 and CHST2_7890 targets.
Experiment 3.2
[0501] The data above showed that the SFMBT2_897 assay reaction produced higher signal, indicating that the reaction is faster. For the purposes of multiplexing these markers, the SFMBT2_897 assay should be refined to match the efficiency of the slower assays, (i.e., to match the signal output of the VAV3 and CHST2_7890 assays). The following experiment tested whether modifying the concentration of forward primer of the SFMBT2_897 would achieve this.
Protocol:
[0502] Assays were run as described in Experiment 3.1, above. 10× oligonucleotide mixes were assembled comprising the components listed above, but having the SFMBT2_897 forward primer in amounts reduced to produce final assay concentrations of 200 nM (as in Experiment 3.1), 100 nM, or 50 nM. The concentration of all other assay primers was 200 nM in the final reaction mixtures, and the Light Cycler protocol was as described in Exp. 3.1.
[0503] Results showed that reducing the SFMBT2_897 forward primer concentration seemed to have no effect on the slope or intercept of the signal curve reflecting of PCR efficiency (data not shown). In addition, the Cp value did not change, thus the number of strands calculated for SFMBT2_897 did not match the calculated number of strands of the other marker targets.
Experiment 3.3:
[0504] The following experiment tested whether modifying the concentration of the SFMBT2_897 probe would reduce the efficiency of the SFMBT2_897 assay, to match the signal output of the CHST2_7890 and VAV3 amplification reactions.
[0505] Assays were run as described above in Experiment 3.1. 10× oligonucleotide mixes were assembled comprising the components listed above, but having the SFMBT2_897 probe oligonucleotide in amounts to produce final assay concentrations of 250 nM or 100 nM, with the CHST2_7890 and VAV3 probes present at 500 nM (as described in Experiment 3.1). The Light Cycler protocol was as described for Experiment 3.1.
Results:
[0506] Strand counts using VAV3/BTACT Plasmid Calibrator Standard Curve:
TABLE-US-00040 VAV3/BTACT Plasmid Calibrator Standard Curve Slope −3.12175 Intercept 31.55241 Efficiency 109.1%
TABLE-US-00041 VAV3/BTACT Plasmid Calibrator Calibrator Strands/Rxn Average Cp Average Strands 200000 14.95 207,537 20000 18.24 18,377 2000 21.17 2,120 200 24.38 198
TABLE-US-00042 SFMBT2_897/BTACT Plasmid Calibrator Calibrator Strands/Rxn Average Cp Average Strands 200000 15.30 161,043 20000 18.50 15,172 2000 21.20 2,065 200 24.01 260
TABLE-US-00043 CHST2_7890/BTACT Plasmid Calibrator Calibrator Strands/Rxn Average Cp Average Strands 200000 14.83 226,551 20000 18.08 20,670 2000 21.05 2,318 200 24.30 210
Strand counts using SFMBT2 897/BTACT Plasmid Calibrator Standard Curve:
TABLE-US-00044 SFMBT2_897/BTACT Plasmid Calibrator Standard Curve Slope −2.885069564 Intercept 30.72006211 Efficiency 122.1%
TABLE-US-00045 VAV3/BTACT Plasmid Calibrator Calibrator Strands/Rxn Average Cp Average Strands 200000 14.95 291,595 20000 18.24 21,164 2000 21.17 2,045 200 24.38 157
TABLE-US-00046 SFMBT2_897/BTACT Plasmid Calibrator Calibrator Strands/Rxn Average Cp Average Strands 200000 15.30 221,611 20000 18.50 17,200 2000 21.20 1,988 200 24.01 211
TABLE-US-00047 CHST2_7890/BTACT Plasmid Calibrator Calibrator Strands/Rxn Average Cp Average Strands 200000 14.83 320,609 20000 18.08 24,036 2000 21.05 2,252 200 24.30 168
Strand counts using CHST2 7890/BTACT Plasmid Calibrator Standard Curve:
TABLE-US-00048 CHST2_7890/BTACT Plasmid Calibrator Standard Curve Slope −3.136297934 Intercept 31.48713495 Efficiency 108.4%
TABLE-US-00049 VAV3/BTACT Plasmid Calibrator Calibrator Strands/Rxn Average Cp Average Strands 200000 14.95 186,901 20000 18.24 16,737 2000 21.17 1,950 200 24.38 184
TABLE-US-00050 SFMBT2_897/BTACT Plasmid Calibrator Calibrator Strands/Rxn Average Cp Average Strands 200000 15.30 145,201 20000 18.50 13,830 2000 21.20 1,900 200 24.01 242
TABLE-US-00051 CHST2_7890/BTACT Plasmid Calibrator Calibrator Strands/Rxn Average Cp Average Strands 200000 14.83 203,942 20000 18.08 18,815 2000 21.05 2,131 200 24.30 196
Results:
[0507] These data show that adjusting the probe concentrations lower caused the intercept to increase slightly and the PCR % efficiency to increase slightly. The Cp values also increased and therefore the calculation of strand counts gave values similar to the results calculated using the other markers as calibration standards.
[0508] The 250 nM SFMBT2_897 probe concentration made the three markers produce similar calculated strand counts, with the SFMBT2_897 strand count values being slightly higher than the other markers. The 50 nM concentration of the probe produced calculated results that slightly underestimated strand counts, but gave some improvement. Therefore, a SFMBT2_897 probe concentration of 200 nM probe was selected for further testing.
