METHODS FOR AMPLIFYING NUCLEIC ACID USING TAG-MEDIATED DISPLACEMENT
20210214759 · 2021-07-15
Assignee
Inventors
Cpc classification
International classification
Abstract
Disclosed are methods for amplifying a nucleic acid target region using an amplification oligomer comprising a target-binding segment and a heterologous displacer tag situated 5′ to the target-binding segment. Initiation of an amplification reaction from the tagged amplification oligomer produces an amplicon comprising the displacer tag, such that once the complement of the displacer tag has been incorporated into a second amplicon, a displacer oligonucleotide having a sequence substantially corresponding to the displacer tag sequence is used to participate in subsequent rounds of amplification for displacement of an extension product primed from a site within the second amplicon 5′ to the displacer priming site. Also disclosed are related kits and reaction mixtures comprising the displacer-tagged amplification oligomer and corresponding displacer oligonucleotide.
Claims
1-65. (canceled)
66. A kit for amplifying a nucleic acid target region, the kit comprising: (1) a first amplification oligomer comprising (a) a target-binding priming segment (T1) complementary to a 3′-end of the target region; and (b) optionally, a heterologous universal tag (U2) located 5′ to T1; whereby the target nucleic acid can serves as a template for extension from the first amplification oligomer to produce a first amplicon; (2) a second amplification oligomer comprising (a) a target-binding segment T2 complementary to a region of the first amplicon that is the complement of a 5′-end of the target region; (b) a heterologous universal tag (U1) located 5′ to T2; and (c) a first heterologous displacer tag (D1) located 5′ to U1; whereby the first amplicon can serve as a template for amplification from the second amplification oligomer to produce a second amplicon comprising U1 and D1; and whereby the second amplicon can serve as a template for extension from the first amplification oligomer to produce a third amplicon comprising segments cU1 and cD1, complementary to U1 and D1, respectively; (3) a third amplification oligomer comprising a universal priming segment U1.sub.p having a nucleotide sequence complementary to U1; (4) a fourth amplification oligomer comprising a displacer priming segment D1.sub.p having a nucleotide sequence complementary to D1; and (5) if the first amplification oligomer comprises U2, a fifth amplification oligomer comprising a second universal priming segment U2.sub.p having a nucleotide sequence complementary to U2; whereby the third amplicon can serve as a template for extension from both the third and fourth amplification oligomers, wherein extension of U1.sub.p from a U1.sub.p:cU1 hybrid produces a fourth amplicon, and wherein extension of D1.sub.p from a D1.sub.p:cD1 hybrid produces a fifth amplicon while displacing the fourth amplicon.
67. The kit of claim 66, wherein the second amplification oligomer further comprises an intervening spacer segment (S1) between U1 and D1.
68. The kit of claim 66, wherein the second amplification oligomer further comprises a second heterologous displacer tag (D2) located 5′ to D1, whereby the second amplicon further comprises D2 and the third amplicon further comprises segment cD2, complementary to D2; and wherein the kit further comprises (6) a sixth amplification oligomer comprising a second displacer priming segment D2.sub.p having a nucleotide sequence complementary to D2; whereby (i) the third amplicon can serve as a template for extension from the sixth amplification oligomer, wherein extension of D2.sub.p from a D2.sub.p:cD2 hybrid produces a sixth amplicon comprising U1, D1, and D2.sub.p; and (ii) the sixth amplicon can serve as a template for extension from the first or fifth amplification oligomer to produce a seventh amplicon comprising cU1, cD1, and cD2.sub.p.
69. The kit of claim 68, wherein the second amplification oligomer further comprises a second intervening spacer segment (S2) between D1 and D2.
70. The kit of claim 66, further comprising (6) a sixth amplification oligomer comprising (a) priming segment D1.sub.p and (b) displacer tag D2 located 5′ to D1.sub.p; and (7) a seventh amplification oligomer comprising a second displacer priming segment D2.sub.p having a nucleotide sequence complementary to D2; whereby (i) the fifth amplicon can serve as a template for extension from the first or fifth amplification oligomer to produce a sixth amplicon comprising segments cU2 and cD1.sub.p; (ii) at least one of the third and sixth amplicons can serve as a template for extension from the sixth amplification oligomer, wherein extension of D1.sub.p from a D1.sub.p:cD1 or D1.sub.p:cD1.sub.p hybrid produces a seventh amplicon comprising U2, D1.sub.p, and D2; (iii) the seventh amplicon can serve as a template for extension from the first amplification oligomer to produce an eighth amplicon comprising cU2, cD1.sub.p, and cD2; (iv) the eighth amplicon can serve as a template for extension from the seventh amplification oligomer, wherein extension of D2.sub.p from a D2.sub.p:cD2 hybrid produces a ninth amplicon comprising U2, D1, and D2.sub.p; and (v) the ninth amplicon can serve as a template for extension from the first or fifth amplification oligomer to produce a tenth amplicon comprising cU2, cD1, and cD2.sub.p.
71. The kit of claim 68, further comprising (7) a seventh amplification oligomer comprising (a) priming segment D1.sub.p and (b) displacer tag D2 located 5′ to D1.sub.p; whereby (i) the fifth amplicon can serve as a template for extension from the first or fifth amplification oligomer to produce an eighth amplicon comprising segments cU1 and cD1.sub.p; (ii) at least one of the third, seventh, and eighth amplicons can serve as a template for extension from the seventh amplification oligomer, wherein extension of D1.sub.p from a D1.sub.p:cD1 or D1.sub.p:cD1.sub.p hybrid produces a ninth amplicon comprising U1, D1.sub.p, and D2; and (iii) the ninth amplicon can serve as a template for extension from the first or fifth amplification oligomer to produce a tenth amplicon comprising cU1, cD1.sub.p, and cD2.
72. The kit of claim 71, wherein the second amplification oligomer further comprises a third heterologous displacer tag (D3) located 5′ to D2, whereby the second amplicon further comprises D3 and the third amplicon further comprises a segment cD3, complementary to D3; and the kit further comprises (8) an eighth amplification oligomer comprising a third displacer priming segment D3.sub.p having a nucleotide sequence complementary to D3; whereby (i) the third amplicon can serve as a template for extension from the eighth amplification oligomer, wherein extension of D3.sub.p from a D3.sub.p:cD3 hybrid produces an eleventh amplicon comprising U1, D1, D2, and D3.sub.p; and (ii) the eleventh amplicon can serve as a template for extension from the first or fifth amplification oligomer to produce an twelfth amplicon comprising cU1, cD1, cD2, and cD3.sub.p.
73. The kit of claim 72, wherein the first amplification oligomer further comprises a third intervening spacer segment (S3) between D2 and D3.
74. The kit of claim 70, further comprising (8) an eighth amplification oligomer comprising (a) priming segment D2.sub.p and (b) displacer tag D3 located 5′ to D2.sub.p; and (9) a ninth amplification oligomer comprising a third displacer priming segment D3.sub.p having a nucleotide sequence complementary to D3; whereby (i) at least one of the eighth and tenth amplicons can serve as a template for extension from the eighth amplification oligomer, wherein extension of D2.sub.p from a D2.sub.p:cD2 or D2.sub.p:cD2.sub.p hybrid produces an eleventh amplicon comprising U2, D1/D1.sub.p, D2.sub.p, and D3; (ii) the eleventh amplicon can serve as a template for extension from the first or fifth amplification oligomer to produce a twelfth amplicon comprising cU2, cD1/cD1.sub.p, cD2.sub.p, and cD3; (iii) the twelfth amplicon can serve as a template for extension from the ninth amplification oligomer, wherein extension of D3.sub.p from a D3.sub.p:cD3 hybrid produces an thirteenth amplicon comprising U2, D1, D2, and D3.sub.p; and (iv) the thirteenth amplicon can serve as a template for extension from the first or fifth amplification oligomer to produce a fourteenth amplicon comprising cU2, cD1, cD2, and cD3.sub.p.
