Exponential base-3 nucleic acid amplification using nested overlapping primers with reduced amplification time

Abstract

Provide a method and primer set composition for efficient nucleic acid amplification. In some embodiments, compared with conventional PCR, this allows to achieve 3 times or more increase of the amplified product of each amplification cycle, and thus achieves increased sensitivity and speed. The method is based on using a set of nested primers, the outer primers include modified bases, for example, 2-aminoadenine or 2-thiothymine, and the inner primers include a single-stranded target binding region and a double-stranded region, and the double-stranded region includes a flap sequence that is not complementary to the target and a region complementary to the outer primer. Compared with those PCR cycle times observed in the absence of modified bases, the modified base improves the PCR cycle time by allowing the outer primer to bind to the target. The method and composition can also be used and include a third intermediate primer having a structure as an inner primer, respectively.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 752,269, filed on October 29, 2018, and U.S. Provisional Patent Application No. 62 / 752,276, filed on October 29, 2018, which are incorporated herein by reference in their entirety.

[0003] STATEMENT OF RIGHTS TO INVENTS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0004] not applicable. Technical Field

[0005] The methods and compositions described herein generally relate to the field of nucleic acid amplification. In particular, described herein are methods and compositions for improving amplification efficiency. Background Art

[0006] A variety of nucleic acid amplification methods are available, and many methods have been used to achieve sensitive diagnostic assays based on nucleic acid detection. Polymerase chain reaction (PCR) remains the most widely used DNA amplification and quantitative method. Nested PCR (a two-step PCR) is used to improve the specificity and sensitivity of PCR (U.S. Patent No. 4,683,195). The nested primers used for PCR amplification are oligonucleotides with sequences complementary to the region between the reverse primer and the forward primer targeting site on the target sequence. However, PCR generally has several limitations. PCR amplification can only achieve an increase of less than twice the amount of target sequence in each cycle. It is still relatively slow. In addition, the sensitivity of the method is generally limited, making it difficult to detect targets that are only present in a few molecules in a single reaction. Summary of the invention

[0007] Described herein are methods and compositions based on the use of new primers (eg, new inner primers) designed such that outer primer binding sites are maintained in the amplicons generated upon amplification.

[0008] Various embodiments contemplated herein may include, but are not necessarily limited to, one or more of the following:

[0009] Embodiment 1: A nucleic acid primer set for amplifying a target nucleic acid in a sample, wherein the target nucleic acid comprises a first template strand and an optional second template strand, wherein the second template strand is complementary to the first template strand, and the primer set comprises such oligonucleotides, which are in the form of at least two first primers or are capable of forming at least two first primers, the first primers are capable of hybridizing with the first template strand, wherein the at least two first primers comprise a first outer primer and a first inner primer, the first outer primer comprises a primer sequence a that specifically hybridizes with a first template strand sequence a', the primer sequence a comprises one or more first modified bases; and the first inner primer comprises a single-stranded primer sequence b that specifically hybridizes with a first template strand sequence b', wherein b' is adjacent to a' and is 5' of a', and wherein the single-stranded primer sequence b is connected at its 5' end to a first strand of a double-stranded primer sequence, the first strand comprising: a primer sequence a that is adjacent to the single-stranded primer sequence b and is 5' of the single-stranded primer sequence b; and a clamp sequence c (clamp sequence c), which is adjacent to the primer sequence a and is 5' of the primer sequence a, wherein the clamping sequence c is not complementary to the first strand template sequence d', said sequence d' is adjacent to the first strand template sequence a' and is 3' of the first strand template sequence a'; wherein the second strand of the double-stranded primer sequence includes a primer sequence c' that is adjacent to the primer sequence a' and is 3' of the primer sequence a', wherein the joint sequence c'-a' is complementary to the joint sequence ca, and the primer sequence a' includes one or more second modified bases; and wherein the unmodified forms of the first and second modified bases are complementary, and the first and second modified bases preferentially pair with the unmodified forms compared to pairing between the first and second modified bases.

[0010] Embodiment 2: The primer set described in Embodiment 1, wherein the primer set further comprises at least one second primer capable of specifically hybridizing to the second template strand.

[0011] Embodiment 3: A method for amplifying a target nucleic acid in a sample, wherein the target nucleic acid comprises a first template strand and optionally a second template strand, wherein the second template strand is complementary to the first template strand, the method comprising: (a) contacting the sample with: (i) at least two first primers capable of hybridizing to the first template strand, wherein the at least two first primers comprise a first outer primer and a first inner primer, the first outer primer comprises a primer sequence a that specifically hybridizes to a first template strand sequence a', the primer sequence a comprising one or more first modified bases; and the first inner primer comprises a single-stranded primer sequence b that specifically hybridizes to a first template strand sequence b', wherein b' is adjacent to a' and is 5' of a', and wherein the single-stranded primer sequence b is linked at its 5' end to a first strand of a double-stranded primer sequence, the first strand comprising: a primer sequence a that is adjacent to the single-stranded primer sequence b and is 5' of the single-stranded primer sequence b; and a clamp sequence c that is adjacent to the primer sequence a and is 5' of the primer sequence a, wherein the clamp sequence c does not hybridize to the first strand template strand; wherein the first and second modified bases are complementary to each other and the first and second modified bases are preferentially paired with the unmodified forms compared to the pairing between the first and second modified bases; and (ii) at least one second primer capable of specifically hybridizing to the second template strand, wherein the contacting is performed under conditions where the primers anneal to their template strands (if any); and (b) under conditions where strand displacement occurs, (if any) amplifying the target nucleic acid using a DNA polymerase lacking 5'-3' exonuclease activity to produce an amplicon, wherein the amplicon comprises a sequence extending from the template sequence a' to the second primer binding site.

[0012] Embodiment 4: The method of Embodiment 3, wherein the DNA polymerase is stable above 85 degrees Celsius.

[0013] Embodiment 5: The primer set or method of any one of the preceding embodiments, wherein the T of the combined sequence ca in the double-stranded form m T higher than the combined sequence ab in double-stranded form m .

[0014] Embodiment 6: The primer set or method according to any one of the preceding embodiments, wherein the combined sequence ca is more GC-rich and / or contains more stable bases than the combined sequence ab.

[0015] Embodiment 7: The primer set or method of any one of the preceding embodiments, wherein the primer set is capable of amplifying the amplification product with at least 3 循环数 The target nucleic acid is amplified at a rate of at least 3 循环数 Amplify the target nucleic acid at a rate of

[0016] Embodiment 8: The primer set or method described in any of the preceding embodiments, wherein the primer set or method allows detection of a single copy nucleic acid in a biological sample within such amplification cycles: the amplification cycles are approximately 12%-42% fewer than the amplification cycles required for the detection using only a single forward primer and a single reverse primer.

[0017] Embodiment 9: The primer set or method of any one of Embodiments 2-8, wherein the second primer comprises an oligonucleotide in the form of at least two second primers or capable of forming at least two second primers, the second primer being capable of hybridizing with the second template strand, wherein the at least two second primers comprise a second outer primer and a second inner primer, the second outer primer comprises a primer sequence e that specifically hybridizes with a second template strand sequence e', the primer sequence e comprises one or more third modified bases; and the second inner primer comprises a single-stranded primer sequence f that specifically hybridizes with a second template strand sequence f', wherein f' is adjacent to e' and is 5' of e', and wherein the single-stranded primer sequence f is connected at its 5' end to a first strand of a double-stranded primer sequence, the first strand comprising: a primer sequence e, It is adjacent to the single-stranded primer sequence f and is 5' of the single-stranded primer sequence f; and a clamping sequence g, which is adjacent to the primer sequence e and is 5' of the primer sequence e, wherein the clamping sequence g is not complementary to the second strand template sequence h', and the sequence h' is adjacent to the second template strand sequence e' and is 3' of the second template strand sequence e'; wherein the second strand of the double-stranded primer sequence includes a primer sequence g', which is adjacent to the primer sequence e' and is 3' of the primer sequence e', wherein the joint sequence g'-e' is complementary to the joint sequence ge, and the primer sequence e' includes one or more fourth modified bases; and wherein the unmodified forms of the third and fourth modified bases are complementary, and the third and fourth modified bases preferentially pair with the unmodified forms compared to pairing between the third and fourth modified bases.

[0018] Embodiment 10: The primer set or method of embodiment 9, wherein the T of the combined sequence ge in the double-stranded form m T higher than the combined sequence ef in double-stranded form m .

[0019] Embodiment 11: The primer set or method according to any one of Embodiments 9-10, wherein the combined sequence ge is more GC-rich and / or contains more stable bases than the combined sequence ef.

[0020] Embodiment 12: The primer set or method of any one of Embodiments 9-11, wherein the primer set is capable of amplifying at least 6 循环数 The target nucleic acid is amplified at a rate of at least 6 循环数 Amplify the target nucleic acid at a rate of

[0021] Embodiment 13: The primer set or method of any one of Embodiments 9-12, wherein the primer set or method allows detection of a single copy nucleic acid in a biological sample within such amplification cycles: the amplification cycles are approximately 36%-66% fewer than the amplification cycles required for the detection using only a single forward primer and a single reverse primer.

[0022] Embodiment 14: The primer set or method of any of the preceding embodiments, wherein the clamping sequences c and g (if present) cannot be replicated during amplification.

[0023] Embodiment 15: The primer set or method of Embodiment 14, wherein the clamping sequence c and / or g (if present) comprises 2'-O-methyl RNA.

[0024] Embodiment 16: The primer set or method of any one of the preceding embodiments, wherein the double-stranded primer sequence of the first inner primer and / or the second inner primer (if present) does not contain a hairpin sequence.

[0025] Embodiment 17: The primer set or method described in any one of Embodiments 1-15, wherein the double-stranded primer sequence of the first inner primer includes a hairpin sequence in which the clamping sequence c is connected to the complementary sequence c', and / or the double-stranded primer sequence of the second inner primer (if present) includes a hairpin sequence in which the clamping sequence g is connected to the complementary sequence g'.

[0026] Embodiment 18: A nucleic acid primer set for amplifying a target nucleic acid in a sample, wherein the target nucleic acid comprises a first template strand and an optional second template strand, wherein the second template strand is complementary to the first template strand, and the primer set comprises an oligonucleotide in the form of at least three first primers or capable of forming at least three first primers, wherein the first primers are capable of hybridizing with the first template strand, wherein the at least three first primers comprise a first outer primer, a first middle primer, and a first inner primer, wherein the first outer primer comprises a primer sequence d that specifically hybridizes with a first template strand sequence d', wherein the primer sequence d comprises one or more first modified bases; and the first middle primer comprises a single-stranded primer sequence a that specifically hybridizes with a first template strand sequence a', wherein the first middle primer comprises a single-stranded primer sequence a that specifically hybridizes with a first template strand sequence a'. wherein a' is adjacent to d' and is 5' of d', the primer sequence a includes one or more second modified bases, wherein the single-stranded primer sequence a is connected at its 5' end to the first strand of the double-stranded primer sequence, the first strand including: a primer sequence d, which is adjacent to the single-stranded primer sequence a and is 5' of the single-stranded primer sequence a; and a clamping sequence c1, which is adjacent to the primer sequence d and is 5' of the primer sequence d, wherein the clamping sequence c1 is not complementary to the first template strand sequence i', the template sequence i' is adjacent to the first template strand sequence d' and is 3' of the first template strand sequence d'; wherein the second strand of the double-stranded primer sequence includes a primer sequence c1', which is adjacent to the primer sequence d' and is 3' of the primer sequence d', wherein the combined sequence c1'-d' and Combined sequence c1-d, primer sequence d' includes one or more third modified bases; and the first inner primer includes a single-stranded primer sequence b that specifically hybridizes to the first template strand sequence b', wherein b' is adjacent to a' and is 5' of a', and wherein the single-stranded primer sequence b is connected at its 5' end to the first strand of the double-stranded primer sequence, the first strand including: primer sequence a, which is adjacent to the single-stranded primer sequence b and is 5' of the single-stranded primer sequence b; primer sequence d, which is adjacent to the primer sequence a and is 5' of the primer sequence a; and a clamping sequence c2, which is adjacent to the primer sequence d and is 5' of the primer sequence d, wherein the clamping sequence c2 is not complementary to the first strand template sequence i'; wherein the second strand of the double-stranded primer sequence of the inner primer includes A primer sequence c2', which is adjacent to the primer sequence d' and is 3' to the primer sequence d', wherein the primer sequence d' is adjacent to the primer sequence a' and is 3' to the primer sequence a', and the primer sequence a' includes one or more fourth modified bases, wherein the combined sequence c2'-d'-a' is complementary to the combined sequence c2-da; wherein the unmodified forms of the first and third modified bases are complementary, and the first and third modified bases preferentially pair with the unmodified forms compared to the pairing between the first and third modified bases; and wherein the unmodified forms of the second and fourth modified bases are complementary, and the second and fourth modified bases preferentially pair with the unmodified forms compared to the pairing between the second and fourth modified bases.

[0027] Embodiment 19: The primer set of Embodiment 18, wherein the primer set further comprises at least one second primer capable of specifically hybridizing to the second template strand.

[0028] Embodiment 20: A method for amplifying a target nucleic acid in a sample, wherein the target nucleic acid comprises a first template strand and an optional second template strand, wherein the second template strand (if present) is complementary to the first template strand, the method comprising: (a) contacting the sample with: (i) at least three first primers capable of hybridizing to the first template strand, wherein the at least three first primers comprise a first outer primer, a first middle primer, and a first inner primer, the first outer primer comprising a primer sequence d that specifically hybridizes to a first template strand sequence d', the primer sequence d comprising one or more first modified bases; the first middle primer comprising a single-stranded primer sequence a that specifically hybridizes to a first template strand sequence a', wherein a' is adjacent to The first strand of the double-stranded primer sequence comprises a primer sequence d adjacent to the first strand of the double-stranded primer sequence, wherein the first strand comprises: a primer sequence d adjacent to the first strand of the single-stranded primer sequence a and being 5' of the single-stranded primer sequence a; and a clamping sequence c1 adjacent to the primer sequence d and being 5' of the primer sequence d, wherein the clamping sequence c1 is not complementary to the first template strand sequence i', which is adjacent to the first template strand sequence d' and being 3' of the first template strand sequence d'; wherein the second strand of the double-stranded primer sequence comprises a primer sequence c1' adjacent to the primer sequence d' and being 3' of the primer sequence d', wherein the union sequence c1'-d' is complementary to the union sequence c1-d are complementary, the primer sequence d' comprises one or more third modified bases; and the first inner primer comprises a single-stranded primer sequence b that specifically hybridizes to the first template strand sequence b', wherein b' is adjacent to a' and is 5' to a', and wherein the single-stranded primer sequence b is connected at its 5' end to the first strand of the double-stranded primer sequence, the first strand comprising: a primer sequence a, which is adjacent to the single-stranded primer sequence b and is 5' to the single-stranded primer sequence b; a primer sequence d, which is adjacent to the primer sequence a and is 5' to the primer sequence a; and a clamping sequence c2, which is adjacent to the primer sequence d and is 5' to the primer sequence d, wherein the clamping sequence c2 is not complementary to the first strand template sequence i'; wherein the second strand of the double-stranded primer sequence comprises a primer sequence a, which is adjacent to the single-stranded primer sequence b and is 5' to the single-stranded primer sequence b; a primer sequence d, which is adjacent to the primer sequence a and is 5' to the primer sequence a; and a clamping sequence c2, which is adjacent to the primer sequence d and is 5' to the primer sequence d, wherein the clamping sequence c2 is not complementary to the first strand template sequence i'; and a sequence c2' adjacent to and 3' of a primer sequence d', wherein the sequence d' is adjacent to and 3' of a primer sequence a', wherein the primer sequence a' comprises one or more fourth modified bases, wherein the joint sequence c2'-d'-a' is complementary to the joint sequence c2-da; wherein the unmodified forms of the first and third modified bases are complementary, and the first and third modified bases preferentially pair with the unmodified forms over the pairing between the first and third modified bases; and wherein the unmodified forms of the second and fourth modified bases are complementary, and the second and fourth modified bases preferentially pair with the unmodified forms over the pairing between the second and fourth modified bases;and (ii) at least one second primer capable of specifically hybridizing to the second template strand, wherein the contacting is performed under conditions in which the primers anneal to their template strands (if present); and (b) amplifying the target nucleic acid using a DNA polymerase lacking 5'-3' exonuclease activity (if present) under conditions in which strand displacement occurs to produce an amplicon comprising a sequence extending from the template sequence a' to the second primer binding site.;

[0029] Embodiment 21: The method of Embodiment 20, wherein the DNA polymerase is stable above 85 degrees.