Experiment 3.4:
[0509] This experiment tested the standard conditions described in Experiment 3.1 (all marker probes used at 500 nM) against the 10× oligonucleotide mix that provides 200 nM SFMBT2_897 probe, with the other probes at 500 nM. This experiment will also determine whether there is an additive effect of having multiple targets in single reaction that all report signal using the same FRET cassette and dye. Single, biplex and triplex combinations of the plasmid targets were used, with all target combinations including the BTACT target as a control.
Plasmid Dilutions for One Marker Plus Control:
[0510] For reactions with a single marker plasmid plus a BTACT control plasmid, mixtures were made containing 1.00E+04 copies/μL of each plasmid in a diluent of 20 ng/μL fish DNA in 10 mM Tris, 0.1 mM EDTA. The marker plasmids are described the Reagent Table in Experiment 3.1. The targets in the plasmid mixtures were as follows: [0511] SFMBT2_897/BTACT [0512] CHST2_7890/BTACT [0513] VAV3/BTACT
Plasmid Dilutions for Two Markers Plus Control:
[0514] For reactions with two marker plasmids plus a BTACT control plasmid, mixtures were made containing 1.00E+04 copies/μL of each plasmid in a diluent of 20 ng/μL fish DNA in 10 mM Tris, 0.1 mM EDTA. The targets in the plasmid mixtures were as follows: [0515] SFMBT2_897/VAV3/BTACT [0516] CHST2_7890/VAV3/BTACT [0517] CHST2_7890/SFMBT2_897/BTACT
Plasmid Dilutions for Three Markers Plus Control:
[0518] For reactions with three marker plasmids plus a BTACT control plasmid, a mixture was made containing 1.00E+04 copies/μL of each plasmid in a diluent of 20 ng/μL fish DNA in 10 mM Tris, 0.1 mM EDTA. The plasmid mixture was as follows: [0519] VAV3/CHST2_7890/SFMBT2_897/BTACT
[0520] Each of the plasmid mixtures was used to prepare solutions having 1.00E+03 copies/μL and 1.00E+02 copies/μL of each of the plasmids, in fish DNA diluent.
[0521] A 10× oligonucleotide mix containing the primers and probes for all 3 markers and for the BTACT control plasmid, and having concentrations of probes to produce 500 nM probe in each QuARTS assay reaction except for the SFMBT2_897 probe, which was provided in an amount to produce a concentration of 200 nM SFMBT2_897 probe in each reaction. The QuARTS assay components were mixed and the assay was performed on a Light Cycler as described in Experiment 3.1.
Results:
Strand Counts Using VAV3/BTACT Plasmid Calibrator Standard Curve:
[0522]
TABLE-US-00052 VAV3/BTACT Plasmid Calibrator Standard Curve Slope −3.164 Intercept 31.977 % Efficiency 107%
Strand Counts for Single Markers, Plus Control Plasmids:
[0523]
TABLE-US-00053 VAV3/BTACT Plasmid Calibrator Calibrator Strands/Rxn Average Cp Average Strands 200000 15.23 195,918 20000 18.39 19,763 2000 21.42 2,179 200 24.77 190
TABLE-US-00054 SFMBT2_897/BTACT Plasmid Calibrator Calibrator Strands/Rxn Average Cp Average Strands 200000 15.08 219,449 20000 18.00 26,151 2000 20.51 4,223 200 23.27 564
TABLE-US-00055 CHST2_7890/BTACT Plasmid Calibrator Calibrator Strands/Rxn Average Cp Average Strands 200000 15.05 224,915 20000 17.89 28,288 2000 20.41 4,532 200 23.02 680
Strand Counts for Two Markers, Plus Control Plasmids:
[0524]
TABLE-US-00056 VAV3/CHST2_7890/BTACT Plasmid Calibrator Calibrator Strands/Rxn Average Cp Average Strands 200000 14.19 417,946 20000 17.33 42,756 2000 20.09 5,716 200 22.89 743
TABLE-US-00057 Additive Expected Strands VAV3/CHST2 Strands 420,833 48,051 6,711 870
TABLE-US-00058 VAV3/SFMBT2_897/BTACT Plasmid Calibrator Calibrator Strands/Rxn Average Cp Average Strands 200000 14.16 429,911 20000 17.27 44,611 2000 20.08 5,744 200 22.75 823
TABLE-US-00059 VAV3/SFMBT2 Strands 415,367 45,914 6,401 754
TABLE-US-00060 CHST2_7890/SFMBT2_897/BTACT Plasmid Calibrator Calibrator Strands/Rxn Average Cp Average Strands 200000 13.99 485,917 20000 17.17 47,863 2000 19.80 7,068 200 22.34 1,113
TABLE-US-00061 Additive Expected Strands CHST2/SFMBT2 Strands 444,364 54,439 8,755 1,244
Strand Counts for Three Markers, Plus Control Plasmids:
[0525]
TABLE-US-00062 VAV3/CHST2_7890/SFMBT2_897/BTACT Plasmid Calibrator Calibrator Strands/Rxn Average Cp Average Strands 200000 13.44 722,434 20000 16.54 76,045 2000 19.21 10,847 200 21.85 1,589
TABLE-US-00063 Additive Expected Strands VAV3/CHST2/SFMBT2 Strands 640,282 74,202 10,934 1,434
Strand Counts Using SFMBT2 897/BTACT Plasmid Calibrator Standard Curve:
[0526]
TABLE-US-00064 SFMBT2_897/BTACT Plasmid Calibrator Standard Curve Slope −2.705 Intercept 29.369 % Efficiency 134%
Strand Counts for Single Markers, Plus Control Plasmids:
[0527]