75. The kit of claim 73, further comprising (9) a ninth amplification oligomer comprising (a) priming segment D2.sub.p and (b) displacer tag D3 located 5′ to D2.sub.p; (i) at least one of the third, seventh, tenth, and twelfth amplicons can serve as a template for extension from the eighth amplification oligomer, wherein extension of D2.sub.p from a D2.sub.p:cD2 or D2.sub.p:cD2.sub.p hybrid produces a thirteenth amplicon comprising U1, D1/D1.sub.p, D2.sub.p, and D3; and (ii) the thirteenth amplicon can serve as a template for extension from the first or fifth amplification oligomer to produce a fourteenth amplicon comprising cU1, cD1/cD1.sub.p, cD2.sub.p, and cD3.
76. The kit of claim 75, wherein the first amplification oligomer further comprises a fourth heterologous displacer tag (D4) located 5′ to T1, whereby the first amplicon comprises T1 and D4; and whereby the second amplicon comprises segments cT1 and cD4, complementary to T1 and D4, respectively; wherein the kit further comprises (10) a tenth amplification oligomer comprising a priming segment T1.sub.p having a nucleotide sequence complementary to T1, or complementary to the complement of a second amplicon target sequence cT1′ near or overlapping with cT1 and situated 5′ to cD4; and (11) an eleventh amplification oligomer comprising a fourth displacer priming segment D4.sub.p having a nucleotide sequence complementary to D4; and whereby the second amplicon can serve as a template for extension from both the tenth and eleventh amplification oligomers, wherein extension of T1.sub.p from a T1.sub.p:cT1/cT1′ hybrid produces a fifteenth amplicon, and wherein extension of D4.sub.p from a D4.sub.p:cD4 hybrid produces a sixteenth amplicon while displacing the fifteenth amplicon.
77. The kit of claim 76, wherein the first amplification oligomer further comprises a fourth intervening spacer segment (S4) between T1 and D4.
78. The kit of claim 66, wherein the first amplification oligomer comprises U2 and further comprises a fourth heterologous displacer tag (D4) located 5′ to U2, whereby the first amplicon comprises U2 and D4; and whereby the second amplicon comprises segments cU2 and cD4, complementary to U2 and D4, respectively; wherein the kit further comprises (10) an eleventh amplification oligomer comprising a fourth displacer priming segment D4.sub.p having a nucleotide sequence complementary to D4; and whereby the second amplicon can serve as a template for extension from both the fifth and tenth amplification oligomers, wherein extension of U2.sub.p from a U2.sub.p:cU2 hybrid produces a fifteenth amplicon, and wherein extension of D4.sub.p from a D4.sub.p:cD4 hybrid produces a sixteenth amplicon while displacing the fifteenth amplicon.
79. The kit of claim 78, wherein the first amplification oligomer further comprises a fourth intervening spacer segment (S4) between U2 and D4.
80. The kit of claim 66, wherein the affinity of D1.sub.p for its complement is lower than that of U1.
81. The kit of claim 68, wherein at least one of the following conditions is present: (a) the affinity of D1.sub.p for its complement is lower than that of U1; (b) the affinity of D2.sub.p for its complement is lower than that of D1.sub.p.
82. The kit of claim 72, wherein at least one of the following conditions is present: (a) the affinity of D1.sub.p for its complement is lower than that of U1; (b) the affinity of D2.sub.p for its complement is lower than that of D1.sub.p; (c) the affinity of D3.sub.p for its complement is lower than that of D2.sub.p.
83. (canceled)
84. The kit of claim 83, further comprises a reverse transcriptase (RT).
85. The kit of claim 84, wherein the first amplification oligomer further comprises an RNA polymerase promoter sequence (P) located 5′ to T1, whereby the second amplicon comprises a segment cP, complementary to the promoter sequence; and whereby an RNA polymerase can initiates transcription upon recognizing a double-stranded promoter sequence (P:cP) formed by extension of the second amplification oligomer on the first amplicon, thereby producing an RNA amplicon.
86-138. (canceled)
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
[0108] The present invention provides methods, kits, and reaction mixtures for amplification of nucleic acids using a tag-mediated displacement strategy. An amplification oligonucleotide is equipped with a heterologous displacer tag situated 5′ to a target-binding segment and having a sequence that substantially corresponds to the priming sequence of a displacer oligonucleotide. In this manner, once the complement of the displacer tag has been incorporated into an amplification product, thereby providing a displacer priming site, the displacer oligonucleotide can participate in subsequent rounds of amplification for displacement of an extension product primed from a site within the amplification product 5′ to the displacer priming site, thereby increasing overall amplification out put and increasing assay kinetics and sensitivity.
[0109] This strategy can be used to improve the kinetics of amplification reactions as well as amplification capacity. For example, an amplification oligomer can be designed to include one or more additional displacer tags, each situated in succession 5′ to the initial displacer tag. Additional displacer oligomers may then be included in the amp reaction, each corresponding to at least one additional tag and designed to bind an amplicon at a site 3′ to at least one other displacer oligomer. Using multiple displacer tags and oligomers in this manner, additional amplification products can be efficiently produced in each round of the amp reaction, thereby increasing overall amplification output and increasing assay kinetics and sensitivity.
[0110] Tag-mediated displacement as described herein is also advantageous, for example, when using heterologous tags to introduce universal priming sites. A non-target-specific “universal” priming site can be incorporated into an amplification product using an amplification oligomer equipped with a universal tag substantially corresponding to the universal tag and located 5′ to an initial target-specific priming segment. Once a complement of the universal tag has been incorporated into an amplification product, a universal priming oligonucleotide can be used to participate in subsequent rounds of amplification. Such a scenario would preclude the use of known displacement methods, which rely on the use of target-specific displacer priming sites. Using, however, a tag-mediated displacement strategy, a heterologous displacer tag can be incorporated into an amplification oligomer at a site 5′ to the universal tag, such that the amplification product comprising the universal priming site further comprises a 3′ displacer priming site, thereby allowing the use of a displacer oligomer in subsequent amplification. Not only will extension of a universal primer from the universal priming site be displaced, but extension of the displacer oligomer will produce an additional amplification product, thereby increasing overall amplification output and increasing assay kinetics and sensitivity.
[0111] The present invention can be adapted for use in essentially any amplification procedure requiring a template-binding priming oligonucleotide capable of extension in the presence of a nucleic acid polymerase. Incorporation of heterologous displacer tags into such amplification procedures can be achieved without substantially modifying the reagents and reaction conditions of such procedures. Any needed modifications would be well within the knowledge and capabilities of a skilled molecular biologist in view of the instant description. Descriptions of various amplification procedures adopting tag-mediated displacement are further provided herein.
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[0114] In certain embodiments of the method, multiple displacer tags are incorporated in a tagged oligonucleotide. For example,
[0115] In other embodiments, an additional displacer oligonucleotide comprising a second heterologous displacer tag is added to the reaction to further increase amplification capacity. For example, in some variations, an amplification oligonucleotide comprising displacer priming segment D1.sub.p and displacer tag D2, situated 5′ to D1.sub.p, may be added to an amplification reaction as depicted in
[0116] In certain variations, an additional displacer oligonucleotide comprising displacer priming segment D1.sub.p and a second displacer tag D2, as described above, is used in conjunction with an initial amplification oligomer comprising priming segment T1 and both displacer tags D1 and D2. In such variations, the D1.sub.p-D2 oligomer may increase the generation of amplicons comprising the D2 tag. For example, as indicated above, the D1.sub.p-D2 can prime from amplicons comprising a cD1.sub.p priming site but lacking cD2 (in addition to the capability of priming from amplicons comprising both cD1/cD1.sub.p and cD2).
[0117] Alternatively, a displacer oligomer comprising D1.sub.p and D2 is used together with an initial amplification oligomer that includes priming segment T1 and displacer tag D1 in the absence of D2. In such variations, the D1.sub.p-D2 oligomer is also useful for the initial incorporation of the D2 tag into an amplification product. For example, the D1.sub.p-D2 oligomer may utilize an amplicon such as that shown in Step 6 of
[0118] In variations of the method utilizing multiple displacer tags, three or more displacer tags are employed, together with corresponding displacer priming oligonucleotide(s), to still further increase amplification capacity.