[0030] Embodiment 22: The method of embodiment 20 or embodiment 21, wherein the time required to complete each amplification cycle is reduced by at least 10-95% compared to the time per cycle of the same primer set not including the modified base.

[0031] Embodiment 23: The method of Embodiment 22, wherein the time required to complete each amplification cycle is reduced by 50-85% compared to the time per cycle for the same primer set not including the modified base.

[0032] Embodiment 24: The primer set or method of any one of Embodiments 18-23, wherein c1 has a sequence different from c2.

[0033] Embodiment 25: The primer set or method of any one of Embodiments 18-24, wherein the T of the combined sequence c1-d in the double-stranded form m T higher than the combined sequence da of the double-stranded form m , and the combined sequence of the double-stranded form is c2-da T m Higher than the T of the combined sequence dab in double-stranded form m .

[0034] Embodiment 26: The primer set or method described in any one of Embodiments 18-25, wherein the combined sequence c1-d is richer in GC and / or contains more stable bases than the combined sequence da, and the combined sequence c2-da is richer in GC and / or contains more stable bases than the combined sequence dab.

[0035] Embodiment 27: The primer set or method of any one of Embodiments 18-26, wherein the primer set is capable of amplifying at least 4 循环数 The target nucleic acid is amplified at a rate of at least 4 循环数 Amplify the target nucleic acid at a rate of

[0036] Embodiment 28: The primer set or method of any one of Embodiments 18-27, wherein the primer set or method allows detection of a single copy nucleic acid in a biological sample within such amplification cycles: the amplification cycles are approximately 25%-55% fewer than the amplification cycles required for the detection using only a single forward primer and a single reverse primer.

[0037] Embodiment 29: The primer set or method of any one of Embodiments 18-28, wherein the second primer comprises such an oligonucleotide, which is in the form of at least three of the second primers or is capable of forming at least three of the second primers, and the second primer is capable of hybridizing with the second template strand, wherein the at least three second primers include a second outer primer, a second middle primer, and a second inner primer, the second outer primer comprises a primer sequence h that specifically hybridizes with a second template strand sequence h', and the primer sequence h comprises one or more fifth modified bases; the second middle primer comprises a single-stranded primer sequence e that specifically hybridizes with a second template strand sequence e', wherein e' is adjacent to h' and is 5' of h', and the primer sequence e includes one or more sixth modified bases, wherein the single-stranded primer sequence e is connected at its 5' end to the first strand of the double-stranded primer sequence, the first strand comprising: a primer sequence h, which is adjacent to the single-stranded primer sequence e and is 5' of the single-stranded primer sequence e; and a clamping sequence g1, which is adjacent to the primer sequence h and is 5' of the primer sequence h, wherein the clamping sequence g1 is not complementary to the second template strand sequence j', the sequence j' is adjacent to the second template strand sequence h' and is 3' of the second template strand sequence h'; wherein the second strand of the double-stranded primer sequence comprises a primer sequence g1' adjacent to the primer sequence h' and is 3' of the primer sequence h', wherein the union sequence g1'-h' is complementary to the union sequence g1-h, and the primer sequence The sequence h' comprises one or more seventh modified bases; and the second inner primer comprises a single-stranded primer sequence f that specifically hybridizes to the second template strand sequence f', wherein the sequence f' is adjacent to e' and is 5' of e', and wherein the single-stranded primer sequence f is connected at its 5' end to the first strand of the double-stranded primer sequence, the first strand comprising: a primer sequence e that is adjacent to the single-stranded primer sequence f and is 5' of the single-stranded primer sequence f; a primer sequence h that is adjacent to the primer sequence e and is 5' of the primer sequence e; and a clamping sequence g2 that is adjacent to the primer sequence h and is 5' of the primer sequence h, wherein the clamping sequence g2 is not complementary to the second template strand sequence j'; wherein the second strand of the double-stranded primer sequence of the inner primer comprises an adjacent A primer sequence g2' that is close to primer sequence h' and is 3' of primer sequence h', wherein sequence h' is adjacent to primer sequence e' and is 3' of primer sequence e', and primer sequence e' includes one or more eighth modified bases, wherein the combined sequence g2'-h'-e' is the combined sequence g2-he; and wherein the unmodified forms of the fifth and seventh modified bases are complementary, and the fifth and seventh modified bases are preferentially paired with the unmodified forms compared to the pairing between the fifth and seventh modified bases; and wherein the unmodified forms of the sixth and eighth modified bases are complementary, and the sixth and eighth modified bases are preferentially paired with the unmodified forms compared to the pairing between the sixth and eighth modified bases.

[0038] Embodiment 30: The primer set or method of Embodiment 29, wherein the T of the combined sequence g1-h in the double-stranded form m T higher than the combined sequence he in double-stranded form m , and the combined sequence g2-he T in the double-stranded form m Higher than the T of the combined sequence hef in double-stranded form m .

[0039] Embodiment 31: The primer set or method described in any one of Embodiments 29 or 30, wherein the combined sequence g1-h is richer in GC and / or contains more stable bases than the combined sequence he, and the combined sequence g2-he is richer in GC and / or contains more stable bases than the combined sequence hef.

[0040] Embodiment 32: The method of any one of Embodiments 29-31, wherein the primer set is capable of amplifying the method at least 8 循环数 The target nucleic acid is amplified at a rate of at least 8 循环数 Amplify the target nucleic acid at a rate of

[0041] Embodiment 33: The method of any of Embodiments 29-32, wherein the amplification allows detection of a single copy nucleic acid in a biological sample within such amplification cycles: the amplification cycles are approximately 42%-72% fewer than the amplification cycles required for the detection using only a single forward primer and a single reverse primer.

[0042] Embodiment 34: The primer set or method of any one of Embodiments 18-33, wherein the clamping sequences c1 and c2 and g1 and g2 (if present) cannot be copied during amplification.

[0043] Embodiment 35: The primer set or method of Embodiment 34, wherein the clamping sequences c1 and c2 and g1 and g2 (if present) comprise 2'-O-methyl RNA.

[0044] Embodiment 36: The primer set or method of any one of Embodiments 20-35, wherein the double-stranded primer sequences of the first inner primer and the first middle primer; and / or the second inner primer and the second middle primer (if present) do not contain a hairpin sequence.

[0045] Embodiment 37: The primer set or method described in any one of Embodiments 20-35, wherein: the double-stranded primer sequence of the first inner primer comprises a hairpin sequence in which the clamping sequence c2 is connected to the complementary sequence c2'; and / or the double-stranded primer sequence of the first intermediate primer comprises a hairpin sequence in which the clamping sequence c1 is connected to the complementary sequence c1'; and / or the double-stranded primer sequence of the second inner primer (if present) comprises a hairpin sequence in which the clamping sequence g2 is connected to the complementary sequence g2'; and / or the double-stranded primer sequence of the second intermediate primer (if present) comprises a hairpin sequence in which the clamping sequence g1 is connected to the complementary sequence g1'.

[0046] Embodiment 38: The method of any one of Embodiments 3-17 or 20-37, wherein the amplification comprises PCR.

[0047] Embodiment 39: The method of any one of Embodiments 3-17 or 20-38, wherein the method comprises detecting and optionally quantifying the target nucleic acid.

[0048] Embodiment 40: The method of any one of Embodiments 3-17 or 20-39, wherein the sample consists of nucleic acid from a single cell.

[0049] Embodiment 41: The primer set or method of any one of Embodiments 1-8, wherein the combined sequence ab contains more unstable bases than the combined sequence ca.

[0050] Embodiment 42: The primer set or method of any one of Embodiments 9-17, wherein the combined sequence ef contains more unstable bases than the combined sequence ge.

[0051] Embodiment 43: The primer set or method of any one of Embodiments 18-28, wherein the combined sequence da contains more unstable bases than the combined sequence c1-d, and / or the combined sequence dab contains more unstable bases than the combined sequence c2-da.

[0052] Embodiment 44: The primer set or method of any one of Embodiments 29-43, wherein the combined sequence he contains more unstable bases than the combined sequence g1-h, and / or the combined sequence hef contains more unstable bases than the combined sequence g2-he.

[0053] Embodiment 45: The primer set or method of any one of the preceding embodiments, wherein the modified complementary bases form fewer hydrogen bonds with each other than the unmodified complementary bases.

[0054] Embodiment 46: The primer set or method of Embodiment 45, wherein the T of the base pair formed between the modified complementary bases is m Below 40℃.

[0055] Embodiment 47: The primer set or method of any one of Embodiments 9-46, wherein at least one modified base is the same as at least one other modified base.

[0056] Embodiment 48: The primer set or method of any one of the preceding embodiments, wherein at least one pair of modified bases comprises modified forms of adenine and thymine.

[0057] Embodiment 49: The primer set or method of Embodiment 48, wherein the modified forms of adenine and thymine are 2-aminoadenine and 2-thiothymine, respectively.

[0058] Embodiment 50: The primer set or method of any one of the preceding embodiments, wherein at least one pair of modified bases comprises modified forms of guanine and cytosine.

[0059] Embodiment 51: The primer set or method described in Embodiment 50, wherein the modified form of guanine includes deoxyinosine, 7-alkyl-7-deazaguanine, 2'-hypoxanthine or 7-nitro-7-deazahypoxanthine (deazahypoxanthine), and the modified form of cytosine includes 3-(2'-deoxy-β-D-ribofuranosyl)pyrrolo-[2,3-d]-pyrimidin-2-(3H)-one, N4-alkylcytosine (e.g., N4-ethylcytosine) or 2-thiocytosine.

[0060] Embodiment 52: The primer set or method of any of the preceding embodiments, wherein one or more of the primer sequences comprising modified bases comprises at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 modified bases.

[0061] Embodiment 53: The primer set or method of any of the preceding embodiments, wherein the primer set comprises a probe or the method uses a probe.

[0062] Embodiment 54: The primer set or method of any of the preceding embodiments, wherein the primer set comprises a probe or the method uses a probe comprising one or more modified bases, wherein the modified bases preferentially pair with unmodified bases. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 : Schematic diagram showing complete nested PCR performed on a double-stranded DNA template. Flanking primers such as Figure 2 and Figure 3As recorded.

[0064] Figure 2 : Schematic diagram showing an exemplary double primer set that hybridizes to one end of a target nucleotide sequence. The set can be, for example, a forward primer set. Different fragments of the primer sequence are shown (a, b, c); complementary sequences are represented as (a', b', c'). The template sequence 3'-5' is represented as d', a' and b'. The outer primer (a) is single-stranded. The inner primer has a single-stranded portion (b) and a double-stranded portion (ac).

[0065] Figure 3 : The schematic diagram shows the Figure 2 An exemplary double primer set hybridizing to the opposite ends of the target nucleotide sequence shown in . The set can be, for example, a reverse primer set. Different fragments of the primer sequence are shown as (e, f, g); complementary sequences are shown as (e', f', g'). The template sequence 3'-5' is shown as h', e' and f'. The outer primer (e) is single-stranded. The inner primer has a single-stranded portion (f) and a double-stranded portion (ag).

[0066] Figure 4 : Schematic diagram shows an exemplary three-primer set that hybridizes to one end of a target nucleotide sequence. The set can be, for example, a forward primer set. Different fragments of the primer sequence are shown as (a, b, c1, c2, d); complementary sequences are represented as (a', b', c1', c2', d'). Template sequences 3'-5' are represented as i', d', a' and b'. The outer primer (d) is single-stranded. The middle primer has a single-stranded portion (a) and a double-stranded portion (d-c1). The inner primer has a single-stranded portion (b) and a double-stranded portion (ad-c2).

[0067] Figure 5 : The schematic diagram shows the Figure 4 An exemplary three-primer set hybridizing to the opposite ends of the target nucleotide sequence shown in . The set can be, for example, a reverse primer set. Different fragments of the primer sequence are shown as (e, f, g1, g2, h); complementary sequences are shown as (e', f', g1', g2', h'). Template sequences 3'-5' are shown as j', h', e' and f'. The outer primer (d) is single-stranded. The middle primer has a single-stranded portion (e) and a double-stranded portion (h-g1). The inner primer has a single-stranded portion (f) and a double-stranded portion (eh-g2).

[0068] Figure 6A-B : Schematic diagram showing two alternative structures of an exemplary primer with a clamp sequence when the primer is allowed to hybridize to a template. A fluorescent quencher (Q) is present in the primer at a position where it quenches the corresponding fluorescent label (F) in the template strand. In Example 1, an experiment was performed in which the T of the primer and the complementary target sequence was measured in the presence or absence of the clamp. m(A) In double-stranded form, the combined sequence ca T m T higher than the combined sequence ab in double-stranded form m (B) In the double-stranded form, the T m Less than the T of the combined sequence ab in double-stranded form m The structure formed when.

[0069] Figure 7A-B : (A) Schematic diagram showing an exemplary dual primer set, where a fluorescence quencher (Q) is present at a position in the inner primer where it quenches the corresponding fluorescent label (F) in the template strand. (B) Fluorescence intensity as a function of time in the primer extension reaction of Example 2. The three ascending traces are separate reactions with slightly different clamps; the horizontal traces have no outer (flanking) displacement primers present.

[0070] Fig. 8A : Base pairing schemes of Watson-Crick doublets between thymine and adenine (Formula 1a), thymine and 2-aminoadenine (Formula 1b), 2-thiothymine and adenine (Formula 2b), and 2-thiothymine and 2-aminoadenine (Formula 2b). 2-Thiothymine and 2-aminoadenine base pairs are unstable, while thymine and 2-aminoadenine and 2-thiothymine and adenine base pairs are stable.

[0071] Figure 8B : The base pairing schemes of the Watson-Crick doublets between cytosine and guanine (Formula 3a), cytosine and inosine (Formula 3b), dP and guanine (Formula 4a), and dP and inosine (Formula 4b). dP and inosine base pairs are unstable, while cytosine and inosine and dP and guanine base pairs are stable.

[0072] Fig. 9 : The use of modified bases can make the desired configuration for primer annealing (top) in base-3 amplification more stable. The bottom shows an undesirable configuration for primer annealing. The larger arrow pointing upward indicates that the undesirable configuration is more unstable than the desired configuration. In other words, the stability of the joint sequence ca (i.e., ca / c'-a') in double-stranded form (a hyphen is used in this context to represent a joint nucleic acid sequence consisting of sequences c and a) is higher than the stability of the joint sequence ab (i.e., ab / a'-b') in double-stranded form.

[0073] Figures 10A-10B: Panels A and B compare real-time PCR growth curves for a modified test primer set ("8 series;" Panel A) and an unmodified test primer set ("6 series;" Panel B). Fluorescence (y-axis) is plotted versus PCR cycle number using a logarithmic y-axis scale. See Example 3.

[0074] Fig.11A : Real-time PCR fluorescence growth curve generated by base-6 (about 6 replicates per cycle) PCR amplification starting from a reduced number of template DNA molecules. See Example 4.

[0075] Fig. 11B : Fig. 11B Shown is a plot of the number of amplification cycles (Ct) required to reach a threshold level of fluorescence versus the log 10 number of starting DNA template molecules for the studies described in Example 4. DETAILED DESCRIPTION

[0076] definition

[0077] Unless otherwise stated, the terms used in the claims and specification are defined as set forth below.

[0078] The term "nucleic acid" refers to a polymer of nucleotides and, unless otherwise limited, includes analogs of natural nucleotides that are capable of functioning (eg, hybridizing) in a manner similar to naturally occurring nucleotides.

[0079] The term nucleic acid includes any form of DNA or RNA, including, for example, genomic DNA; complementary DNA (cDNA), which is a DNA representation of mRNA, typically obtained by reverse transcription of messenger RNA (mRNA) or by amplification; DNA molecules produced synthetically or by amplification; mRNA; and non-coding RNA.

[0080] The term nucleic acid encompasses double-stranded or triple-stranded nucleic acid complexes, as well as single-stranded molecules. In double-stranded or triple-stranded nucleic acid complexes, the nucleic acid strands need not be coextensive (ie, a double-stranded nucleic acid need not be double-stranded over the entire length of both strands).