TABLE-US-00065 VAV3/BTACT Plasmid Calibrator Calibrator Strands/Rxn Average Cp Average Strands 200000 15.12 185,009 20000 18.26 12,793 2000 21.28 980 200 24.57 60
TABLE-US-00066 SFMBT2_897/BTACT Plasmid Calibrator Calibrator Strands/Rxn Average Cp Average Strands 200000 14.98 209,356 20000 17.84 18,236 2000 20.38 2,097 200 23.15 200
TABLE-US-00067 CHST2_7890/BTACT Plasmid Calibrator Calibrator Strands/Rxn Average Cp Average Strands 200000 14.89 225,658 20000 17.72 20,240 2000 20.29 2,275 200 22.86 256
Strand Counts for Two Markers, Plus Control Plasmids:
[0528]
TABLE-US-00068 VAV3/CHST2_7890/BTACT Plasmid Calibrator Calibrator Strands/Rxn Average Cp Average Strands 200000 14.09 446,402 20000 17.22 31,148 2000 19.99 2,926 200 22.72 288
TABLE-US-00069 Additive Expected Strands VAV3/CHST2 Strands 410,667 33,033 3,255 315
TABLE-US-00070 VAV3/SFMBT2_897/BTACT Plasmid Calibrator Calibrator Strands/Rxn Average Cp Average Strands 200000 14.05 460,951 20000 17.17 32,470 2000 19.97 2,983 200 22.60 319
TABLE-US-00071 Additive Expected Strands VAV3/SFMBT2 Strands 394,365 31,029 3,077 260
TABLE-US-00072 CHST2_7890/SFMBT2_897/BTACT Plasmid Calibrator Calibrator Strands/Rxn Average Cp Average Strands 200000 13.84 552,761 20000 17.08 34,990 2000 19.65 3,908 200 22.22 439
TABLE-US-00073 Additive Expected Strands CHST2/SFMBT2 Strands 435,015 38,476 4,372 455
Strand Counts for Three Markers, Plus Control Plasmids:
[0529]
TABLE-US-00074 VAV3/CHST2_7890/SFMBT2_897/BTACT Plasmid Calibrator Calibrator Strands/Rxn Average Cp Average Strands 200000 13.31 863,327 20000 16.40 62,334 2000 19.12 6,171 200 21.69 692
TABLE-US-00075 Additive Expected Strands VAV3/CHST2/SFMBT2 Strands 620,024 51,269 5,353 515
Strand Counts Using CHST2 7890/BTACT Plasmid Calibrator Standard Curve:
[0530]
TABLE-US-00076 CHST2_7890/BTACT Plasmid Calibrator Standard Curve Slope −2.644 Intercept 29.02 % Efficiency 139%
Strand Counts for Single Markers, Plus Control Plasmids:
[0531]
TABLE-US-00077 VAV3/BTACT Plasmid Calibrator Calibrator Strands/Rxn Average Cp Average Strands 200000 15.14 177,035 20000 18.28 11,490 2000 21.30 828 200 24.60 47
TABLE-US-00078 SFMBT2_897/BTACT Plasmid Calibrator Calibrator Strands/Rxn Average Cp Average Strands 200000 15.01 199,391 20000 17.88 16,382 2000 20.41 1,808 200 23.17 162
TABLE-US-00079 CHST2_7890/BTACT Plasmid Calibrator Calibrator Strands/Rxn Average Cp Average Strands 200000 14.93 213,922 20000 17.75 18,236 2000 20.31 1,966 200 22.89 209
Strand Counts for Two Markers, Plus Control Plasmids:
[0532]
TABLE-US-00080 VAV3/CHST2_7890/BTACT Plasmid Calibrator Calibrator Strands/Rxn Average Cp Average Strands 200000 14.11 436,308 20000 17.24 28,620 2000 20.02 2,542 200 22.75 235
TABLE-US-00081 Additive Expected Strands VAV3/CHST2 Strands 390,956 29,726 2,794 255
TABLE-US-00082 VAV3/SFMBT2_897/BTACT Plasmid Calibrator Calibrator Strands/Rxn Average Cp Average Strands 200000 14.07 448,748 20000 17.18 29,908 2000 19.99 2,596 200 22.62 262
TABLE-US-00083 Additive Expected Strands VAV3/SFMBT2 Strands 376,425 27,872 2,637 209
TABLE-US-00084 CHST2_7890/SFMBT2_897/BTACT Plasmid Calibrator Calibrator Strands/Rxn Average Cp Average Strands 200000 13.87 535,611 20000 17.10 32,329 2000 19.68 3,405 200 22.24 365
TABLE-US-00085 Additive Expected Strands CHST2/SFMBT2 Strands 413,312 34,618 3,774 371
Strand Counts for Three Markers, Plus Control Plasmids:
[0533]
TABLE-US-00086 VAV3/CHST2_7890/SFMBT2_897/BTACT Plasmid Calibrator Calibrator Strands/Rxn Average Cp Average Strands 200000 13.34 853,557 20000 16.42 57,973 2000 19.13 5,479 200 21.72 578
TABLE-US-00087 Additive Expected Strands VAV3/CHST2/SFMBT2 Strands 590,347 46,108 4,602 418
[0534] These data confirm the results shown in Experiment 3.2, showing that adjustment of the SFMBT2_897 probe concentration down to 200 nM aligns the efficiency of this assay reaction with the efficiencies of the reactions for detecting VAV3 and CHST2_7890. They also show that when multiple targets in a reaction report signal to the same FRET cassette and dye channel, the result shows an additive effect on the amount of fluorescence signal produced in the reaction. Surprisingly, no increase in background or cross reactivity is observed. The data further show that, when the VAV3 dilution series is used as the calibration standard, the strand counts of SFMBT2_897 and CHST2_7890 DNAs calculated from the data at the low end of the curve are overestimates of the amounts actually added to these reactions. The VAV3 amplification curves are more variable at the lower end of the standard curve, causing overestimates of strand counts for the other markers.
