Exponential base-x amplification with hairpin nested primers and universal flanking primers

Abstract

Described herein are methods and compositions that provide highly efficient nucleic acid amplification. In some embodiments, this allows a 3-fold or greater increase of amplification product for each amplification cycle and therefore increased sensitivity and speed over conventional PCR. Modified bases can be employed in primers to provide this base-3 or greater amplification with satisfactory PCR cycle times, which are improved, as compared to those observed in the absence of modified bases.

Description

[0001] Attorney Docket No.: 6010-0013WO01 EXPONENTIAL BASE-X AMPLIFICATION WITH HAIRPIN NESTED PRIMERS AND UNIVERSAL FLANKING PRIMERS SPECIFICATION This application claims benefit of U.S. Serial Number 63 / 673,257, filed July 19, 2024, which is incorporated herein by reference in its entirety. INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A TEXT FILE A Sequence Listing is provided herewith as file, “60100013PV01_Seq_Listing_ST26_07192024.xml” created on 07 / 19 / 2024 and having a size of 20 kilobytes. The contents of the text file are incorporated by reference herein in their entirety. STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT Not applicable. FIELD The methods and compositions described herein relate generally to the area of nucleic acid amplification. In particular, described herein are methods and compositions for increasing amplification efficiency. BACKGROUND A wide variety of nucleic acid amplification methods are available, and many have been employed in the implementation of sensitive diagnostic assays based on nucleic acid detection. Polymerase chain reaction (PCR) remains the most widely used DNA amplification and quantitation method. Nested PCR, a two-stage PCR, is used to increase the specificity and sensitivity of the PCR (U.S. Pat. No.4,683,195). Nested primers for use in the PCR amplification are oligonucleotides having a sequence complementary to a region on a target sequence between reverse and forward primer targeting sites. However, PCR in general has several limitations. PCR amplification can only achieve less than two-fold increase of the amount of target sequence at each cycle. It is still relatively slow. In addition, the sensitivity of this method is typically limited, making it difficult to detect target that may be present at only a few molecules in a single reaction. BRIEF SUMMARY Attorney Docket No.: 6010-0013WO01 Various embodiments contemplated herein may include, but need not be limited to, one or more of the following: 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, optionally, a second template strand, wherein the second template strand is complementary to the first template strand, the primer set comprising oligonucleotides in the form of, or capable of forming, 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 comprising outer primer sequence a that specifically hybridizes to first template strand sequence a′, outer primer sequence a optionally comprising at least one first modified base; and the first inner primer comprising a single-stranded primer sequence b that specifically hybridizes to first template strand sequence b′, wherein b′ is adjacent to, and 5′ of, a′, and wherein single-stranded primer sequence b is linked at its 5′ end to a double-stranded hairpin primer sequence comprising: primer sequence a adjacent to, and 5′ of, single-stranded primer sequence b; and a spacer sequence which is adjacent to, and 5' of primer sequence a; a primer sequence a', which is adjacent to, and 5' of the spacer sequence, wherein primer sequence a' is complementary to primer sequence a, wherein primer sequence a′ comprises at least one second modified base. Embodiment 2. 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, optionally, a second template strand, wherein the second template strand is complementary to the first template strand, the primer set comprising oligonucleotides in the form of, or capable of forming, 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 comprising outer primer sequence e that is capable of specifically hybridizing to the first inner primer sequence e′, wherein outer primer sequence e comprises universal flanking primer sequence e, further wherein outer primer sequence e optionally comprises at least one first modified base; and the first inner primer comprising a single-stranded primer sequence b that specifically hybridizes to first template strand sequence b′, wherein b′ is adjacent to, and 5′ of, a′, and wherein single-stranded primer sequence b is linked at its 5′ end to a double-stranded hairpin primer sequence comprising: primer sequence e adjacent to, and 5′ of, single-stranded primer sequence b; and a spacer sequence which is adjacent to, and 5' of primer sequence e; a primer sequence e', which is adjacent to, and Attorney Docket No.: 6010-0013WO01 5' of the spacer sequence, wherein primer sequence e' is complementary to primer sequence e, wherein primer sequence e′ comprises at least one second modified base. Embodiment 3. The nucleic acid primer set of embodiment 2, wherein the universal flanking primer sequence is from about 12 to about 50 bases in length. Embodiment 4. The nucleic acid primer set of any one of embodiments 2 to 3, wherein the universal flanking primer sequence has no significant homology to any segment in the template strand. Embodiment 5. The nucleic acid primer set of any one of embodiments 2 to 4, wherein the universal flanking primer sequence lacks secondary structure. Embodiment 6. The nucleic acid primer set of any one of embodiments 2 to 5, wherein the universal flanking primer sequence is optimized for Tm. Embodiment 7. The nucleic acid primer set of any one of embodiments 2 to 6, wherein the universal flanking primer sequence is optimized for GC content. Embodiment 8. The nucleic acid primer set of any one of embodiments 2 to 7, wherein the universal flanking primer sequence comprises a sequence that is at least 90% identical or complementary to at least 12 contiguous nucleotides of ATGTCCGCCTACTTTA (SEQ ID NO: 1). Embodiment 9. The nucleic acid primer set of any one of embodiments 1 to 8, wherein the spacer sequence comprises an alkylene or a heteroalkylene group. Embodiment 10. The nucleic acid primer set of any one of embodiments 1 to 9, wherein the spacer sequence comprises a C10 to C30 alkylene or a heteroalkylene group. Embodiment 11. The nucleic acid primer set of any one of embodiments 1 to 10, wherein the hairpin primer sequence is double stranded and allows the outer primer to hybridize with the template. Embodiment 12. The nucleic acid primer set of any one of embodiments 1 to 11, wherein the hairpin primer sequence is double stranded and allows the universal flanking primer to hybridize to the amplicon. Embodiment 13. The nucleic acid primer set of any one of embodiments 1 to 12, wherein each of the first inner primer sequences a′ and e′ comprises at least one first modified base. Embodiment 14. The nucleic acid primer set of any one of embodiments 1 to 13, wherein the at least one first modified base is a stabilizing base. Attorney Docket No.: 6010-0013WO01 Embodiment 15. The nucleic acid primer set of any one of embodiments 1 to 14, wherein the at least one first modified base is a stabilizing base selected from the group consisting of Locked nucleic acids (LNAs), unlocked nucleic acids (UNAs), AP-dC (G-clamp), 2-aminoadenine, 5- methylcytosine, C(5)-propynylcytosine, C(5)-propynyluracil, 2-aminoadenine, 2-thiothymine, deoxyinosine, 7-alkyl-7-deazaguanine, 2′-hypoxanthine, or 7-nitro-7-deazahypoxanthine, 3-(2′- deoxy-beta-D-ribofuranosyl)pyrrolo-[2,3-d]-pyrimidine-2-(3H)-one, N4-alkylcytosine, 2- thiocytosine, and combinations thereof. Embodiment 16. The nucleic acid primer set of any one of embodiments 1 to 15, wherein the at least one first modified base is a stabilizing base and provides added stability to the primer, thereby facilitating the primer’s access to a target sequence. Embodiment 17. The nucleic acid primer set of any one of embodiments 1 to 16, wherein the at least one first modified base is a stabilizing base, wherein this added stability gives the primer access to a target sequence and wherein the added stability does not prevent displacement of the hairpin sequence by a DNA polymerase and / or copying of the flanking primer region. Embodiment 18. The nucleic acid primer set of any one of embodiments 1 to 17, wherein the at least one second modified base is a stabilizing base. Embodiment 19. The nucleic acid primer set of any one of embodiments 1 to 18, wherein the at least one second modified base which is a stabilizing base is selected from the group consisting of Locked nucleic acids (LNAs), unlocked nucleic acids (UNAs), AP-dC (G-clamp), 2- aminoadenine, 5-methylcytosine, C(5)-propynylcytosine, C(5)-propynyluracil, 2-aminoadenine, 2-thiothymine, deoxyinosine, 7-alkyl-7-deazaguanine, 2′-hypoxanthine, or 7-nitro-7- deazahypoxanthine, 3-(2′-deoxy-beta-D-ribofuranosyl)pyrrolo-[2,3-d]-pyrimidine-2-(3H)-one, N4-alkylcytosine, 2-thiocytosine, and combinations thereof. Embodiment 20. The nucleic acid primer set of any one of embodiments 1 to 19, wherein the at least one second modified base which is a stabilizing base provides added stability to the primer, thereby facilitating the primer’s access to a target sequence. Embodiment 21. The nucleic acid primer set of any one of embodiments 1 to 20, wherein the at least one second modified base which is a stabilizing base, wherein this added stability gives the primer access to a target sequence and wherein the added stability does not prevent displacement of the hairpin sequence by a DNA polymerase and / or copying of the flanking primer region. Attorney Docket No.: 6010-0013WO01 Embodiment 22. The nucleic acid primer set of any one of embodiments 1 to 21, wherein each of the outer primer sequence a and the universal flanking primer sequence e comprises at least one second modified base which is stabilizing base. Embodiment 23. The nucleic acid primer set of any one of embodiments 1 to 22, wherein at least one second modified base which is a stabilizing base selected from the group consisting of Locked nucleic acids (LNAs), unlocked nucleic acids (UNAs), AP-dC (G-clamp), 2-aminoadenine, 5- methylcytosine, C(5)-propynylcytosine, C(5)-propynyluracil, 2-aminoadenine, 2-thiothymine, deoxyinosine, 7-alkyl-7-deazaguanine, 2′-hypoxanthine, or 7-nitro-7-deazahypoxanthine, 3-(2′- deoxy-beta-D-ribofuranosyl)pyrrolo-[2,3-d]-pyrimidine-2-(3H)-one, N4-alkylcytosine, 2- thiocytosine, and combinations thereof. Embodiment 24. The nucleic acid primer set of any one of embodiments 1 to 23, 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, as compared to pairing between the first and second modified bases. Embodiment 25. The nucleic acid primer set of any one of embodiments 1 to 24, wherein the primer set is capable of amplifying the target nucleic acid at the rate of at least 3number of cycles during an exponential phase of amplification. Embodiment 26. The nucleic acid primer set of any one of embodiments 1 to 25, wherein the primer set permits detection of a single-copy nucleic acid in a biological sample within about 12%-42% fewer amplification cycles than would be required for said detection using only a single forward and a single reverse primer. Embodiment 27. The nucleic acid primer set of any one of embodiments 1 to 26, wherein the primer set additionally comprises at least one second primer capable of specifically hybridizing to the second template strand. Embodiment 28. The nucleic acid primer set of embodiment 27, wherein the second primer comprises oligonucleotides in the form of, or capable of forming, at least two second primers 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 comprising a primer sequence f that specifically hybridizes to second template strand sequence f′, primer sequence f optionally comprising at least one first modified base(s); and the second inner primer comprising a single-stranded primer sequence g that specifically hybridizes to second template Attorney Docket No.: 6010-0013WO01 strand sequence g′, wherein g′ is adjacent to, and 5′ of, f′, and wherein single-stranded primer sequence g is linked at its 5′ end to a double-stranded hairpin primer sequence comprising: a primer sequence f adjacent to, and 5′ of, single-stranded primer sequence g; and a spacer sequence which is adjacent to, and 5' of primer sequence f; a primer sequence f' which is adjacent to, and 5' of the spacer sequence, wherein primer sequence f' is complementary to primer sequence f, wherein primer sequence f′ comprises at least one second modified base. Embodiment 29. The nucleic acid primer set of any one of embodiments 27 to 28, wherein the second primer comprises oligonucleotides in the form of, or capable of forming, at least two second primers 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 comprising an outer primer sequence j that is capable of specifically hybridizing to primer sequence j′, wherein outer primer sequence j comprises a universal flanking primer sequence j, further wherein outer primer sequence j optionally comprises at least one first modified base; and the second inner primer comprising a single-stranded primer sequence g that specifically hybridizes to second template strand sequence g′, wherein g′ is adjacent to, and 5′ of, f′, and wherein single-stranded primer sequence g is linked at its 5′ end to a double-stranded hairpin primer sequence comprising: a primer sequence j adjacent to, and 5′ of, single-stranded primer sequence g; and a spacer sequence which is adjacent to, and 5' of primer sequence j; a primer sequence j' which is adjacent to, and 5' of the spacer sequence, wherein primer sequence j' is complementary to primer sequence j, wherein primer sequence j′ comprises at least one second modified base. Embodiment 30. The nucleic acid primer set of any one of embodiments 27 to 29, wherein the universal flanking primer sequence e and the universal flanking primer sequence j are the same. Embodiment 31. The nucleic acid primer set of any one of embodiments 27 to 30, wherein universal flanking primer sequence e is from about 12 to about 50 bases in length, and universal flanking primer sequence j is from about 12 to about 50 bases in length. Embodiment 32. The nucleic acid primer set of any one of embodiments 27 to 31, wherein the universal flanking primer sequence has no significant homology to any segment in the template strand. Embodiment 33. The nucleic acid primer set of any one of embodiments 27 to 32, wherein the universal flanking primer sequence lacks secondary structure. Attorney Docket No.: 6010-0013WO01 Embodiment 34. The nucleic acid primer set of any one of embodiments 27 to 33, wherein the universal flanking primer sequence is optimized for Tm. Embodiment 35. The nucleic acid primer set of any one of embodiments 27 to 34, wherein the universal flanking primer sequence is optimized for GC content. Embodiment 36. The nucleic acid primer set of any one of embodiments 27 to 3528, wherein the universal flanking primer sequence e comprises a sequence that is at least 90% identical or complementary to at least 12 contiguous nucleotides of ATGTCCGCCTACTTTA (SEQ ID NO: 1). Embodiment 37. The nucleic acid primer set of any one of embodiments 27 to 36, wherein the universal flanking primer sequence j comprises a sequence that is at least 90% identical or complementary to at least 12 contiguous nucleotides of ATGTCCGCCTACTTTA (SEQ ID NO 1). Embodiment 38. The nucleic acid primer set of any one of embodiments 27 to 37, wherein the spacer sequence comprises an alkylene or a heteroalkylene group. Embodiment 39. The nucleic acid primer set of any one of embodiments 27 to 38, wherein the spacer sequence comprises a C10 to C30 alkylene or a heteroalkylene group. Embodiment 40. The nucleic acid primer set of any one of embodiments 27 to 39, wherein the hairpin primer sequence is double stranded and allows the outer primer to hybridize with the template. Embodiment 41. The nucleic acid primer set of any one of embodiments 27 to 40, wherein the hairpin primer sequence is double stranded and allows the universal flanking primer to hybridize to the amplicon. Embodiment 42. The nucleic acid primer set of any one of embodiments 27 to 41, wherein each of the first inner primer sequences a′ and e′ comprises at least one first modified base. Embodiment 43. The nucleic acid primer set of any one of embodiments 27 to 42, wherein the at least one first modified base is a stabilizing base. Embodiment 44. The nucleic acid primer set of any one of embodiments 27 to 43, wherein the at least one modified base which is a stabilizing base selected from the group consisting of Locked nucleic acids (LNAs), unlocked nucleic acids (UNAs), AP-dC (G-clamp), 2-aminoadenine, 5- methylcytosine, C(5)-propynylcytosine, C(5)-propynyluracil, 2-aminoadenine, 2-thiothymine, deoxyinosine, 7-alkyl-7-deazaguanine, 2′-hypoxanthine, or 7-nitro-7-deazahypoxanthine, 3-(2′- Attorney Docket No.: 6010-0013WO01 deoxy-beta-D-ribofuranosyl)pyrrolo-[2,3-d]-pyrimidine-2-(3H)-one, N4-alkylcytosine, 2- thiocytosine, and combinations thereof. Embodiment 45. The nucleic acid primer set of any one of embodiments 27 to 44, wherein the at least one first modified base which is a stabilizing base provides added stability to the primer, thereby facilitating the primer’s access to a target sequence. Embodiment 46. The nucleic acid primer set of any one of embodiments 27 to 45, wherein the at least one first modified base which is a stabilizing base, wherein this added stability gives the primer access to a target sequence and wherein the added stability does not prevent displacement of the hairpin sequence by a DNA polymerase and / or copying of the flanking primer region. Embodiment 47. The nucleic acid primer set of any one of embodiments 27 to 46, wherein each of the first inner primer sequences f′ and j′ comprises at least one first modified base. Embodiment 48. The nucleic acid primer set of any one of embodiments 27 to 47, wherein the at least one second modified base is a stabilizing base. Embodiment 49. The nucleic acid primer set of any one of embodiments 27 to 48, wherein the at least one second modified base which is a stabilizing base selected from the group consisting of Locked nucleic acids (LNAs), unlocked nucleic acids (UNAs), AP-dC (G-clamp), 2- aminoadenine, 5-methylcytosine, C(5)-propynylcytosine, C(5)-propynyluracil, 2-aminoadenine, 2-thiothymine, deoxyinosine, 7-alkyl-7-deazaguanine, 2′-hypoxanthine, or 7-nitro-7- deazahypoxanthine, 3-(2′-deoxy-beta-D-ribofuranosyl)pyrrolo-[2,3-d]-pyrimidine-2-(3H)-one, N4-alkylcytosine, 2-thiocytosine, and combinations thereof. Embodiment 50. The nucleic acid primer set of any one of embodiments 27 to 49, wherein the at least one second modified base which is a stabilizing base provides added stability to the primer, thereby facilitating the primer’s access to a target sequence. Embodiment 51. The nucleic acid primer set of any one of embodiments 27 to 50, wherein the at least one second modified base which is a stabilizing base, wherein this added stability gives the primer access to a target sequence and wherein the added stability does not prevent displacement of the hairpin sequence by a DNA polymerase and / or copying of the flanking primer region. Embodiment 52. The nucleic acid primer set of any one of embodiments 27 to 51, 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, as compared to pairing between the first and second modified bases. Attorney Docket No.: 6010-0013WO01 Embodiment 53. The nucleic acid primer set of any one of embodiments 27 to 52, wherein the primer set is capable of amplifying the target nucleic acid at the rate of at least 6number of cycles during an exponential phase of amplification. Embodiment 54. The nucleic acid primer set of any one of embodiments 27 to 53, wherein the primer set permits detection of a single-copy nucleic acid in a biological sample within about 36%-66% fewer amplification cycles than would be required for said detection using only a single forward and a single reverse primer. Embodiment 55. The nucleic acid primer set of any one of embodiments 27 to 54, wherein the primer set is free of GC-rich regions. Embodiment 56. A kit comprising the nucleic acid primer set of any one of embodiments 27 to 55, an amplification buffer comprising a DNA polymerase lacking 5′-3′ exonuclease activity, a mixture of nucleotides, and optionally polyethylene glycol (PEG) 8000. Embodiment 57. 