[0119] In other embodiments employing the use of three or more displacer tags, a third or further displacer tag(s) may be incorporated into an additional displacer oligonucleotide in a similar manner previously depicted in
[0120] In various embodiments of the method, the tag-mediated displacement strategy as described herein may be employed using either a forward or reverse amplification oligonucleotide comprising one or more displacer tags. In other embodiments, both a forward and a reverse amplification oligonucleotide include one or more displacer tags. For example, in the amplification reaction previously depicted in
[0121] Some preferred variations of the amplification method utilize an isothermal, transcription-based amplification reaction known as transcription-based amplification (TMA), various aspects of which are disclosed in Becker et al., U.S. Pat. No. 7,374,885. As previously discussed herein, TMA employs an RNA polymerase to produce multiple RNA copies of a target region. A promoter primer or promoter provider oligonucleotide is utilized to incorporate a promoter sequence for the RNA polymerase. Upon formation of a double-stranded promoter, produced by a primer extension reaction on the initial promoter sequence as a template, the RNA polymerase binds to the promoter and produces multiple RNA transcripts, which can become templates for further rounds of amplification in the presence of a priming oligonucleotide capable of hybridizing to the RNA transcripts.
[0122] Accordingly, particular variations of the present method include the use of a promoter primer in the amplification reaction. For example, in the amplification reaction previously depicted in
[0123] In other embodiments employing TMA, a promoter provider is used is used in the amplification reaction.
[0124] In more particular variations of embodiments employing TMA, a promoter primer or promoter primer further includes one or more displacer tags situated 5′ to a target-binding segment. In such embodiments, the one or more displacer tag(s) of a promoter primer or promoter primer are preferably situated 3′ to the promoter sequence. In this manner, extension of the 3′-end of the template amplicon, upon hybridization of target-binding segment T2, produces a template comprising segments complementary to the displacer tag(s) and incorporation of the displacer tag(s) into the resulting RNA amplicon. Subsequent amplification of the RNA amplicon may then further include the use of displacer oligonucleotides to initiate displacement reactions in accordance with the present invention.
[0125] In some embodiments of the method, amplification of target region utilizes a target-specific priming site situated downstream from (5′ to) a displacer priming site. For example, in particular variations utilizing a T1-D1 first amplification oligomer, a third amplification oligomer comprises a priming segment T1.sub.p having a nucleotide sequence substantially corresponding to T1 (and hence configured to hybridize to cT1, see, e.g.,
[0126] As previously discussed, certain variations of the method incorporate the use of universal priming sites and primers following initial amplification of a target nucleic acid. In such embodiments, an amplification oligomer comprising a target-specific hybridizing sequence (e.g., target-binding segment T1 of a first amplification oligonucleotide, or target-binding segment T2 of a second amplification oligonucleotide) includes a heterologous universal tag segment situated 5′ to the target-specific segment T1. In this manner, subsequent rounds of amplification can employ the use of an amplification oligonucleotide comprising a universal priming segment (e.g., “U1.sub.p”) corresponding to the universal tag, in place of an oligonucleotide comprising a target-binding priming segment (e.g., “T1.sub.p”) specific for the target region of the target nucleic acid. In certain embodiments, both a forward and a reverse amplification oligomer (e.g., first and second amplification oligonucleotides as discussed herein) include a universal tag situated 5′ to a target-binding segment. In particular embodiments employing a promoter primer or promoter primer for TMA, a universal tag is included 5′ to the target-binding segment and 3′ to the promoter sequence. Any variation of the methods discussed herein can be adapted for use with universal priming sites and primers following initial amplification of a target nucleic acid and such embodiments are within the scope of the present invention.
[0127] In each of the embodiments described herein, a wide variety of identities and functionalities can be designed into the displacer tag sequences. For example, in some embodiments, where two or more displacer sequences are used, at least two of these sequence can be different from each other. In other embodiments, at least two of the multiple displacer tags can be the same as each other. Similarly, in some variations where a universal priming sequence is used, one or more displacer tag sequence(s) is the same as the universal sequence; in other variations, one or more of the displacer tag sequence(s) are different from the universal sequence. Thus, in some embodiments, wherever two or more heterologous sequences (universal and displacer) are employed, there can be at least two unique sequences, or more depending on the number of displacer sequences added. In a more specific variation employing multiple displacer tags with a universal tag, each of the multiple displacer tags sequences are the same but different from the universal sequence.
[0128] In other more particular variations, sequences are designed to have different affinities for their complements. For example, in certain embodiments the affinity of a first displacer tag D1 for its complement is lower than that of a target-specific site T1 (or lower than that of a universal site U1 if a universal tag is used). Similarly, where multiple displacers are used, each successive displacer may be designed to have a lower affinity for its complement that the one situate 3′ to it (e.g., D2 can be designed to have a lower affinity for its complement than D1; D3 can be designed to have a lower affinity for its complement than D2; etc.). In this way, the displacing potential of the oligonucleotide constructs can be increased by increasing, for example, the potential that a target-specific or universal priming site is available for binding before binding and extension of a D1 displacer oligomer (e.g., allowing a D1 displacer oligomer to bind to its priming site “after” binding of a target-specific or universal primer having a higher affinity), thereby maximizing the potential for binding and extension from both priming oligonucleotides together with a displacement reaction. The skilled artisan will appreciate that affinities of various displacer and universal sequences may be varied as desired based upon such known factors as, e.g., GC content and length of the hybridizing sequence.
[0129] In addition to improving the kinetics of an amplification reaction via displacement of extension products from their template strands, tag-mediated displacement reactions can also increase amplification capacity. This advantage of the present invention is illustrated in
[0130] The methods of the present invention are useful in assays for detecting and/or quantitating specific target nucleic acids in clinical, water, environmental, industrial, beverage, food, seed stocks, and other samples or to produce large numbers of nucleic acid amplification products from a specific target sequence for a variety of uses. For example, the present invention is useful to screen clinical samples (e.g., blood, urine, feces, saliva, semen, or spinal fluid), food, water, laboratory and/or industrial samples for the presence of specific nucleic acids, specific organisms (e.g., using species-specific oligonucleotides) and/or specific classes of organisms (e.g., using class-specific oligonucleotides) in applications such as in sterility testing. The present invention can be used to detect the presence of, for example, viruses, bacteria, fungi, or parasites.
[0131] Samples suspected of containing a target nucleic acid are prepared for subsequent amplification as described herein using methods generally known in the art. Typically, a target nucleic acid is separated or purified from one or more other components of a sample. Such purification may include may include methods of separating and/or concentrating organisms contained in a sample from other sample components. In particular embodiments, purifying the target nucleic acid includes capturing the target nucleic acid to specifically or non-specifically separate the target nucleic acid from other sample components. Non-specific target capture methods may involve selective precipitation of nucleic acids from a substantially aqueous mixture, adherence of nucleic acids to a support that is washed to remove other sample components, or other means of physically separating nucleic acids from a mixture that contains target nucleic acid and other sample components.
[0132] In some embodiments, a target nucleic acid is selectively separated from other sample components by specifically hybridizing the target nucleic acid to a capture probe oligomer. The capture probe comprises a target-binding segment configured to specifically hybridize to a target sequence so as to form a target-nucleic-acid:capture-probe (“target:capture-probe”) complex that is separated from sample components. In a preferred variation, the specific target capture further comprises binding the target:capture-probe complex to an immobilized probe to form a target:capture-probe:immobilized-probe complex that is separated from the sample and, optionally, washed to remove non-target sample components (see, e.g., U.S. Pat. Nos. 6,110,678; 6,280,952; and 6,534,273; each incorporated by reference herein). In such variations, the capture probe oligomer further comprises a sequence or moiety that binds attaches the capture probe, with its bound target-binding segment, to an immobilized probe attached to a solid support, thereby permitting the hybridized target nucleic acid to be separated from other sample components. In more specific embodiments, the capture probe oligomer includes a tail portion (e.g., a 3′ tail) that is not complementary to the target nucleic acid but that specifically hybridizes to a nuclei acid sequence on the immobilized probe, thereby serving as the moiety allowing the target nucleic acid to be separated from other sample components, such as previously described in, e.g., U.S. Pat. No. 6,110,678, incorporated herein by reference. Any sequence may be used in a tail region, which is generally about 5 to 50 nt long, and preferred embodiments include a substantially homopolymeric tail of about 10 to 40 nt (e.g., A.sub.10 to A.sub.40), more preferably about 14 to 33 nt (e.g., A.sub.14 to A.sub.30 or T.sub.3A.sub.14 to T.sub.3A.sub.30), that bind to a complementary immobilized sequence (e.g., poly-T) attached to a solid support, e.g., a matrix or particle.