[0081] The term nucleic acid also encompasses any modification thereof, for example by methylation and / or by capping. Nucleic acid modifications may include the addition of chemical groups that introduce additional charge, polarizability, hydrogen bonding, electrostatic interactions, and functionality to individual nucleic acid bases or to the nucleic acid as a whole. Such modifications may include base modifications, such as sugar modifications at the 2' position, pyrimidine modifications at the 5' position, purine modifications at the 8' position, modifications at the amine outside the cytosine ring, displacement of 5-bromo-uracil, sugar-phosphate backbone modifications, rare base pairing combinations such as the isobases isocytidine and isoguanidine, etc.

[0082] More specifically, in some embodiments, nucleic acids can include polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (including D-ribose), and nucleic acids of any other type of N- or C-glycoside of a purine or pyrimidine base, as well as other polymers containing non-nucleotide backbones, such as polyamides (e.g., peptide nucleic acids (PNA)) and polymorpholino polymers (see, e.g., Summerton and Weller (1997) "Morpholino Antisense Oligomers: Design, Preparation, and Properties," Antisense & Nucleic Acid Drug Dev. 7: 1817-195; Okamoto et al. (20020) "Development of electrochemically gene-analyzing method using DNA-modified electrodes," Nucleic Acids Res. Supplement No. 2: 171-172), and other synthetic sequence-specific nucleic acid polymers, provided that the polymer contains nucleobases in a configuration that allows base pairing and base stacking, such as found in DNA and RNA. The term nucleic acid also encompasses locked nucleic acids (LNAs), which are described in U.S. Pat. Nos. 6,794,499, 6,670,461, 6,262,490, and 6,770,748, the LNA disclosures of which are incorporated herein by reference in their entirety.

[0083] Nucleic acids can be obtained from completely chemical synthesis methods, such as solid phase-mediated chemical synthesis; can be obtained from biological sources, such as by isolation from any species that produces nucleic acids; or can be obtained from methods involving the manipulation of nucleic acids by molecular biology tools, such as DNA replication, PCR amplification, reverse transcription; or can be obtained from a combination of these methods.

[0084] As used herein, the term "complementarity" refers to the ability of two nucleotides to pair precisely; that is, if a nucleotide at a given position in a nucleic acid is able to hydrogen bond with a nucleotide in another nucleic acid to form a standard base pair, the two nucleic acids are considered to be complementary to each other at that position. Complementarity between two single-stranded nucleic acid molecules can be "partial", in which only some of the nucleotides bind, or can be complete when there is complete complementarity between the two single-stranded molecules. The degree of complementarity between nucleic acid chains has a significant effect on the efficiency and intensity of hybridization between nucleic acid chains.

[0085] "Specific hybridization" refers to the binding of a nucleic acid to a target nucleotide sequence under defined stringent conditions in the absence of substantial binding to other nucleotide sequences present in the hybridization mixture. It is understood by those skilled in the art that relaxing the stringency of hybridization conditions allows tolerance of sequence mismatches.

[0086] In some embodiments, hybridization is performed under stringent hybridization conditions. The term "stringent hybridization conditions" generally refers to conditions below the melting temperature (T) of a specific sequence under defined ionic strength and pH. m ) is a temperature range of about 5°C to about 20°C or 25°C. As used herein, T m It is the temperature at which half of a population of double-stranded nucleic acid molecules dissociates into single strands. m The method of stringent hybridization conditions is well known in the art (see, for example, Berger and Kimmel (1987) METHODS IN ENZYMOLOGY, VOL. 152: GUIDE TO MOLECULAR CLONING TECHNIQUES, San Diego: Academic Press, Inc. and Sambrook et al. (1989) MOLECULAR CLONING: A LABORATORY MANUAL, 2ND ED., VOLS. 1-3, Cold Spring Harbor Laboratory, both of which are incorporated herein by reference for descriptions of stringent hybridization conditions). As described in standard references, when nucleic acids are in 1 M NaCl aqueous solution, T m A simple estimate of the value can be calculated using the following equation: m =81.5+0.41(%G+C) (see, e.g., Anderson and Young, Quantitative Filter Hybridization in NUCLEIC ACID HYBRIDIZATION (1985)). The melting temperature of the hybrid (and the conditions for stringent hybridization) is affected by a variety of factors, such as the length and nature of the primer or probe (DNA, RNA, base composition) and the nature of the target nucleic acid (DNA, RNA, base composition, present in solution or fixed, etc.), as well as the concentration of salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol). The effects of these factors are well known and discussed in standard reference texts in the art. Exemplary stringent conditions suitable for achieving specific hybridization of most sequences are: a temperature of at least about 60°C and a salt concentration of about 0.2 molar at pH 7. T values ​​for oligonucleotide sequences based on nearest neighbor thermodynamics mThe calculation of can be performed as described in “Aunified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics” John SantaLucia, Jr., PNAS February 17, 1998 vol.95 no.41460-1465 (the description of which is incorporated herein by reference).

[0087] The term "oligonucleotide" is used to refer to a relatively short nucleic acid, which is typically shorter than 200 nucleotides, more particularly shorter than 100 nucleotides, and most particularly shorter than 50 nucleotides. Typically, an oligonucleotide is a single-stranded DNA molecule.

[0088] The term "primer" refers to such oligonucleotides, which can be hybridized with nucleic acids (also referred to as "annealing"), and can be used as the starting site of nucleotide (RNA or DNA) polymerization reaction under suitable conditions (i.e., in the presence of four different nucleoside triphosphates and polymerization reagents (such as DNA or RNA polymerase or reverse transcriptase)) in a suitable buffer and at a suitable temperature. The suitable length of primer depends on the intended use of primer, but the length of primer is generally at least 7 nucleotides, and in some embodiments, the primer length is 10 to 30 nucleotides, or in some embodiments, the primer length is 10 to 60 nucleotides. In some embodiments, the primer length can be, for example, 15 to 50 nucleotides. Short primer molecules usually require lower temperatures to form sufficiently stable hybrid complexes with templates. Primers do not need to reflect the accurate sequence of templates, but must be fully complementary to hybridize with templates.

[0089] A primer is said to anneal to another nucleic acid if a primer or a portion thereof hybridizes to a nucleotide sequence within a nucleic acid. The statement that a primer hybridizes to a particular nucleotide sequence is not intended to imply that the primer hybridizes completely or exclusively to that nucleotide sequence. For example, in some embodiments, an amplification primer used herein is referred to as "annealing to" or "specific for" a nucleotide sequence. This description encompasses primers that anneal in their entirety to a nucleotide sequence, as well as primers that anneal in part to a nucleotide sequence.

[0090] The term "primer pair" refers to a set of primers that includes a 5' "upstream primer" or "forward primer" that hybridizes to the complementary sequence at the 5' end of the DNA sequence to be amplified, and a 3' "downstream primer" or "reverse primer" that hybridizes to the 3' end of the sequence to be amplified. Those skilled in the art will appreciate that the terms "upstream" and "downstream" or "forward" and "reverse" are not intended to be limiting, but rather provide exemplary directions in some embodiments.

[0091] "Probe" is a nucleic acid that can bind to a target nucleic acid with a complementary sequence by one or more types of chemical bonds, generally by complementary base pairing, usually by hydrogen bond formation, thereby forming a double-stranded structure. The probe can be labeled with a detectable marker to make the probe easy to detect, particularly once the probe hybridizes with its complementary target. Alternatively, however, the probe can be unlabeled, but can be detected by specific binding directly or indirectly to a labeled ligand. The size of the probe can be significantly different. Typically, the probe length is at least 7 to 15 nucleotides. Other probes are at least 20, 30 or 40 nucleotides in length. Still other probes are longer, with a length of at least 50, 60, 70, 80 or 90 nucleotides. Still other probes are longer, with a length of at least 100, 150, 200 or more nucleotides. The probe can also be any length (e.g., a length of 15-20 nucleotides) within any range defined by any of the above numerical values.

[0092] Primer or probe can be fully complementary or not yet fully complementary to the target nucleotide sequence. In some embodiments, primer has at least 65% identity with the complementary sequence of the target nucleotide sequence on a sequence of at least 7 nucleotides, more typically on a sequence of 10-30 nucleotides and in some embodiments on a sequence of at least 14-25 nucleotides, in some embodiments, has at least 75% identity, at least 85% identity, at least 90% identity or at least 95%, 96%, 97%, 98% or 99% identity. It should be understood that it is usually desired that certain bases (e.g., 3' bases of primers) are fully complementary to the corresponding bases of the target nucleotide sequence. Under stringent hybridization conditions, primers and probes are usually annealed to the target sequence.

[0093] As used herein, the term "specific for" a nucleic acid when referring to a portion of a primer or a nucleotide sequence within a primer refers to a primer or a nucleotide sequence that can specifically anneal to a target nucleic acid under appropriate annealing conditions.

[0094] Amplification according to the teachings of the present invention encompasses any method that generally replicates at least a portion of at least one target nucleic acid in a template-dependent manner, including but not limited to a wide range of techniques for linearly or exponentially amplifying nucleic acid sequences. Exemplary methods for performing the amplification step include PCR, nucleic acid chain-based amplification (NASBA), two-step multiplex amplification, rolling circle amplification (RCA), etc., including multiple versions thereof and combinations thereof, such as but not limited to OLA / PCR, PCR / OLA, LDR / PCR, PCR / PCR / LDR, PCR / LDR, LCR / PCR, PCR / LCR (also known as combined chain reaction-CCR), helicase-dependent amplification (HDA), etc. Such techniques are described in Ausbel et al.; PCR Primer: A Laboratory Manual, Diffenbach, Ed., Cold Spring Harbor Press (1995); The Electronic Protocol Book, Chang Bioscience (2002); Msuih et al., J. Clin. Micro. 34:501-07 (1996); The Nucleic Acid Protocols Handbook, R. Rapley, ed., Humana Press, Totowa, NJ (2002); Abramson et al., Curr Opin Biotechnol. 1993 Feb.; 4(1):41-7, U.S. Pat. No. 6,027,998; U.S. Pat. No. 6,605,451, Barany et al., PCT Publication No. WO 97 / 31256; Wenz et al., PCT Publication No. WO 01 / 112579; Day et al., PCT Publication No. WO 01 / 112579; al., Genomics, 29(1):152-162(1995), Ehrlich et al., Science 252:1643-50(1991); Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press(1990); Favis et al., Nature Biotechnology 18:561-64(2000); andRabenau et al. al., Infection 28:97-102 (2000); Belgrader, Barany, and Lubin, Development of a Multiplex Ligation Detection Reaction DNA Typing Assay, Sixth International Symposium on Human Identification, 1995 (available on the World Wide Web: promega.com / geneticidproc / ussymp6proc / blegrad.html); LCR Kit Instruction Manual, Cat. #200520, Rev. #050002, Stratagene, 2002; Barany, Proc. Natl. Acad. Sci. USA 88: 188-93 (1991); Bi and Sambrook, Nucl. Acids Res. 25: 2924-2951 (1997); Zirvi et al., Nucl. Acid Res.27:e40i-viii(1999); Dean et al. al., Proc Natl Acad Sci USA 99:5261-66 (2002); Barany and Gelfand, Gene 109:1-11 (1991); Walker et al., Nucl. AcidRes.20:1691-96 (1992); Polstra et al., BMC Inf. Dis. 2:18-(2002); Lage et al. al.,Genome Res.2003Feb.;13(2):294-307,and Landegren et al.,Science 241:1077-80(1988),Demidov,V.,Expert Rev Mol Diagn.2002Nov.;2(6):542-8.,Cook et al.,JMicrobiol Methods.2003May;53(2):165-74,Schweitzer et al. al., Curr Opin Biotechnol. 2001 Feb.; 12(1): 21-7; U.S. Patent No. 5,830,711; U.S. Patent No. 6,027,889; U.S. Patent No. 5,686,243; PCT Publication No. WO0056927A3 and PCT Publication No. WO9803673A1, etc.

[0095] In some embodiments, amplification includes at least one cycle of the following consecutive steps: annealing at least one primer to a complementary sequence or a substantially complementary sequence in at least one target nucleic acid; synthesizing at least one nucleotide chain in a template-dependent manner using a polymerase; denaturing the newly formed nucleic acid duplex to separate the chains. The cycles may or may not be repeated. Amplification may include thermal cycling or may be performed isothermally.

[0096] "Nested amplification" refers to the use of more than two primers to amplify a target nucleic acid.

[0097] "Semi-nested amplification" refers to the use of more than one (eg, two or three) primers that anneal to one end of a target nucleotide sequence.

[0098] "Completely nested amplification" refers to the use of more than one primer that anneals to each end of the target nucleotide sequence.

[0099] With respect to nested amplification, the terms "inner," "outer," and "middle" are used to distinguish between the multiple primers that anneal at one end of the amplicon.

[0100] "External primer" refers to a primer that anneals to a sequence closer to the end of a target nucleotide sequence than another primer that anneals at the same end of the target nucleotide sequence. In some embodiments, the external primer sequence defines the end of the amplicon generated from the target nucleic acid. In this article, "external primer" is also referred to as "flanking primer".

[0101] An "inner primer" refers to a primer that anneals to a sequence closer to the middle of a target nucleotide sequence than another primer that anneals to the same end of the target nucleotide sequence.

[0102] The term "middle primer" is used herein for nested amplification in which at least three primers annealing to one end of a target nucleotide sequence are used. The middle primer is a primer annealing to a sequence between an inner primer and an outer primer.

[0103] As used herein, the term "adjacent to" is used to refer to sequences that are close enough for the method to work. In some embodiments, sequences that are adjacent to each other are directly adjacent, with no intervening nucleotides.

[0104] A "multiplex amplification reaction" is a reaction in which two or more nucleic acids that are distinguishable by sequence are amplified simultaneously.

[0105] The term "qPCR" is used herein to designate quantitative real-time polymerase chain reaction (PCR), which is also known as "real-time PCR" or "kinetic polymerase chain reaction"; all terms refer to PCR with real-time signal detection.

[0106] "Reagent" refers broadly to any reagent used in a reaction other than an analyte (e.g., a nucleic acid being analyzed). Exemplary reagents for nucleic acid amplification reactions include, but are not limited to, buffers, metal ions, polymerases, reverse transcriptases, primers, template nucleic acids, nucleotides, markers, dyes, nucleases, dNTPs, etc. Reagents for enzyme reactions include, for example, substrates, cofactors, buffers, metal ions, inhibitors, and activators.

[0107] The term "label" as used herein refers to any atom or molecule that can be used to provide a detectable and / or quantifiable signal. Specifically, the label can be directly or indirectly attached to a nucleic acid or protein. Suitable labels that can be attached to the probe include, but are not limited to, radioisotopes, fluorophores, chromophores, mass markers, electron-dense particles, magnetic particles, spin labels, chemiluminescent molecules, electrochemically active molecules, enzymes, cofactors, and enzyme substrates.

[0108] As used herein, the term "dye" generally refers to any organic or inorganic molecule that absorbs electromagnetic radiation.

[0109] The natural bases adenine, thymine, uracil, guanine, and cytosine that make up DNA and RNA are described herein as "unmodified bases" or "unmodified forms."

[0110] As used herein, the term "modified base" refers to a base that is not a standard naturally occurring base (eg, adenine, cytosine, guanine, thymine, or uracil). Examples of modified bases are 2-thiothymine and 2-aminoadenine.

[0111] Nucleotides comprising modified bases are referred to herein as "modified nucleotides".

[0112] A DNA polymerase is said to be "stable" at a particular temperature if it provides a satisfactory extension rate in a nucleic acid amplification reaction.

[0113] General methods for improving amplification efficiency

[0114] U.S. Pat. No. 8,252,558 and Harris et al., BioTechniques 54:93-97 (February 2013) teach a nested PCR format, called "polymerase chain displacement reaction" (PCDR) (the descriptions of both documents are incorporated herein by reference). In PCDR, when extension occurs from the outer primer, the outer primer displaces the extended chain generated from the inner primer because the reaction uses a polymerase with chain displacement activity. In theory, this allows the increase in amplification product of each amplification cycle to be greater than 2 times, so the sensitivity and speed are improved relative to conventional PCR. In practice, each amplicon generated from the nested primer no longer contains the primer annealing site of the outer primer. Therefore, PCDR cannot maintain an increase of more than 2 times the amplification product of each amplification cycle for a very large number of cycles. For this reason, PCDR only provides a moderate reduction in the number of amplification cycles required to detect the target nucleic acid (e.g., from about 23 to about 20). In contrast, Table 1 below shows that the continuous quadrupling of each cycle (4 循环数 ) should halve the number of cycles required for the same amplification, since each cycle doubles the activity. A 6-fold repeat per cycle should be achieved in 15 cycles, which requires 40 normal PCR cycles.