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) adding the sample to a polymerase chain reaction (PCR) reaction mixture comprising: (i) a primer set comprising oligonucleotides in the form of, or capable of forming, 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 comprising outer primer sequence a that specifically hybridizes to first template strand sequence a′, outer primer sequence a optionally comprising at least one first modified base; and the first inner primer comprising a single-stranded primer sequence b that specifically hybridizes to first template strand sequence b′, wherein b′ is adjacent to, and 5′ of, a′, and wherein single-stranded primer sequence b is linked at its 5′ end to a double-stranded hairpin primer sequence comprising: primer sequence a adjacent to, and 5′ of, single-stranded primer sequence b; and a spacer sequence which is adjacent to, and 5' of primer sequence a; a primer sequence a', which is adjacent to, and 5' of the spacer sequence, wherein primer sequence a' is complementary to primer sequence a, wherein primer sequence a′ comprises at least one second modified base, wherein the hybridization is carried out under conditions wherein the primers anneal to their template strands, if present; and (ii) an amplification buffer comprising a DNA polymerase lacking 5′-3′ exonuclease activity, and a mixture of nucleotides; (b) amplifying the target nucleic acid, if present, under conditions where Attorney Docket No.: 6010-0013WO01 strand displacement occurs, thereby producing amplicons that include the sequences of all primers employed in the amplification reaction. Embodiment 58. The method of embodiment 57 wherein said PCR reaction mixture comprises polyethylene glycol (PEG). Embodiment 59. 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) adding the sample to a polymerase chain reaction (PCR) reaction mixture comprising: (i) a primer set comprising oligonucleotides in the form of, or capable of forming, 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 comprising outer primer sequence e that is capable of specifically hybridizing to the first inner primer sequence e′, wherein outer primer sequence e comprises a universal flanking primer sequence e, further wherein outer primer sequence e optionally comprises at least one first modified base; the first inner primer comprising a single-stranded primer sequence b that specifically hybridizes to first template strand sequence b′, wherein b′ is adjacent to, and 5′ of, a′, and wherein single-stranded primer sequence b is linked at its 5′ end to a double-stranded hairpin primer sequence comprising: primer sequence e adjacent to, and 5′ of, single-stranded primer sequence b; and a spacer sequence which is adjacent to, and 5' of primer sequence e; a primer sequence e', which is adjacent to, and 5' of the spacer sequence, wherein primer sequence e' is complementary to primer sequence e, wherein primer sequence e′ comprises at least one second modified base; and (ii) an amplification buffer comprising a DNA polymerase lacking 5′-3′ exonuclease activity, and a mixture of nucleotides, (b) amplifying the target nucleic acid, if present, under conditions where strand displacement occurs, thereby producing amplicons that include the sequences of all primers employed in the amplification reaction. Embodiment 60. The method of claim 59 wherein said PCR reaction mixture comprises polyethylene glycol (PEG). Embodiment 61. The method of any one of embodiments 59 to 60, wherein the universal flanking primer sequence is from about 12 to about 50 bases in length. Embodiment 62. The method of any one of embodiments 59 to 61, wherein the universal flanking primer sequence has no significant homology to any segment in the template strand. Attorney Docket No.: 6010-0013WO01 Embodiment 63. The method of any one of embodiments 59 to 62, wherein the universal flanking primer sequence lacks secondary structure. Embodiment 64. The method of any one of embodiments 59 to 63, wherein the universal flanking primer sequence is optimized for Tm. Embodiment 65. The method of any one of embodiments 59 to 64, wherein the universal flanking primer sequence is optimized for GC content. Embodiment 66. The method of any one of embodiments 59 to 65, wherein the universal flanking primer sequence has no significant homology to any segment in the template strand. Embodiment 67. The method of any one of embodiments 59 to 66, wherein the universal flanking primer sequence comprises a sequence that is at least 90% identical or complementary to at least 12 contiguous nucleotides of ATGTCCGCCTACTTTA (SEQ ID NO: 1). Embodiment 68. The method of any one of embodiments 57 to 67, wherein the spacer sequence comprises an alkylene or a heteroalkylene group. Embodiment 69. The method of any one of embodiments 57 to 68, wherein the spacer sequence comprises a C10 to C30 alkylene or a heteroalkylene group. Embodiment 70. The method of any one of embodiments 57 to 69, wherein the hairpin primer sequence is double stranded and allows the outer primer to hybridize with the template. Embodiment 71. The method of any one of embodiments 57 to 70, wherein the hairpin primer sequence is double stranded and allows the universal flanking primer to hybridize to the amplicon. Embodiment 72. The method of any one of embodiments 57 to 71, wherein each of the first inner primer sequences a′ and e′ comprises at least one first modified base. Embodiment 73. The method of any one of embodiments 57 to 72, wherein the at least one first modified base is a stabilizing base. Embodiment 74. The method of any one of embodiments 57 to 73, wherein the at least one first modified base which is a stabilizing base is selected from the group consisting of Locked nucleic acids (LNAs), unlocked nucleic acids (UNAs), AP-dC (G-clamp), 2-aminoadenine, 5- methylcytosine, C(5)-propynylcytosine, C(5)-propynyluracil, 2-aminoadenine, 2-thiothymine, deoxyinosine, 7-alkyl-7-deazaguanine, 2′-hypoxanthine, or 7-nitro-7-deazahypoxanthine, 3-(2′- deoxy-beta-D-ribofuranosyl)pyrrolo-[2,3-d]-pyrimidine-2-(3H)-one, N4-alkylcytosine, 2- thiocytosine, and combinations thereof. Attorney Docket No.: 6010-0013WO01 Embodiment 75. The method of any one of embodiments 57 to 74, wherein the at least one first modified base which is a stabilizing base provides added stability to the primer, thereby facilitating the primer’s access to a target sequence. Embodiment 76. The method of any one of embodiments 57 to 75, wherein the at least one first modified base which is a stabilizing base, wherein this added stability gives the primer access to a target sequence and wherein the added stability does not prevent displacement of the hairpin sequence by a DNA polymerase and / or copying of the flanking primer region. Embodiment 77. The method of any one of embodiments 57 to 76, wherein the at least one second modified base is a stabilizing base. Embodiment 78. The method of any one of embodiments 57 to 77, wherein the at least one second modified base which is a stabilizing base is selected from the group consisting of Locked nucleic acids (LNAs), unlocked nucleic acids (UNAs), AP-dC (G-clamp), 2-aminoadenine, 5- methylcytosine, C(5)-propynylcytosine, C(5)-propynyluracil, 2-aminoadenine, 2-thiothymine, deoxyinosine, 7-alkyl-7-deazaguanine, 2′-hypoxanthine, or 7-nitro-7-deazahypoxanthine, 3-(2′- deoxy-beta-D-ribofuranosyl)pyrrolo-[2,3-d]-pyrimidine-2-(3H)-one, N4-alkylcytosine, 2- thiocytosine, and combinations thereof. Embodiment 79. The method of any one of embodiments 57 to 78, wherein the at least one second modified base which is a stabilizing base provides added stability to the primer, thereby facilitating the primer’s access to a target sequence. Embodiment 80. The method of any one of embodiments 57 to 79, wherein the at least one second modified base which is a stabilizing base, wherein this added stability gives the primer access to a target sequence and wherein the added stability does not prevent displacement of the hairpin sequence by a DNA polymerase and / or copying of the flanking primer region. Embodiment 81. The method of any one of embodiments 57 to 80, wherein the universal flanking primer sequence e is optimized for primer-artifact reduction. Embodiment 82. The method of any one of embodiments 57 to 81, wherein each of the outer primer sequence a and the universal flanking primer sequence e comprises at least one second modified base which is stabilizing base. Embodiment 83. The method of any one of embodiments 57 to 82, wherein at least one second modified base which is a stabilizing base selected from the group consisting of Locked nucleic acids (LNAs), unlocked nucleic acids (UNAs), AP-dC (G-clamp), 2-aminoadenine, 5- Attorney Docket No.: 6010-0013WO01 methylcytosine, C(5)-propynylcytosine, C(5)-propynyluracil, 2-aminoadenine, 2-thiothymine, deoxyinosine, 7-alkyl-7-deazaguanine, 2′-hypoxanthine, or 7-nitro-7-deazahypoxanthine, 3-(2′- deoxy-beta-D-ribofuranosyl)pyrrolo-[2,3-d]-pyrimidine-2-(3H)-one, N4-alkylcytosine, 2- thiocytosine, and combinations thereof. Embodiment 84. The method of any one of embodiments 57 to 83, 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, as compared to pairing between the first and second modified bases. Embodiment 85. The method of any one of embodiments 57 to 84, wherein the primer set is capable of amplifying the target nucleic acid at the rate of at least 3number of cycles during an exponential phase of amplification. Embodiment 86. The method of any one of embodiments 57 to 85, wherein the primer set permits detection of a single-copy nucleic acid in a biological sample within about 12%-42% fewer amplification cycles than would be required for said detection using only a single forward and a single reverse primer. Embodiment 87. The method of any one of embodiments 57 to 86, wherein the primer set additionally comprises at least one second primer capable of specifically hybridizing to the second template strand. Embodiment 88. The method of embodiment 87 wherein the second primer comprises oligonucleotides in the form of, or capable of forming, at least two second primers 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 comprising a primer sequence f that specifically hybridizes to second template strand sequence f′, primer sequence f optionally comprising at least one first modified base; and the second inner primer comprising a single-stranded primer sequence g that specifically hybridizes to second template strand sequence g′, wherein g′ is adjacent to, and 5′ of, f′, and wherein single-stranded primer sequence g is linked at its 5′ end to a double-stranded hairpin primer sequence comprising: a primer sequence f adjacent to, and 5′ of, single-stranded primer sequence g; and a spacer sequence which is adjacent to, and 5' of primer sequence f; a primer sequence f' which is adjacent to, and 5' of the spacer sequence, wherein primer sequence f' is complementary to primer sequence f, wherein primer sequence f′ comprises at least one second modified base, wherein the hybridization is carried out Attorney Docket No.: 6010-0013WO01 under conditions wherein the primers anneal to their template strands, if present; and (ii) an amplification buffer comprising a DNA polymerase lacking 5′-3′ exonuclease activity, and a mixture of nucleotides; (b) amplifying the target nucleic acid, if present, under conditions where strand displacement occurs, thereby producing amplicons that include the sequences of all primers employed in the amplification reaction. Embodiment 89. The method of any one of embodiments 59 to 88 wherein the second primer comprises oligonucleotides in the form of, or capable of forming, at least two second primers 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 comprising an outer primer sequence j that is capable of specifically hybridizing to primer sequence j′, wherein outer primer sequence j comprises a universal flanking primer sequence j, further wherein outer primer sequence j optionally comprises at least one first modified base, the second inner primer comprising a single-stranded primer sequence g that specifically hybridizes to second template strand sequence g′, wherein g′ is adjacent to, and 5′ of, f′, and wherein single-stranded primer sequence g is linked at its 5′ end to a double-stranded hairpin primer sequence comprising: a primer sequence j adjacent to, and 5′ of, single-stranded primer sequence g; and a spacer sequence which is adjacent to, and 5' of primer sequence j; a primer sequence j' which is adjacent to, and 5' of the spacer sequence, wherein primer sequence j' is complementary to primer sequence j, wherein primer sequence j′ comprises at least one modified base, wherein the hybridization is carried out under conditions wherein the primers anneal to their template strands, if present; and (ii) an amplification buffer comprising a DNA polymerase lacking 5′-3′ exonuclease activity, and a mixture of nucleotides; (b) amplifying the target nucleic acid, if present, under conditions where strand displacement occurs, thereby producing amplicons that include the sequences of all primers employed in the amplification reaction. Embodiment 90. The method of any one of embodiments 59 to 89, wherein the spacer universal flanking primer sequence e and the universal flanking primer sequence j are the same. Embodiment 91. The method of any one of embodiments 59 to 90, wherein universal flanking primer sequence e is from about 12 to about 50 bases in length, and universal flanking primer sequence j is from about 12 to about 50 bases in length. Embodiment 92. The method of any one of embodiments 59 to 91, wherein the universal flanking primer sequence has no significant homology to any segment in the template strand. Attorney Docket No.: 6010-0013WO01 Embodiment 93. The method of any one of embodiments 59 to, wherein the universal flanking primer sequence lacks secondary structure. Embodiment 94. The method of any one of embodiments 59 to 93, wherein the universal flanking primer sequence is optimized for Tm. Embodiment 95. The method of any one of embodiments 59 to 94, wherein the universal flanking primer sequence is optimized for GC content. Embodiment 96. The method of any one of embodiments 59 to 95, wherein the universal flanking primer sequence e comprises a sequence that is at least 90% identical or complementary to at least 12 contiguous nucleotides of ATGTCCGCCTACTTTA (SEQ ID NO: 1). Embodiment 97. The method of any one of embodiments 59 to 96, wherein the universal flanking primer sequence j comprises a sequence that is at least 90% identical or complementary to at least 12 contiguous nucleotides of ATGTCCGCCTACTTTA (SEQ ID NO: 1). Embodiment 98. The method of any one of embodiments 59 to 97, wherein the spacer sequence comprises an alkylene or a heteroalkylene group. Embodiment 99. The method of any one of embodiments 59 to 98, wherein the spacer sequence comprises a C10 to C30 alkylene or a heteroalkylene group. Embodiment 100. The method of any one of embodiments 59 to 991 to 6, wherein the hairpin primer sequence is double stranded and allows the outer primer to hybridize with the template. Embodiment 101. The method of any one of embodiments 59 to 100, wherein the hairpin primer sequence is double stranded and allows the universal flanking primer to hybridize to the amplicon. Embodiment 102. The method of any one of embodiments 59 to 101, wherein each of the first inner primer sequences a′ and e′ comprises at least one first modified base. Embodiment 103. The method of any one of embodiments 59 to 102, wherein the at least one first modified base is a stabilizing base. Embodiment 104. The method of any one of embodiments 59 to 103, wherein the at least one modified base which is a stabilizing base selected from the group consisting of Locked nucleic acids (LNAs), unlocked nucleic acids (UNAs), AP-dC (G-clamp), 2-aminoadenine, 5- methylcytosine, C(5)-propynylcytosine, C(5)-propynyluracil, 2-aminoadenine, 2-thiothymine, deoxyinosine, 7-alkyl-7-deazaguanine, 2′-hypoxanthine, or 7-nitro-7-deazahypoxanthine, 3-(2′- deoxy-beta-D-ribofuranosyl)pyrrolo-[2,3-d]-pyrimidine-2-(3H)-one, N4-alkylcytosine, 2- thiocytosine, and combinations thereof. Attorney Docket No.: 6010-0013WO01 Embodiment 105. The method of any one of embodiments 59 to 104, wherein the at least one first modified base which is a stabilizing base provides added stability to the primer, thereby facilitating the primer’s access to a target sequence. Embodiment 106. The method of any one of embodiments 59 to 105, wherein the at least one first modified base which is a stabilizing base, wherein this added stability gives the primer access to a target sequence and wherein the added stability does not prevent displacement of the hairpin sequence by a DNA polymerase and / or copying of the flanking primer region. Embodiment 107. The method of any one of embodiments 59 to 106, wherein each of the first inner primer sequences a′ and e′ comprises at least one first modified base. Embodiment 108. The method of any one of embodiments 59 to 107, wherein the at least one second modified base is a stabilizing base. Embodiment 109. The method of any one of embodiments 59 to 108, wherein the at least one second modified base which is a stabilizing base selected from the group consisting of Locked nucleic acids (LNAs), unlocked nucleic acids (UNAs), AP-dC (G-clamp), 2-aminoadenine, 5- methylcytosine, C(5)-propynylcytosine, C(5)-propynyluracil, 2-aminoadenine, 2-thiothymine, deoxyinosine, 7-alkyl-7-deazaguanine, 2′-hypoxanthine, or 7-nitro-7-deazahypoxanthine, 3-(2′- deoxy-beta-D-ribofuranosyl)pyrrolo-[2,3-d]-pyrimidine-2-(3H)-one, N4-alkylcytosine, 2- thiocytosine, and combinations thereof. Embodiment 110. The method of any one of embodiments 59 to 109, wherein the at least one second modified base which is a stabilizing base provides added stability to the primer, thereby facilitating the primer’s access to a target sequence. Embodiment 111. The method of any one of embodiments 59 to 110, wherein the at least one second modified base which is a stabilizing base, wherein this added stability gives the primer access to a target sequence and wherein the added stability does not prevent displacement of the hairpin sequence by a DNA polymerase and / or copying of the flanking primer region. Embodiment 112. The method of any one of embodiments 59 to 111, wherein the universal flanking primer sequence e is optimized for primer-artifact reduction. Embodiment 113. The method of any one of embodiments 59 to 113, wherein the universal flanking primer sequence has no significant homology to any segment in the template strand. Embodiment 114. The method of any one of embodiments 59 to 113, wherein the universal flanking primer sequence lacks secondary structure. Attorney Docket No.: 6010-0013WO01 Embodiment 115. The method of any one of embodiments 59 to 114, wherein the universal flanking primer sequence is optimized for Tm. Embodiment 116. The method of any one of embodiments 59 to 115, wherein the universal flanking primer sequence is optimized for GC content. Embodiment 117. The method of any one of embodiments 59 to 116, wherein the universal flanking primer sequence comprises a sequence that is at least 90% identical or complementary to at least 12 contiguous nucleotides of ATGTCCGCCTACTTTA (SEQ ID NO: 1). Embodiment 118. The method of any one of embodiments 59 to 117, 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, as compared to pairing between the first and second modified bases. Embodiment 119. The method of any one of embodiments 59 to 118, wherein the primer set is capable of amplifying the target nucleic acid at the rate of at least 6number of cycles during an exponential phase of amplification. Embodiment 120. The method of any one of embodiments 59 to 119, wherein the primer set permits detection of a single-copy nucleic acid in a biological sample within about 36%-66% fewer amplification cycles than would be required for said detection using only a single forward and a single reverse primer. Embodiment 121. The method of any one of embodiments 59 to 120, wherein the primer set is free of GC-rich regions. Embodiment 122. The method of any one of embodiments 59 to 121, wherein the primer set is capable of amplifying the target nucleic acid at the rate of at least 6number of cycles during an exponential phase of amplification. Embodiment 123. The method of any one of embodiments 59 to 122wherein said PCR reaction mixture comprises polyethylene glycol (PEG). Embodiment 124. The method of any one of embodiments 59 to 123, wherein said PCR reaction mixture comprises PEG at a concentration of at least about 2 percent. Embodiment 125. The method of any one of embodiments 59 to 124, wherein said PCR reaction