[0133] Target capture typically occurs in a solution phase mixture that contains one or more capture probes that hybridize specifically to the target nucleic acid under hybridizing conditions, usually at a temperature higher than the T.sub.m of the tail-sequence:immobilized-probe-sequence duplex. For embodiments comprising a capture probe tail, the target:capture-probe complex is captured by adjusting the hybridization conditions so that the capture probe tail hybridizes to the immobilized probe, and the entire complex on the solid support is then separated from other sample components. The support with the attached target:capture-probe:immobilized-probe may be washed one or more times to further remove other sample components. Certain embodiments use a particulate solid support, such as paramagnetic beads, so that particles with the attached target:capture-probe:immobilized-probe complex may be suspended in a washing solution and retrieved from the washing solution, preferably by using magnetic attraction. To limit the number of handling steps, the nucleic acid target region may be amplified by simply mixing the target nucleic acid in the complex on the support with amplification oligomers and proceeding with amplification steps.
[0134] For amplification of a target nucleic acid, a variety of nucleic acid amplification methods are known and may be readily adapted for use to incorporate a tag-mediated displacement strategy in accordance with the present invention (see above). Generally, certain amplification steps as described herein are “extension reactions” in which the 3′-end of a priming oligonucleotide is extended by the addition of nucleotides complementary to a nucleic acid template to which the priming oligonucleotide is hybridized, thereby synthesizing a complementary copy of the template. Conditions for extension reactions are well-known and generally utilize a polymerization agent (e.g., DNA polymerase) to synthesize the complementary DNA copy. A DNA polymerase may be characterized as “DNA-dependent” or “RNA-dependent,” depending on whether the polymerase utilizes a DNA template or RNA template, respectively. Examples of DNA-dependent DNA polymerases are DNA polymerase I from E. coli, bacteriophage T7 DNA polymerase, or DNA polymerases from bacteriophages T4, Phi-29, M2, or T5. DNA-dependent DNA polymerases may be the naturally occurring enzymes isolated from bacteria or bacteriophages or expressed recombinantly, or may be modified or “evolved” forms which have been engineered to possess certain desirable characteristics, e.g., thermostability, or the ability to recognize or synthesize a DNA strand from various modified templates. It is known that under suitable conditions a DNA-dependent DNA polymerase may synthesize a complementary DNA copy from an RNA template. Alternatively, in some variations, an RNA-dependent DNA polymerase, or “reverse transcriptase” (“RT”), is utilized in a primer extension reaction. A reverse transcriptase synthesizes a complementary DNA copy from an RNA template. All known reverse transcriptases also have the ability to make a complementary DNA copy from a DNA template, and are thus both RNA- and DNA-dependent. RTs may also have an RNAse H activity.
[0135] Other amplification steps as described herein do not necessarily require an extension reaction. For example, certain amplification reactions may be catalyzed using a DNA-dependent RNA polymerase or “transcriptase,” which synthesizes multiple RNA copies from a double-stranded or partially double-stranded DNA molecule having a promoter sequence that is usually double-stranded. The RNA molecules (“transcripts”) are synthesized in the 5′-to-3′ direction beginning at a specific position just downstream of the promoter. Examples of transcriptases are the DNA-dependent RNA polymerase from E. coli and bacteriophages T7, T3, and SP6. As previously discussed, in certain embodiments, a promoter region is introduced into an amplification reaction by the use of an amplification oligomer further comprising a promoter sequence situated 5′ to a target-binding segment.
[0136] Certain embodiments of the present invention relate to amplification of a target nucleic acid utilizing transcription-mediated amplification (TMA). Some such embodiments relate more specifically to amplification of a target nucleic acid comprising an RNA target region. In certain cases, the target nucleic acid has indeterminate 3′- and 5′-ends relative to the desired RNA target region. The target nucleic acid is treated with a priming oligonucleotide which has a base region sufficiently complementary to a 3′-end of the RNA target region to hybridize therewith and, as discussed above, further comprises at least one heterologous displacer tag and optionally a universal tag in the first primer extension reaction. Priming oligonucleotides are designed to hybridize to a suitable region of any desired target sequence, according to primer design methods well-known to those of ordinary skill in the art. While the presence of the displacer tag sequence in a priming oligonucleotide may alter the binding characteristics of a target hybridizing region to a target nucleic acid sequence, the artisan skilled in the molecular arts can readily design priming oligonucleotides which contain both a target-binding segment and tag segments that can be used in accordance with the methods described herein. Additionally, the 5′-end of a priming oligonucleotide (preferably not a tagged priming oligonucleotide) may include one or modifications which improve the binding properties (e.g., hybridization or base stacking) of the priming oligonucleotide to a DNA extension product or to an RNA amplification product, provided the modifications do not substantially interfere with the priming function of the priming oligonucleotide or cleavage of an RNA amplification product to which the priming oligonucleotide is hybridized. The 3′-end of the priming oligonucleotide is extended by an appropriate DNA polymerase, e.g., an RNA-dependent DNA polymerase (reverse transcriptase) in an extension reaction using the RNA target region or amplification product as a template to give a DNA primer extension product which is complementary to the RNA template or amplification product.
[0137] DNA primer extension products may be separated (at least partially) from an RNA template using an enzyme which degrades the RNA template or amplification product. Suitable enzymes, i.e., “selective RNAses,” are those which act on the RNA strand of an RNA:DNA complex, and include enzymes which comprise an RNAse H activity. Some reverse transcriptases include an RNAse H activity, including those derived from Moloney murine leukemia virus and avian myeloblastosis virus. According to preferred amplification embodiments, the selective RNAse may be provided as an RNAse H activity of a reverse transcriptase, or may be provided as a separate enzyme, e.g., as an E. coli RNAse H or a T. thermophilus RNAse H. Other enzymes which selectively degrade RNA present in an RNA:DNA duplex may also be used.
[0138] Following initial amplification of an RNA target region so as to incorporate one or more displacer tags into an amplification product, subsequent strand separation may be achieved using displacer oligonucleotides corresponding to the displacer tag sequences as described herein. For example, referring
[0139] When the initial target nucleic acid is DNA, then a first forward amplification oligomer can be a DNA primer comprising a 3′ target-binding priming segment and a 5′ first heterologous displacer tag. The first forward amplification oligomer can be used to make a first DNA primer extension product. When the amplification reaction is a PCR amplification reaction, then the first DNA primer extension product can be separated from the target nucleic acid by a number of methods, including using the temperature cycling parameters of a PCR reaction. Alternatively, and also for use with isothermal amplification reactions, the first DNA primer extension product can be separated from the from the target nucleic acid using a displacer primer comprising a target-specific priming segment that hybridizes to the DNA target nucleic acid at a position upstream from the forward priming oligonucleotide binding site (a first amplification oligomer such as described herein). In this manner, the first amplicon produced by extension of the first amplification oligomer can be displaced from the template strand by extension of the target-specific displacer primer, thereby making it available for hybridization to an amplification oligonucleotide (e.g., a promoter primer or promoter provider, or other amplification oligomer as described herein) for further amplification to produce a second amplicon. In a further alternative approach, conditions can be established whereby an oligonucleotide gains access to the first DNA primer extension product through strand invasion facilitated by, for example, DNA breathing (e.g., AT rich regions), low salt conditions, and/or the use of DMSO and/or osmolytes, such as betaine. A particularly suitable amplification oligomer for this embodiment is a promoter provider oligonucleotide such as that described herein, which is modified to prevent the promoter oligonucleotide from functioning as a priming oligonucleotide for a DNA polymerase (e.g., the promoter oligonucleotide includes a blocking moiety at its 3′-terminus). Irrespective of the method used to separate the first amplicon from a DNA template, once the first amplicon is made available to produce a second amplicon and with the use of heterologous displacer tags to incorporate heterologous displacer priming sites in amplicons as described herein, tag-mediated displacement can be utilized in subsequent amplification rounds to separate nucleic acid strands.