Experiment 3.5:
[0535] In this experiment, the probe and primer concentrations of the VAV3 marker were adjusted to reduce overestimation of low-level targets when the VAV3 calibrator curve is used for as the reference curve for calculating DNA concentrations.
[0536] For the VAV3 calibration curve, a dilution series having the VAV3 plasmid combined with the BTACT plasmid was as described in Experiment 3.4. Plasmid dilutions having all three markers plus the BTACT control were used.
[0537] 10× oligonucleotide mixes containing the primers and probes for all 3 markers and for the BTACT control plasmid were made, having primers and probes provided to produce the concentrations shown below: [0538] 1. VAV3 (400 nM Primers)/SFMBT2_897 (200 nM Probe)ICHST2_7890/BTACT [0539] 2. VAV3 (750 nM Probe)/SFMBT2_897 (200 nM Probe)ICHST2_7890/BTACT [0540] 3. VAV3/SFMBT2_897 (200 nM Probe)ICHST2_7890/BTACT
[0541] With the exception of the variations in primer and probe concentrations indicated above, the final reaction concentrations of all other primers was 200nM each primer, and of all other probes was 500 nM for each probe. The QuARTS assay reactions were mixed and the assay was performed on a Light Cycler as described in Experiment 3.1.
[0542] Both condition modifications improve the slope of the low calibrator in the VAV3 assay, but these conditions do produce signal that is the same as the single marker oligonucleotide mix. The data show that the single marker mix does not have the issue of over-estimation of strand counts at the low end of the standard curve. Based on these data, 400 nM each VAV3 primer with 500 nM probe was selected for investigation of testing the assay on clinical samples.
Experiment 3.6
[0543] This experiment tests the multiple marker/1 dye sample configuration on human clinical plasma samples. Plasma samples were previously tested using the standard one marker:one dye method, as described in Example 2. The same samples were re-tested using an oligonucleotide mix that has VAV3, SFMBT2_897 and CHST2_7890 reporting to one fluorescent channel (FAM).
[0544] In Example 2, DNA was prepared from a series of plasma samples and the target DNAs were amplified QuARTS assays. Amplicon material produced in Example 2 from the samples 105-120 (see
[0545] The single marker/BTACT plasmid calibrator dilutions were as described in Experiment 3.1. A 10× oligonucleotide mix comprising primers and probes for all three markers and for the BTACT control DNA, and configured to produce reactions having the 400 nM each VAV3 primer and 200 nM SFMBT2_897 probe, and having all other primers at 200 nM and all other probes at 500 nM, as described in Experiment 3.5, was used. The QuARTS assays were mixed and the assay was performed on a Light Cycler as described in Experiment 3.1. Each reaction was run in duplicate.
[0546] The counts of target strands for each of the samples were separately calculated using each of the three different marker calibration curves. The resulting strand count values were similar, regardless of which standard curve was used. In addition, the strand counts for each of the samples using the single-dye configuration were close to the combined strand counts for this set of markers measured in Example 2 using separate FRET cassettes and dye channels. Further, samples that had zero strands detected, i.e., that produced no signal in the Example 2 experiment, stayed at zero when using the multiple markers reporting to one dye configuration, showing that background signal is not increased when the multiplexed reactions report to a FRET cassette/single dye channel.
[0547] These results show that using multiple different target sites, e.g., multiple different marker genes, reporting to one FRET cassette and the same dye can increase the sensitivity of detection, and also show that multiplex combinations need not be limited by the number of available dye channels for signal detection. In addition, the use of this approach is not limited to having a single dye per reaction well. For example, an assay could be configured having three (or more) markers reporting to a first dye (e.g., FAM) and three (or more) markers reporting to a second dye (e.g., HEX), doubling the number of markers that may be tested in a single reaction, on a single preparation of nucleic acid sample. Additional dye channels may be used for additional sets of markers and/or for one or more internal control targets.
Example 4
Multiple Regions of a Marker Reporting to One Dye
[0548] For three methylation markers VAV3 (877), SFMBT2 (897), and CHST2 (7890), that showed low to zero strand counts in normal plasma using the methods described herein above, additional QuARTS assay oligonucleotide sets targeting other regions within each of the markers were designed and tested, to see whether detecting additional regions of the markers in the same reaction and reporting to the same dye channel would increase the signal-to-noise ratio for each marker, thus increasing the sensitivity of the assay, e.g., in detection of cancer.