mixture comprises PEG 8000 at a concentration of at least about 2 percent. Embodiment 126. The method of any one of embodiments 59 to 125, wherein the primer set permits detection of a single-copy nucleic acid in a biological sample within about 36%-66% Attorney Docket No.: 6010-0013WO01 fewer amplification cycles than would be required for said detection using only a single forward and a single reverse primer. BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS The invention will be described in conjunction with the following drawings in which like reference numerals designate like elements and wherein: Fig. 1 is a schematic drawing showing fully nested PCR being carried out on a double- stranded DNA template. The flanking primers are as described in Fig.2 and Fig.4. Fig.2 is a schematic drawing showing an illustrative two-primer set hybridized to one end of a target nucleotide sequence. This set can be, e.g., a forward primer set. Different segments of primer sequence are shown (a, a', b); target sequences are indicated as (a′, b′). Template sequences are indicated 3′-5′ 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 (a-spacer-a'). Fig.3 is a schematic drawing showing an exemplary amplification reaction. The left-hand side shows a forward primer set wherein the outer primer sequence is shown e; different segments of inner primer sequence are shown (b, e, e'); template sequences are indicated 3′-5′ as a′, and b′. The outer primer e is single-stranded. The inner primer has a single stranded portion b and a double-stranded portion (e-spacer-e'). The right-hand side of the figure shows the result after one round of amplification. The amplicon sequences are indicated 5' - 3' as e'-b', i.e., sequence e' has been incorporated into the amplicon. In certain embodiments, primer sequence e may be a universal flanking primer sequence. Fig. 4 is a schematic drawing showing an illustrative two-primer set hybridized to the opposite end of a target nucleotide sequence from that shown in Fig. 2. This set can be, e.g., a reverse primer set. Different segments of primer sequence are shown (g, f, f'); complementary sequences are indicated as (f′, g′). Template sequences are indicated 3′-5′ as h′, f′, and g′. The outer primer f is single-stranded. The inner primer has a single stranded portion g and a double- stranded portion (f-spacer-f'). Fig.5 is a schematic drawing showing an exemplary amplification reaction. The left-hand side shows a reverse primer set wherein the outer primer sequence is shown j; different segments of inner primer sequence are shown (g, j, j'); template sequences are indicated 3′-5′ as f′, and g′. The outer primer j is single-stranded. The inner primer has a single stranded portion g and a double-stranded portion (j-spacer-j'). The right-hand side of the figure shows the result after one Attorney Docket No.: 6010-0013WO01 round of amplification. The amplicon sequences are indicated 5' - 3' as j'-g', i.e., sequence j' has been incorporated into the amplicon. In certain embodiments, primer sequence j may be a universal flanking primer sequence. Fig. 6 is a schematic drawing showing an exemplary inner primer comprising a hairpin loop sequence and stabilized bases. Fig. 7 is a chart showing that stabilized hairpin primers have equivalent performance to GC- clamped primers in base 3 PCR. Fig.8 is a schematic drawing showing a universal, non-complementary flanking sequence that is incorporated into an amplicon after a first PCR cycle. Fig.9 is an example of impact of varying percentages of PEG 8000 on different types of nucleic acid amplification: base-3, base-3 and base-6. PEG 8000 had the greatest effect on base- 6 PCR. (See Example 3). Fig.10 is a chart showing real-time PCR fluorescence growth curves generated by varying PEG 8000 concentration, TaqG46E vs. a polymerase, Flu A target. Fig. 11 is a chart showing the impact on threshold of varying PEG8000 concentration, TaqG46E, Flu A target. Fig.12 is a chart showing the impact on end point fluorescence of varying PEG8000 vs. a polymerase, Flu A target. Fig. 13 is a chart showing is a chart showing real-time PCR fluorescence growth curves generated in the presence of 10% PEG with varying molecular weight N2 target. Fig. 14 is a chart showing is a chart showing real-time PCR fluorescence growth curves generated in the presence of 10% PEG by varying molecular weight E target. Fig.15 is a chart showing the impact on N2 cycle threshold in the presence of 10% PEG of varying molecular weight. Fig.16 is a chart showing the impact on E cycle threshold in the presence of 10% PEG of varying molecular weight. DETAILED DESCRIPTION Terms used in the claims and specification are defined as set forth below unless otherwise specified. The term “nucleic acid” refers to a nucleotide polymer, and unless otherwise limited, includes analogs of natural nucleotides that can function in a similar manner (e.g., hybridize) to Attorney Docket No.: 6010-0013WO01 naturally occurring nucleotides. The term “nucleic acid” encompasses multi-stranded, as well as single-stranded molecules. In double- or triple-stranded nucleic acids, the nucleic acid strands need not be coextensive (i.e., a double-stranded nucleic acid need not be double-stranded along the entire length of both strands). Nucleic acid templates described herein may be any size depending on the sample (from small cell-free DNA fragments to entire genomes), including but not limited to 50-300 bases, 100-2000 bases, 100-750 bases, 170-500 bases, 100-5000 bases, 50- 10,000 bases, or 50-2000 bases in length. In some instances, templates are at least 50, 100, 200, 500, 1000, 2000, 5000, 10,000, 20,00050,000, 100,000, 200,000, 500,000, 1,000,000 or more than 1,000,000 bases in length. Methods described herein provide for the amplification of nucleic acids, such as nucleic acid templates. Methods described herein additionally provide for the generation of isolated and at least partially purified nucleic acids and libraries of nucleic acids. Nucleic acids include but are not limited to those comprising DNA, RNA, circular RNA, cfDNA (cell free DNA), cfRNA (cell free RNA), siRNA (small interfering RNA), cffDNA (cell free fetal DNA), mRNA, tRNA, rRNA, miRNA (microRNA), synthetic polynucleotides, polynucleotide analogues, any other nucleic acid consistent with the specification, or any combinations thereof. The length of polynucleotides, when provided, are described as the number of bases and abbreviated, such as nt (nucleotides), bp (bases), kb (kilobases), or Gb (gigabases). 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, usually obtained by reverse transcription of messenger RNA (mRNA) or by amplification; DNA molecules produced synthetically or by amplification; mRNA; and non-coding RNA. The term nucleic acid encompasses double- or triple-stranded nucleic acid complexes, as well as single-stranded molecules. In double- or triple-stranded nucleic acid complexes, the nucleic acid strands need not be coextensive (i.e, a double-stranded nucleic acid need not be double-stranded along the entire length of both strands). The term nucleic acid also encompasses any modifications thereof, such as by methylation and / or by capping. Nucleic acid modifications can include addition of chemical groups that incorporate additional charge, polarizability, hydrogen bonding, electrostatic interaction, and functionality to the individual nucleic acid bases or to the nucleic acid as a whole. Such modifications may include base modifications such as 2′-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at cytosine exocyclic Attorney Docket No.: 6010-0013WO01 amines, substitutions of 5-bromo-uracil, sugar-phosphate backbone modifications, unusual base pairing combinations such as the isobases isocytidine and isoguanidine, and the like. More particularly, in some embodiments, nucleic acids, can include polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), and any other type of nucleic acid that is an N- or C-glycoside of a purine or pyrimidine base, as well as other polymers containing nonnucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids (PNAs)) 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 providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is 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, which are incorporated herein by reference in their entirety for their disclosure of LNAs. The nucleic acid(s) can be derived from a completely chemical synthesis process, such as a solid phase-mediated chemical synthesis, from a biological source, such as through isolation from any species that produces nucleic acid, or from processes that involve the manipulation of nucleic acids by molecular biology tools, such as DNA replication, PCR amplification, reverse transcription, or from a combination of those processes. As used herein, the term “complementary” refers to the capacity for precise pairing between two nucleotides, i.e., if a nucleotide at a given position of a nucleic acid is capable of hydrogen bonding with a nucleotide of another nucleic acid to form a canonical base pair, then the two nucleic acids are considered to be complementary to one another at that position. Complementarity between two single-stranded nucleic acid molecules may be “partial,” in which only some of the nucleotides bind, or it may be complete when total complementarity exists between the single-stranded molecules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. “Specific hybridization” refers to the binding of a nucleic acid to a target nucleotide sequence in the absence of substantial binding to other nucleotide sequences present in the Attorney Docket No.: 6010-0013WO01 hybridization mixture under defined stringency conditions. Those of skill in the art recognize that relaxing the stringency of the hybridization conditions allows sequence mismatches to be tolerated. In some embodiments, hybridizations are carried out under stringent hybridization conditions. The phrase “stringent hybridization conditions” generally refers to a temperature in a range from about 5°C to about 20°C or 25°C below than the melting temperature (Tm) for a specific sequence at a defined ionic strength and pH. As used herein, the Tm is the temperature at which a population of double-stranded nucleic acid molecules becomes half-dissociated into single strands. Methods for calculating the Tmof nucleic acids are well known in the art (see, e.g., 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 incorporated herein by reference for their descriptions of stringent hybridization conditions). As indicated by standard references, a simple estimate of the Tmvalue may be calculated by the equation: Tm=81.5+0.41(% G+C), when a nucleic acid is in aqueous solution at 1 M NaCl (see, e.g., Anderson and Young, Quantitative Filter Hybridization in NUCLEIC ACID HYBRIDIZATION (1985)). The melting temperature of a hybrid (and thus the conditions for stringent hybridization) is affected by various factors such as the length and nature (DNA, RNA, base composition) of the primer or probe and nature of the target nucleic acid (DNA, RNA, base composition, present in solution or immobilized, and the like), 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 are discussed in standard references in the art. Illustrative 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 pH7. Tmcalculation for oligonucleotide sequences based on nearest-neighbors thermodynamics can be carried out as described in “A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics” John SantaLucia, Jr., PNAS Feb.17, 1998 vol.95 no.41460-1465 (which is incorporated by reference herein for this description). The term “oligonucleotide” is used to refer to a nucleic acid that is relatively short, generally shorter than 200 nucleotides, more particularly, shorter than 100 nucleotides, most Attorney Docket No.: 6010-0013WO01 particularly, shorter than 50 nucleotides. Typically, oligonucleotides are single-stranded DNA molecules. The term “primer” refers to an oligonucleotide that is capable of hybridizing (also termed “annealing”) with a nucleic acid and serving as an initiation site for nucleotide (RNA or DNA) polymerization under appropriate conditions (i.e., in the presence of four different nucleoside triphosphates and an agent for polymerization, such as DNA or RNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature. The appropriate length of a primer depends on the intended use of the primer, but primers are typically at least 7 nucleotides long and, in some embodiments, range from 10 to 30 nucleotides, or, in some embodiments, from 10 to 60 nucleotides, in length. In some embodiments, primers can be, e.g., 15 to 50 nucleotides long. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. A primer need not reflect the exact sequence of the template but must be sufficiently complementary to hybridize with a template. A primer is said to anneal to another nucleic acid if the primer, or a portion thereof, hybridizes to a nucleotide sequence within the nucleic acid. The statement that a primer hybridizes to a particular nucleotide sequence is not intended to imply that the primer hybridizes either completely or exclusively to that nucleotide sequence. For example, in some embodiments, amplification primers used herein are said to “anneal to” or be “specific for” a nucleotide sequence.” This description encompasses primers that anneal wholly to the nucleotide sequence, as well as primers that anneal partially to the nucleotide sequence. The term “primer pair” refers to a set of primers including a 5′ “upstream primer” or “forward primer” that hybridizes with the complement of the 5′ end of the DNA sequence to be amplified and a 3′ “downstream primer” or “reverse primer” that hybridizes with the 3′ end of the sequence to be amplified. As will be recognized by those of skill in the art, the terms “upstream” and “downstream” or “forward” and “reverse” are not intended to be limiting, but rather provide illustrative orientations in some embodiments. A “probe” is a nucleic acid capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, generally through complementary base pairing, usually through hydrogen bond formation, thus forming a duplex structure. The probe can be labeled with a detectable label to permit facile detection of the probe, particularly once the probe has hybridized to its complementary target. Alternatively, however, the probe may be Attorney Docket No.: 6010-0013WO01 unlabeled, but may be detectable by specific binding with a ligand that is labeled, either directly or indirectly. Probes can vary significantly in size. Generally, probes are at least 7 to 15 nucleotides in length. Other probes are at least 20, 30, or 40 nucleotides long. Still other probes are somewhat longer, being at least 50, 60, 70, 80, or 90 nucleotides long. Yet other probes are longer still, and are at least 100, 150, 200 or more nucleotides long. Probes can also be of any length that is within any range bounded by any of the above values (e.g., 15-20 nucleotides in length). The primer or probe can be perfectly complementary to the target nucleotide sequence or can be less than perfectly complementary. In some embodiments, the primer has at least 65% identity to the complement of the target nucleotide sequence over a sequence of at least 7 nucleotides, more typically over a sequence in the range of 10-30 nucleotides, and, in some embodiments, over a sequence of at least 14-25 nucleotides, and, 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 will be understood that certain bases (e.g., the 3′ base of a primer) are generally desirably perfectly complementary to corresponding bases of the target nucleotide sequence. Primer and probes typically anneal to the target sequence under stringent hybridization conditions. As used herein with reference to a portion of a primer or a nucleotide sequence within the primer, the term “specific for” a nucleic acid, refers to a primer or nucleotide sequence that can specifically anneal to the target nucleic acid under suitable annealing conditions. Amplification according to the present teachings encompasses any means by which at least a part of at least one target nucleic acid is reproduced, typically in a template-dependent manner, including without limitation, a broad range of techniques for amplifying nucleic acid sequences, either linearly or exponentially. Illustrative means for performing an amplifying step include PCR, nucleic acid strand-based amplification (NASBA), two-step multiplexed amplifications, rolling circle amplification (RCA), and the like, including multiplex versions and combinations thereof, for example 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), and the like. Descriptions of such techniques can be found in, among other sources, Ausubel et al.; PCR Primer: A Laboratory Manual, Diffenbach, Ed., Cold Spring Harbor Press (1995); The Electronic Protocol Book, Chang Bioscience (2002); Attorney Docket No.: 6010-0013WO01 Msuih et al., J. Clin. Micro.34:501-07 (1996); The Nucleic Acid Protocols Handbook, R. Rapley, ed., Humana Press, Totowa, N.J. (2002); Abramson et al., Curr Opin Biotechnol.1993 February; 4(1):41-7, U.S. Pat. Nos.6,027,998; 6,605,451, Barany et al., PCT Publication No. WO 97 / 31256; Wenz et al., PCT Publication No. WO 01 / 112579; Day et 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); and Rabenau et 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 at: 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., Proc Natl Acad Sci USA 99:5261-66 (2002); Barany and Gelfand, Gene 109:1-11 (1991); Walker et al., Nucl. Acid Res.20:1691-96 (1992); Polstra et al., BMC Inf. Dis. 2:18-(2002); Lage et al., Genome Res. 2003 February; 13(2):294-307, and Landegren et al., Science 241:1077-80 (1988), Demidov, V., Expert Rev Mol Diagn. 2002 November; 2(6):542- 8., Cook et al., J Microbiol Methods. 2003 May; 53(2):165-74, Schweitzer et al., Curr Opin Biotechnol. 