[0140] In certain embodiments, the methods of the present invention further comprise treating a target nucleic acid as described above to limit the length of a primer extension product to a certain desired length. Such length limitation is typically carried out through use of a “binding molecule” which hybridizes to or otherwise binds to the target nucleic acid adjacent to or near the 5′-end of the desired target sequence. In certain embodiments, a binding molecule comprises a base region. The base region may be DNA, RNA, a DNA:RNA chimeric molecule, or an analog thereof Binding molecules comprising a base region may be modified in one or more ways, as described elsewhere herein. Suitable binding molecules include, but are not limited to, a binding molecule comprising a terminating oligonucleotide or a terminating protein that binds RNA and/or DNA and prevents primer extension past its binding region, or a binding molecule comprising a modifying molecule, for example, a modifying oligonucleotide such as a “digestion” oligonucleotide that directs hydrolysis of that portion of the RNA and/or DNA target hybridized to the digestion oligonucleotide, or a sequence-specific nuclease that cuts the RNA and/or DNA target.
[0141] Illustrative terminating oligonucleotides of the present invention have a 5′-base region sufficiently complementary to the target nucleic acid at a region adjacent to, near to, or overlapping with the 5′-end of the target sequence, to hybridize therewith. In certain embodiments, a terminating oligonucleotide is synthesized to include one or more modified nucleotides. For example, certain terminating oligonucleotides of the present invention comprise one or more 2′-O-ME ribonucleotides, or are synthesized entirely of 2′-O-ME ribonucleotides. See, e.g., Majlessi et al., Nucleic Acids Res., 26, 2224-2229, 1998. A terminating oligonucleotide of the present invention typically also comprises a blocking moiety at its 3′-end to prevent the terminating oligonucleotide from functioning as a primer for a DNA polymerase. In some embodiments, the 5′-end of a terminating oligonucleotide of the present invention overlaps with and is complementary to at least about 2 nucleotides of the 5′-end of the target region. Typically, the 5′-end of a terminating oligonucleotide of the present invention overlaps with and is complementary to at least 3, 4, 5, 6, 7, or 8 nucleotides of the 5′-end of the target sequence, but no more than about 10 nucleotides of the 5′-end of the target region. (As used herein, the term “end” refers to a 5′- or 3′-region of an oligonucleotide, nucleic acid or nucleic acid region which includes, respectively, the 5′- or 3′-terminal base of the oligonucleotide, nucleic acid or nucleic acid region.)
[0142] In particular embodiment employing transcription-mediated amplification, a single-stranded DNA primer extension product, or “first” DNA primer extension product, which has either a defined 3′-end or an indeterminate 3′-end, is treated with a promoter oligonucleotide (a promoter primer or promoter provider) that comprises a target-binding segment substantially complementary to a 3′-region of the DNA primer extension product to hybridize therewith, and a second segment comprising a promoter for an RNA polymerase, e.g., T7 polymerase, which is situated 5′ to the first segment (e.g., immediately 5′ to or spaced from the first region). In some variations, the promoter oligonucleotide is a promoter provider modified to prevent the promoter oligonucleotide from functioning as a primer for a DNA polymerase (e.g., the promoter oligonucleotide includes a blocking moiety attached at its 3′-terminus). In particular variations, a promoter oligonucleotide further includes one or more heterologous displacer tags situated 5′ to the target-binding segment and, most typically, 3′ to the promoter region. In other embodiments, a promoter oligonucleotide includes a universal tag segment 5′ to the target-binding segment and 3′ to the promoter and any displacer tag(s). Upon identifying a desired target-binding segment and any desired displacer or universal tags, suitable promoter oligonucleotides can be constructed by one of ordinary skill in the art using only routine procedures. Those of ordinary skill in the art will readily understand that a promoter region has certain nucleotides which are required for recognition by a given RNA polymerase. In addition, certain nucleotide variations in a promoter sequence might improve the functioning of the promoter with a given enzyme, including the use of an intervening spacer segment between the promoter sequence and the target-binding segment (also referred to as an “insertion sequence”). Insertion sequences may be positioned between the target-binding and promoter segments of promoter oligonucleotides and function to increase amplification rates. (A displacer or universal tag segment of a tagged promoter oligonucleotide may provide this beneficial effect.)
[0143] Assaying promoter oligonucleotides with variations in the promoter sequences is easily carried out by the skilled artisan using routine methods. Furthermore, if it is desired to utilize a different RNA polymerase, the promoter sequence in the promoter oligonucleotide is easily substituted by a different promoter. Substituting different promoter sequences is well within the understanding and capabilities of those of ordinary skill in the art. For real-time TMA, promoter oligonucleotides provided to the amplification reaction mixture are modified to prevent efficient initiation of DNA synthesis from their 3′-termini, and preferably comprise a blocking moiety attached at their 3′-termini. Furthermore, terminating oligonucleotides and capping oligonucleotides, and even probes used in certain embodiments of the present invention also optionally comprise a blocking moiety attached at their 3′-termini.
[0144] Where a terminating oligonucleotide is used in a TMA reaction, the first, target-binding segment of a promoter oligonucleotide is designed to hybridize with a desired 3′-end of the first DNA primer extension product with substantial, but not necessarily exact, precision. Subsequently, the second segment of the promoter oligonucleotide may act as a template, allowing the first DNA primer extension product to be further extended to add a base region complementary to the second segment of the promoter oligonucleotide, i.e., the segment comprising the promoter sequence, rendering the promoter double-stranded. Alternatively, where a terminating oligonucleotide or other binding molecule is not used in a TMA reaction, a promoter primer may be used as the promoter oligonucleotide, thereby allowing the incorporation of a promoter sequence into a second extension product initiated from and thus comprising the promoter primer. In this case, priming of a third extension product using the second extension product as a template produces a double-stranded DNA that includes the double-stranded promoter. An RNA polymerase which recognizes the promoter then binds to the promoter sequence, and initiates transcription of multiple RNA copies complementary to the DNA primer extension product, which copies are substantially identical to the target region. By “substantially identical” it is meant that the multiple RNA copies may have additional nucleotides either 5′ or 3′ relative to the target sequence, or may have fewer nucleotides either 5′ or 3′ relative to the target sequence, depending on, e.g., the boundaries of the target region, the transcription initiation point, or whether the priming oligonucleotide comprises additional nucleotides 5′ of the primer region. Where a target region is DNA, the sequence of the RNA copies is described herein as being “substantially identical” to the target region. It is to be understood, however, that an RNA sequence which has uridine residues in place of the thymidine residues of the DNA target region still has a “substantially identical” sequence. The RNA transcripts so produced may automatically recycle in the above system without further manipulation. Thus, this reaction is autocatalytic.
[0145] Promoters or promoter sequences suitable for incorporation in promoter oligonucleotides used in the methods of the present invention are nucleic acid sequences (either naturally occurring, produced synthetically or a product of a restriction digest) that are specifically recognized by an RNA polymerase that recognizes and binds to that sequence and initiates the process of transcription, whereby RNA transcripts are produced. Typical, known and useful promoters include those which are recognized by certain bacteriophage polymerases, such as those from bacteriophage T3, T7, and SP6, and a promoter from E. coli. The sequence may optionally include nucleotide bases extending beyond the actual recognition site for the RNA polymerase which may impart added stability or susceptibility to degradation processes or increased transcription efficiency. Promoter sequences for which there is a known and available polymerase that is capable of recognizing the initiation sequence are particularly suitable to be employed.