[0549] For each of these markers, two different regions determined by RRBS to have differential methylation between cancer tissue and normal tissue were identified. Those regions are: [0550] VAV3 region 877: chr1: 108507618-108507675 [0551] VAV3 region 11878: chr1: 108507406-108507499 [0552] SFMBT2 region 895: chr10: 7452337-7452406 [0553] SFMBT2 region 897: chr10: 7452865-7452922 [0554] CHST2 region 7890: chr3:142838847-142839000 [0555] CHST2 region 7889: chr3: 142838300-142838388
Experiment 4.1
[0556] The CHST2 regions (7889 and 7890) reporting to the HEX dye were tested both individually and in a combined reaction to evaluate any synergy between the two regions when combined. A calibrator plasmid containing CHST2 insert was diluted as described in Experiment 3.1 to produce a dilution series of 1E4 to 1E0 copies per μL. For individual detection of region 7889, assay reactions contained the forward and reverse primers and the arm 1 probe for CHST2_7889, the Arm 1 HEX FRET cassette, and the primers and the arm 3 probe for the BTACT control, along with the Arm 3 Quasar 670 FRET cassette. For individual detection of region 7890, assay reactions contained the forward and reverse primers and the arm 1 probe for CHST2_7890, the Arm 1 HEX FRET cassette, and the primers and arm 3 probe for the BTACT control, along with the Arm 3 Quasar 670 FRET cassette. The combined reaction contained the complete set of arm 1 probes and primers for both CHST2_7889 and 7890, along with the oligonucleotides for detection of BTACT and the same two FRET cassettes.
[0557] 10× oligonucleotide mixes contained the primers and probes at concentrations to produce 500 nM of each probe and 200 nM of each primer in each QuARTS assay reaction. The QuARTS assay components were mixed and the assay was performed on a Light Cycler as described in Experiment 3.1.
[0558] It was found that in the combined reaction, having these two regions report to the same dye using a single FRET cassette did not result in any increase in signal. The CHST2_7889 amplification was substantially more efficient and appeared to dominate the resulting signal, suggesting that the different reactions should be modified to have more similar efficiencies, as discussed above in Example 3.
[0559] Experiment 4.2 Experiments were conducted to determine what probe concentration should be used for each pair of regions in each marker {CHST2 (7889 and 7890), SFMBT2 (895 and 897) and VAV3 (877 and 11878)} to balance the reaction kinetics between the different regions. 10× oligonucleotide mixes were made to provide the following mixtures of assay oligonucleotides at the indicated final concentrations:
TABLE-US-00088 CHST2_7890A (1xProbe) Final 1X Marker Oligo Conditions (μM) CHST2_7890 CHST2_7890 FP 0.2 CHST2_7890 CHST2_7890 RP 0.2 CHST2_7890 Probe A5 CHST2_7890 0.5 A5 FAM FRET 0.5 BTACT ACTB_BT_FP65 0.2 BTACT ACTB_BT_RP65 0.2 BTACT ACTB BT Pb A3 0.5 A3 Quasar670 FRET 0.5 dNTPs 250 water NA
TABLE-US-00089 CHST2_7889A (1xProbe) Final 1X Marker Oligo Conditions (μM) CHST2_7889 F Primer CHST2_7889 0.2 CHST2_7889 R Primer CHST2_7889 0.2 CHST2_7889 Probe A5 CHST2_7889 0.5 A5 FAM FRET 0.5 BTACT ACTB_BT_FP65 0.2 BTACT ACTB_BT_RP65 0.2 BTACT ACTB BT Pb A3 0.5 A3 Quasar670 FRET 0.5 dNTPs 250 water NA
TABLE-US-00090 CHST2_7890A (3xProbe) Final 1X Marker Oligo Conditions (μM) CHST2_7890 CHST2_7890 FP 0.2 CHST2_7890 RP 0.2 Probe A5 CHST2_7890 1.5 A5 FAM FRET 0.5 ACTB ACTB_BT_FP65 0.2 ACTB_BT_RP65 0.2 ACTB BT Pb A3 0.5 A3 Quasar670 FRET 0.5 dNTPs 250 water NA
TABLE-US-00091 CHST2_7890A (2xProbe) Final 1X Marker Oligo Conditions (μM) CHST2_7890 CHST2_7890 FP 0.2 CHST2_7890 RP 0.2 Probe A5 CHST2_7890 1 A5 FAM FRET 0.5 ACTB ACTB_BT_FP65 0.2 ACTB_BT_RP65 0.2 ACTB BT Pb A3 0.5 A3 Quasar670 FRET 0.5 dNTPs 250 water NA