2001 February; 12(1):21-7, U.S. Pat. Nos. 5,830,711, 6,027,889, 5,686,243, PCT Publication No. WO0056927A3, and PCT Publication No. WO9803673A1. In some embodiments, amplification comprises at least one cycle of the sequential procedures of: annealing at least one primer with complementary or substantially complementary sequences in at least one target nucleic acid; synthesizing at least one strand of nucleotides in a template-dependent manner using a polymerase; and denaturing the newly-formed nucleic acid duplex to separate the strands. The cycle may or may not be repeated. Amplification can comprise thermocycling or can be performed isothermally. As used herein, the term "microbead" refers to a bead having a diameter that is less than 1 mM (i.e., less than 1000 microns). Micro beads may be microscopic or near microscopic and may have diameters of about 0.005 to 100 μm, about 0.1 to 50 μm, or about 0.5 to 30 μm. “Nested amplification” refers the use of more than two primers to amplify a target nucleic acid. Attorney Docket No.: 6010-0013WO01 “Hemi-nested amplification” refers to the use of more than one primer (e.g., two or three) that anneal at one end of a target nucleotide sequence. “Fully nested amplification” refers to the use of more than one primer that anneal at each end of a target nucleotide sequence. With reference to nested amplification, the multiple primers that anneal at one end of an amplicon are differentiated by using the terms “inner,” “outer,” and “intermediate.” An “outer primer” refers to a primer that anneals to a sequence closer to the end of the target nucleotide sequence than another primer that anneals at that same end of the target nucleotide sequence. In some embodiments, the outer primer sequence defines the end of the amplicon produced from the target nucleic acid. The “outer primer” is also referred to herein as a “flanking primer.” An “inner primer” refers to a primer that anneals to a sequence closer to the middle of the target nucleotide sequence than another primer that anneals at that same end of the target nucleotide sequence. The term “intermediate primer” is used herein with reference to nest amplification in which at least three primers that anneal at one end of a target nucleotide sequence are used. An intermediate primer is one that anneals to a sequence in between an inner primer and an outer primer. As used herein, the term “adjacent to” is used to refer to sequences that are in sufficiently close proximity for the methods to work. In some embodiments, sequences that are adjacent to one another are immediately adjacent, with no intervening nucleotides. A “multiplex amplification reaction” is one in which two or more nucleic acids distinguishable by sequence are amplified simultaneously. The term “qPCR” is used herein to refer to 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. A “reagent” refers broadly to any agent used in a reaction, other than the analyte (e.g., nucleic acid being analyzed). Illustrative reagents for a nucleic acid amplification reaction include, but are not limited to, buffer, metal ions, polymerase, reverse transcriptase, primers, template nucleic acid, nucleotides, labels, dyes, nucleases, dNTPs, and the like. Reagents for Attorney Docket No.: 6010-0013WO01 enzyme reactions include, for example, substrates, cofactors, buffer, metal ions, inhibitors, and activators. The term “label,” as used herein, refers to any atom or molecule that can be used to provide a detectable and / or quantifiable signal. In particular, the label can be attached, directly or indirectly, to a nucleic acid or protein. Suitable labels that can be attached to probes include, but are not limited to, radioisotopes, fluorophores, chromophores, mass labels, electron dense particles, magnetic particles, spin labels, molecules that emit chemiluminescence, electrochemically active molecules, enzymes, cofactors, and enzyme substrates. As used herein, the term label refers to an agent capable of detection, for example by ELISA, spectrophotometry, flow cytometry, immunohistochemistry, immunofluorescence, microscopy, Northern analysis or Southern analysis. For example, a marker can be attached to a nucleic acid molecule or protein, thereby permitting detection of the nucleic acid molecule or protein. Examples of labels include, but are not limited to, radioactive isotopes, nitorimidazoles, enzyme substrates, co-factors, ligands, chemiluminescent agents, fluorophores, haptens, enzymes, and combinations thereof. Methods for labeling and guidance in the choice of markers appropriate for various purposes are discussed for example in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998). As used herein, the term Marker or Label is an agent capable of detection, for example by ELISA, spectrophotometry, flow cytometry, immunohistochemistry, immunofluorescence, microscopy, Northern analysis or Southern analysis. For example, a marker can be attached to a nucleic acid molecule or protein, thereby permitting detection of the nucleic acid molecule or protein. Examples of markers include, but are not limited to, radioactive isotopes, nitorimidazoles, enzyme substrates, co-factors, ligands, chemiluminescent agents, fluorophores, haptens, enzymes, and combinations thereof. Methods for labeling and guidance in the choice of markers appropriate for various purposes are discussed for example in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998). The term “dye,” as used herein, generally refers to any organic or inorganic molecule that absorbs electromagnetic radiation. Attorney Docket No.: 6010-0013WO01 The naturally occurring bases adenine, thymine, uracil, guanine, and cytosine, which make up DNA and RNA, are described herein as “unmodified bases” or “unmodified forms.” The term “modified base” is used herein to refer to a base that is not a canonical, naturally occurring base (e.g., adenine, cytosine, guanine, thymine, or uracil). Examples of modified bases are 2-thiothymine and 2-aminoadenine. Nucleotides comprising modified bases are referred to herein as “modified nucleotides.” 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. Oligonucleotides including one or more "modified nucleotides" (e.g., pseudo- complementary nucleotides) are referred to herein as pseudo-complementary oligonucleotides (e.g., pseudo-complementary blocker oligonucleotide). As used herein, the terms "subject" and "patient" are used interchangeably. As used herein, the term "patient" refers to an animal, preferably a mammal such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats etc.) and a primate (e.g., monkey and human), and most preferably a human. In some embodiments, the subject is a non-human animal such as a farm animal (e.g., a horse, pig, or cow) or a pet (e.g., a dog or cat). In a specific embodiment, the subject is an elderly human. In another embodiment, the subject is a human adult. In another embodiment, the subject is a human child. In yet another embodiment, the subject is a human infant. The patient or subject to be medicated according to the compositions and methods as disclosed herein may be any animal or human. In certain embodiments, animals may include vertebrates. The terms vertebrate or animals in this context is understood to comprise, for example fish, amphibians, reptiles, birds, and mammals including humans. One preferred group of vertebrates or animals according to the invention comprises warm-blooded animals including farm animals, such as cattle, horses, pigs, sheep and goats, poultry such as chickens, turkeys, guinea fowls and geese, fur-bearing animals such as mink, foxes, chinchillas, rabbits and the like, as well as companion animals such as ferrets, guinea pigs, rats, hamster, cats and dogs. A further group of preferred vertebrates or animals according to the disclosure comprises fish including salmonids, for example salmon, trout or whitefish. The subject is preferably mammalian. In some embodiments the subject is a human. In other embodiments the subject is an animal, more preferably a non-human mammal. The non- human mammal may be a domestic pet, or animal kept for commercial purposes, e.g., a racehorse, or farming livestock or animals such as pigs, sheep or cattle. As such the disclosure may have Attorney Docket No.: 6010-0013WO01 veterinary applications. Non-human mammals include rabbits, guinea pigs, rats, mice or other rodents (including any animal in the order Rodentia), cats, dogs, pigs, sheep, goats, cattle (including cows or any animal in the order Bos), horse (including any animal in the order Equidae), donkey, and non-human primates. The subject may be male or female. The subject may be an adult or a child. The subject may be a patient. As used herein, the term "about" when used in conjunction with a stated numerical value or range has the meaning reasonably ascribed to it by a person skilled in the art, i.e., denoting somewhat more or somewhat less than the stated value or range. When a group of substituents is disclosed herein, it is understood that all individual members of those groups and all subgroups and classes that can be formed using the substituents are disclosed separately. When a Markush group or other grouping is used herein, all individual members of the group and all combinations and subcombinations possible of the group are intended to be individually included in the disclosure. As used herein, “and / or” means that one, all, or any combination of items in a list separated by “and / or” are included in the list; for example, “1, 2 and / or 3” is equivalent to “1, 2, 3, 1 and 2, 1 and 3, 2 and 3, or 1, 2, and 3”. As used herein, “comprising” is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, “consisting of” excludes any element, step, or ingredient not specified in the claim element. As used herein, “consisting essentially of” does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Any recitation herein of the term “comprising”, particularly in a description of components of a composition, in a description of a method, or in a description of elements of a device, is understood to encompass those compositions, methods, or devices consisting essentially of and consisting of the recited components or elements, optionally in addition to other components or elements. The disclosure as illustratively described herein suitably may be practiced in the absence of any element, elements, limitation, or limitations which is not specifically disclosed herein. As used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a method” includes a plurality of such methods and reference to “the nanoparticle” includes reference to one or more nanoparticles and equivalents thereof known to those skilled in the art, Attorney Docket No.: 6010-0013WO01 and so forth. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope as disclosed herein claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims. All references throughout this application, for example patent documents, including issued or granted patents or equivalents and patent application publications, and non-patent literature documents or other source material are hereby incorporated by reference herein in their entireties, as though individually incorporated by reference. None is admitted to being prior art. When a group of substituents is disclosed herein, it is understood that all individual members of those groups and all subgroups and classes that can be formed using the substituents are disclosed separately. When a Markush group or other grouping is used herein, all individual members of the group and all combinations and sub-combinations possible of the group are intended to be individually included in the disclosure. As used herein, “and / or” means that one, all, or any combination of items in a list separated by “and / or” are included in the list; for example, “1, 2 and / or 3” is equivalent to “1, 2, 3, 1 and 2, 1 and 3, 2 and 3, or 1, 2, and 3”. The term “template” and variations thereof refer to a nucleic acid sequence that is a target of nucleic acid sequencing reactions. A template sequence can comprise a naturally-occurring or synthetic nucleic acid sequence. A template sequence also can include a known or unknown nucleic acid sequence from a sample of interest. In various exemplary embodiments herein, a template sequence can be attached to a solid support, such as, for example, a bead, microparticle, flow cell, or any other surface or object. As used herein, the term sample may be, for example, selected from tissue(s) samples, cells, biological fluid samples (e.g., blood, urine, saliva, lymphatic fluid, cerebrospinal fluid (CSF), amniotic fluid, pleural fluid, pericardial fluid, ascites, aqueous humor), bone marrow samples, semen samples, biopsy samples, cancer samples, tumor samples, cell lysate samples, forensic samples, archaeological samples, paleontological samples, infection samples, production samples, whole plants, plant parts, microbiota samples, viral preparations, soil samples, marine Attorney Docket No.: 6010-0013WO01 samples, freshwater samples, household or industrial samples, and combinations and isolates thereof. In one embodiment of the systems and methods as disclosed herein, the sample is a cell (e.g., an animal cell [e.g., a human cell], a plant cell, a fungal cell, a bacterial cell, and a protozoal cell). In one specific embodiment, the cell is lysed prior to the replication. In one specific embodiment, cell lysis is accompanied by proteolysis. In one specific embodiment, the cell is selected from a cell from a preimplantation embryo, a stem cell, a fetal cell, a tumor cell, a suspected cancer cell, a cancer cell, a cell subjected to a gene editing procedure, a cell from a pathogenic organism, a cell obtained from a forensic sample, a cell obtained from an archeological sample, and a cell obtained from a paleontological sample. In one embodiment of any of the systems and methods as disclosed herein, the sample is a cell from a preimplantation embryo (e.g., a blastomere. In one specific embodiment, the method further comprises determining the presence of disease predisposing germline or somatic variants in the embryo cell. In one embodiment of any of the systems and methods as disclosed herein, the sample is a cell from a pathogenic organism (e.g., a bacterium, a fungus, a protozoan). In one specific embodiment, the pathogenic organism cell is obtained from fluid taken from a patient, microbiota sample (e.g., GI microbiota sample, vaginal microbiota sample, skin microbiota sample, etc.) or an indwelling medical device (e.g., an intravenous catheter, a urethral catheter, a cerebrospinal shunt, a prosthetic valve, an artificial joint, an endotracheal tube, etc.). In one specific embodiment, the method further comprises the step of determining the identity of the pathogenic organism. In one specific embodiment, the method further comprises determining the presence of genetic variants responsible for resistance of the pathogenic organism to a treatment. In one embodiment of any of the systems and methods as disclosed herein, the sample is a tumor cell, a suspected cancer cell, or a cancer cell. In one specific embodiment, the method further comprises determining the presence of one or more diagnostic or prognostic mutations. In one specific embodiment, the method further comprises determining the presence of germline or somatic variants responsible for resistance to a treatment. In one embodiment of any of the systems and methods as disclosed herein, the sample is a cell subjected to a gene editing procedure. In one specific embodiment, the method further comprises determining the presence of unplanned mutations caused by the gene editing process. In one embodiment of any of the systems and methods as disclosed herein, the method further comprises determining the history of a cell lineage. In a related aspect, the Attorney Docket No.: 6010-0013WO01 invention provides a use of any of the systems and methods as disclosed herein for identifying low frequency sequence variants (e.g., variants which constitute ≥0.01% of the total sequences). The phrase “fragment library” refers to a collection of nucleic acid fragments, wherein one or more fragments are used as a sequencing template. A fragment library can be generated in numerous ways that are known in the art. As an example, a fragment library can be generated by cutting, shearing, restricting, or otherwise subdividing a larger nucleic acid into smaller fragments. Fragment libraries can be generated from naturally occurring nucleic acids, such as, for example, from bacteria, cancer cells, normal cells, or solid tissue. Libraries comprising synthetic nucleic acid sequences can also be generated to create a synthetic fragment library. The phrase “synthetic nucleic acid sequence” and variations thereof refers to a designed and synthesized sequence of nucleic acid. For example, a synthetic nucleic acid sequence can be designed to follow rules or guidelines. Every formulation or combination of components described or exemplified can be used to practice the invention, unless otherwise stated. Specific names of materials are intended to be exemplary, as it is known that one of ordinary skill in the art can name the same material differently. It will be appreciated that methods, device elements, starting materials, and synthetic methods other than those specifically exemplified can be employed in the practice of the invention without resort to undue experimentation. All art-known functional equivalents, of any such methods, device elements, starting materials, and synthetic methods are intended to be included in this invention. Whenever a range is given in the specification, for example, a temperature range, a time range, or a composition range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure. It is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither, or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included. Attorney Docket No.: 6010-0013WO01 General Approach for Increasing Amplification Efficiency U.S. Pat. No.8,252,558 and Harris et al., BioTechniques 54:93-97 (February 2013) teach a form of nested PCR, termed “Polymerase Chain Displacement Reaction” (PCDR) (both documents are incorporated by reference herein for this description). In PCDR, when extension occurs from an outer primer, it displaces the extension strand produced from an inner primer because the reaction employs a polymerase that has strand displacement activity. In theory, this allows a greater than 2-fold increase of amplification product for each amplification cycle and therefore increased sensitivity and speed over conventional PCR. In practice, every amplicon created from a nested primer no longer contains a primer annealing site for the outer primer. Accordingly, PCDR cannot sustain a greater than 2-fold increase of amplification product for each amplification cycle for very many cycles. For this reason, PCDR offers only modest reduction in the number of amplification cycles (e.g., from about 23 to about 20) needed to detect a target nucleic acid. By contrast, Table 1 below shows that a sustained quadrupling per cycle (4number of cycles) should halve the number of cycles needed to have the same amplification as a doubling per cycle. A sustained 6-fold replication per cycle should achieve in 15 cycles what would take 40 normal PCR cycles. TABLE 1 Degree of Amplification with Different "Bases" B Attorney Docket No.: 6010-0013WO01 17 131072 1.29E+08 1.72E+10 7.63E+11 1.69E+13 18 262144 3.87E+08 6.87E+10 3.81E+12 1.02E+14 In certain embodiments as disclosed herein, sustaining a greater than 2-fold increase of amplification product for each amplification cycle is provided by designing the inner (nested) primer so that the extension product of the inner (nested) primer contains the outer (flanking) primer sequence. Fig.1 shows a scheme in which fully nested PCR is carried out using a forward inner and outer primer and a reverse inner and outer primer. In this scheme the nested primer comprises a complementary primer which binds to the inner primer anneals to template, which may comprise GC-rich DNA to improve stability. However, a disadvantage of this scheme is the necessity of using excess, single-strand GC-rich DNA which may negatively impact the ability to multiplex, for example, in order to ensure there is no “unclamped” nested primer, 1.5 X excess “clamp” oligo is included in PCR. A solution to this problem is to use, for example, a stabilized hairpin primer as the nested (inner) primer. In certain embodiments, the hairpin loop sequence is within or adjacent to a spacer sequence. In certain embodiments, for example as shown in Fig.2, the nested primer comprises a single sequence which comprises a hairpin loop. In this embodiment, the nested primer Attorney Docket No.: 6010-0013WO01 comprises a'-spacer-a-b sequences. There are several advantages of using a nested primer containing a hairpin loop. For example, use of a nested primer containing a hairpin loop allows replacement by shorter hairpin that is more efficiently synthesized. In addition, there are fewer primers to design, for example, a hairpin primer is “one thing” to make and qualify as compared to two primers. In certain embodiments as disclosed herein, 2-thiodT is placed opposite to 2- aminoadenine in a corresponding complement sequence, for example, comprising the 5' end of a nested primer. Additionally, clamp to primer stoichiometry automatically maintained, i.e., there is no excess ss GC-rich DNA. Furthermore, long GC sequences more likely to interact w / non- homologous long GC sequences (each base 3 primer set requires its own GC clamp sequence) which may make higher order multiplexing difficult. A ~40 bp stretch of dsDNA in primers may inhibit DNA polymerase – the more “plex” the more the potential inhibition. As shown in Example 1, in various embodiments, the more stabilized hairpin primers have equivalent performance to GC-clamped primers in base 3 PCR. This avoids the necessity of using excess, single-strand GC-rich DNA which may negatively impact the ability to multiplex, for example, in order to ensure there is no “unclamped” nested primer, 1.5 X excess “clamp” oligo is included in PCR. In a further embodiment, the outer primer may comprise a universal, non-complimentary flanking sequence. Because the universal flanking primer is only complementary to a region of the inner primer (and not the template), the universal, non-complimentary flanking sequence would be incorporated into an amplicon, for example, after the 1st PCR cycle. (See Example 2; Fig.3). The term universal flanking primer used here refers to an oligonucleotide primer that has no significant homology to any segment in the template strand. The universal flanking primer preferably has all the requirements for a normal oligonucleotide primer, such as lack of secondary structure, an appropriate Tm, and an appropriate GC content. The incorporation of a universal flanking sequence has several advantages, for example, reducing the overall number of primers used for multiplexing reactions, since the same universal left and same universal right flanking primers can be used for all amplicons in the reaction system. Another advantage is that only two additional primers for each new amplicon are required instead of four. The use of universal flanking primers allows for selection of optimal flanking primer for Tm, secondary structure & primer-artifact