[0146] Suitable DNA polymerases for use in accordance with the methods of the invention, particularly for use with embodiments employing TMA, include reverse transcriptases. Particularly suitable DNA polymerases include AMV reverse transcriptase and MMLV reverse transcriptase. Some of the reverse transcriptases suitable for use in the methods of the present invention, such as AMV and MMLV reverse transcriptases, have an RNAse H activity. Indeed, according to certain embodiments of the present invention, the only selective RNAse activity in the amplification reaction is provided by the reverse transcriptase—no additional selective RNAse is added. However, in some situations it may also be useful to add an exogenous selective RNAse, such as E. coli RNAse H. Although the addition of an exogenous selective RNAse is not required, under certain conditions, the RNAse H activity present in, e.g., AMV reverse transcriptase may be inhibited or inactivated by other components present in the reaction mixture. In such situations, addition of an exogenous selective RNAse may be desirable. For example, where relatively large amounts of heterologous DNA are present in the reaction mixture, the native RNAse H activity of the AMV reverse transcriptase may be somewhat inhibited and thus the number of copies of the target sequence produced accordingly reduced. In situations where the target nucleic acid comprises only a small portion of the nucleic acid present (e.g., where the sample contains significant amounts of heterologous DNA and/or RNA), it is particularly useful to add an exogenous selective RNAse. See, e.g., Kacian et al, U.S. Pat. No. 5,399,491, incorporated by reference herein.
[0147] RNA amplification products produced by TMA methods may serve as templates to produce additional amplification products related to the target sequence through mechanisms described herein. The system is autocatalytic and amplification by the methods of the present invention occurs without the need for repeatedly modifying or changing reaction conditions such as temperature, pH, ionic strength and the like. These methods do not require an expensive thermal cycling apparatus, nor do they require several additions of enzymes or other reagents during the course of an amplification reaction.
[0148] The amplification product can be detected by any conventional means. For example, amplification product can be detected by hybridization with a detectably labeled probe and measurement of the resulting hybrids. Design criteria in selecting probes for detecting particular target sequences are well-known in the art and are described in, for example, Hogan et al., U.S. Pat. No. 6,150,517, incorporated by reference herein. Generally, probes should be designed to maximize homology for the target sequence(s) and minimize homology for possible non-target sequences. To minimize stability with non-target sequences, guanine and cytosine rich regions should be avoided, the probe should span as many destabilizing mismatches as possible, and the length of perfect complementarity to a non-target sequence should be minimized. Contrariwise, stability of the probe with the target sequence(s) should be maximized, adenine and thymine rich regions should be avoided, probe:target hybrids are preferably terminated with guanine and cytosine base pairs, extensive self-complementarity is generally to be avoided, and the melting temperature of probe:target hybrids should be about 2-10° C. higher than the assay temperature.
[0149] In a particular embodiment, the amplification product is assayed by the Hybridization Protection Assay (“HPA”), which involves hybridizing a chemiluminescent oligonucleotide probe (e.g., an acridinium ester-labeled (“AE”) probe) to its target sequence, selectively hydrolyzing the chemiluminescent label present on unhybridized probe, and measuring the chemiluminescence produced from the remaining probe in a luminometer. See, e.g., Arnold et al., U.S. Pat. No. 5,283,174 and Norman C. Nelson et al., Nonisotopic Probing, Blotting, and Sequencing, Ch. 17 (Larry J. Kricka ed., 2d ed. 1995), each incorporated by reference herein.
[0150] In further embodiments, the present invention provides quantitative evaluation of the amplification process in real-time. Evaluation of an amplification process in “real-time” involves determining the amount of amplicon in the reaction mixture either continuously or periodically during the amplification reaction, and the determined values are used to calculate the amount of target sequence initially present in the sample. There are a variety of known methods for determining the amount of initial target sequence present in a sample based on real-time amplification. These include those disclosed by Wittwer et al., U.S. Pat. No. 6,303,305, and Yokoyama et al., U.S. Pat. No. 6,541,205, each incorporated by reference herein. Another method for determining the quantity of target sequence initially present in a sample, but which is not based on a real-time amplification, is disclosed by Ryder et al., U.S. Pat. No. 5,710,029, incorporated by reference herein.
[0151] Amplification products may be detected in real-time through the use of various self-hybridizing detection probes, most of which have a stem-loop structure. Such self-hybridizing probes are labeled so that they emit differently detectable signals, depending on whether the detection probes are in a self-hybridized state or an altered state through hybridization to a target sequence. By way of example, “molecular torches” are a type of self-hybridizing detection probe that includes distinct regions of self-complementarity (referred to as “the target binding domain” and “the target closing domain”) that are connected by a joining region (e.g., non-nucleotide linker) and hybridize to each other under predetermined hybridization assay conditions. In a preferred embodiment, molecular torches contain single-stranded base regions in the target-binding domain that are from 1 to about 20 bases in length and are accessible for hybridization to a target sequence present in an amplification product under strand displacement conditions. Under strand displacement conditions, hybridization of the two complementary regions (which may be fully or partially complementary) of the molecular torch is favored, except in the presence of the target sequence, which will bind to the single-stranded region present in the target-binding domain and displace all or a portion of the target closing domain. The target binding domain and the target closing domain of a molecular torch include a detectable label or a pair of interacting labels (e g., luminescent/quencher) positioned so that a different signal is produced when the molecular torch is self-hybridized than when the molecular torch is hybridized to the target sequence, thereby permitting detection of probe:target duplexes in a test sample in the presence of unhybridized molecular torches. Molecular torches and a variety of types of interacting label pairs are disclosed by Becker et al., U.S. Pat. No. 6,534,274, incorporated by reference herein.
[0152] Another example of a detection probe having self-complementarity is a “molecular beacon.” Molecular beacons include nucleic acid molecules having a target complement sequence, an affinity pair (or nucleic acid arms) holding the probe in a closed conformation in the absence of a target sequence present in an amplification product, and a label pair that interacts when the probe is in a closed conformation. Hybridization of the target sequence and the target complement sequence separates the members of the affinity pair, thereby shifting the probe to an open conformation. The shift to the open conformation is detectable due to reduced interaction of the label pair, which may be, for example, a fluorophore and a quencher (e.g., DABCYL and EDANS). Molecular beacons are disclosed by Tyagi et al., U.S. Pat. No. 5,925,517, and Tyagi et al., U.S. Pat. No. 6,150,097, each incorporated by reference herein.
[0153] Other self-hybridizing probes for use in the present invention are well-known to those of ordinary skill in the art. By way of example, probe binding pairs having interacting labels, such as those disclosed by Morrison, U.S. Pat. No. 5,928,862, and Gelfand et al., U.S. Pat. No. 5,804,375 for PCR reactions (each incorporated by reference herein), might be adapted for use in the present invention. Additional detection systems include “molecular switches,” as disclosed by Arnold et al., U.S. Pat. Appln. Pub. No. US 2005-0042638 A1, incorporated by reference herein. And other probes, such as those comprising intercalating dyes and/or fluorochromes, might be useful for detection of amplification products in the present invention. See, e.g., Ishiguro et al., U.S. Pat. No. 5,814,447, incorporated by reference herein.
[0154] In those methods of the present invention where the initial target sequence and the RNA transcription product share the same sense, it may be desirable to initiate amplification before adding probe for real-time detection. Adding probe prior to initiating an amplification reaction may slow the rate of amplification since probe which binds to the initial target sequence has to be displaced or otherwise remove during the primer extension step to complete a primer extension product having the complement of the target sequence. The initiation of amplification is judged by the addition of amplification enzymes (e.g., a reverse transcriptase and an RNA polymerase).