[0115] Table 1 - Amplification degree with different "bases"

[0116]

[0117] The key to maintaining a greater than 2-fold increase in amplification product per amplification cycle is to design the inner (nested) primers so that the extension products of the inner (nested) primers encompass the outer (flanking) primer sequences. Figure 1 A scheme for a fully nested PCR using forward inner and outer primers and reverse inner and outer primers is shown. The "flap" formed when the inner primers anneal to the template contains the outer primer sequence so that each of the four new chains generated from the two template chains extends from (and includes) the forward outer primer sequence (or its complementary sequence), through (and includes) the reverse outer primer sequence. However, more is needed than simply adding the outer primer sequence to the 5' end of the inner primer, because when the inner primer anneals, the added sequence will also anneal immediately and prevent the outer primer from annealing. The solution to this problem is to use an additional 5' addition to the inner primer (i.e., a sequence other than the outer primer sequence), and an oligonucleotide complementary to these two sequences, which is referred to herein as a "clamp." For ease of discussion, the added sequence is referred to as a "clamping sequence." This configuration is shown in FIG. Figure 2 shown.

[0118] Clamping sequence c is not homologous to template (d' zone). Herein ca / c'-a' is more stable than ab / a'-b'. In some embodiments, this can be achieved by using sequence c, which is longer, rich in GC (i.e., richer in GC) or comprises one or more stable bases (when a does not comprise such bases) or more stable bases than a relative to a. In some embodiments, this can be achieved by using sequence c, which is longer, rich in GC (i.e., richer in GC) or comprises one or more stable bases (when b does not comprise such bases) or more stable bases than b relative to b. In some embodiments, stable bases can be included in the a district of cab, and in the a' district of c'-a' to increase the stability of ca / c'-a' relative to ab / a'-b'. Alternatively or additionally, b can comprise one or more unstable bases, such as inosine. The outer primer retains sequence a. In this case, the outer primer still can contact the sequence a' in the template. At higher temperatures, the c'-a' clamp and the 5' end of the inner primer will anneal faster than any other sequence, so the c'-a' clamp does not have to be attached to the inner primer to form a hairpin structure. However, in some embodiments, using an inner primer with this type of hairpin structure can increase the reaction speed.

[0119] In some embodiments, the c sequence in the inner primer (highlighted in red) is preferably not replicated during PCR. If it replicates during PCR, these new templates will have c'-a' tails, which will produce cab / c'-a'-b' duplexes with the inner primers, which will "outperform" other possible structures in the strand displacement competition and again prevent the flanking primer sequence a from annealing. In order to prevent this replication, the red sequence can be prepared from RNA that DNA polymerases cannot replicate well (or 2'-O-methyl RNA, which is relatively easy to prepare by synthetic methods). The red sequence can be prepared from any base that can base pair but cannot be replicated.

[0120] In some embodiments, the clamping oligonucleotides (a'-c') are blocked from extension at the 3' end, for example due to the absence of a 3' hydroxyl group or the use of a chemical blocking group, which can improve the specificity of amplification.

[0121] sample

[0122] Samples containing nucleic acids can be obtained and prepared from biological sources using conventional methods known in the art. In particular, nucleic acids useful in the methods described herein can be obtained from any source, including unicellular organisms and higher organisms such as plants or non-human animals, such as dogs, cats, horses, primates and other non-human mammals, and humans. In some embodiments, samples can be obtained from individuals suspected or known to be infected with pathogens, individuals suspected of or known to have a disease (e.g., cancer), or pregnant individuals.

[0123] Nucleic acids can be obtained from cells, body fluids (e.g., blood, blood fractions, urine, etc.), or tissue samples by any of a variety of standard techniques. In some embodiments, the method utilizes samples of plasma, serum, spinal fluid, lymph, peritoneal fluid, pleural fluid, oral fluid, and external sections of skin; samples from the respiratory tract, intestinal tract, reproductive tract, or urinary tract; samples of tears, saliva, blood cells, stem cells, or tumors. Samples can be obtained from living or dead organisms, or from in vitro cultures. Exemplary samples may include single cells, paraffin-embedded tissue samples, and needle biopsy samples. In some embodiments, the nucleic acid analyzed is obtained from a single cell.

[0124] The nucleic acid of interest can be isolated using methods well known in the art.The sample nucleic acid need not be in pure form, but is generally sufficiently pure to allow the steps of the methods described herein to be performed.

[0125] Target nucleic acid

[0126] Any target nucleic acid that can be detected by nucleic acid amplification can be detected using the methods described herein. In typical embodiments, at least some nucleotide sequence information of the target nucleic acid will be known. For example, if the amplification reaction used is PCR, sufficient sequence information at each end of a given target nucleic acid is usually available to allow for the design of suitable amplification primers.

[0127] Targets may include, for example, nucleic acids associated with pathogens (e.g., viruses, bacteria, protozoa, or fungi); RNA, such as those whose overexpression or underexpression indicates disease, those RNAs expressed in a tissue-specific manner or development-specific manner; or those induced by specific stimuli; genomic DNA, which can be analyzed for specific polymorphisms (e.g., SNPs), alleles, or haplotypes, such as in genotyping. Of particular interest are genomic DNA that is altered (e.g., amplified, deleted, and / or mutated) in genetic diseases or other pathologies; sequences associated with desired or undesirable traits; and / or sequences that uniquely identify an individual (e.g., in forensics or parentage determination).

[0128] Primer design

[0129] Primers suitable for nucleic acid amplification are long enough to start the synthesis of extension products in the presence of a suitable nucleic acid polymerase. The exact length and composition of primers will depend on many factors, including the temperature of the annealing reaction, the source and composition of the primers, and in the case of using probes, the proximity of the probe annealing site to the primer annealing site, and primer: probe concentration ratio. For example, according to the complexity of the target nucleic acid sequence, oligonucleotide primers generally comprise about 10 to about 60 nucleotides, although it may comprise more or less nucleotides. Primers should be fully complementary to selectively anneal to their respective chains and form stable duplexes.

[0130] Generally, those skilled in the art know how to design suitable primers that can amplify the target nucleic acid of interest. For example, PCR primers can be designed by using any commercially available software or open source software such as Primer3 (see, for example, Rozen and Skaletsky (2000) Meth. Mol. Biol., 132: 365-386; www.broad.mit.edu / node / 1060, etc.), or by accessing the Roche UPL website. The amplicon sequence is input into the Primer3 program, wherein the UPL probe sequence is in brackets to ensure that the Primer3 program will design primers on either side of the probe sequence in brackets.

[0131] Primers will be prepared by any suitable method, including, for example, by direct chemical synthesis using, for example, the phosphotriester method of Narang et al. (1979) Meth. Enzymol. 68: 90-99; the phosphodiester method of Brown et al. (1979) Meth. Enzymol. 68: 109-151; the diethylphosphoramidite method of Beaucage et al. (1981) Tetra. Lett., 22: 1859-1862; the solid support method of U.S. Pat. No. 4,458,066, etc., or primers may be provided by commercial sources. Primers may be purified by using Sephadex columns (Amersham Biosciences, Inc., Piscataway, NJ) or other methods known to those skilled in the art. Primer purification may improve the sensitivity of the methods described herein.

[0132] Outer Primers

[0133] Figure 2 It shows how the dual primer set anneals to the first template strand at one end of the target nucleotide sequence. For ease of discussion, this primer set can be considered a "forward" primer set. The outer primer contains a sequence a that specifically hybridizes to the first template strand sequence a'. Figure 3Shown is how the dual primer set anneals to the second template strand at the opposite end of the target nucleotide sequence. For ease of discussion, this primer set can be considered a "reverse" primer set. Here, the outer primer contains a sequence e that specifically hybridizes to the first template strand sequence e'. Figure 4 and 5 Exemplary "forward" and "reverse" three-primer sets are shown. Figure 4 In , the forward outer primer comprises a sequence d that specifically hybridizes to the first template strand sequence d'. Figure 5 In the example of FIG. 1 , the forward outer primer comprises a sequence d that specifically hybridizes to the first template strand sequence d'. In general, the considerations for designing suitable outer primers are no different than those for designing outer primers for conventional nested PCR. It is noteworthy that in some embodiments, the T of any primer sequence "outside" relative to another primer sequence (e.g., an inner primer sequence or an intermediate primer sequence) is preferably greater than or equal to 0. m Preferably, the T of the inner (or middle) primer sequence is m Thus, for example, during the temperature ramp down of PCR, the inner primers may anneal and begin to extend before the outer primers; or premature extension of the outer primers will block the target site of the inner primers and prevent the inner primers from annealing. More specifically, during the temperature ramp down of PCR, the inner primers may anneal and begin to extend before the outer primers; or premature extension of the outer primers may block the target site of the inner primers and prevent the inner primers from annealing. Figure 2 In the embodiment shown, primer sequence a will have a lower T value than primer sequence b. m . Similarly, in Figure 3 In the embodiment shown, the T of the primer sequence e m The primer sequence f will have a lower T m In some embodiments, T m The difference is at least about 4°C, typically ranging from about 4°C to about 20°C. In some embodiments, T m The difference ranged from about 4°C to about 15°C. However, the T m Typically high enough to maintain efficient PCR, for example, in some embodiments, the T of the outer primers m The T can be adjusted by adjusting the sequence length, GC content and / or by including stable or unstable bases in the sequence. m .

[0134] "Stable bases" include, for example, peptide nucleic acid (PNA) segments (stretches) that can be incorporated into DNA oligonucleotides to increase duplex stability. Locked nucleic acids (LNA) and unlocked nucleic acids (UNA) are RNA analogs that can be easily incorporated into DNA oligonucleotides in solid phase oligonucleotide synthesis, and increase and decrease duplex stability, respectively. Suitable stable bases also include modified DNA bases that increase the stability of base pairs (and therefore the entire duplex). These modified bases can be incorporated into oligonucleotides during solid phase synthesis and provide a more predictable method for increasing DNA duplex stability. Examples include AP-dC (G-clamp) and 2-aminoadenine and 5-methylcytosine and C(5)-propynylcytosine (instead of cytosine) and C(5)-propynyluracil (instead of thymine).

[0135] "Unstable bases" are those bases that destabilize double-stranded DNA due to the formation of base pairs that are more unstable than typical AT and / or GC base pairs. Inosine (I) is an unstable base because it pairs with cytosine (C), but the IC base is more unstable than the GC base pair. This lower stability is due to the fact that inosine is a purine that can only form two hydrogen bonds compared to the three hydrogen bonds of the GC base pair. Other unstable bases are known or easily identified by those skilled in the art.

[0136] Inner primer of dual primer set

[0137] refer to Figure 2 , the inner primer in the forward double primer set comprises a single-stranded primer sequence b, which specifically hybridizes with the first template strand sequence b', wherein b' is adjacent to a' and is 5' to a', and wherein the single-stranded primer sequence b is connected to the first strand of the double-stranded primer sequence at its 5' end. The first strand comprises: a primer sequence a, which is adjacent to the single-stranded primer sequence b and is 5' to the single-stranded primer sequence b; and a clamping sequence c, which is adjacent to the primer sequence a and is 5' to the primer sequence a, wherein the clamping sequence c is not complementary to the first strand template sequence d', which is adjacent to the first strand template sequence a' and is 3' to the first strand template sequence a'. In some embodiments, the T of the double-stranded form of the combined sequence ca (a hyphen is used herein to represent a combined nucleic acid sequence consisting of sequences c and a) (i.e., ca / c'-a') m Higher than the T of the combined sequence ab (i.e. ab / a'-b') in the double-stranded form mThis is easily achieved, for example, by preparing a longer and / or more GC-rich joint sequence ca than the joint sequence ab, and / or designing the joint sequence ca to contain more stable bases than the joint sequence ab (the "more" requirement includes the case where the sequence ab contains no GC base pairs and / or contains no stable bases). Alternatively or additionally, the joint sequence ab can be designed to contain more unstable bases than the joint sequence ca (the "more" requirement includes the case where the sequence ca contains no unstable bases). In some embodiments, a'-c' is blocked from extending at its 3' end.

[0138] The forward dual primer set can be used with a simple conventional reverse primer for semi-nested amplification or with a reverse dual primer set.

[0139] refer to Figure 3 , the inner primer in the reverse double primer set comprises a single-stranded primer sequence f, which specifically hybridizes with the first template strand sequence f', wherein f' is adjacent to e' and is 5' to e', and wherein the single-stranded primer sequence f is connected to the first strand of the double-stranded primer sequence at its 5' end. The first strand comprises: a primer sequence e, which is adjacent to the single-stranded primer sequence f and is 5' to the single-stranded primer sequence f; and a clamping sequence g, which is adjacent to the primer sequence e and is 5' to the primer sequence e, wherein the clamping sequence g is not complementary to the first strand template sequence h', which is adjacent to the first strand template sequence e' and is 3' to the first strand template sequence e'. In some embodiments, the T of the combined sequence ge (i.e., ge / g'-e') in the double-stranded form m Higher than the T of the combined sequence ef (ie ef / e'-f') in the double-stranded form m This can be easily achieved, for example, by preparing a joint sequence ge that is longer and / or more GC-rich than the joint sequence ef, and / or by designing a joint sequence (T of the double-stranded form of the joint sequence ge) that is m T higher than the combined sequence ef in double-stranded form m ) to contain more stable bases than the joint sequence ef (the "more" requirement includes the case where the sequence ef does not contain GC base pairs and / or does not contain stable bases). Alternatively or additionally, the joint sequence ef can be designed to contain more unstable bases than the joint sequence ge (the "more" requirement includes the case where the sequence ge does not contain unstable bases). In some embodiments, e'-g' is blocked at its 3' end.

[0140] In some embodiments, the clamping sequences c and g (if present) cannot be replicated during amplification. RNA or RNA analogs (e.g., RNA analogs resistant to hydrolysis) can be used to provide the required base pairs to form double-stranded clamping sequences without being replicated by DNA-dependent polymerases during amplification. The most common RNA analog is 2'-O-methyl substituted RNA. Other nucleic acid analogs that can specifically base pair but cannot be replicated include locked nucleic acids (LNA) or BNA (bridging nucleic acids), morpholinos, and peptide nucleic acids (PNA). Although these oligonucleotides have different backbone sugars, or in the case of PNA, have amino acid residues that replace ribose phosphates, they still bind to RNA or DNA according to Watson and Crick pairing, but are not affected by nuclease activity. They cannot be synthesized enzymatically and can only be obtained by synthesis using the phosphoramidite strategy, or for PNA, by peptide synthesis.

[0141] If desired, the clamp sequence c can be covalently linked to the complementary sequence c' to form ac / a-c' from the hairpin structure; however, this is not necessary for efficient formation of the double-stranded clamp portion of the primer. Similarly, the clamp sequence g can but need not be covalently linked to the complementary sequence g' to form eg / e'-g' from the hairpin structure.

[0142] Primers for the three-primer set

[0143] In some embodiments, a third primer can be used at one or both ends of the target nucleic acid sequence to further increase the number of copies produced in each cycle of amplification. Figure 4 and Figure 5 Exemplary "forward" and "reverse" three-primer sets are shown. The three-primer set includes outer primers as discussed above and a middle primer that is substantially identical in structure to the inner primers discussed above. The additional primer is an inner primer that is designed to hybridize to the template strand 5' of the middle primer.

[0144] The inner primer in the forward triple primer set contains a single-stranded primer sequence b that specifically hybridizes with the first template strand sequence b', where b' is adjacent to a' and is the 5' of a'. The single-stranded primer sequence b is linked at its 5' end to the first strand of the double-stranded primer sequence, which first strand contains: a primer sequence a, which is adjacent to the single-stranded primer sequence b and is the 5' of the single-stranded primer sequence b; a primer sequence d, which is adjacent to the primer sequence a and is the 5' of the primer sequence a; and a clamping sequence c2, which is adjacent to the primer sequence d and is the 5' of the primer sequence d, where the clamping sequence c2 is not complementary to the first strand template sequence i'. The clamping sequence c2 can be the same as or different from the clamping sequence (c1) for the inner primer. In a preferred embodiment, c1 and c2 are different sequences. As discussed above for the inner primer in the double primer set, similar considerations apply to the design of the inner primer in the triple primer set, and the inner primer in the reverse triple primer set ( Figure 5 as shown) has the same structure as the inner primer in the forward triple primer set. Extension can be blocked at the 3' end of one or more (or all) of the clamping oligonucleotides ( Figure 4 d'-c1' and a'-d'-c2' in, and Figure 5 h'-g1' and e'-h'-g2' in). The forward triple primer set can be used with a simple conventional reverse primer for semi-nested amplification, with a reverse double primer set, or with a reverse triple primer set.