reduction. Furthermore, use of universal flanking primers allows segregation of Attorney Docket No.: 6010-0013WO01 thio-dT away from bases requiring harsh de-protection (special A, C42). Gain of about a cycle made up for by reduced Ct at low target concentration due to specificity enhancement. Exemplary ways the universal flanking primer sequence could be optimized for Tm include primer length, base composition, selective base pair mismatches and by using modified bases with different base pair stabilities. Optimizing for secondary structure in this context usually means reducing secondary structure. The simplest way to reduce secondary structure is by selecting sequences (the UFP is an arbitrary sequence) that lack secondary structure. This can be determined by looking for internal base pairing (GC & AT base pairs, etc.) which can be most easily done by using available software programs that calculate the stability of secondary structures within oligo sequences. Less stability is better. A less easy way would be to use “pseudo-complementary” or self‐avoiding molecular recognition system (SAMRS) bases within the sequence to destabilize any internal base pairs. In certain embodiments as disclosed herein primers such as UFPs can be optimized for primer dimer reduction which may be done by choosing the two UFP sequences such that they 1) do not have 3’ “stable” matches to each other (“stable” is relative, but one could use “more than three base pair matches in a row” as a rule of thumb) and that they 2) do not have such 3’ matches to any of all the other oligos in the PCR. Certain embodiments as disclosed herein provide for the use of SAMRS bases to destabilize such matches between oligos. Samples Nucleic acid-containing samples can be obtained from biological sources and prepared using conventional methods known in the art. In particular, nucleic acids useful in the methods and systems as described herein can be obtained from any source, including unicellular organisms and higher organisms such as plants or non-human animals, e.g., canines, felines, equines, primates, and other non-human mammals, as well as humans. In some embodiments, samples may be obtained from an individual suspected of being, or known to be, infected with a pathogen, an individual suspected of having, or known to have, a disease, such as cancer, or a pregnant individual. Nucleic acids can be obtained from cells, bodily fluids (e.g., blood, a blood fraction, urine, etc.), or tissue samples by any of a variety of standard techniques. In some embodiments, the method employs samples of plasma, serum, spinal fluid, lymph fluid, peritoneal fluid, pleural Attorney Docket No.: 6010-0013WO01 fluid, oral fluid, and external sections of the skin; samples from the respiratory, intestinal genital, or urinary tracts; samples of tears, saliva, blood cells, stem cells, or tumors. Samples can be obtained from live or dead organisms or from in vitro cultures. Illustrative samples can include single cells, paraffin-embedded tissue samples, and needle biopsies. In some embodiments, the nucleic acids analyzed are obtained from a single cell. Nucleic acids of interest can be isolated using methods well known in the art. The sample nucleic acids need not be in pure form but are typically sufficiently pure to allow the steps of the methods described herein to be performed. Target Nucleic Acids 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 will be known for the target nucleic acids. For example, if the amplification reaction employed is PCR, sufficient sequence information is generally available for each end of a given target nucleic acid to permit design of suitable amplification primers. The targets can include, for example, nucleic acids associated with pathogens, such as viruses, bacteria, protozoa, or fungi; RNAs, e.g., those for which over- or under-expression is indicative of disease, those that are expressed in a tissue- or developmental-specific manner; or those that are induced by particular stimuli; genomic DNA, which can be analyzed for specific polymorphisms (such as SNPs), alleles, or haplotypes, e.g., in genotyping. Of particular interest are genomic DNAs that are altered (e.g., amplified, deleted, and / or mutated) in genetic diseases or other pathologies; sequences that are associated with desirable or undesirable traits; and / or sequences that uniquely identify an individual (e.g., in forensic or paternity determinations). Primer Design Primers suitable for nucleic acid amplification are sufficiently long to prime the synthesis of extension products in the presence of a suitable nucleic acid polymerase. The exact length and composition of the primer will depend on many factors, including, for example, temperature of the annealing reaction, source and composition of the primer, and where a probe is employed, proximity of the probe annealing site to the primer annealing site and ratio of primer:probe concentration. For example, depending on the complexity of the target nucleic acid sequence, an oligonucleotide primer typically contains in the range of about 10 to about 60 nucleotides, Attorney Docket No.: 6010-0013WO01 although it may contain more or fewer nucleotides. The primers should be sufficiently complementary to selectively anneal to their respective strands and form stable duplexes. In general, one skilled in the art knows how to design suitable primers capable of amplifying a 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, e.g., Rozen and Skaletsky (2000) Meth. Mol. Biol., 132: 365-386; www.broad.mit.edu / node / 1060, and the like) or by accessing the Roche UPL website. The amplicon sequences are input into the Primer3 program with the UPL probe sequences in brackets to ensure that the Primer3 program will design primers on either side of the bracketed probe sequence. Primers may be prepared by any suitable method, including, for example, direct chemical synthesis by methods such as 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 and the like or can be provided from a commercial source. Primers may be purified by using a Sephadex column (Amersham Biosciences, Inc., Piscataway, N.J.) or other methods known to those skilled in the art. Primer purification may improve the sensitivity of the methods described herein. Outer Primer FIG.2 shows how a two-primer set anneals to a first template strand at one end of a target nucleotide sequence. For exemplary purposes, this primer set can be considered to be a "forward" primer set. The outer primer includes a sequence a that specifically hybridizes to first template strand sequence a'. FIG.4 shows how a two-primer set anneals to a second template strand at the opposite end of the target nucleotide sequence. For exemplary purposes, this primer set can be considered to be a "reverse" primer set. Here, the outer primer includes a sequence f that specifically hybridizes to first template strand sequence f'. In general, the considerations for designing suitable outer primers do not differ from those for designing outer primers for use in conventional nested PCR. In certain embodiments, the outer primer may include at least one universal flanking primer sequence. Notably, in some embodiments, the Tmof any primer sequence in double strand form that is “outer” relative to another primer sequence (e.g., an inner or intermediate primer sequence) is preferably lower than the Tm of the inner (or intermediate) primer sequence in double strand form. Attorney Docket No.: 6010-0013WO01 Thus, for example, during the down temperature ramp of PCR, the inner primer can anneal and begin extension before the outer primer; otherwise premature extension of the outer primer may block the target site of the inner primer and prevent its annealing. For example, in the embodiment shown in Fig.2, primer sequence a would have a Tm less than that of primer sequence b. Similarly, in the embodiment shown in FIG. 4, primer sequence f would have a lower Tmthan primer sequence g. In some embodiments, the Tm differences are at least about 4 degrees, generally in the range of about 4 to about 20°C. In some embodiments, the Tm differences are in the range of about 4 to about 15°C. However, the Tmof the outer primer is generally high enough to maintain efficient PCR, e.g., in some embodiments, the Tmof the outer primer is at least 40°C. Tmcan be adjusted by adjusting the length of a sequence, the G-C content, and / or by including stabilizing or destabilizing base(s) in the sequence. Accordingly, in certain embodiments, the outer primer sequence a may optionally comprise at least one first modified base. "Stabilizing bases" include, e.g., stretches of peptide nucleic acids (PNAs) that can be incorporated into DNA oligonucleotides to increase duplex stability. Locked nucleic acids (LNAs) and unlocked nucleic acids (UNAs) are analogues of RNA that can be easily incorporated into DNA oligonucleotides during solid-phase oligonucleotide synthesis, and respectively increase and decrease duplex stability. Suitable stabilizing bases also include modified DNA bases that increase the stability of base pairs (and therefore the duplex as a whole). These modified bases can be incorporated into oligonucleotides during solid-phase synthesis and offer a more predictable method of increasing DNA duplex stability. Examples include AP-dC (G-clamp) and 2-aminoadenine, as well as 5-methylcytosine and C(5)-propynylcytosine (replacing cytosine), and C(5)-propynyluracil (replacing thymine). “Destabilizing bases” are those that destabilize double-stranded DNA by virtue of forming less stable base pairs than the typical A-T and / or G-C base pairs. Inosine (I) is a destabilizing base because it pairs with cytosine (C), but an I-C base pair is less stable than a G-C base pair. This lower stability results from the fact that inosine is a purine that can make only two hydrogen bonds, compared to the three hydrogen bonds of a G-C base pair. Other destabilizing bases are known to, or readily identified by, those of skill in the art. Some modified bases functions as both stabilizing and destabilizing bases. For example, the 2-thiothymine and 2-aminoadenine base pair is destabilizing, whereas the thymine and 2- aminoadenine or the 2-thiothymine and adenine base pairs are stabilizing. In exemplary Attorney Docket No.: 6010-0013WO01 embodiments as disclosed herein, for example referring to Fig.2, the outer primer sequence a comprises 2-aminoadenine and inner primer sequence a' comprises 2-thiothymine. This prevents or reduces hybridization between outer primer sequence a and inner primer sequence a'. In certain embodiments as disclosed herein, when outer primer sequence a, for example, hybridizes with the template, the presence of the modified 2'aminoadenine provides a stabilizing effect. In certain embodiments as disclosed herein, when inner primer sequence a' hybridizes with inner primer sequence a (no modifications), the presence of the modified 2'thiothymine provides a stabilizing effect. In certain embodiments as disclosed herein, 2-thiodT is placed opposite to 2-aminoadenine in a corresponding complement sequence, for example, comprising the 5' end of a nested primer. Inner Primer Referring to FIG. 2, the inner primer in a forward two-primer set includes a single- stranded primer sequence b that specifically hybridizes to first template strand sequence b′, wherein b′ is adjacent to, and 5′ of, a′, and wherein single-stranded primer sequence b is linked at its 5′ end to a double-stranded hairpin primer sequence comprising primer sequence a adjacent to, and 5′ of, single-stranded primer sequence b; and a spacer sequence which is adjacent to, and 5' of primer sequence a; a primer sequence a', which is adjacent to, and 5' of the spacer sequence, wherein primer sequence a' is complementary to primer sequence a. In certain embodiments, primer sequence a′ may comprise at least one modified base. In a further embodiment, the inner primer may comprise primer sequence a' adjacent to and 5' of the spacer sequence. In some embodiments, the Tm of combined sequence a-spacer-a' comprises a hairpin loop, wherein the hairpin loop comprises stabilizing bases. (See Fig. 6). "Stabilizing bases" include, e.g., stretches of peptide nucleic acids (PNAs) that can be incorporated into DNA oligonucleotides to increase duplex stability. Locked nucleic acids (LNAs) and unlocked nucleic acids (UNAs) are analogues of RNA that can be easily incorporated into DNA oligonucleotides during solid-phase oligonucleotide synthesis, and respectively increase and decrease duplex stability. Suitable stabilizing bases also include modified DNA bases that increase the stability of base pairs (and therefore the duplex as a whole). These modified bases can be incorporated into oligonucleotides during solid-phase synthesis and offer a more predictable method of increasing DNA duplex stability. Examples include AP-dC (G-clamp) and 2-aminoadenine, as well as 5-methylcytosine and C(5)-propynylcytosine (replacing cytosine), and C(5)-propynyluracil (replacing thymine). Attorney Docket No.: 6010-0013WO01 Alternatively, or in addition, combined sequence a-spacer-a' can be designed to include more destabilizing bases. “Destabilizing bases” are those that destabilize double-stranded DNA by virtue of forming less stable base pairs than the typical A-T and / or G-C base pairs. Inosine (I) is a destabilizing base because it pairs with cytosine (C), but an I-C base pair is less stable than a G-C base pair. This lower stability results from the fact that inosine is a purine that can make only two hydrogen bonds, compared to the three hydrogen bonds of a G-C base pair. Other destabilizing bases are known to, or readily identified by, those of skill in the art. The forward two-primer set can be employed with a simple conventional reverse primer for a hemi-nested amplification or with a reverse two-primer set. Referring to Fig.3, the primer set comprising oligonucleotides in the form of, or capable of forming, 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 comprising outer primer sequence e that is capable of specifically hybridizing to the first inner primer sequence e′, wherein outer primer sequence e comprises universal flanking primer sequence e, further wherein outer primer sequence e optionally comprises at least one first modified base; and the first inner primer comprising a single-stranded primer sequence b that specifically hybridizes to first template strand sequence b′, wherein b′ is adjacent to, and 5′ of, a′, and wherein single-stranded primer sequence b is linked at its 5′ end to a double-stranded hairpin primer sequence comprising: primer sequence e adjacent to, and 5′ of, single-stranded primer sequence b; and a spacer sequence which is adjacent to, and 5' of primer sequence e; a primer sequence e', which is adjacent to, and 5' of the spacer sequence, wherein primer sequence e' is complementary to primer sequence e, wherein primer sequence e′ comprises at least one second modified base. After the first round of amplification, the amplicon would comprise an amplicon sequence e', which is complementary to the universal flanking primer sequence comprised in outer primer sequence e, as well as template sequence b'. Referring to Fig.4 the second primer comprises oligonucleotides in the form of, or capable of forming, at least two second primers 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 comprising a primer sequence f that specifically hybridizes to second template strand sequence f′, primer sequence f optionally comprising at least one first modified base(s); and the second inner primer comprising a single-stranded primer sequence g that Attorney Docket No.: 6010-0013WO01 specifically hybridizes to second template strand sequence g′, wherein g′ is adjacent to, and 5′ of, f′, and wherein single-stranded primer sequence g is linked at its 5′ end to a double-stranded hairpin primer sequence comprising: a primer sequence f adjacent to, and 5′ of, single-stranded primer sequence g; and a spacer sequence which is adjacent to, and 5' of primer sequence f; a primer sequence f' which is adjacent to, and 5' of the spacer sequence, wherein primer sequence f' is complementary to primer sequence f, wherein primer sequence f′ comprises at least one second modified base. Referring to Fig.5 wherein the at least two second primers comprise a second outer primer and a second inner primer, the second outer primer comprising an outer primer sequence j that is capable of specifically hybridizing to primer sequence j′, wherein outer primer sequence j comprises a universal flanking primer sequence j, further wherein outer primer sequence j optionally comprises at least one first modified base; and the second inner primer comprising a single-stranded primer sequence g that specifically hybridizes to second template strand sequence g′, wherein g′ is adjacent to, and 5′ of, f′, and wherein single-stranded primer sequence g is linked at its 5′ end to a double-stranded hairpin primer sequence comprising: a primer sequence j adjacent to, and 5′ of, single-stranded primer sequence g; and a spacer sequence which is adjacent to, and 5' of primer sequence j; a primer sequence j' which is adjacent to, and 5' of the spacer sequence, wherein primer sequence j' is complementary to primer sequence j, wherein primer sequence j′ comprises at least one second modified base. In certain embodiments, the universal flanking primer sequence e and the universal flanking primer sequence j are the same. Modified Bases Modified bases useful in the primers and other oligonucleotides described herein include those wherein the modified base forms stable hydrogen-bonded base pairs with the natural complementary base but does not form stable hydrogen-bonded base pairs with its modified complementary base (e.g., pseudo-complementary bases). (For ease of discussion, complementary bases are also referred to herein as “partners.” Also, for ease of discussion, the following description relates to primers and primer pairs, but, as those of skill in the art, readily appreciate, this description also applies to the other oligonucleotides and oligonucleotide pairs described herein.) In some embodiments, this is accomplished when the modified base can form two or more hydrogen bonds with its natural partner, but only one or no hydrogen bonds with its modified partner. This allows the production of primer and other oligonucleotide pairs that do not Attorney Docket No.: 6010-0013WO01 form substantially stable hydrogen-bonded hybrids with one another, as manifested in a melting temperature (under physiological or substantially physiological conditions) of less than about 40° C. The primers of the primer pair, however, form substantially stable hybrids with the complementary nucleotide sequence in a template strand (e.g., first template strand) of a single- or double-stranded target nucleic acid and with a strand complementary to the template strand (e.g., second template strand). In some embodiments, due to the increased (in some embodiments, double) number of hydrogen bonds in such hybrids, the hybrids formed with the primers of the present invention are more stable than hybrids that would be formed using primers with unmodified bases. In accordance with well-established convention, the naturally occurring nucleotides of nucleic acids have the designation 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 otherwise requires, no distinction needs to be made in this description between A and dA, U and dT, etc. Analogs of A that are modified in the base portion to form a stable hydrogen-bonded pair with T, (or U in the case of RNA) but not with a modified T are designated A*. Analogs of T that are modified in the base portion to form a stable hydrogen-bonded pair with A, hut not with A* are designated T*. Analogs of G that are modified in the base portion to form a stable hydrogen- bonded pair with C, but not with a modified C are designated G*. Analogs of C that are modified in the base portion to form a stable hydrogen-bonded pair with G, but not with G* are designated C*, In some embodiments, the foregoing conditions are satisfied when each of the A*, T*, G*, and C* nucleotides (collectively, the modified nucleotides) form two or more hydrogen bonds with their natural partner, but only one or no hydrogen bonds with their modified partner. This is illustrated by Formulas 1a, 1b, 2a, 2b, 3a, 3b, 4a and 4b below (and in FIG.8A-8B), where the hydrogen bonding between natural A-T (or A-U in case of RNA) and G-C pairs, and hydrogen bonding between exemplary A*-T, T*-A, G*-C, C*-G, A*-T* and G*-C* pairs are illustrated. Attorney Docket No.: 6010-0013WO01 1b Attorney Docket No.: 6010-0013WO01 5