TABLE-US-00092 CHST2_7889A (0.5xProbe) Final 1X Marker Oligo Conditions (μM) CHST2_7889 F Primer CHST2_7889 0.2 R Primer CHST2_7889 0.2 Probe A5 CHST2_7889 0.25 A5 FAM FRET 0.5 ACTB ACTB_BT_FP65 0.2 ACTB_BT_RP65 0.2 ACTB BT Pb A3 0.5 A3 Quasar670 FRET 0.5 dNTPs 250 water NA
TABLE-US-00093 SFMBT2_895A (1xProbe) Final 1X Marker Oligo Conditions (μM) SFMBT2_895v2 FP SFMBT2_895_v2 0.2 RP SFMBT2_895_v2 0.2 Prb A1 SFMBT2_895 v2 0.5 A1 HEX FRET 0.5 ACTB ACTB_BT_FP65 0.2 ACTB_BT_RP65 0.2 ACTB BT Pb A3 0.5 A3 Quasar670 FRET 0.5 dNTPs 250 water NA
TABLE-US-00094 SFMBT2_897/BTACT SFMBT2_897A (1xProbe) Final 1X Marker Oligo Conditions (μM) SFMBT2_897 F Primer SFMBT2_897v5 0.2 R Primer SFMBT2_897v4 0.2 Probe A1 SFMBT2_897v5 0.5 A1 HEX FRET 0.5 ACTB ACTB_BT_FP65 0.2 ACTB_BT_RP65 0.2 ACTB BT Pb A3 0.5 A3 Quasar670 FRET 0.5 dNTPs 250 water NA
TABLE-US-00095 SFMBT2_897A (0.5xProbe) Final 1X Marker Oligo Conditions (μM) SFMBT2_897 F Primer SFMBT2_897v5 0.2 R Primer SFMBT2_897v4 0.2 Probe A1 SFMBT2_897v5 0.25 A1 HEX FRET 0.5 ACTB ACTB_BT_FP65 0.2 ACTB_BT_RP65 0.2 ACTB BT Pb A3 0.5 A3 Quasar670 FRET 0.5 dNTPs 250 water NA
TABLE-US-00096 SFMBT2_895A (2xProbe) Final 1X Marker Oligo Conditions (μM) SFMBT2_895v2 FP SFMBT2_895_v2 0.2 RP SFMBT2_895_v2 0.2 Prb A1 SFMBT2_895 v2 1 A1 HEX FRET 0.5 ACTB ACTB_BT_FP65 0.2 ACTB_BT_RP65 0.2 ACTB BT Pb A3 0.5 A3 Quasar670 FRET 0.5 dNTPs 250 water NA
TABLE-US-00097 SFMBT2_897A (0.25xProbe) Final 1X Marker Oligo Conditions (μM) SFMBT2_897 F Primer SFMBT2_897v5 0.2 R Primer SFMBT2_897v4 0.2 Probe A1 SFMBT2_897v5 0.125 A1 HEX FRET 0.5 ACTB ACTB_BT_FP65 0.2 ACTB_BT_RP65 0.2 ACTB BT Pb A3 0.5 A3 Quasar670 FRET 0.5 dNTPs 250 water NA
TABLE-US-00098 VAV3 877A (1xProbe) Final 1X Marker Oligo Conditions (μM) VAV3_877 F Primer VAV3 0.2 VAV3_877 R Primer VAV3 ver 2 0.2 VAV3_877 Probe A5 VAV3 0.5 A5 FAM FRET 0.5 BTACT ACTB_BT_FP65 0.2 BTACT ACTB_BT_RP65 0.2 BTACT ACTB BT Pb A3 0.5 A3 Quasar670 FRET 0.5 dNTPs 250 water NA
TABLE-US-00099 VAV3_878A (1xProbe) Final 1X Marker Oligo Conditions (μM) VAV3_11878 F Primer VAV3_11878 0.2 VAV3_11878 R Primer VAV3_11878 0.2 VAV3_11878 Probe A5 VAV3_11878 0.5 A5 FAM FRET 0.5 BTACT ACTB_BT_FP65 0.2 BTACT ACTB_BT_RP65 0.2 BTACT ACTB BT Pb A3 0.5 A3 Quasar670 FRET 0.5 dNTPs 250 water NA
TABLE-US-00100 VAV3_877A(1.5xProbe) Final 1X Marker Oligo Conditions (μM) VAV3_877 F Primer VAV3 0.2 VAV3_877 R Primer VAV3 ver 2 0.2 VAV3_877 Probe A5 VAV3 0.75 A5 FAM FRET 0.5 BTACT ACTB_BT_FP65 0.2 BTACT ACTB_BT_RP65 0.2 BTACT ACTB BT Pb A3 0.5 A3 Quasar670 FRET 0.5 dNTPs 250 water NA
TABLE-US-00101 VAV3_877A(2xProbe) Final 1X Marker Oligo Conditions (μM) VAV3_877 F Primer VAV3 0.2 VAV3_877 R Primer VAV3 ver 2 0.2 VAV3_877 Probe A5 VAV3 1 A5 FAM FRET 0.5 BTACT ACTB_BT_FP65 0.2 BTACT ACTB_BT_RP65 0.2 BTACT ACTB BT Pb A3 0.5 A3 Quasar670 FRET 0.5 dNTPs 250 water NA
TABLE-US-00102 VAV3_878(0.75xProbe) Final 1X Marker Oligo Conditions (μM) VAV3_11878 F Primer VAV3_11878 0.2 VAV3_11878 R Primer VAV3_11878 0.2 VAV3_11878 Probe A5 VAV3_11878 0.375 A5 FAM FRET 0.5 BTACT ACTB_BT_FP65 0.2 BTACT ACTB_BT_RP65 0.2 BTACT ACTB BT Pb A3 0.5 A3 Quasar670 FRET 0.5 dNTPs 250 water NA
TABLE-US-00103 VAV3_878(0.5xProbe) Final 1X Marker Oligo Conditions (μM) VAV3_11878 F Primer VAV3_11878 0.2 VAV3_11878 R Primer VAV3_11878 0.2 VAV3_11878 Probe A5 VAV3_11878 0.25 A5 FAM FRET 0.5 BTACT ACTB_BT_FP65 0.2 BTACT ACTB_BT_RP65 0.2 BTACT ACTB BT Pb A3 0.5 A3 Quasar670 FRET 0.5 dNTPs 250 water NA
[0560] The QuARTS assay components were mixed and the assays were performed on a Light Cycler as described in Experiment 3.1 The average Cp values achieved under the different reaction conditions are as follows:
TABLE-US-00104 Average Cp Values Plasmid CHST2_7890 CHST2_7890 CHST2_7890 CHST2_7889 CHST2_7889 Calibrator 1XProbe 2XProbe 3XProbe 1XProbe 0.5XProbe Concentration Conc. Conc. Conc. Conc. Conc. 200,000 15.4 14.8 14.2 13.9 14.7 20,000 18.6 18.0 17.4 17.1 18.1 2,000 22.1 21.4 21.0 20.6 21.2 200 25.2 24.9 24.2 24.0 24.7 20 28.7 27.8 27.0 27.2 28.1