[0145] In some embodiments, the order of primer annealing and extension is controlled according to the T m of the primer sequences such that any primer that is "inner" relative to another primer anneals and begins extension before that other primer. Thus, for example, in the double primer set, the inner primer anneals and begins extension before the outer primer, and in the triple primer set, the inner primer anneals and begins extension before the middle primer, and the middle primer anneals and begins extension before the outer primer. For example, in the Figure 4 embodiment shown, the T m of the primer sequences will have the following relationship: the T m of d < m the T m of a < the T Figure 5 of b. In the m embodiment shown, the T m of the primer sequences will have the following relationship: the T m of h < m the T m of e < the T

[0146] of f. As noted above, T

[0147] Use of modified bases in primers

[0148] Fig. 9 The upper panel of the figure shows the desired primer annealing configuration for base-3 amplification, while the lower panel shows an alternative primer annealing configuration that does not produce base-3 amplification. In order to effectively perform base-3 amplification, the upper configuration should be more stable than the lower configuration. One way to achieve this is to design primers so that the T of the combined sequence ca (i.e., ca / c'-a') in the double-stranded form is m Higher than the T of the combined sequence ab (i.e. ab / a'-b') in the double-stranded form m .like Fig. 9 As shown, the use of modified bases in these primers provides a new method for enhancing this stability difference. The A* and T* bases are modified so that each modified base forms a stable hydrogen-bonded base pair with the natural (standard) complementary base, but does not form a stable hydrogen-bonded base pair with its modified complementary base. This ensures that the stability of the bottom primer annealing configuration is significantly lower than the upper primer annealing configuration. Fig. 9 The larger arrow pointing upward indicates that the desired configuration above is more stable than the undesired configuration because the above configuration has more duplex structures. In fact, the modified base is stable in terms of its pairing with its natural complementary sequence, but is unstable in terms of its pairing with its modified complementary sequence.

[0149] The advantage of using modified base in primer sets as described herein is that, compared with the time of each cycle of the same primer sets not including modified base, it reduces the time amount required for completing each amplification cycle.In various embodiments, as described herein in primer, using modified base can reduce the time of each cycle by for example 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% or 95% or more. The cycle time percentage reduction can fall within a range bounded by any of these values, such as 10-95%, 20-95%, 30-95%, 40-95%, 50-95%, 60-95%, 70-95%, 80-95%, 85-95%, 10-90%, 20-90%, 30-90%, 40-90%, 50-90%, 60-90%, 70-90%, 80-90%, 85-90%, 10-85%, 20-85%, 30-85%, 40-85%, 50-85%, 60-85%, 70-85%, 80-85%, etc.

[0150] The next section describes in detail modified bases suitable for use in primers.The remainder of this section describes the positioning of modified bases in the primers described herein in the various embodiments discussed above.

[0151] Dual primer sets with modified bases

[0152] Fig. 9 (Top configuration) shows how a double primer set with modified bases anneals to the first template strand at one end of the target nucleotide sequence. The design of this primer set is similar to Figure 2 The same as shown. For ease of discussion, the primer set can be regarded as a "forward" primer set. The outer primer includes a sequence a that specifically hybridizes with the first template strand sequence a'. In the embodiment of modified bases, the primer sequence a can include one or more first modified bases. In the embodiment of modified bases, the primer sequence a can include one or more first modified bases. If the primer sequence a includes more than one first modified base, the first modified base can be the same or different (the terms "first", "second", "third", etc. are used in this article for ease of discussion, but do not mean that all first, second or third modified bases are the same).

[0153] As in Figure 2 middle, Fig. 9 The primer set includes an inner primer having a double-stranded portion, one strand of which includes a primer sequence c' that is adjacent to the primer sequence a' and is 3' of the primer sequence a', wherein the combined sequence c'-a' is complementary to the combined sequence ca. In the embodiment of modified bases, the primer sequence a' may include one or more second modified bases. The modified bases in the outer primer sequence a are complementary sequences and are located in positions that allow them to base pair with the modified bases in the second strand of the inner primer (primer sequence a'). However, because they are complementary, the modified bases do not base pair in a stable manner (relative to their unmodified form), so the disadvantage is that undesirable primer annealing configurations ( Fig. 9 The formation of the configuration in the middle and lower figure).

[0154] Figure 3 1 shows how the double primer set anneals to the second template strand at the opposite end of the target nucleotide sequence. For ease of discussion, this primer set can be considered a "reverse" primer set. Here, the outer primer includes a sequence e that specifically hybridizes to the first template strand sequence e'. In the modified base embodiment, the primer sequence e can include one or more third modified bases.

[0155] Figure 3The primer set also includes an inner primer having a double-stranded portion, the second strand of the double-stranded primer sequence includes a primer sequence g' that is adjacent to the primer sequence e' and is 3' of the primer sequence e', wherein the joint sequence g'-e' is complementary to the joint sequence ge. In the embodiment of the modified base, the primer sequence e' may include one or more fourth modified bases. The modified bases in the outer primer sequence e are complementary sequences and are located in a position that allows them to base pair with the modified bases (primer sequence e') in the second strand of the inner primer. However, because they are complementary, the modified bases do not base pair in a stable manner (relative to their unmodified form), so the disadvantage is that the primer annealing configuration is not expected ( Fig. 9 The formation of the configuration in the middle and lower figure).

[0156] Triple primer sets with modified bases

[0157] Figure 4 and 5 Exemplary "forward" and "reverse" three-primer sets are shown, which can also be designed with modified bases. Figure 4 In the forward primer set, in the modified base embodiment, the primer sequence d of the outer primer may include one or more first modified bases.

[0158] The middle primer has a single-stranded portion and a double-stranded portion. The single-stranded portion includes a primer sequence a, which may include one or more second modified bases. The double-stranded portion includes a chain that includes a primer sequence c1' adjacent to the primer sequence d' and 3' of the primer sequence d', wherein the combined sequence c1'-d' is complementary to the combined sequence c1-d. In the embodiment of the modified base, the primer sequence d' may include one or more third modified bases.

[0159] The inner primer also has a single-stranded portion and a double-stranded portion. The double-stranded portion includes a chain that includes a primer sequence c2' adjacent to the primer sequence d' and 3' of the primer sequence d', the primer sequence d' is adjacent to the primer sequence a' and 3' of the primer sequence a', wherein the combined sequence c2'-d'-a' is complementary to the combined sequence c2-da. In the embodiment of the modified base, the primer sequence a' may include one or more fourth modified bases.

[0160] The first and third modified bases are complementary sequences and are in positions that would allow them to base pair, but the modifications do not encourage such pairing in favor of base pairing with their natural, unmodified complementary sequences. Similarly, the second and fourth modified bases are complementary sequences and are in positions that would allow them to base pair, but the modifications do not encourage such pairing in favor of base pairing with their natural, unmodified complementary sequences.

[0161] refer to Figure 5 In the reverse primer set, in the modified base embodiment, the primer sequence h of the outer primer may include one or more fifth modified bases.

[0162] The middle primer has a single-stranded portion and a double-stranded portion. The single-stranded portion includes a primer sequence e, which may include one or more sixth modified bases. The double-stranded portion includes a chain that includes a primer sequence g1' adjacent to the primer sequence h' and 3' of the primer sequence h', wherein the combined sequence g1'-h' is complementary to the combined sequence g1-h. In the embodiment of the modified base, the primer sequence h' may include one or more seventh modified bases.

[0163] The inner primer also has a single-stranded portion and a double-stranded portion. The double-stranded portion includes a chain that includes a primer sequence g2' adjacent to the primer sequence h' and 3' of the primer sequence h', and the sequence h' is adjacent to the primer sequence e' and 3' of the primer sequence e', wherein the combined sequence g2'-h'-e' is complementary to the combined sequence g2-he. In the embodiment of the modified base, the primer sequence a' can include one or more eighth modified bases.

[0164] The fifth and seventh modified bases are complementary sequences and are in positions that would allow them to base pair, but the modifications do not encourage such pairing in favor of base pairing with their natural, unmodified complementary sequences. Similarly, the sixth and eighth modified bases are complementary sequences and are in positions that would allow them to base pair, but the modifications do not encourage such pairing in favor of base pairing with their natural, unmodified complementary sequences.

[0165] Modified bases

[0166] The modified bases that can be used in the primers described herein include those in which the modified base forms a stable hydrogen-bonded base pair with a natural complementary base, but does not form a stable hydrogen-bonded base pair with its modified complementary base (for ease of discussion, complementary bases are also referred to herein as "pairing bodies"). In some embodiments, the modified base can form two or more hydrogen bonds with its natural pairing body, but only forms one hydrogen bond or does not form a hydrogen bond with its modified pairing body, and this is achieved. This allows the generation of primer pairs that do not form substantially stable hydrogen-bonded hybrids with each other, as shown in a melting temperature (under physiological or substantially physiological conditions) below about 40°C. However, the primers of the primer pairs form substantially stable hybrids with the complementary nucleotide sequences in the template strand (e.g., the first template strand) of a single-stranded or double-stranded target nucleic acid and with the strand complementary to the template strand (e.g., the second template strand). In some embodiments, hybrids formed with primers of the invention are more stable than hybrids formed using primers with unmodified bases due to the increase (in some embodiments, two-fold) in the number of hydrogen bonds in such hybrids.

[0167] According to generally accepted convention, the designations of the nucleotides of naturally occurring nucleic acids are A, U, G and C (RNA) and dA, dT, dG and dC (DNA). The following description applies to both ribonucleotides and deoxyribonucleotides, and therefore, unless the context requires otherwise, no distinction needs to be made in this specification between A and dA, U and dT, etc.

[0168] An analog of A modified in the base portion to form a stable hydrogen bonded pair with T (or U in the case of RNA) but not with the modified T is referred to as A*. An analog of T modified in the base portion to form a stable hydrogen bonded pair with A but not with A* is referred to as T*. An analog of G modified in the base portion to form a stable hydrogen bonded pair with C but not with the modified C is referred to as G*. An analog of C modified in the base portion to form a stable hydrogen bonded pair with G but not with G* is referred to as C*. In some embodiments, the aforementioned conditions are satisfied when each of the A*, T*, G* and C* nucleotides (collectively, modified nucleotides) forms two or more hydrogen bonds with their natural counterparts, but only forms one hydrogen bond or no hydrogen bond with their modified counterparts. This is provided by Formulas 1a, 1b, 2a, 2b, 3a, 3b, 4a and 4b (and in Figures 8A-8B ), which illustrates hydrogen bonding between natural AT (or AU in the case of RNA) and GC pairs, as well as hydrogen bonding between exemplary A*-T, T*-A, G*-C, C*-G, A*-T*, and G*-C* pairs.

[0169]

[0170]

[0171] Typically, a sufficient number of modified nucleotides are incorporated into the primers described herein to preferentially increase the annealing of the primer to the template strand of the target nucleic acid compared to primer-to-primer annealing. To achieve this, it is not necessary to replace each natural nucleotide of the primer with a modified nucleotide. In some embodiments, in addition to one or more modified nucleotides, the primer also includes one or more naturally occurring nucleotides and / or variants of naturally occurring nucleotides, provided that the changes do not significantly interfere with the complementary binding ability of the primers, as discussed above. For example, a primer including modified nucleotides may include a pentofuranosyl moiety other than ribose or 2-deoxyribose, and derivatives of ribose and 2-deoxyribose, such as 3-amino-2-deoxyribose, 2-fluoro-2-deoxyribose, and 2-OC 1-6 Alkyl or 2-O-allyl ribose, particularly 2-O-methyl ribose. The glycosidic bond may be in alpha or beta configuration. If desired, the phosphate backbone of the primer may include a phosphorothioate bond.

[0172] Formulas 5, 6 and 7 below provide the general structures of suitable classes of modified A analogs A*, shown as 3'-phosphates (or phosphorothioates) incorporated into primers, wherein:

[0173] X is N or CH;

[0174] Y is O or S;

[0175] Z is OH or CH3;

[0176] R is H, F or OR2, where R2 is C 1-6 alkyl or allyl, or in the case of RNA, H; and

[0177] R1 is C 1-4 Alkyl, C 1-4 Alkoxy, C 1-4 alkylthio, F or NHR3, wherein R3 is H, or C 1-4 An exemplary embodiment of A* has 2,6-diaminopurine (2-aminoadenine) as a base, as shown in Formula 1b. If applicable, the latter nucleotide may be abbreviated as 2-amA or d2-amA.

[0178]

[0179]

[0180] Formula 8 provides the general structure of a suitable class of modified T analogs, T*, shown as 3'-phosphates (or phosphorothioates) incorporated into a primer, wherein:

[0181] Y, Z and R are as defined above; and

[0182] R4 is H, C 1-6 Alkyl, C 1-6 Alkenyl or C 1-6 An exemplary embodiment of T* has 2-thio-4-oxo-5-methylpyrimidine (2-thiothymine) as a base, as shown in Formula 2b.

[0183] If applicable, the latter nucleotide may be abbreviated as 2-sT or d2-sT.

[0184]

[0185] Formulas 9, 10 and 11 provide the general structures of suitable classes of modified G analogs G*, shown as 3'-phosphates (or phosphorothioates) incorporated into primers, wherein:

[0186] R1 is H, C 1-4 Alkyl, C 1-4 Alkoxy, C 1-4 alkylthio, F or NHR3, wherein R3 is as defined above; and

[0187] X, Y, Z and R are as defined above. An exemplary embodiment of G* has 6-oxo-purine (hypoxanthine) as the base, as shown in Formula 3b. The latter nucleotide may be abbreviated as I or dI, if applicable.

[0188]

[0189] Formulas 12 and 13 provide the general structures of suitable classes of modified C analogs C*, shown as 3'-phosphates (or phosphorothioates) incorporated into primers, wherein:

[0190] Y, Z, R and R4 are as defined above;

[0191] Z1 is O or NH; and

[0192] R5 is H or C 1-4 An exemplary embodiment of C* has pyrrolo-[2,3-d]pyrimidin-2(3H)-one as the base, as shown in Formula 4b. The latter nucleotide may be abbreviated as P or dP, if applicable.

[0193] The above modified bases and nucleotides are also described in U.S. Pat. No. 5,912,340 (issued to Kutyavin et al. on June 15, 1999), the description of which is incorporated herein by reference. The hybridization properties of d2-amA and d2-sT are described in Kutyavin et al. (1996) Biochemistry 35: 11170-76, the description of which is incorporated herein by reference. The synthesis and hybridization properties of dI and dP are described in Woo et al. (1996) Nucleic Acids Research 25 (13): 2470-75, the description of which is incorporated herein by reference.

[0194] Other examples of G* and C* include 7-alkyl-7-deazaguanine and N 4 -alkylcytosine (where alkyl = methyl or ethyl), as described in Lahoud et al. (2008) Nucleic Acids Research 36(10):3409-19 (the description of which is incorporated herein by reference). The analog tested in this study is shown in Formula 12.

[0195]

[0196] Other examples of G* and C* include 7-nitro-7-deazahypoxanthine (NitrocH) and 2-thiocytosine (sC), respectively, which are described in Lahoud et al. (2008) Nucleic Acids Research 36(22):6999-7008 (the description of which is incorporated herein by reference). Hoshinka et al. (2010) Angew Chem Int Ed Engl. 49(32):5554-5557 describes the use of such bases ("Self-Avoiding Molecular Recognition Systems"), which include 2'-hypoxanthine as G* (the description of this reference is incorporated herein by reference; see in particular Figure 1 ); see also Yang et al. (2015) Chembiochem. 16(9): 1365-1367 (the description of which is incorporated herein by reference; see especially Scheme 1). The analogs tested in this study are shown in Formula 13.

[0197]

[0198] Polymerase

[0199] The disclosed method uses polymerase to amplify. In some embodiments, polymerase is a DNA polymerase lacking 5' to 3' exonuclease activity. The polymerase is used under such conditions so that the chain extended from the first primer can be displaced by the polymerization of the chain being formed by extending from the second primer, and the second primer is "outside" relative to the first primer. Conveniently, polymerase can displace the chain complementary to the template strand, and this characteristic is called "strand displacement". The strand displacement causes each template molecule to synthesize a multi-copy target sequence. In some embodiments, the DNA polymerase used in the disclosed method is highly propellant (processive). Exemplary DNA polymerases include variants of Taq DNA polymerases lacking 5' to 3' exonuclease activity, such as the mutant Taq, Bca polymerase (Takara), Pfx50 polymerase (Invitrogen), Tfu DNA polymerase (Qbiogene) described in the Stoffel fragment (ABI) of Taq DNA polymerase, SD polymerase (Bioron), USPN5474920 lacking 5' to 3' exonuclease activity. If thermal cycling is to be performed (as in PCR), the DNA polymerase is preferably a thermostable DNA polymerase. Table 2 below lists polymerases available from New England Biolabs that do not have 5' to 3' exonuclease activity, but have strand displacement activity with concomitant thermostability.