[0002] Attorney Docket No.: 6010-0013WO01 4b In general, a sufficient number of modified nucleotides are incorporated into the primers described herein to preferentially increase the annealing of the primers to the template strands of a target nucleic acid, as compared to primer-to-primer annealing. It is not necessary to replace each natural nucleotide of the primer with a modified nucleotide in order to accomplish this. In some embodiments, the primers include, in addition to one or more modified nucleotides, one or more naturally occurring nucleotides and / or variants of naturally occurring nucleotides, provided that the variations do not interfere significantly with the complementary binding ability of the primers, as discussed above. For example, primers including modified nucleotides can include pentofuranose moieties other than ribose or 2-deoxyribose, as well as derivatives of ribose and 2- deoxyribose, for example 3-amino-2-deoxyribose, 2-fluoro-2-deoxyribose, and 2—O—C1-6alkyl or 2-O-allyl ribose, particularly 2-O-methyl ribose. The glycosidic linkage can be in the α or β Attorney Docket No.: 6010-0013WO01 configuration. The phosphate backbone of the primer can, if desired, include phosphorothioate linkages. A general structure for a suitable class of the modified A analog, A*, shown as a 3′- phosphate (or phosphorothioate) incorporated into a primer, is provided by Formulas 5, 6, and 7, below, wherein: X is N or CH; Y is O or S; Z is OH or CH3; R is H, F, or OR2, where R2is C1-6alkyl or allyl, or H in case of RNA; and R1 is C1-4alkyl, C1-4 alkoxy, alkylthio, F, or NHR3, where R3 is H, or C1-4 alkyl. An illustrative embodiment of A* has 2,6-diaminopurine (2-aminoadenine) as the base, as shown in Formula 1b. The latter nucleotide can be abbreviated as 2-amA or d2-amA, as applicable. Attorney Docket No.: 6010-0013WO01

