COMPARATIVE ANALYSIS OF MICROSATELLITES BY CAPILLARY ELECTROPHORESIS (CE) DNA PROFILES

Abstract

The present invention is directed to a method for determining of at least one microsatellite instability (MSI) based on a shift in a capillary electrophoresis (CE) profile (CE profile shift), the CE profile shift being determined by a comparison between the capillary electrophoresis (CE) profile of a target sequence of at least one microsatellite (MSI target profile) and the capillary electrophoresis (CE) profile of its specific wild type sequence (MS wild type profile). Further, the invention encompass suitable primer for use in said method, a kit comprising all essential components for performing said method successfully, a complete closed device as a system, namely “MSI Modaplex Analysis System” and a method for diagnosis of MSI phenotypes associated with an inflammation, cancer, inflammation associated cancer and/or auto immune disease, wherein the diagnosis comprises the method for determining of at least one CE profile shift as mentioned above. Finally, the present invention is directed to the use of an improved MSI panel for the determination and preferably diagnosis of MSI tumors, said panel consist of the STR biomarker NR-21, NR-24, Mono27, D2S123, D5S346, D17S250, Bat-25 and Bat-26.

Claims

1. A method for determining of at least one microsatellite instability (MSI) based on a shift in a capillary electrophoresis (CE) profile (CE profile shift), the CE profile shift being determined by a comparison between the capillary electrophoresis (CE) profile of a target sequence of at least one microsatellite (MSI target profile) and the capillary electrophoresis (CE) profile of its specific wild type sequence (MS wild type profile), the method comprising: amplification by means of a real time PCR simultaneously of at least one target sequence and of at least one specific wild type sequence and labeling nucleic acids thereof during amplification; separating the labeled nucleic acids by capillary electrophoresis (CE) and detecting at least one fluorophore labeled amplicon of at least one target sequence and of at least one wild type sequence, respectively, in real time while amplification; computer program-based calculation of the at least one target profile and of the at least one corresponding wild type profile; comparing the at least one target profile and the corresponding wild type profile; and determining a CE profile shift between the target profile and the corresponding wild type profile.

2. The method according to claim 1, wherein at least the steps of amplification, separation, detection and calculation is performed automatically in a closed system.

3. The method according to claim 1, wherein the MS wild type sequence represents an unaffected microsatellite locus of interest and the MSI target sequence represents a microsatellite locus of interest which potentially is affected by an MSI.

4. The method according to claim 1, wherein the shift in the MSI target profile of the at least one microsatellite of interest is an evidence for a medical indication which is associated with the at least one selected microsatellite.

5. The method according to claim 1, wherein the MSI target sequence and its specific wild type sequence are selected from the group consisting of an STR biomarker comprising NR-21, NR-22, NR-24, NR-27, D2S123, D5S346, D17S250, Mono27, CAT-25, HT-17, Bat-52, Bat-55, Bat-56, Bat-57, Bat-59, Bat-25, and/or Bat-26, and combinations thereof.

6. The method according to claim 1, wherein the MSI target sequence and its specific wild type sequences are selected from the group consisting of NR-21, NR-24, Mono27, D2S123, D5S346, D17S250, Bat-25 and Bat-26, and combinations thereof.

7. (canceled)

8. The method according to claim 1, wherein at least four or more different microsatellites are analyzed simultaneously.

9. The method according to claim 1, wherein an MSI target profile of at least one target sequence and/or an MS wild type profile of at least one wild type sequence is achieved.

10. The method according to claim 1, wherein at least one primer pair specific for and suitable to amplify at least one target sequence of at least one microsatellite and its specific wild type sequence is used, and further wherein the at least one primer of the at least one primer pair comprise an artificial tailing sequence for the modulation of the migration behavior in CE and the at least one primer of the primer pair comprises a fluorophore, wherein the artificial tailing sequence comprises a high adenosine and/or thymidine content, a hexaethylene glycol moiety or a twisted intercalating nucleic acid building block covalently linked alone or in combinations with further 5′-end nucleotide extensions.

11. The method according to claim 1, wherein labeled amplicons of the at least one MSI target sequence and of the at least one MS wild type sequence are produced and detected.