[0200] In some embodiments, it may be advantageous to use a blend of two or more polymerases. For example, an exemplary polymerase blend includes a polymerase that is particularly good at extending from a partially double-stranded DNA primer and a polymerase that is particularly good at strand displacement synthesis, because combining these properties can provide a net advantage in some embodiments. Alternatively or additionally, in the case where it is necessary to use a Taqman-type probe to carry out the inventor's real-time PCR, the polymerase mixture may include a polymerase with 5' to 3' exonuclease activity, as long as the primer structure is designed so that it is not susceptible to the "flap" endonuclease activity; in fact, due to the double-stranded nature of the "flap", the structure described herein may be inherently not susceptible to the activity. Taq DNA polymerase can, for example, be used in such a polymerase blend, although Taq DNA polymerase is described as including 5' to 3' exonuclease activity, the way Taq DNA polymerase works is more like a flap endonuclease.

[0201] Table 2 - Thermostable strand displacement polymerases lacking 5' to 3' exonuclease activity

[0202]

[0203] In some embodiments, the DNA polymerase comprises a fusion between Taq polymerase and a portion of a topoisomerase, such as TOPOTAQ TM (Fidelity Systems, Inc.).

[0204] For example, for SD polysaccharase, exemplary polysaccharase concentration range is about 20 to 200 units per reaction. In various embodiments, polysaccharase concentration can be at least: 10,20,30,40,50,60,70,80,90,100,110,120,130,140,150,160,170,180,190 or 200 or more units per reaction. In some embodiments, polysaccharase concentration falls within the range defined by any of these values, for example, units such as 10-200,10-150,10-100,10-50,20-150,20-100,20-50,50-200,50-150,50-100,100-200,100-150 per reaction. When using a polysaccharase blend, the total concentration of total polysaccharase can be any of these values ​​or fall into any of these scopes.

[0205] Strand displacement can also be promoted by using a strand displacement factor (e.g., a helicase). Any DNA polymerase capable of performing strand displacement in the presence of a strand displacement factor is suitable for use in the disclosed methods, even if the DNA polymerase does not perform strand displacement in the absence of such a factor. Strand displacement factors suitable for use in the methods described herein include BMRF1 polymerase auxiliary subunit (Tsurumi et al., J. Virology 67(12):7648-7653 (1993)), adenovirus DNA binding protein (Zijderveld and van der Vliet, J. Virology 68(2):1158-1164 (1994)), herpes simplex virus protein ICP8 (Boehmer and Lehman, J. Virology 67(2):711-715 (1993); Skaliter and Lehman, Proc. Natl. Acad. Sci. USA 91(22):10665-10669 (1994)), single-stranded DNA binding protein (SSB; Rigler and Romano, J. Biol. Chem. 270:8910-8919 (1995)) and calf thymus helicase (Siegel et al., J. Biol. Chem. 267:13629-13635 (1992)). Thermostable forms of helicase and SSB are available and are therefore suitable for PCR.

[0206] Amplification

[0207] The above primer sets are contacted with the sample nucleic acid under such conditions: wherein the primers anneal to their template strands (if present). The desired nucleic acid amplification method is performed using a DNA polymerase lacking 5'-3' exonuclease activity that can perform strand displacement under the reaction conditions employed. The amplification produces an amplicon that contains the sequences of all primers used in the amplification reaction. The primer set can be conveniently added to the amplification mixture in the form of a separate oligonucleotide. For example, a double primer set can be composed of three oligonucleotides (assuming that the inner primer does not contain a hairpin structure), and a triple primer set can be composed of five oligonucleotides (assuming that neither the inner primer nor the middle primer contains a hairpin structure).

[0208] For semi-nested amplification using a dual primer set as described above, at least 3 循环数 Amplification using a semi-nested dual primer set can reduce the number of amplification cycles required to detect a single copy nucleic acid by about 12% to about 42% (e.g., a 37% reduction). This facilitates detection of a single copy nucleic acid in a biological sample within about 23-27 amplification cycles (which otherwise may require 40 or more cycles). In some embodiments, a semi-nested dual primer set PCR facilitates detection of a single copy nucleic acid in a biological sample in 23, 24, 25, 26, or 27 amplification cycles.

[0209] Table 3 below shows the amplification of a single copy nucleic acid to 10 12 For complete nested amplification using a dual primer set as described above, at least 6 cycles can be achieved in the exponential phase of PCR. 循环数 Amplification using a fully nested dual primer set can reduce the number of amplification cycles required to detect a single copy nucleic acid by about 36% to about 66% (e.g., a 61% reduction). This facilitates detection of a single copy nucleic acid in a biological sample within about 13-17 amplification cycles. In some embodiments, a fully nested dual primer set PCR facilitates detection of a single copy nucleic acid in a biological sample in 13, 14, 15, 16, or 17 amplification cycles.

[0210] Table 3 - Reduction in the number of cycles required for amplification as a function of PCR base number

[0211]

[0212] For the semi-nested amplification using the three-primer set as described above, at least 4 循环数Amplification using a semi-nested three-primer set can reduce the number of amplification cycles required to detect a single copy nucleic acid by about 25% to about 55% (e.g., a 50% reduction). This facilitates detection of a single copy nucleic acid in a biological sample within about 20 amplification cycles (which otherwise may require 40 or more cycles). In some embodiments, a semi-nested three-primer set PCR facilitates detection of a single copy nucleic acid in a biological sample in 18, 19, 20, 21, or 22 amplification cycles.

[0213] For complete nested amplification using a three-primer set as described above, at least 8 循环数 Amplification using a fully nested three-primer set can reduce the number of amplification cycles required to detect a single copy nucleic acid by about 42% to about 72% (e.g., a 67% reduction). This facilitates detection of a single copy nucleic acid in a biological sample within about 11-15 amplification cycles. In some embodiments, a fully nested three-primer set PCR facilitates detection of a single copy nucleic acid in a biological sample in 9, 10, 11, 12, or 13 amplification cycles.

[0214] In some embodiments, PCR is used to perform the amplification step. For running a real-time PCR reaction, the reaction mixture typically includes a suitable buffer, magnesium ions (Mg 2+) in a range of about 1 to about 10 mM (e.g., in a range of about 2 to about 8 mM), and a concentration of 100 μM magnesium ions (Mg 2+). 2+ ) source, nucleotides and optional detergents and stabilizers. An example of a suitable buffer is a TRIS buffer having a concentration of about 5mM to about 85mM, preferably a concentration of 10mM to 30mM. In one embodiment, in a double strength (2X) form of the reaction mixture, the TRIS buffer concentration is 20mM. The pH range of the reaction mixture can be about 7.5 to about 9.0, with a typical pH range of about 8.0 to about 8.5. The concentration range of nucleotides can be about 25mM to about 1000mM, with a typical concentration range of about 100mM to about 800mM. Examples of dNTP concentrations are 100, 200, 300, 400, 500, 600, 700 and 800mM. Detergents such as Tween 20, Triton X 100 and Nonidet P40 may also be included in the reaction mixture. Stabilizers such as dithiothreitol (DTT, Cleland's reagent) or mercaptoethanol may also be included. In addition, the master mix may optionally contain dUTP and uracil DNA glycosylase (uracil-N-glycosylase, UNG). The master mix can be purchased from Applied Biosystems (Foster City, California, USA) Universal Master Mix, catalog numbers 4304437, 4318157, and 4326708).

[0215] Tagging strategy

[0216] Any suitable labeling strategy can be used in the methods described herein. When analyzing the presence of a single amplification product in a reaction, a universal detection probe can be used in the amplification mixture. In a specific embodiment, a universal qPCR probe can be used for real-time PCR detection. Suitable universal qPCR probes include double-stranded DNA binding dyes, such as SYBR green, Pico green (Molecular Probes, Inc., Eugene, OR), Eva green (Biotinum), ethidium bromide, etc. (see Zhu et al., 1994, Anal. Chem. 66: 1941-48).

[0217] In some embodiments, one or more target-specific qPCR probes (i.e., specific for the target nucleotide sequence to be detected) are used in the amplification mixture to detect the amplification products. By carefully selecting the labels, an analysis can be performed in which different labels are excited and / or detected at different wavelengths in a single reaction ("multiplexed detection"). See, for example, Fluorescence Spectroscopy (Pesce et al., Eds.), Marcel Dekker, New York, (1971); White et al., Fluorescence Analysis: A Practical Approach, Marcel Dekker, New York, (1970); Berlman, Handbook of Fluorescence Spectra of Aromatic Molecules, 2nd ed., Academic Press, New York York, (1971); Griffiths, Color and Constitution of Organic Molecules, Academic Press, New York, (1976); Indicators (Bishop, Ed.). Pergamon Press, Oxford, 19723; and Haugland, Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Eugene (1992); and Linck et al. al. (2017) "Amultiplex TaqMan qPCR assay for sensitive and “rapid detection of phytoplasmasinfecting Rubus species,” PLOS One 12(5).

[0218] In some embodiments, it may be convenient to include a label on one or more of the primers used in the amplification mixture.

[0219] Exemplary Automation and Systems

[0220] In some embodiments, an automated sample processing and / or analysis platform is used to detect the target nucleic acid. In some embodiments, a commercially available automated analysis platform is utilized. For example, in some embodiments, a System (Cepheid, Sunnyvale, CA).

[0221] The methods described herein are illustrated for use with the GeneXpert system. Exemplary sample preparation and analysis methods are described below. However, the present invention is not limited to a specific detection method or analysis platform. Those skilled in the art will appreciate that any number of platforms and methods may be utilized.

[0222] Utilizing a self-contained, single use cartridge. Sample extraction, amplification, and detection can all be performed within this self-contained "lab-in-a-cartridge" (available from Cepheid, see www.cepheid.com).

[0223] The components of the cartridge include, but are not limited to, a processing chamber containing reagents, a filter, and capture techniques that can be used to extract, purify, and amplify target nucleic acids. Valves allow fluid to be transferred from one chamber to another and contain nucleic acid cleavage and filtering components. Optical windows enable real-time optical detection. Reaction tubes enable very fast thermal cycling.

[0224] In some embodiments, The system includes multiple expandable modules, each of which includes multiple cartridges, as well as sample processing and analysis components.

[0225] After the sample is added to the cartridge, the sample is contacted with a lysis buffer, and the released nucleic acids are bound to a nucleic acid-binding matrix (e.g., a silica or glass matrix). The sample supernatant is then removed and the nucleic acids are eluted in an elution buffer (e.g., a Tris / EDTA buffer). The eluate can then be processed in the cartridge to detect target genes as described herein. In some embodiments, the eluate is used to reconstitute at least some of the reagents that are present in the cartridge as lyophilized particles.

[0226] In some embodiments, PCR is used to amplify and detect the presence of one or more target nucleic acids. In some embodiments, PCR uses a Taq polymerase with a hot start function, such as AptaTaq (Roche).

[0227] In some embodiments, off-line centrifugation is used to improve assay results using samples with low cell content. The sample with or without added buffer is centrifuged and the supernatant is removed. The pellet is then resuspended in a smaller volume of supernatant, buffer, or other liquid. The resuspended pellet is then added to the previously described In the tube.

[0228] Reagent test kit

[0229] Also contemplated are kits for carrying out the methods described herein. Such kits include one or more reagents that can be used to implement any of these methods. The kits typically include packaging with one or more containers for holding the reagents, which are one or more separate compositions, or optionally a mixture where reagent compatibility permits. The kits may also include other materials required from a user's perspective, such as buffers, diluents, standards, and / or any other materials that can be used for sample processing, cleaning, or any other step in performing the assay.

[0230] The kit preferably includes instructions for implementing one or more screening methods described herein. The instructions contained in the kit may be attached to the packaging material or may be included as a packaging insert. Although the instructions are usually written or printed materials, they are not limited to this. Any medium that can store such instructions and spread them to end users can be used. Such media include, but are not limited to, electronic storage media (e.g., disks, tapes, cassettes, chips), optical media (e.g., CD ROMs), etc. The term "instructions" used herein may include an Internet address that provides the instructions.

[0231] Example

[0232] Example 1: Determining the effect of a "clamping" oligonucleotide on primer / target structure

[0233] Experiments were performed in which the Tm of a primer oligonucleotide (although referred to as a "primer", it was not used as such in this experiment) and a complementary target sequence were measured in the presence and absence of a "clamping" oligonucleotide. The synthetic target oligonucleotide had a 5' fluorescent tag (fluorescein) and a fluorescence quenching moiety was incorporated into the primer (see Figure 6). The red color indicates the presence of a 2' O-methyl backbone. The oligonucleotide sequences tested are listed in Table 4 below. The Tm of the rightmost double helix region shown in the structure of Figure 6 was measured by tracking the increase in fluorescence, which results from the separation of the fluorophor and quencher as the temperature increases and by the melting of the double helix region.

[0234] If, as shown in FIG6A , the target binding region of the primer is restricted to b / b′ binding by the clamp, then the T of this region is predicted to be m Compared with the T in the case of ab / a'b' combination in FIG6B m The oligonucleotides used in this and subsequent experiments are listed in Table 4 below. The predicted and measured T values ​​of primers and target oligonucleotides in the presence or absence of the clamping oligonucleotide are shown in Table 5. m .

[0235] Table 4 - Oligonucleotides used

[0236]

[0237]

[0238] A* = 2,6-diaminopurine, U01 = dabcyl quencher labeled uracil, lower case letters are 2'-O-methyl nucleotides. Oligonucleotide 16142 was blocked to prevent extension.

[0239] Table 5 - T m Measurement

[0240] Primers Target Clip <![CDATA[Predicted T m (°C)]]> <![CDATA[Measured T m (°C)]]> 16140 16145 none 76.4(ab / a'b') 78.5 16141 16146 none 74.6(ab / a'b') 78.0 16140 16145 16142 68.3(b / b') 67.0 16141 16146 16142 62.0(b / b') 66.5

[0241] The conditions for all hybrid melt analysis were: 0.01 M tris-HCl, 0.05 M KCl and 0.006 M MgCl2. All oligonucleotides were 1 μM. The oligonucleotide mixture in Table 4 above was heated to 95°C and slowly cooled to 45°C using a Cepheid SmartCycler TM Monitor fluorescein fluorescence. m Measured as the temperature at which the rate of fluorescence change is maximum.

[0242] The T values ​​of b / b' and ab / a'b' were predicted using the software (www.idtdna.com / analyzer / Applications / OligoAnalyzer). m The measured T m This is consistent with a structure in which, in the presence of the clamping oligonucleotide, the d'-a' region in the target remains single-stranded and available for hybridization.

[0243] As illustrated in FIG. 7A , the flanking primers (16147 to 16149), also present at 1 μM, showed no significant difference in the measured T m There was almost no difference in the results.

[0244] Example 2: Extendibility of outer (flanking) primers

[0245] The outer (flanking) primers schematically shown in FIG. 7A were tested for extendibility in PCR reactions under the conditions shown in Table 6.

[0246] Table 6

[0247] reaction Flanking primers 1 16147 2 16148 3 16149 4 none

[0248] All reactions contained 10 mM Tris-HCl; 0.125 mM each of dATP, dTTP, dCTP and dGTP; 0.15 μM primer oligonucleotide 16140 from Table 5 above; 0.125 μM target oligonucleotide 16145 from Table 1; 0.125 μM clamping oligonucleotide 16142 from Table 1; 45 mM KCl; 3.5 mM MgCl2; 14 units of AmpliTaqCS, which has DNA polymerase activity but neither 5' to 3' nor 3' to 5' exonuclease activity; and 15 units of Taq polymerase antibody, which provides a temperature-activated "hot start" for the amplification reaction.

[0249] 0.125 mM or no flanking primers were added according to Table 6 above; the reaction was monitored over time using a SmartCycler, the temperature was raised to 95°C to separate the oligonucleotides, while activating the polymerase, and then the temperature was lowered to 60°C to allow the oligonucleotides to anneal and allow any primer extension to occur. The results are shown in Figure 7B. The three rising traces are separate reactions with slightly different clamps; the horizontal traces do not have the outer (flanking) displacement primer present. These results indicate that when the outer (flanking) primer is present, a strand displacement reaction that displaces the quencher occurs.