[0155] Also provided by the subject invention is a reaction mixture for amplification of a target nucleic acid. A reaction mixture in accordance with the present invention at least comprises a combination of amplification oligomers as described herein for amplification of a nucleic acid target region. In certain embodiments, a reaction mixture also includes a capture probe for purifying the target nucleic acid and/or a detection probe for determining the presence or absence of an amplification product. The reaction mixture may further include a number of optional components such as, for example, arrays of capture probe nucleic acids. For an amplification reaction mixture, the reaction mixture will typically include other reagents suitable for performing in vitro amplification such as, e.g., buffers, salt solutions, appropriate nucleotide triphosphates (e.g., dATP, dCTP, dGTP, dTTP, ATP, CTP, GTP and UTP), and/or enzymes (e.g., reverse transcriptase, and/or RNA polymerase), and will typically include test sample components, in which a target nucleic acid may or may not be present. In addition, for a reaction mixture that includes a detection probe together with an amplification oligomer combination, selection of amplification oligomers and detection probe oligomers for a reaction mixture are linked by a common target region (i.e., the reaction mixture will include a probe that binds to a sequence amplifiable by an amplification oligomer combination of the reaction mixture).
[0156] Also provided by the subject invention are kits for practicing the methods as described herein. A kit in accordance with the present invention at least comprises a combination of amplification oligomers as described herein for amplification of a nucleic acid target region. In certain embodiments, a reaction mixture also includes a capture probe for purifying the target nucleic acid and/or a detection probe for determining the presence or absence of an amplification product. The kits may further include a number of optional components such as, for example, arrays of capture probe nucleic acids. Other reagents that may be present in the kits include reagents suitable for performing in vitro amplification such as, e.g., buffers, salt solutions, appropriate nucleotide triphosphates (e.g., dATP, dCTP, dGTP, dTTP, ATP, CTP, GTP and UTP), and/or enzymes (e.g., reverse transcriptase, and/or RNA polymerase). Oligomers as described herein may be packaged in a variety of different embodiments, and those skilled in the art will appreciate that the invention embraces many different kit configurations. For example, a kit may include amplification oligomers for only one target region, or it may include amplification oligomers for multiple target regions. In addition, for a kit that includes a detection probe together with an amplification oligomer combination, selection of amplification oligomers and detection probe oligomers for a kit are linked by a common target region (i.e., the kit will include a probe that binds to a sequence amplifiable by an amplification oligomer combination of the kit). In certain embodiments, the kit further includes a set of instructions for practicing methods in accordance with the present invention, where the instructions may be associated with a package insert and/or the packaging of the kit or the components thereof.
[0157] The invention is further illustrated by the following non-limiting examples.
Reagents
[0158] Various reagents are identified in the examples below, the formulations and pH values (where relevant) of these reagents were as follows.
[0159] A “Lysis Buffer” contains 15 mM sodium phosphate monobasic monohydrate, 15 mM sodium phosphate dibasic anhydrous, 1.0 mM EDTA disodium dihydrate, 1.0 mM EGTA free acid, and 110 mM lithium lauryl sulfate, pH 6.7.
[0160] A “Urine Lysis Buffer” contains 150 mM HEPES free acid, 294 mM lithium lauryl sulfate, 57 mM lithium hydroxide monohydrate, 100 mM ammonium sulfate, pH 7.5.
[0161] A “Target Capture Reagent” contains 250 mM HEPES free acid dihydrate, 310 mM lithium hydroxide monohydrate, 1.88 M lithium chloride, 100 mM EDTA free acid, 2 M lithium hydroxide to pH 6.4, and 250 μg/ml 1 micron magnetic particles Sera-Mag′ MG-CM Carboxylate Modified (Seradyn, Inc.; Indianapolis, Ind.; Cat. No. 24152105-050450) having oligo(dT).sub.14 covalently bound thereto.
[0162] A “Wash Solution” contains 10 mM HEPES free acid, 6.5 mM sodium hydroxide, 1 mM EDTA free acid, 0.3% (v/v) ethyl alcohol absolute, 0.02% (w/v) methyl paraben, 0.01% (w/v) propyl paraben, 150 mM sodium chloride, 0.1% (w/v) lauryl sulfate, sodium (SDS), and 4 M sodium hydroxide to pH 7.5.
[0163] An “Amplification Reagent” is a lyophilized form of a 3.6 mL solution containing 26.7 mM rATP, 5.0 mM rCTP, 33.3 mM rGTP and 5.0 mM rUTP, 125 mM HEPES free acid, 8% (w/v) trehalose dihydrate, 1.33 mM dATP, 1.33 mM dCTP, 1.33 mM dGTP, 1.33 mM dTTP, and 4 M sodium hydroxide to pH 7.5. The Amplification Reagent is reconstituted in 9.7 mL of “Amplification Reagent Reconstitution Solution” described below.
[0164] An “Amplification Reagent Reconstitution Solution” contains 0.4% (v/v) ethyl alcohol absolute, 0.10% (w/v) methyl paraben, 0.02% (w/v) propyl paraben, 33 mM KCl, 30.6 mM MgCl.sub.2, 0.003% phenol red.
[0165] A “Primer Reagent” contains 1 mM EDTA disodium dihydrate, ACS, 10 mM Trizma7 base, and 6M hydrochloric acid to pH 7.5.
[0166] An “Enzyme Reagent” is a lyophilized form of a 1.45 mL solution containing 20 mM HEPES free acid dihydrate, 125 mM N-acetyl-L-cysteine, 0.1 mM EDTA disodium dihydrate, 0.2% (v/v) TRITON® X-100 detergent, 0.2 M trehalose dihydrate, 0.90 RTU/mL Moloney murine leukemia virus (“MMLV”) reverse transcriptase, 0.20 U/mL T7 RNA polymerase, and 4M sodium hydroxide to pH 7.0. (One “unit” or “RTU” of activity is defined as the synthesis and release of 5.75 fmol cDNA in 15 minutes at 37° C. for MMLV reverse transcriptase, and for T7 RNA polymerase, one “unit” or “U” of activity is defined as the production of 5.0 fmol RNA transcript in 20 minutes at 37° C.) The Enzyme Reagent is reconstituted in 3.6 mL of “Enzyme Reagent Reconstitution Solution” described below.
[0167] An “Enzyme Reagent Reconstitution Solution” contains 50 mM HEPES free acid, 1 mM EDTA free acid, 10% (v/v) TRITON X-100 detergent, 120 mM potassium chloride, 20% (v/v) glycerol anhydrous, and 4 M sodium hydroxide to pH 7.0.
[0168] A “Probe Reagent” is a lyophilized form of a 3.6 mL solution containing 110 mM lithium lauryl sulfate, 10 mM of mercaptoethane sulfonic acid, 100 mM lithium succinate, and 3% PVP. The Probe Reagent is reconstituted in 36 mL of “Probe Reagent Reconstitution Solution” described below.
[0169] A “Probe Reagent Reconstitution Solution” contains 100 mM succinic acid, 73 mM lithium lauryl sulfate, 100 mM lithium hydroxide monohydrate, 15 mM aldrithiol, 1.2 M lithium chloride, 20 mM EDTA, 3% (v/v) ethyl alcohol, and 2M lithium hydroxide to pH 4.7.
[0170] A “Selection Reagent” contains 600 mM boric acid, ACS, 182.5 mM sodium hydroxide, ACS, 1% (v/v) TRITON X-100 detergent, and 4 M sodium hydroxide to pH 8.5.
[0171] A “Detection Reagents” comprises Detect Reagent I, which contains 1 mM nitric acid and 32 mM hydrogen peroxide, 30% (v/v), and Detect Reagent II, which contains 1.5 M sodium hydroxide.
[0172] An “Oil Reagent” is a silicone oil.
Example 1: S-Complex Preparation
[0173] S-Complexes were made in a reagent mixture made up of Target Capture Reagent (minus the magnetic particles), Lysis Buffer and water in a 1:2:2 ratio respectively. T7 oligonucleotide, non-T7 oligonucleotide, and S-Oligo were added to the reagent mixture at 6, 5, and 6 picomoles (pmol) per microliter (ul) respectively. The reagent mixture with the oligonucleotides was incubated at 95° C. for one minute in a hot block, followed by a 4° C. incubation for 5 minutes, followed by a room temperature incubation for approximately 20 minutes. The S-Complex (1 ul) was added to the Target Capture Reagent.