12. The method of claim 1 for in-vitro analysis of nucleic acids comprising in a biological sample for the determination of at least one microsatellite instability (MSI) based on the shift in a capillary electrophoresis (CE) profile.

13. (canceled)

14. The method according to claim 1, for which a kit for analyzing of at least one CE profile shift is provided, the kit comprising: at least one pair of primers specific for and suitable to amplify the at least one target sequence of at least one microsatellite to be analyzed, wherein each of the least one primer pair comprise an artificial tailing sequence and at least one primer of the primer pair comprise a fluorophore; at least one calibration sequence; at least one pair of primers specific for and suitable to amplify the at least one calibration sequence; and a contamination control.

15-20. (canceled)

21. The method according to claim 1, wherein the at least one pair of primer is specific for and suitable to amplify at least one MS locus selected from the group consisting of NR-21, N-R24, Mono27, D2S123, D5S346, D17S250, Bat-25 and Bat-26, and combinations thereof.

22. The method according to claim 21, wherein the specific primer pair is selected from the group consisting of: for NR-21 Seq ID No: 1 and Seq ID No: 2; for NR-24 Seq ID No: 3 and Seq ID No: 4; for Bat-25 Seq ID No: 5 and Seq ID No: 6; for Bat-26 Seq ID No: 7 and Seq ID No: 8; and for Mono27 Seq ID No: 9 and Seq ID No: 10 and combinations thereof.

23. The method according to claim 1, wherein the at least one CE profile shift is associated with an inflammation, a cancer, an inflammation associated cancer, an autoimmune disease, or any combination thereof.

24. The method of claim 1, which is for diagnosing of an MSI phenotype associated with an inflammation, a cancer, an inflammation associated cancer, an autoimmune disease, or any combination thereof.

25. The method according to claim 24, wherein the diagnosis of the MSI phenotype comprises determining of the at least one CE profile shift comprising calculating an MSI score representing the degree of the identified shift of the selected MS locus.

26. The method according to claim 24, wherein the diagnosis of the MSI phenotype further comprises: evaluating the identified CE profile shift and/or of the calculated MSI score by considering further patient-related data associated with the health status of the patient at least at the time in which a biological sample was obtain from the patient and preferably the CE profile shift was identified; and generating a diagnosis report considering and presenting all considered data, and preferably presenting a tailored therapy plan recommendation for the prevention or treatment of an inflammation, cancer, inflammation associated cancer, and/or autoimmune disease.

27. The method according to claim 1, wherein the method is performed in an automated capillary electrophoresis (CE) performed using an automated CE device.

28. The method according to claim 27, wherein the CE-device is a Modaplex device, and further wherein the Modaplex device comprises at least one excitation source and least one detector for detecting the fluorophore labeled nucleic acids, at least one processor, a least one microchip, and at least one interface for digital data transmission, wherein the processor comprises at least one program that controls and operates the method.

Description

EXAMPLE

[0107] Matched pairs (disease and normal) of formalin-fixed, paraffine-embedded (FFPE) tissue intersections which were pre-characterized as colon cancer positive were obtained from Indivumed GmbH (Hamburg, DE). Written informed consent of the patients were given. DNA was isolated with the QIAamp© DNA FFPE Tissue kit (Qiagen GmbH, Hilden, DE). The quality, amount and concentration of DNA was determined with a Qubit™ 3.0 Fluorometer (ThermoFisher Scientific, Waltham, Mass.-US) calibrated with standard DNA of the manufacturer. A pentaplex PCR was set up which comprises the five quasi-monomorphic mononucleotide STR-markers NR-21, NR-24, BAT-25, BAT-26 and Mono-27 as proposed by Bacher et al. (2004) which support the Revised Bethesda Guidelines for MSI diagnosis of gastrointestinal tumors (Umar et al. 2004). The primers used in the Modaplex multiplex assay are depicted in table 3 and within the sequence listing. The Revised Bethesda Guidelines define samples as MSS (microsatellite stabile), MSI-L (instable low) and MSI-H (instable high) if 0, 1-2 and 3-5 or more of quasi-monomorphic mononucleotide STR-markers are visually interpreted as affected, respectively.