TABLE-US-00105 Average Cp Values Plasmid SFMBT2_895 SFMBT2_895 SFMBT2_897 SFMBT2_897 SFMBT2_897 Calibrator 1XProbe 2XProbe 1XProbe 0.25XProbe 0.5XProbe Concentration Conc. Conc. Conc. Conc. Conc. 200,000 16.5 15.2 14.5 16.7 16.0 20,000 20.1 19.1 18.0 20.1 19.3 2,000 23.4 22.6 21.3 23.3 22.5 200 27.1 26.1 24.4 26.5 25.8 20 30.2 29.4 27.4 30.6 29.3
TABLE-US-00106 Average CP Values Plasmid VAV3_877 VAV3_877 VAV3_877 VAV3_11878 VAV3_11878 VAV3_11878 Calibrator 1XProbe 1.5XProbe 2XProbe 1XProbe 0.75XProbe 0.5XProbe Concentration Conc. Conc. Conc. Conc. Conc. Conc. 200,000 15.0 14.5 14.2 13.4 13.8 14.3 20,000 18.2 17.9 17.6 16.9 17.0 17.8 2,000 21.6 21.3 21.0 20.3 20.3 21.1 200 25.2 24.4 24.2 23.4 23.8 24.2 20 27.9 28.1 27.3 26.7 27.5 27.5
[0561] These data show that by varying the probe concentrations, it is possible to adjust the Cp values for the individual assays to the point where each of the five points of the calibration curve are within <1 Cp for each of the two regions for each marker. For the markers tested, use of the following probe concentrations in the QuARTS assay reactions produced balanced reaction efficiencies for the sets of target regions:
TABLE-US-00107 Marker [Probe]-A5-FAM [Probe]-A1-HEX SFMBT2_895 — 0.5 uM SFMBT2_897 — 0.125 uM CHST2_889 0.25 uM — CHST2_7890 1 uM — VAV3_877 1 uM — VAV3_11878 0.25 uM —
Experiment 4.3
[0562] New triplex reactions (see Example 2 for original triplex reaction configurations) were designed to use the multiple region/one dye assay configurations in multiplexed reactions. “Pool 17” below lists a set of 6 markers co-amplified with a β-actin control, then analyzed in triplex QuARTS assays in the groupings shown below. Pool 17+MR-OD is adapted to include the multiple regions/one dye assay configurations for the SFMBT2, VAV3, and CHST2 markers. The JAM3, ZNF671, and ZNF568 assay designs were as shown in
TABLE-US-00108 Pool 17 Pool 17 + MR-OD JSA JAM3 JSSA JAM3 SFMBT2_897 SFMBT2_897 BTACT SFMBT2_895 VZA VAV3_877 BTACT ZNF671 VVZA VAV3_877 BTACT VAV3_11878 CZA1 CHST2_7890 ZNF671 ZNF568 BTACT BTACT CCZA1 CHST2_7890 CHST2_7889 ZNF568 BTACT
[0563] The new triplex formulations were tested on a plasmid calibration dilution series comprising the Pool 17 multiplex, comprising all target regions in the groups listed above, in a series of dilutions providing 2e5 to 2e1 strands of each target per assay reaction. The final concentrations of the probes for the SFMBT2, VAV3, and CHST2 MR-OD were as described in the results of Experiment 4.2. The probes for JAM3, ZNF671, and ZNF568 markers and for the BTACT control were 1 μM. All FRET cassettes were at 500 nM in the final reactions mixtures. The QuARTS assay components were mixed and the assays were performed on a Light Cycler as described in Experiment 3.1 The triplex containing VAV3-877 plus VAV3-11878 performed as expected, giving approximately 2 to 3-fold increase in strand count over the count of target added to the reaction, while the targets having only one region targeted . However, the triplexes containing CHST2-7889_CHST2-7890 and SFMBT2-895_SFMBT2-897 did not show the expected additive signal. Further experiments were conducted using different concentrations of the probes for CHST2-7889_CHST2-7890 and SFMBT2-895_SFMBT2-897, to test them in the multiplex QuARTS assays grouped as shown above. Within the triplex format, it was possible to modify the probe concentration of CHST2_7889 and CHST2_7890 to achieve the expected MR_OD results (i.e., results having the expected additive values of the individual reactions) based on a plasmid calibration curve. However, SFMBT2_895 and SFMBT2_897 assay, while improved using the modified probe concentrations, when used in the triplex format the assay still produced signal below the expected 200% level expected for detection of two regions. Nonetheless, the following modified probe concentrations were selected for testing the triplex assays on plasma samples.