[0003] Attorney Docket No.: 6010-0013WO01 A general structure for a suitable class of the modified T analog, T*, shown as a 3′- phosphate (or phosphorothioate) incorporated into the primer, is provided by Formula 8, wherein: Y, Z, and R are defined as above; and R4 is H, C1-6 alkyl, C1-6 alkenyl, or C1-6 alkynyl. An illustrative embodiment of T* has 2- thio-4-oxo-5-methylpyrimidine (2-thiothymine) as the base, as shown in Formula 2b. The latter nucleotide can be abbreviated as 2-sT or d2-sT, as applicable.

[0004] Attorney Docket No.: 6010-0013WO01 A general structure for a suitable class of the modified G analog, G*, shown as a 3°- phosphate (or phosphorothioate) incorporated into the primer, is provided by Formulas 9, 10 and 11, wherein: R1is H, C1-4alkyl, C1-4alkoxy, C1-4alkylthio, F, or NHR3, where R3is defined as above; and X, Y, Z, and R are defined as above. An illustrative embodiment of G* has 6-oxo-purine (hypoxanthine) as the base, as shown in Formula 3b. The latter nucleotide can be abbreviated as I or dI, as applicable. Attorney Docket No.: 6010-0013WO01 5 Attorney Docket No.: 6010-0013WO01 A general structure for a suitable class of the modified C analog, C*, shown as a 3′- phosphate (or phosphorothioate) incorporated into the primer, is provided by Formulas 12 and 13, wherein: Y, Z, R, and R4are defined as above; Z1 is O or NH; and R5 is H or C1-4 alkyl. An illustrative embodiment of C* has pyrrolo-[2,3-d]pyrimidine- 2(3H)-one as the base, as shown in Formula 4b. The latter nucleotide can be abbreviated as P or dP, as applicable. The above-described modified bases and nucleotides are also described in U.S. Pat. No. 5,912,340 (issued Jun.15, 1999 to Kutyavin et al.), which is hereby incorporated by reference for this description. The hybridization properties of d2-amA and d2-sT are described in Kutyavin, et al. (1996) Biochemistry 35:11170-76, which is also hereby incorporated by reference for this description. The synthesis and hybridization properties of d / I and dP are described in Woo et al. (1996) Nucleic Acids Research 25(13):2470-75, which is also hereby incorporated by reference for this description. Additional examples of G* and C* include 7-alkyl-7-deazaguanine and N4-alkylcytosine (where alkyl methyl or ethyl), respectively, which are described in Lahoud et al. (2008) Nucleic Acids Research 36(10):3409-19 (hereby incorporated by reference for this description). Analogs tested in this study are shown in Formula 12.

[0005] Attorney Docket No.: 6010-0013WO01 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 (hereby incorporated by reference for this description). Hoshinka et al. (2010) Angew Chem Int Ed Engl.49(32):5554-5557 describes the use of such bases (“Self- Avoiding Molecular Recognition Systems”), including 2′-hypoxanthine as G* (this reference is hereby incorporated by reference for this description; see especially, FIG.1 ); see also Yang et al. (2015) Chembiochem. 16(9):1365-1367 (this reference is hereby incorporated by reference for this description; see especially, Scheme 1). The analogs tested in this study are shown in Formula 13.