[0250] Example 3: Use of modified primers to produce base-3 amplification with satisfactory extension times

[0251] As illustrated in this example, by including modified bases, the primer extension time in each amplification cycle can be further shortened.

[0252] Figures 10A and 10B compare the real-time PCR growth curves of the modified test primer set ("8 series") and the unmodified test primer set ("6 series"). The fluorescence (y-axis) is plotted against the PCR cycle number using a logarithmic y-axis scale. All oligonucleotide sequences can be found in Table 7 below, which also shows the positions of the modified nucleotides 2,6-diaminopurine and 2-thiothymine.

[0253] Table 7

[0254]

[0255] A* = 2,6-diaminopurine, C01 = 5-methylcytosine, lowercase letters are 2'-O-methyl nucleotides. Clamp b'c'6 is 3'-blocked to prevent extension. Clamp b'c'8 can also be 3'-blocked to prevent extension.

[0256] All PCRs used the same forward f1 primer (SEQ ID NO: 1), which was included in the master mix. EvaGreen dye (Biotium) is a green fluorescent nucleic acid intercalating dye for real-time PCR. The final concentration of each primer was 0.4 μM, and 1.25E+.07 copies of the template (SEQ ID NO: 9), a synthetic oligonucleotide of 69 nucleotides, or a water control (NTC or "no template control" condition) were added. Strand displacement (SD) polymerase (Bioron) was used, which is a thermostable polymerase that provides strong displacement activity. The PCR master mix solution of reagents common to all PCRs includes 20 units of SD polymerase, 1x SD polymerase buffer, 1xEvagreen dye, 5mM MgCl2, 0.4mM dNTP, and 0.4 μM of f1 primer. All reactions were prepared on ice and then subjected to a 3-temperature thermocycler protocol using a Cepheid SmartCycler real-time PCR machine: 95°C for 15 sec, 68°C for 16 sec, and 56°C for 37 sec for 25 cycles.

[0257] In Figure 10A, the "cba8" and "b8" reactions were used as "double primers", the control condition consisted of forward primer f1 plus reverse primer cba8 (SEQ ID NO:3), used with either the b'c'8 (SEQ ID NO:4) "sandwich" oligonucleotide or reverse primer b8 (SEQ ID NO:8). The "8 Test" combined all three primers, and the "8 Test NTC" was used as a no-template control for the test conditions. At a fluorescence threshold of 50, the three-primer test condition resulted in an earliest Ct of 7.7, approximately 5 cycles earlier than the two-primer control condition. At or near the fluorescence threshold, more than a doubling of fluorescence levels was seen for the "8 Test" PCR, while no more than a doubling was seen for the two primer control PCR.

[0258] In Figure 10B, reactions "cba6", which include primer cba6 (SEQ ID NO:3) and oligonucleotide b'c'6 (SEQID NO:5), "b6", which includes primer b6 (SEQ ID NO:7), "6 test" and "6 test NTC" (including cba6, b'c'6 and b6) are similar to "cba8", "b8", "8 test" and "8 test NTC" of Figure 10A, respectively, except that the modified nucleotides 2,6-diaminopurine and 2-thiothymine are not present in any oligonucleotide.

[0259] The use of modified bases in these primers resulted in threefold growth per cycle, with extension times of less than 60 seconds per cycle.

[0260] Example 4: Using modified primers at both ends of the amplicon to generate base-6 amplification, the number of cycles required for effective amplification Significantly reduced number

[0261] Fig.11A Real-time PCR fluorescence growth curves generated by base-6 (approximately 6 replicates per cycle) PCR amplification starting with decreasing numbers of template DNA molecules are shown. Log 10 dilutions of S. pyogenes genomic DNA were used as DNA template input. Fig. 11B Shown is a graph of the number of amplification cycles (Ct) required to reach a threshold level of fluorescence plotted against the log 10 of the number of starting DNA template molecules. All oligonucleotide sequences can be found in Table 8.

[0262] Table 8

[0263]

[0264] The PCR master mix solution of reagents used for all reactions in this experiment included 80 units of strand displacement (SD) polymerase, 1x SD polymerase buffer, 1x Evagreen fluorescent dye, 5 mM MgCl2, 0.4 mM dNTP, 0.5 μM of each oligonucleotide in Table 8.

[0265] S. pyogenes genomic DNA (ATCC 12433D-5) was diluted with water to 1.27E7 copies / μL, 1.27E6 copies / μL, 1.27E5 copies / μL, 1.27E4 copies / μL and 1.27E3 copies / μL. 1 μL of each diluted template solution was added to the PCR master mix solution and water was added to a total volume of 25 μL. This resulted in a 10-fold decrease in 5 template reactions, from 1.27E7 copies to 1270 copies, and no template control reaction. All reactions were prepared on ice and then placed in a Cepheid SmartCycler real-time PCR instrument for a 2-temperature thermal cycler protocol: 92°C for 1 second, 62°C for 30 seconds, and 25 cycles.

[0266] Fig.11A The number of cycles required to pass the fluorescence threshold (dashed line) is shown to increase for decreasing amounts of template. This increased fluorescence is caused by an increase in the amount of double-stranded DNA amplicons in the presence of the fluorescent, dsDNA binding dye, Evagreen. Fig. 11B The cycle number (Ct) is plotted against the log 10 of the starting copy number of the template. The inverse of the negative slope of this line is approximately 1.3 cycles per log 10 dilution, which is consistent with approximately 6 replications per cycle. In standard base-2 PCR, there are approximately 3.3 cycles per log 10 dilution. Sequence Listing <110> Cepheid <120> Exponential base-3 nucleic acid amplification using nested overlapping primers with reduced amplification time <130> CPHDP015WO <140> PCT / US2019 / 057958 <141> 2019-10-24 <150> 62 / 752,269 <151> 2018-10-29 <150> 62 / 752,276 <151> 2018-10-29 <160> twenty three <170> PatentIn version 3.5 <210> 1 <211> 49 <212> DNA <213> Artificial <220> <223> Primers <400> 1 ggcgcuccgg accggcgtag gctggtaacc aaccgctgaa ggcauacgg 49 <210> 2 <211> 46 <212> DNA <213> Artificial <220> <223> Primers <400> 2 ggcgcuccgg accggcgtag gctggtaacc aaccgctgaa ggcaua 46 <210> 3 <211> 31 <212> DNA <213> Artificial <220> <223> Clip <400> 3 tggttaccag cctacgccgg tccggagcgc c 31 <210> 4 <211> 38 <212> DNA <213> Artificial <220> <223> Target <400> 4 ccgtatgcct tcagcggttg gttaccagcc tacgcatt 38 <210> 5 <211> 35 <212> DNA <213> Artificial <220> <223> Target <400> 5 tatgccttca gcggttggtt accagcctac gcatt 35 <210> 6 <211> 15 <212> DNA <213> Artificial <220> <223> Flanking primers <400> 6 cgtaggctgg taacc 15 <210> 7 <211> 16 <212> DNA <213> Artificial <220> <223> Flanking primers <400> 7 gcgtaggctg gtaacc 16 <210> 8 <211> 16 <212> DNA <213> Artificial <220> <223> Flanking primers <400> 8 gcgtaggctg gtaacc 16 <210> 9 <211> twenty two <212> DNA <213> Artificial <220> <223> f1 <400> 9 ttcagaggat aaaggtaagc aa 22 <210> 10 <211> 22 <212> DNA <213> artificial <220> <223> f7 <400> 10 ttcagaggat aaaggtaagc aa 22 <210> 11 <211> 62 <212> DNA <213> artificial <220> <223> cba6 <400> 11 ccgcgggacc ggcgccagct gaccttaact tcgaatatca atactctgac caagtgactg 60 aa 62 <210> 12 <211> 41 <212> DNA <213> artificial <220> <223> b'c'8 <400> 12 ttgatattcg aagttaaggt cagctggcgc cggtcccgcg g 41 <210> 13 <211> 41 <212> DNA <213> artificial <220> <223> b'c'6 <400> 13 ttgatattcg aagttaaggt cagctggcgc cggtcccgcg g 41 <210> 14 <211> 16 <212> DNA <213> artificial <220> <223> b1 <400> 14 tgaccttaac ttcgaa 16 <210> 15 <211> 16 <212> DNA <213> Artificial <220> <223> b6 <400> 15 tgaccttaac ttcgaa 16 <210> 16 <211> 16 <212> DNA <213> Artificial <220> <223> b8 <400> 16 tgaccttaac ttcgaa 16 <210> 17 <211> 69 <212> DNA <213> Artificial <220> <223> stencil <400> 17 ttcagaggat aaaggtaagc aatgggttca gtcacttggt cagagtattg atattcgaag 60 ttaaggtca 69 <210> 18 <211> 59 <212> DNA <213> Artificial <220> <223> cba4 <400> 18 ccgcgggacc ggcgccagcg caccatcgat aacaaaggca tgtccgccta ctttaccga 59 <210> 19 <211> 39 <212> DNA <213> Artificial <220> <223> b'c'2 <400> 19 gcctttgtta tcgatggtgc gctggcgccg gtcccgcgg 39 <210> 20 <211> 16 <212> DNA <213> Artificial <220> <223> b6 <400> 20 caccatcgat aacaaa 16 <210> 21 <211> 61 <212> DNA <213> Artificial <220> <223> def1 <400> 21 cggccgcggc cagggcgccg accaaatcaa ccgtagcgac tttagcaaac aagattggga 60 a 61 <210> 22 <211> 37 <212> DNA <213> Artificial <220> <223> e'd'1 <400> 22 gctacggttg atttggtcgg cgccctggcc gcggccg 37 <210> 23 <211> 15 <212> DNA <213> Artificial <220> <223> e1 <400> 23 accaaatcaa ccgta 15

Claims

1. A nucleic acid primer set for amplifying a target nucleic acid in a sample, wherein the target nucleic acid comprises a first template strand and an optional second template strand, wherein the second template strand is complementary to the first template strand, the primer set comprises such oligonucleotides, which are in the form of at least two first primers capable of hybridizing with the first template strand or capable of forming at least two first primers capable of hybridizing with the first template strand, wherein the at least two first primers comprise a first outer primer and a first inner primer, The first outer primer comprises a primer sequence a, which specifically hybridizes with a first template strand sequence a', and the primer sequence a comprises one or more first modified bases; and The first inner primer comprises a single-stranded primer sequence b and a double-stranded primer sequence, wherein the single-stranded primer sequence b specifically hybridizes to the first template strand sequence b', wherein b' is adjacent to a' and is 5' to a', and wherein the double-stranded primer sequence comprises a first strand and a second strand, wherein the first strand comprises: a primer sequence a, which is adjacent to and 5' of the single-stranded primer sequence b; and a clamping sequence c, which is adjacent to and 5' of the primer sequence a, wherein the clamping sequence c is not complementary to a first-strand template sequence d', which is adjacent to and 3' of the first-strand template sequence a'; wherein the second strand comprises a primer sequence c', which is adjacent to and 3' to the primer sequence a', wherein the association sequence c'-a' is complementary to the association sequence ca, and the primer sequence a' comprises one or more second modified bases; wherein the single-stranded primer sequence b is connected at its 5′ end to the first strand of the double-stranded primer sequence; wherein unmodified forms of the first and second modified bases are complementary, and the first and second modified bases preferentially pair with the unmodified forms compared to pairing between the first and second modified bases; The T of the combined sequence ca in the double-stranded form m T higher than the combined sequence ab in double-stranded form m , the clamping sequence c cannot be copied during amplification; and The primer set further comprises at least one second primer capable of specifically hybridizing to the second template strand, and wherein the first primer and the at least one second primer are a forward primer and at least one reverse primer, respectively, or the first primer and the at least one second primer are a reverse primer and at least one forward primer, respectively.

2. A method for amplifying a target nucleic acid in a sample, wherein the target nucleic acid comprises a first template strand and optionally a second template strand, wherein the second template strand is complementary to the first template strand, the method comprising: (a) contacting the sample with: (i) at least two first primers, the first primers being capable of hybridizing to the first template strand, wherein the at least two first primers include a first outer primer and a first inner primer, The first outer primer comprises a primer sequence a, which specifically hybridizes with the first template strand sequence a', and the primer sequence a includes one or more first modified bases; and The first inner primer comprises a single-stranded primer sequence b and a double-stranded primer sequence, wherein the single-stranded primer sequence b specifically hybridizes to the first template strand sequence b', wherein b' is adjacent to a' and is 5' to a', and wherein the double-stranded primer sequence comprises a first strand and a second strand, wherein the first strand comprises: a primer sequence a, which is adjacent to and 5' of the single-stranded primer sequence b; and a clamping sequence c, which is adjacent to and 5' of the primer sequence a, wherein the clamping sequence c is not complementary to a first-strand template sequence d', which is adjacent to and 3' of the first-strand template sequence a'; wherein the second strand comprises a primer sequence c', which is adjacent to and 3' to the primer sequence a', wherein the association sequence c'-a' is complementary to the association sequence ca, and the primer sequence a' comprises one or more second modified bases; wherein the single-stranded primer sequence b is connected at its 5' end to the first strand of the double-stranded primer sequence; wherein the unmodified forms of the first and second modified bases are complementary, and the first and second modified bases preferentially pair with the unmodified forms compared to pairing between the first and second modified bases; and (ii) at least one second primer capable of specifically hybridizing to the second template strand, wherein the contacting is performed under conditions wherein the primers are able to anneal to their template strands; and (b) subjecting the sample and primers to appropriate amplification conditions using a DNA polymerase lacking 5'-3' exonuclease activity, wherein strand displacement occurs to produce an amplicon comprising a sequence extending from the template sequence a' to the second primer binding site, And the T of the combined sequence ca in the double-stranded form m T higher than the combined sequence ab in double-stranded form m , and the clamping sequence c cannot be copied during amplification.

3. The method of claim 2, wherein the DNA polymerase is stable above 85 degrees Celsius.

4. The primer set according to claim 1, or the method according to claim 2 or 3, wherein the joint sequence ca is more GC-rich and / or contains more stable bases than the joint sequence ab.

5. The primer set according to claim 1, or the method according to claim 2 or 3, wherein the primer set is capable of amplifying the exponential phase of the amplification with at least 3 循环数 The target nucleic acid is amplified at a rate of at least 3 循环数 Amplify the target nucleic acid at a rate of 6. A primer set according to claim 1, or a method according to claim 2 or 3, wherein the primer set or method allows detection of a single copy nucleic acid in a biological sample with an amplification cycle that is 12%-42% fewer than the amplification cycles required for the detection using only a single forward primer and a single reverse primer.

7. The primer set according to claim 1, or the method according to claim 2 or 3, wherein the joint sequence ab contains more unstable bases than the joint sequence ca.

8. The primer set according to claim 1, or the method according to claim 2 or 3, wherein the second primer comprises oligonucleotides forming at least two second primers, the second primers being capable of hybridizing to the second template strand, wherein the at least two second primers comprise a second outer primer and a second inner primer, The second outer primer comprises a primer sequence e, which specifically hybridizes with a second template strand sequence e', wherein the primer sequence e includes one or more third modified bases; and The second inner primer comprises a single-stranded primer sequence f and a double-stranded primer sequence, wherein the single-stranded primer sequence f specifically hybridizes to the second template strand sequence f', wherein f' is adjacent to e' and is 5' to e', and wherein the double-stranded primer sequence comprises a first strand and a second strand, wherein the first strand comprises: a primer sequence e, which is adjacent to and 5' of the single-stranded primer sequence f; and a clamping sequence g, which is adjacent to and 5' of the primer sequence e, wherein the clamping sequence g is not complementary to a second-strand template sequence h', which is adjacent to and 3' of the second template strand sequence e'; wherein the second strand comprises a primer sequence g', which is adjacent to and 3' of the primer sequence e', wherein the union sequence g'-e' is complementary to the union sequence ge, and the primer sequence e' comprises one or more fourth modified bases; wherein the single-stranded primer sequence f is linked at its 5′ end to the first strand of the double-stranded primer sequence; wherein the unmodified forms of the third and fourth modified bases are complementary, and the third and fourth modified bases preferentially pair with the unmodified forms as compared to pairing between the third and fourth modified bases, And the T of the combined sequence ge in the double-stranded form m T higher than the combined sequence ef in double-stranded form m , and the clamping sequence g cannot be copied during amplification.