TABLE-US-00109 Revised Final Probe Concentrations for MR-OD Reactions Marker_region [Probe]-Arm5-FAM [Probe]-Arm 1-HEX SFMBT2_895 — 1 uM SFMBT2_897 — 0.25 uM CHST2_7889 0.5 uM — CHST2_7890 1.5 uM — VAV3_877 1 uM — VAV3_11878 0.25 uM —
Experiment 4.4
[0564] This experiment examined the effect of combining multiplex pre-amplification and triplex QuARTS assay detection using the multiple regions-one dye assay designs to test human plasma samples from both normal and cancer patients. The experiment compared detection of 13 methylation markers (plus Process Control, ZF_RASSF1) of Pool 17 to detection using the Pool 17+MR_OD configuration on 63 normal plasma samples and 12 colon cancer plasma samples. The markers of Pool 17 were co-amplified together in a pre-amplification, then the pre-amplified DNA was detected in the list of grouped reactions listed below, and as described in detail in Example 1.
TABLE-US-00110 Pool 17 Pool 17 + MR-OD JSA JAM3 JSSA JAM3 SFMBT2 SFMBT2_897 BTACT SFMBT2_895 PDA PDGFD BTACT DTX1 PDA PDGFD BTACT DTX1 GQA GRIN2D BTACT QKI GQA GRIN2D BTACT QKI VZA VAV3 BTACT ZNF671 VVZA VAV3_877 BTACT VAV3_11878 CZA1 CHST2 ZNF671 ZNF568 BTACT BTACT CCZA1 CHST2_7890 AFA ANKRD13B CHST2_7889 FER1L4 ZNF568 BTACT BTACT CZA2 CNNM1 AFA ANKRD13B ZFRASSF1 FER1L4 BTACT BTACT CZA2 CNNM1 ZFRASSF1 BTACT
[0565] The triplex names comprise the first letter of each included marker, plus ‘A’ for the β-actin control. Double letters in the triplex names (e.g., “JSSA”) in the right-hand column indicate single markers tested at two different regions.
[0566] DNA was isolated from plasma samples as described in Example 1. Bisulfite conversion, multiplex pre-amplification, and QuARTS assay on multiplex-amplified DNA were conducted as described in Example 1. Prior to bisulfite conversion, aliquots of the isolated DNA were saved for testing KRAS 38A and 35C mutations on unconverted DNA. The amplification primers and detection probes used for each marker were as shown in
[0567] A logistic linear regression fit using strands-per-reaction for VAV3, SFMBT2, CHST2, and ZNF671 showed a considerable advantage when QuARTS is used in combination with MR_OD (multiple regions_one dye) as compared to the standard QuARTS assay configuration, as shown below. In these analysis, the marker ZNF671 was a major contributor to the detection results, and was included in the logistic fit for both QuARTS only and QuARTS+MR_OD. As noted above, KRAS 38A and 35C mutations the unconverted DNA were also tested.
[0568] The following sensitivity and specificity was obtained for using the multiplex pre-amplification with the standard triplex assays:
TABLE-US-00111 Multiplex with standard QuARTS assay Prediction Stage N Tested Cancer Normal Sensitivity I 4 2 2 50% II 3 2 1 67% III 3 2 1 67% IV 2 2 0 100% Prediction % Sensitivity/ Pathology N Tested Cancer Normal Specificity Cancer 12 8 4 67% Normal 62 0 62 100%
[0569] When the multiple region/one dye configuration was used, the sensitivity and specificity were as follows:
TABLE-US-00112 Multiplex with QuARTS assay using Multiple Regions_one Dye (MR_OD) Prediction Stage N Tested Cancer Normal Sensitivity I 4 4 0 100% II 3 3 0 100% III 3 2 1 67% IV 2 2 0 100%
TABLE-US-00113 Prediction % Sensitivity/ Pathology N Tested Cancer Normal Specificity Cancer 12 11 1 92% Normal 62 6 56 90%
[0570] Although the sample size is small, the use of this multiple region-to-one dye (FRET cassette) configuration shows substantial improvement in sensitivity, but may result in some loss of specificity.
[0571] It should be noted that, while this example detected DNA isolated from plasma samples, this panel of markers and use of the multiplex QuARTS assay modified as described above can be applied to stool or other blood or bodily fluid-based testing, and find application in, e.g., colon cancer and other cancer screening.
[0572] not limited to, patents, patent applications, articles, books, treatises, and internet web pages are expressly incorporated by reference in their entirety for any purpose. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which the various embodiments described herein belongs. When definitions of terms in incorporated references appear to differ from the definitions provided in the present teachings, the definition provided in the present teachings shall control.
[0573] Various modifications and variations of the described compositions, methods, and uses of the technology will be apparent to those skilled in the art without departing from the scope and spirit of the technology as described. Although the technology has been described in connection with specific exemplary embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in pharmacology, biochemistry, medical science, or related fields are intended to be within the scope of the following claims.