[0108] The Multiplex-PCR consisted of a total volume of 25 μl with one fold Modaplex buffer 3 (containing Tris/HCl buffer pH 8.0, 250 mM dNTPs, monovalent cations, enhancers and stabilizers; Biotype GmbH, Dresden, DE), 4 mM MgCl.sub.2, 1 unit Modaplex Taq DNA polymerase T (Biotype GmbH, Dresden, DE), 0.5-1.0 μM of each primer pair and 2 ng genomic DNA. All reactions, samples, negative and positive controls, further included Modaplex calibrator mix 2 in 1fold final concentration.

[0109] The PCR was performed within the Modaplex detection system without a lid and without lid-heating to allow periodical injection into the capillary electrophoresis. Therefore, the reaction wells were finally overlaid with a drop of mineral oil to avoid evaporation.

[0110] The automated Modaplex detection system was setup according to the instructions of the manufacturer. A three step (for 40 s each) thermocycling protocol was used with a DNA denaturation step of 96-98° C., a DNA annealing step of 54-64° C. and a DNA extension step of 72° C. During PCR, amplification products were injected electrokinetically and separated in an automated capillary electrophoresis. Electrophoresis was performed periodically every second cycle at the end of the extension step, starting from PCR cycle 19 and ending with PCR cycle 41. Raw data were collected and imported into the Modaplex Result Analyzer (Moda-RA) software for DNA profile display which was then used for visual analysis as recommended by clinical guidelines for MSI analysis. The latter software can automatically superpose the amplicon profiles of the matched pairs of the patient samples. In FIGS. 1 and 2 DNA profiles of the STR amplicons at the end of PCR cycle 39 are depicted in doted lines for a normal tissue (wild type sequence) and in through lines for the matched disease tissue (target sequence). It is important to emphasize that the plotted profiles of the FIGS. 1A-B and 2A-B does not represent the real sizes or length of the respective amplicon. The definition of the x-axis [bp] is calculated based on the calibrators as defined above and could also been expressed as percent in respect to the calibrators or as retention time. Consequently, only relative sizes of each amplicon can be estimated compared to the calibrators.

[0111] Depending on the signal strength the raw data of other PCR cycles can be used for an overview or the presentation of individual biomarkers. The software allows to get at a first glance an overview of the multiplex PCR results (FIGS. 1, A and B). The STR specific detection regions can then be individually enlarged by a computer mouse click. In doing so the relative fluorescent units (RFU, y axis) of the biomarker profiles are normalized to get comparable presentations for weakly and strongly amplified STRs (see FIGS. 2, A and B). This facilitates the visual data analysis according to the current clinical guidelines.

[0112] As shown, the patient sample could be clearly interpreted as MSI-H (high MSI, Ulmar et al. 2004) because 5 out of the 5 markers could be analyzed to be affected by MSI.

FIGURE LEGENDS

[0113] FIG. 1: Electropherogram of the 6-FAM (FIG. 1A) labeled and of the 5′-TYE™ 665 (FIG. 1B) labeled biomarkers of the MSI multiplex PCR.

[0114] Raw data of matched pairs (disease tissue in through and normal tissue in doted lines) of FFPE tissue intersections were superposed with Modaplex Result Analyzer (Moda-RA) software and visualized as DNA profiles. The ranges which were used for the detection of the biomarkers are depicted as rectangles and labeled by the biomarker name. C1 and C2 are the profiles of calibrator 1 and 2 of the Modaplex calibrator Mix 2.

[0115] FIG. 2: Enlarged electropherograms of the 6-FAM labeled biomarkers NR-21 and Mono-27 of the MSI multiplex PCR. Raw data of matched pairs (disease tissue in through and normal tissue in doted lines) of FFPE tissue intersections were superposed with Modaplex Result Analyzer (Moda-RA) software, visualized as DNA profiles and individually enlarged by a computer mouse click. The RFU scale of the week MONO-27 signal (FIG. 2B, compare to FIG. 1) was adjusted to NR-21 (FIG. 2A, compare to FIG. 1) to facilitate the visual data analysis according to the current clinical guidelines. The ranges which were used for the detection of the biomarkers are depicted as rectangles and labeled by the biomarker name.

[0116] FIG. 3: flow sheet of one embodiment of the inventive methods: the steps according to claim 1 and according to claim 17 are summarized showing that according to the invention the methods are applicable in one passage without interruption in case of a complete validated in-vitro-diagnostic system and/or device.

REFERENCES

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