Example 2: Universal Tagged TMA with and without Displacer Oligonucleotides
[0174] In this example, several non-T7 amplification oligonucleotide variations, with and without displacer oligonucleotides were compared. The non-T7 sequence variations were (5′ to 3′): displacer tag:spacer:tag:target specific (SEQ ID NO:6); tag:spacer:tag:target specific (SEQ ID NO:7); displacer tag:tag:target specific (SEQ ID NO:8); and tag:target specific (SEQ ID NO:9). A T7 promoter-based amplification oligomer (SEQ ID NO:11) was used with each of the 4 nonT7 amplification oligomers. The amplification oligomers (nonT7 and T7 pairs) were joined using the s-oligo SEQ ID NO:12 (see, e.g., Brentano et al. WO 2008/080029 describing reagents and methods for amplification using forward and reverse amplification oligomers joined with an s-oligo, incorporated herein by reference). The amplification oligonucleotide combinations were analyzed using target capture to extract target nucleic acid from a sample; single primer transcription mediated amplification (spTMA) to amplify the target nucleic acid; and molecular torches to detect the amplification product (i.e. amplicons) in “real-time” (i.e. continuous monitoring of fluorescent levels over time). Target Capture is described in Weisburg et al, U.S. Pat. No. 6,110,678 (the contents of which are incorporated by reference), spTMA is described in Becker et al., U.S. Pat. No. 7,374,885 and U.S. App. No. 20060046265A1 (the contents of which are incorporated by reference), and molecular torches are described in Becker et al, U.S. Pat. Nos. 6,849,412, 6,835,542, 6,534,274, and 6,361,945, US App. No. 20060068417A1; and Arnold et al. US App. No. US20060194240A1 (the contents of which are incorporated by reference). The protocols for each method are briefly described below.
[0175] The prostate specific antigen (PSA) gene was cloned and transected into competent cells. In vitro transcript (IVT) from the cloned PSA was used as the target nucleic acid. PSA IVT was spiked into a 1:1 mixture of Lysis Buffer and water at 0, 10.sup.2, and 10.sup.4 copies per 400 microliters (ul) and 400 ul of the spiked, diluted Lysis Buffer was transferred to a 96 deep well plate. Target Capture Reagent (100 ul) containing 5 pmol of SEQ ID NO:1, 1 ul of the S-Complex prepared according to Example 1 and using the combinations of nonT7, T7 and s-oligomer described directly above (see Table 1 for S-Complex primer combinations), and 2 pmol of SEQ ID NO:3 blocker was added to each well. The 96 well plate was sealed, a 90° C. heat block was placed on top of the plate, and the plate was vortexed on a low setting for 10 seconds. The 96 well plate was incubated at 60° C. for 25 and then cooled to room temperature for 25 minutes. The magnetic beads were pelleted using a KingFisher® instrument (Thermo Scientific) and the supernatant was removed. The magnetic beads were resuspended in 400 ul of Wash Solution, repelletted, and the wash solution was removed. Following capture and wash, the remaining complex was—a magnetic beadimmobilized probe:capture probe:target nucleic acid:s-oligo complex according to each of the combinations in Table 1. The magnetic beads were then resuspended in 60 ul of Amplification Reagent containing 10 pmol of SEQ ID NO:4, 15 pmol of each of SEQ ID NO:10, 10 pmol of SEQ ID NO:2 (labeled with ROX and FAM), and for half of the samples, 15 pmol of SEQ ID NO:5. The 96 well plate was placed in a Chromo4® instrument (Bio-Rad Laboratories, Inc., Hercules, Calif.) pre-warmed to 42° C. for 5 minutes. Enzyme Reagent (20 ul) was added to each well, the plate was briefly vortexed for 20 seconds, and returned to the Chromo4 instrument to start the fluorescence monitoring.
[0176] Four replicates were run for each assay condition. The results were measured by the amount of fluorescence over time. The reported Ct is the cycle time when the fluorescence level becomes higher than the background level. The results are summarized in Table 2, below and indicate that the use of tag-mediated displacer technology reduced the average Ct for each condition. That is, a fluorescent signal was detected earlier when using displacers than when not indicating that the amount of amplification product generated in a condition is greater when using the tag-mediated displacer than when not.
TABLE-US-00001 TABLE 1 S-Oligomer Complexes T7 SEQ ID NO: Non-T7 SEQ ID NO: S-oligo SEQ ID NO: 11 6 12 11 7 12 11 8 12 11 9 12
TABLE-US-00002 TABLE 2 Amt. of PSA IVT S-Complex Displacer Ave. Ct (minutes) 0 11, 6, 12 No No signal 100 11, 6, 12 No 50 10,000 11, 6, 12 No 49.5 0 11, 6, 12 Yes No signal 100 11, 6, 12 Yes 41.5 10,000 11, 6, 12 Yes 39 0 11, 7, 12 No No signal 100 11, 7, 12 No 45 10,000 11, 7, 12 No 43 0 11, 7, 12 Yes No signal 100 11, 7, 12 Yes 42.5 10,000 11, 7, 12 Yes 41 0 11, 8, 12 No No signal 100 11, 8, 12 No 47 10,000 11, 8, 12 No 44 0 11, 8, 12 Yes No signal 100 11, 8, 12 Yes 45 10,000 11, 8, 12 Yes 41 0 11, 9, 12 No No signal 100 11, 9, 12 No 46.5 10,000 11, 9, 12 No 46
Example 3: Sensitivity of Universal Tagged TMA with and without Displacer Oligonucleotides
[0177] In this example, the sensitivity of a non-T7 amplification oligonucleotide (SEQ ID NO:6, displacer:spacer:tag:target specific) with and without displacer oligonucleotides was evaluated. The procedures and oligonucleotide concentrations were the same as those described in Example 2 with the following changes. The PSA IVT was tested at 0, 100, 1000, 10000, 100000, and 1000000 copies per reaction. Four replicates were run for each assay condition. The results are summarized in Table 3, below and indicate that the use of displacers reduced the average Ct for a condition. Tag-mediated displacer technology increases the number of amplification products generated in a reaction to provide an earlier emergence of fluorescent signal.
TABLE-US-00003 TABLE 3 Amt. of PSA IVT Displacer Ct (minutes) 0 No No signal 100 No 60 1000 No 56 10000 No 50 100000 No 46 1000000 No 39 0 Yes No signal 100 Yes 52 1000 Yes 50 10000 Yes 43 100000 Yes 40 1000000 Yes 34
[0178] These examples show that tag-mediated displacement increased overall output and increased assay kinetics and sensitivity.
TABLE-US-00004 TABLE 4 SEQ ID Preferred NO Sequence 5′ .fwdarw. 3′ Function 1 CGAACUUGCGCACACACGUCAUUGGAtttaaa Target aaaaaaaaaaaaaaaaaaaaaaaaaaa Capture 2 UGUGUCUUCAGGAUGAAACACACA Torch 3 GAUGCAGUGGGCAGCUGUGAGGA Blocker 4 aatttaatacgactcactatagggagaCCACA T7 ACGGTTT 5 GAGGTCGTGGCTGGAGTCAT Displacer 6 GAGGTCGTGGCTGGAGTCATatgtcaacgtGT Non-T7 CATATGCGACGATCTCAGGCTGTGGCTGACCT GAAATACC 7 GTCATATGCGACGATCTCAGatgtcaacgtGT Non-T7 CATATGCGACGATCTCAGGCTGTGGCTGACCT GAAATACC 8 GAGGTCGTGGCTGGAGTCATGTCATATGCGAC Non-T7 GATCTCAGGCTGTGGCTGACCTGAAATACC 9 GTCATATGCGACGATCTCAGGCTGTGGCTGAC Non-T7 CTGAAATACC 10 GTCATATGCGACGATCTCAG Non-T7 11 aatttaatacgactcactatagggagaCCACA T7 ACGGTTTACCCAGCAAGATCACGCTTTTG 12 AAACCGTTGTGGTCTCCCTATACTGAGATCGT s-oligo CGCATATGAC
[0179] Table 4 illustrates oligonucleotide sequences such as those used in the examples. The legend for Table 4 is as follows. Bold=target specific; Italics=displacer tag; Underline=tag non-T7; Underline italics=tag T7; lowercase=promoter or capture tail.
[0180] From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entireties for all purposes.