9. The primer set or method according to claim 8, wherein the joint sequence ge is more GC-rich and / or contains more stable bases than the joint sequence ef.

10. The primer set or method according to claim 8, wherein the primer set is capable of increasing the expression of the target gene by at least 6 folds in the exponential phase of amplification. 循环数 The target nucleic acid is amplified at a rate of at least 6 循环数 Amplify the target nucleic acid at a rate of 11. The primer set or method of claim 8, wherein the primer set or method allows detection of a single copy nucleic acid in a biological sample within an amplification cycle that is 36%-66% fewer than the amplification cycles required for the detection using only a single forward primer and a single reverse primer.

12. The primer set or method of claim 8, wherein the clamping sequence c and / or g comprises 2'-O-methyl RNA.

13. The primer set or method according to claim 8, wherein the double-stranded primer sequence of the first inner primer and / or the second inner primer does not contain a hairpin sequence.

14. The primer set or method according to claim 8, wherein the double-stranded primer sequence of the first inner primer comprises a hairpin sequence in which a clamping sequence c is connected to a complementary sequence c', and / or the double-stranded primer sequence of the second inner primer comprises a hairpin sequence in which a clamping sequence g is connected to a complementary sequence g'.

15. The primer set or method according to claim 8, wherein the joint sequence ef contains more unstable bases than the joint sequence ge.

16. The primer set or method of claim 8, wherein at least one modified base is identical to at least one other modified base.

17. A nucleic acid primer set for amplifying a target nucleic acid in a sample, wherein the target nucleic acid comprises a first template strand and an optional second template strand, wherein the second template strand is complementary to the first template strand, the primer set comprises at least three oligonucleotides in the form of first primers or capable of forming at least three first primers, the first primers are capable of hybridizing with the first template strand, wherein the at least three first primers include a first outer primer, a first middle primer and a first inner primer, The first outer primer comprises a primer sequence d, which specifically hybridizes with the first template strand sequence d', and the primer sequence d comprises one or more first modified bases; The first intermediate primer comprises a single-stranded primer sequence a and a double-stranded primer sequence, wherein the single-stranded primer sequence a specifically hybridizes to the first template strand sequence a', wherein a' is adjacent to d' and is 5' to d', and the single-stranded primer sequence a comprises one or more second modified bases, wherein the double-stranded primer sequence comprises a first strand and a second strand, wherein the first strand comprises: a primer sequence d, which is adjacent to and 5' of the single-stranded primer sequence a; and a clamping sequence c1, which is adjacent to and 5' of the primer sequence d, wherein the clamping sequence c1 is not complementary to a first template strand sequence i', which is adjacent to and 3' of the first template strand sequence d'; wherein the second strand comprises a primer sequence c1′, which is adjacent to and 3′ of the primer sequence d′, wherein the union sequence c1′-d′ is complementary to the union sequence c1-d, and the primer sequence d′ comprises one or more third modified bases; wherein the single-stranded primer sequence a is linked at its 5' end to the first strand of the double-stranded primer sequence; and The first inner primer comprises a single-stranded primer sequence b and a double-stranded primer sequence, wherein the single-stranded primer sequence b specifically hybridizes to the first template strand sequence b', wherein b' is adjacent to a' and is 5' to a', and wherein the double-stranded primer sequence comprises a first strand and a second strand, wherein the first strand comprises: a primer sequence a, which is adjacent to and 5' of the single-stranded primer sequence b; a primer sequence d, which is adjacent to and 5' of primer sequence a; and a clamping sequence c2, which is adjacent to the primer sequence d and is 5' to the primer sequence d, wherein the clamping sequence c2 is not complementary to the first strand template sequence i'; wherein the second strand comprises a primer sequence c2' adjacent to and 3' of primer sequence d', wherein primer sequence d' is adjacent to and 3' of primer sequence a', wherein primer sequence a' comprises one or more fourth modified bases, wherein the union sequence c2'-d'-a' is complementary to the union sequence c2-da; wherein the single-stranded primer sequence b is connected at its 5' end to the first strand of the double-stranded primer sequence; wherein unmodified forms of the first and third modified bases are complementary, and the first and third modified bases preferentially pair with the unmodified forms as compared to pairing between the first and third modified bases; and wherein the unmodified forms of the second and fourth modified bases are complementary, and the second and fourth modified bases preferentially pair with the unmodified forms over pairing between the second and fourth modified bases, The T of the combined sequence c1-d in the double-stranded form m T higher than the combined sequence da of the double-stranded form m , and the combined sequence of the double-stranded form is c2-da T m Higher than the T of the combined sequence dab in double-stranded form m , and the clamping sequences c1 and c2 cannot be copied during amplification; and The primer set further comprises at least one second primer capable of specifically hybridizing to the second template strand, and wherein the first primer and the at least one second primer are a forward primer and at least one reverse primer, respectively, or the first primer and the at least one second primer are a reverse primer and at least one forward primer, respectively. The primer set according to claim 17 , wherein c1 has a sequence different from that of c2.

19. The primer set according to claim 17, wherein the joint sequence c1-d is more GC-rich and / or contains more stable bases than the joint sequence da, and the joint sequence c2-da is more GC-rich and / or contains more stable bases than the joint sequence dab.

20. The primer set of claim 17, wherein the primer set is capable of increasing the expression of the target gene by at least 4 fold in the exponential phase of amplification. 循环数 Amplify the target nucleic acid at a rate of 21. The primer set of claim 17, wherein the primer set allows detection of a single copy nucleic acid in a biological sample within 25%-55% fewer amplification cycles than required for the detection using only a single forward primer and a single reverse primer.

22. The primer set according to claim 17, wherein the joint sequence da contains more unstable bases than the joint sequence c1-d, and the joint sequence dab contains more unstable bases than the joint sequence c2-da.

23. The primer set of claim 1 or 17, or the method of claim 2 or 3, wherein at least one pair of modified bases comprises modified forms of adenine and thymine.

24. The primer set or method according to claim 23, wherein the modified forms of adenine and thymine are 2-aminoadenine and 2-thiothymine, respectively.

25. The primer set of claim 1 or 17, or the method of claim 2 or 3, wherein at least one pair of modified bases comprises modified forms of guanine and cytosine.

26. The primer set or method of claim 25, wherein the modified form of guanine comprises deoxyinosine, 7-alkyl-7-deazaguanine, 2'-hypoxanthine or 7-nitro-7-deazahypoxanthine, and the modified form of cytosine comprises 3-(2'-deoxy-β-D-ribofuranosyl)pyrrolo-[2,3-d]-pyrimidin-2-(3H)-one, N4-alkylcytosine or 2-thiocytosine.

27. The primer set of claim 1 or 17, or the method of claim 2 or 3, wherein one or more of the primer sequences comprising modified bases comprises at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 modified bases.

28. The primer set according to claim 1 or 17, or the method according to claim 2 or 3, wherein the primer set comprises a probe or the method uses a probe.

29. The primer set of claim 1 or 17, or the method of claim 2 or 3, wherein the primer set comprises a probe or the method uses a probe comprising one or more modified bases, wherein the modified bases preferentially pair with unmodified bases.

30. The primer set of claim 1 or 17, or the method of claim 2 or 3, wherein the modified complementary bases form fewer hydrogen bonds with each other than unmodified complementary bases.

31. The primer set or method of claim 30, wherein the T of the base pair formed between the modified complementary bases is m Below 40℃.

32. The primer set according to claim 17, wherein the second primer comprises an oligonucleotide in the form of at least three second primers or capable of forming at least three second primers, the second primer being capable of hybridizing with the second template strand, wherein the at least three second primers comprise a second outer primer, a second middle primer and a second inner primer, The second outer primer comprises a primer sequence h, which specifically hybridizes with the second template strand sequence h', and the primer sequence h includes one or more fifth modified bases; The second intermediate primer comprises a single-stranded primer sequence e and a double-stranded primer sequence, wherein the single-stranded primer sequence e specifically hybridizes to the second template strand sequence e', wherein e' is adjacent to h' and is 5' to h', and the single-stranded primer sequence e comprises one or more sixth modified bases, wherein the double-stranded primer sequence comprises a first strand and a second strand, wherein the first strand comprises: a primer sequence h, which is adjacent to and 5' of the single-stranded primer sequence e; and a clamping sequence g1, which is adjacent to the primer sequence h and is 5' to the primer sequence h, wherein the clamping sequence g1 is not complementary to the second template strand sequence j', which is adjacent to the second template strand sequence h' and is 3' to the second template strand sequence h'; wherein the second strand comprises a primer sequence g1′ adjacent to and 3′ of the primer sequence h′, wherein the union sequence g1′-h′ is complementary to the union sequence g1-h, and the primer sequence h′ comprises one or more seventh modified bases; wherein the single-stranded primer sequence e is connected at its 5′ end to the first strand of the double-stranded primer sequence; The second inner primer comprises a single-stranded primer sequence f and a double-stranded primer sequence, wherein the single-stranded primer sequence f specifically hybridizes to the second template strand sequence f', wherein sequence f' is adjacent to e' and is 5' to e', and wherein the double-stranded primer sequence comprises a first strand and a second strand, wherein the first strand comprises: a primer sequence e, which is adjacent to and 5' of the single-stranded primer sequence f; a primer sequence h, which is adjacent to and 5' of primer sequence e; and a clamping sequence g2, which is adjacent to the primer sequence h and is 5' to the primer sequence h, wherein the clamping sequence g2 is not complementary to the second template strand sequence j'; wherein the second strand comprises a primer sequence g2' adjacent to and 3' of primer sequence h', wherein sequence h' is adjacent to and 3' of primer sequence e', wherein primer sequence e' comprises one or more eighth modified bases, wherein the union sequence g2'-h'-e' is complementary to the union sequence g2-he; wherein the single-stranded primer sequence f is linked at its 5′ end to the first strand of the double-stranded primer sequence; wherein the unmodified forms of the fifth and seventh modified bases are complementary, and the fifth and seventh modified bases preferentially pair with the unmodified forms as compared to pairing between the fifth and seventh modified bases; and wherein the unmodified forms of the sixth and eighth modified bases are complementary, and the sixth and eighth modified bases preferentially pair with the unmodified forms as compared to pairing between the sixth and eighth modified bases, and wherein the T of the combined sequence g1-h in the double-stranded form m T higher than the combined sequence he in double-stranded form m , and the combined sequence g2-he T in the double-stranded form m Higher than the T of the combined sequence hef in double-stranded form m , and the clamping sequences g1 and g2 cannot be copied during amplification.

33. The primer set according to claim 32, wherein the combined sequence g1-h is richer in GC and / or contains more stable bases than the combined sequence he, and the combined sequence g2-he is richer in GC and / or contains more stable bases than the combined sequence hef.

34. The primer set of claim 32, wherein the primer set is capable of increasing the expression of the target gene by at least 8 循环数 Amplify the target nucleic acid at a rate of 35. The primer set of claim 32, wherein amplification allows detection of a single copy nucleic acid in a biological sample within 42%-72% fewer amplification cycles than required for said detection using only a single forward primer and a single reverse primer.

36. The primer set of claim 32, wherein the clamping sequences c1 and c2 and g1 and g2 comprise 2'-O-methyl RNA.

37. The primer set according to claim 32, wherein The double-stranded primer sequences of the first inner primer and the first intermediate primer; and / or the second inner primer and the second intermediate primer do not contain a hairpin sequence.

38. The primer set of claim 32, wherein: The double-stranded primer sequence of the first inner primer comprises a hairpin sequence in which the clamping sequence c2 is connected to the complementary sequence c2'; and / or The double-stranded primer sequence of the first intermediate primer comprises a hairpin sequence in which the clamping sequence c1 is connected to the complementary sequence c1'; and / or The double-stranded primer sequence of the second inner primer comprises a hairpin sequence in which the clamping sequence g2 is connected to the complementary sequence g2'; and / or The double-stranded primer sequence of the second intermediate primer comprises a hairpin sequence in which the clamping sequence g1 is connected to the complementary sequence g1'.

39. The primer set of claim 32, wherein the joint sequence he contains more unstable bases than the joint sequence g1-h, and the joint sequence hef contains more unstable bases than the joint sequence g2-he.

40. The primer set of claim 32, wherein at least one modified base is identical to at least one other modified base.

41. A method for amplifying a target nucleic acid in a sample, wherein the target nucleic acid comprises a first template strand and optionally a second template strand, wherein the second template strand is complementary to the first template strand, the method comprising: (a) contacting the sample with: (i) at least three first primers capable of hybridizing to the first template strand, wherein the at least three first primers include a first outer primer, a first middle primer and a first inner primer, The first outer primer comprises a primer sequence d, which specifically hybridizes with the first template strand sequence d', and the primer sequence d comprises one or more first modified bases; The first intermediate primer comprises a single-stranded primer sequence a and a double-stranded primer sequence, wherein the single-stranded primer sequence a specifically hybridizes to the first template strand sequence a', wherein a' is adjacent to d' and is 5' to d', and the single-stranded primer sequence a includes one or more second modified bases, wherein the double-stranded primer sequence comprises a first strand and a second strand, wherein the first strand comprises: a primer sequence d, which is adjacent to and 5' of the single-stranded primer sequence a; and a clamping sequence c1, which is adjacent to and 5' of the primer sequence d, wherein the clamping sequence c1 is not complementary to a first template strand sequence i', which is adjacent to and 3' of the first template strand sequence d'; wherein the second strand comprises a primer sequence c1′, which is adjacent to and 3′ of the primer sequence d′, wherein the union sequence c1′-d′ is complementary to the union sequence c1-d, and the primer sequence d′ comprises one or more third modified bases; wherein the single-stranded primer sequence a is linked at its 5' end to the first strand of the double-stranded primer sequence; and The first inner primer comprises a single-stranded primer sequence b and a double-stranded primer sequence, wherein the single-stranded primer sequence b specifically hybridizes to the first template strand sequence b', wherein b' is adjacent to a' and is 5' to a', and wherein the double-stranded primer sequence comprises a first strand and a second strand, wherein the first strand comprises: a primer sequence a, which is adjacent to and 5' of the single-stranded primer sequence b; a primer sequence d, which is adjacent to and 5' of primer sequence a; and a clamping sequence c2, which is adjacent to the primer sequence d and is 5' to the primer sequence d, wherein the clamping sequence c2 is not complementary to the first strand template sequence i'; wherein the second strand comprises a primer sequence c2' adjacent to and 3' of primer sequence d', wherein sequence d' is adjacent to and 3' of primer sequence a', wherein primer sequence a' comprises one or more fourth modified bases, wherein the combined sequence c2'-d'-a' is complementary to the combined sequence c2-da, wherein the single-stranded primer sequence b is linked at its 5' end to the first strand of the double-stranded primer sequence; and wherein unmodified forms of the first and third modified bases are complementary, and the first and third modified bases preferentially pair with the unmodified forms as compared to pairing between the first and third modified bases; and wherein the unmodified forms of the second and fourth modified bases are complementary, and the second and fourth modified bases preferentially pair with the unmodified forms over pairing between the second and fourth modified bases; and (ii) at least one second primer capable of specifically hybridizing to the second template strand, wherein the contacting is performed under conditions wherein the primers are able to anneal to their template strands; and (b) subjecting the sample and primers to appropriate amplification conditions using a DNA polymerase lacking 5'-3' exonuclease activity, wherein strand displacement occurs to produce an amplicon comprising a sequence extending from the template sequence a' to the second primer binding site, and wherein the T of the combined sequence c1-d in the double-stranded form m T higher than the combined sequence da of the double-stranded form m , and the combined sequence of the double-stranded form is c2-da T m Higher than the T of the combined sequence dab in double-stranded form m , and the clamping sequences c1 and c2 cannot be copied during amplification.

42. The method of claim 41, wherein the DNA polymerase is stable above 85 degrees.

43. The method of any one of claims 41-42, wherein the amount of time required to complete each amplification cycle is reduced by at least 10-95% compared to the time for each cycle of the same primer set not including the modified base.

44. The method of claim 43, wherein the amount of time required to complete each amplification cycle is reduced by 50-85% compared to the time per cycle for the same primer set not including the modified base.

45. The method of claim 2, 3, 41 or 42, wherein the amplification comprises PCR.

46. ​​The method of claim 2, 3, 41 or 42, wherein the method comprises detecting and optionally quantifying the target nucleic acid.

47. The method of claim 2, 3, 41 or 42, wherein the sample consists of nucleic acid from a single cell.

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