[0006] Attorney Docket No.: 6010-0013WO01 For amplification in any of the methods described herein, primers and any other appropriate oligonucleotides are contacted with sample nucleic acids under conditions wherein the primers anneal to their template strands, if present. In some embodiments, the amplification step is performed using PCR. Illustrative PCR reaction mixtures generally contain an appropriate Attorney Docket No.: 6010-0013WO01 buffer, a source of magnesium ions (Mg2+) in the range of about 1 to about 10 mM, e.g., in the range of about 2 to about 8 mM, nucleotides, and optionally, detergents, and stabilizers. An example of one suitable buffer is TRIS buffer at a concentration of about 5 mM to about 85 mM, with a concentration of 10 mM to 30 mM preferred. In one embodiment, the TRIS buffer concentration is 20 mM in the reaction mix double-strength (2X) form. The reaction mix can have a pH range of from about 7.5 to about 9,0, with a pH range of about 8.0 to about 8.5 as typical. Concentration of nucleotides can be in the range of about 25 mM to about 1000 mM, typically in the range of about 100 mM to about 800 mM. Examples of dNTP concentrations are 100, 200, 300, 400, 500, 600, 700, and 800 mM. Detergents such as Tween 20, Triton X 100, and Nonidet P40 may also be included in the reaction mixture. Stabilizing agents such as dithiothreitol (DTT, Cleland's reagent) or 2-mercaptoethanol may also be included. In addition, master mixes may optionally contain dUTP as well as uracil DNA glycosylase (uracil-N-glycosylase, UNG). A master mix is commercially available from Applied Biosystems, Foster City, Calif., (TaqMan® Universal Master Mix, cat. nos.4304437, 4318157, and 4326708). Labeling Strategies Any suitable labeling strategy can be employed in the methods described herein. Where the reaction is analyzed for presence of a single amplification product, a universal detection probe can be employed in the amplification mixture. In particular embodiments, real-time PCR detection can be carried out using a universal qPCR probe. Suitable universal qPCR probes include double-stranded DNA-binding dyes, such as SYBR Green, Pico Green (Molecular Probes, Inc., Eugene, Oreg.), Eva Green (Biotium), ethidium bromide, and the like (see Zhu et al., 1994, Anal. Chem.66:1941-48). In some embodiments, one or more target-specific qPCR probes (i.e., specific for a target nucleotide sequence to be detected) is employed in the amplification mixtures to detect amplification products. By judicious choice of labels, analyses can be conducted in which the different labels are excited and / or detected at different wavelengths in a single reaction (“multiplex detection”). See, e.g., Fluorescence Spectroscopy (Pence 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, (1971); Griffiths, Colour and Constitution of Organic Molecules, Academic Press, New York, (1976); Indicators (Bishop, Ed.). Pergamon Press, Oxford, 19723; Attorney Docket No.: 6010-0013WO01 and Haugland, Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Eugene (1992); and Linck et al. (2017) “A multiplex TaqMan qPCR assay for sensitive and rapid detection of phytoplasmas infecting Rubus species,” PLOS One 12(5). In some embodiments, probes are designed so that annealing of the probe to a target nucleic acid leads to Fluorescence Resonance Energy Transfer (FRET). FRET is a quantum phenomenon occurring between two dye molecules. Excitation is transferred from a donor to an acceptor fluorophore, whereby the donor molecule fluorescence is quenched, and the acceptor molecule becomes excited. In certain embodiments, parts of a fluorophore-labeled DNA probe can participate in collisional and static fluorescence quenching. These non-FRET-based mechanisms can mimic the fluorescence quenching effects of FRET. The design of FRET and other fluorescence-based probes useful in real-time PCR reactions is well-known and reviewed, for example, in Didenko, Biotechniques (2001) 31:5, 1106-1121, which is incorporated by reference herein for this description. In some embodiments, it may be convenient to include labels on one or more of the primers employed in in amplification mixture. Exemplary Automation and Systems In some embodiments, a target nucleic acid is detected using an automated sample handling and / or analysis platform. In some embodiments, commercially available automated analysis platforms are utilized. For example, in some embodiments, the GeneXpert® system (Cepheid, Sunnyvale, Calif.) is utilized. 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 particular detection method or analysis platform. One of skill in the art recognizes that any number of platforms and methods may be utilized. The GeneXpert® utilizes a self-contained, single use cartridge. Sample extraction, amplification, and detection may all be carried out within this self-contained “laboratory in a cartridge” (available from Cepheid—see www.cepheid.com). Components of the cartridge include, but are not limited to, processing chambers containing reagents, filters, and capture technologies useful to extract, purify, and amplify target nucleic acids. A valve enables fluid transfer from chamber to chamber and contains nucleic acids lysis Attorney Docket No.: 6010-0013WO01 and filtration components. An optical window enables real-time optical detection. A reaction tube enables very rapid thermal cycling. In some embodiments, the GeneXpert® system includes a plurality of modules for scalability. Each module includes a plurality of cartridges, along with sample handling and analysis components. After the sample is added to the cartridge, the sample is contacted with lysis buffer and released nucleic acid is bound to a nucleic acid-binding substrate such as a silica or glass substrate. The sample supernatant is then removed and the nucleic acid eluted in an elution buffer such as a Tris / EDTA buffer. The eluate may 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, which are present in the cartridge as lyophilized particles. In some embodiments, PCR is used to amplify and detect the presence of one or more target nucleic acids. In some embodiments, the PCR uses Taq polymerase with hot start function, such as AptaTaq (Roche). In some embodiments, an off-line centrifugation is used to improve assay results with samples with low cellular content. The sample, with or without the buffer added, is centrifuged and the supernatant removed. The pellet is then resuspended in a smaller volume of supernatant, buffer, or other liquid. The resuspended pellet is then added to a GeneXpert® cartridge as previously described. Kits Also contemplated is a kit for carrying out the methods described herein. Such kits include one or more reagents useful for practicing any of these methods. A kit generally includes a package with one or more containers holding the reagents, as one or more separate compositions or, optionally, as an admixture where the compatibility of the reagents will allow. The kit can also include other material(s) that may be desirable from a user standpoint, such as a buffer(s), a diluent(s), a standard(s), and / or any other material useful in sample processing, washing, or conducting any other step of the assay. Kits preferably include instructions for carrying out one or more of the methods described herein. Instructions included in kits can be affixed to packaging material or can be included as a package insert. While the instructions are typically written or printed materials, they are not limited to such. Any medium capable of storing such instructions and communicating them to an Attorney Docket No.: 6010-0013WO01 end user can be employed. Such media include, hut are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. As used herein, the term “instructions” can include the address of an internet site that provides the instructions. The invention will be illustrated in more detail with reference to the following Examples, but it should be understood that the present invention is not deemed to be limited thereto. EXAMPLES Example 1: Stabilized Hairpin Containing Primer The number of PCR thermocycles can be reduced using either by using GC-clamped primers or by including a stabilized hairpin loop sequence. Fig.7 shows a comparison of stabilized hairpin loop containing primers with GC-clamed primers in base-3 PCR. An experiment was performed in which the effect on Ct was measured of a primer oligo comprising a 0, 1, 2, or 3 stabilizing bases stabilized hairpin loop sequences and a complimentary target sequence. The target oligo was 8,000 copies of SARS-Cov-2 N2 gene. The Ct of the hairpin loop sequences comprising 3 stabilizing bases was comparable to the Ct of a control GC-claim containing sequence. The results demonstrated that the more stabilized hairpin primers have equivalent performance to GC- clamped primers in base 3 PCR. Example 2: Universal, Non-Complementary Flanking Sequence that is Incorporated into Amplicon After the First PCR cycle An experiment was performed to test for PCR cycle reduction by optimizing the PCR process to achieve the desired amplification of DNA in fewer cycles while maintaining or improving the quality of the results. In this experiment, nested (inner) primers comprised universal, non-complementary flanking sequences that are incorporated into the amplicon after the first PCR cycle. This experiment made use of the CDC Influenza SARS-CoV-2 (Flu SC2) Multiplex Assay which is a real-time reverse-transcription polymerase chain reaction (rRT-PCR) laboratory test that can simultaneously detect and differentiate between influenza A, influenza B, and SARS-CoV-2. Attorney Docket No.: 6010-0013WO01 Nested primers w / 3' ends from GX Fluvid+ primers & 5' ends from selected UFP pairs were designed and used to amplify target nucleic acids. In certain embodiments, GX Fluvid+ primers & probes, may be optimized for primer-artifact suppression. Table 2 shows that nested (inner) primers comprising universal, non-complementary flanking sequences that are incorporated into amplicon after the first PCR cycle produced Base 6 amplification and reduced the number of PCR cycles. Non-limiting examples of universal flanking primers are set forth in Table 2: Table 2 Name Sequence Cov forward unfl 1 CGAAGAC(2-thiodT)A(2-thiodT)CGTG(2-thiodT)AA SEQ ID NO: 10 Further non-limiting examples of universal flanking primers include the complements of the sequences as set forth in Table 2, and in certain embodiments, the complementary sequences comprise 2-aminoadenine as the complement of 2-thiodT. The results demonstrate that the use of universal, non-complementary flanking sequence that is incorporated into amplicon after the first PCR cycle produces base-6 amplification with greatly reduced number of cycles required for efficient amplification. (See Figs. 3, 5, and 8). Systematically optimizing the primers can achieve efficient PCR amplification with a reduced number of cycles, saving time and resources while maintaining the integrity of the results. Table 3 Organism Gene Base 6 vs. Base 2 Attorney Docket No.: 6010-0013WO01 Flu B NS 36% RSV B N 0% , mer artifact contribution in combination w / GX Fluvid+ primers & probes, for example in an EvaGreen assay. In certain embodiments, nested primers w / 3' ends from GX Fluvid+ primers & 5' ends from selected UFP pair can be designed. Additionally, RNAse H2 activated probes based on GX Fluvid+ Taqman probes can also be designed. Example 3: Effect of Different Concentrations of PEG 8000 In an effort to further reduce the number of amplification cycles needed for detection, high molecular weight polyethylene glycol (PEG) was included in base greater than-three amplification reactions. Macromolecules can increase molecular crowding in solutions which can increase the effective concentration of reactants, thereby favoring intermolecular associate in reactions, such as nucleic acid amplification. PEG 8000 includes molecules large enough to produce significant molecular crowding under appropriate conditions. Accordingly, the effect of including different concentrations of PEG 8000 in a base-6 amplification reaction was tested. To evaluate the impact of PEG 8000 on target Ct in different types of reactions, we added PEG 8000 at 0%, 2%, 6%, 8% and 10% concentrations into base-2, base-3 and base-6 nucleic acid amplification reactions, respectively. All the reactions targeted the same region of N gene of SARSCo V-2. Detection of target amplification is achieved using an oligonucleotide probe labelled with dye and quencher. The oligonucleotide probe also harbored a single ribonucleotide in the middle. Upon its annealing to complementary target DNA strand, the probe was digested by RNase H2 enzyme and released the fluorescent dye (i.e., a cycling probe). The results showed that the Ct of the base-2 reaction did not change with incremental increase in PEG 8000 concentration. However, the Ct values of base-3 and base-6 reactions improved significantly and gradually with incremental increase in PEG 8000 concentration. The base-6 reaction had the most obvious improvement in Ct among the reaction types. (See Fig.9) Attorney Docket No.: 6010-0013WO01 Example 4: Effect of PEG with Different Polymerases An experiment was performed to test for the effect of varying the amount of PEG8000 when using two strand displacement enzymes, TaqG46E and a polymerase. The precent of PEG8000 varied from 10% to 5% to 1% and then no PEG8000 for each enzyme. The Reaction mix included the following: 1000 RNA copies per 80uL RT volume 1000 copies / 80uL = 12.5 copies / uL 12.5 copies / uL X 40uL = 500 copies for PCR 500 copies / 80uL = 6.25 copies / uL 6.25 copies / uL X 50uL tube = ~313 copies in tube for PCR The Results are shown in Table 4: Table 4 Sample ID (N=3) Flu A (FAM) Attorney Docket No.: 6010-0013WO01 As shown in Fig.10, the best results (earliest Ct) came from using 10% PEG8000 with delayed results as the amount of PEG8000 was decreased. The difference between 10% and 5% when using TaqG46E is striking as there is almost a nine Ct difference. The impact on threshold of varying PEG8000 concentration, TaqG46E, Flu A target is shown in Fig.11. The impact on end point fluorescence of varying PEG8000 vs. a polymerase, Flu A target is shown in Fig.12. Example 5: Effect of Different Molecular Weights of PEG An experiment was performed to test for the effect of 10% PEG but with different molecular weights. PEG with varying molecular weights, was tested as a molecular crowder with Base 6 N2 simplex beads, 2-step RT-PCR. The sample used was Asuragen COVID Panel, 1000 copies per 80uL RT volume. The Reaction mix included the following: 1000 RNA copies in 80uL RT volume = 12.5 copies / uL 50uL RT sample = 625 RT copies 625uL in 80uL = 7.81 copies / uL 7.81 copies / uL X 54uL = 422 RT copies fo rPCR 422 copies in 80uL = 5.28 copies / uL 5.28 copies X 50uL tube = 264 copies at PCR The results are shown in Table 5. Table 5 Sample ID (N=3) N2 (FAM) E (CF4) Attorney Docket No.: 6010-0013WO01 The 10,000 and 8,000 MWs gave the best cycle thresholds and end point fluorescence for two SARS targets (E and N2) with the 8,000 MW doing a little better with the end point fluorescence. Real-time PCR fluorescence growth curves generated in the presence of 10% PEG with varying molecular weight N2 target are shown in Fig. 13. Real-time PCR fluorescence growth curves generated in the presence of 10% PEG by varying molecular weight E target are shown in Fig. 14. The impact on N2 cycle threshold in the presence of 10% PEG of varying molecular weight is shown in Fig. 15. The impact on E cycle threshold in the presence of 10% PEG of varying molecular weight is shown in Fig.16. While the invention has been described in detail and with reference to specific examples thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.

Claims

Attorney Docket No.: 6010-0013WO01 CLAIMS WHAT IS CLAIMED IS:

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, optionally, a second template strand, wherein the second template strand is complementary to the first template strand, the primer set comprising oligonucleotides in the form of, or capable of forming, 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 comprising outer primer sequence a that specifically hybridizes to first template strand sequence a′, outer primer sequence a optionally comprising at least one first modified base; and the first inner primer comprising a single-stranded primer sequence b that specifically hybridizes to first template strand sequence b′, wherein b′ is adjacent to, and 5′ of, a′, and wherein single-stranded primer sequence b is linked at its 5′ end to a double-stranded hairpin primer sequence comprising: primer sequence a adjacent to, and 5′ of, single-stranded primer sequence b; and a spacer sequence which is adjacent to, and 5' of primer sequence a; a primer sequence a', which is adjacent to, and 5' of the spacer sequence, wherein primer sequence a' is complementary to primer sequence a, wherein primer sequence a′ comprises at least one second modified base.

2. 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, optionally, a second template strand, wherein the second template strand is complementary to the first template strand, the primer set comprising oligonucleotides in the form of, or capable of forming, 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 comprising outer primer sequence e that is capable of specifically hybridizing to the first inner primer sequence e′, wherein outer primer sequence eAttorney Docket No.: 6010-0013WO01 comprises universal flanking primer sequence e, further wherein outer primer sequence e optionally comprises at least one first modified base; and the first inner primer comprising a single-stranded primer sequence b that specifically hybridizes to first template strand sequence b′, wherein b′ is adjacent to, and 5′ of, a′, and wherein single-stranded primer sequence b is linked at its 5′ end to a double-stranded hairpin primer sequence comprising: primer sequence e adjacent to, and 5′ of, single-stranded primer sequence b; and a spacer sequence which is adjacent to, and 5' of primer sequence e; a primer sequence e', which is adjacent to, and 5' of the spacer sequence, wherein primer sequence e' is complementary to primer sequence e, wherein primer sequence e′ comprises at least one second modified base.

3. The nucleic acid primer set of claim 2, wherein the universal flanking primer sequence is from about 12 to about 50 bases in length.

4. The nucleic acid primer set of any one of claims 2 to 3, wherein the universal flanking primer sequence has no significant homology to any segment in the template strand.

5. The nucleic acid primer set of any one of claims 2 to 4, wherein the universal flanking primer sequence lacks secondary structure.

6. The nucleic acid primer set of any one of claims 2 to 5, wherein the universal flanking primer sequence is optimized for Tm.

7. The nucleic acid primer set of any one of claims 2 to 6, wherein the universal flanking primer sequence is optimized for GC content.

8. The nucleic acid primer set of any one of claims 2 to 7, wherein the universal flanking primer sequence comprises a sequence that is at least 90% identical or complementary to at least 12 contiguous nucleotides of ATGTCCGCCTACTTTA (SEQ ID NO: 1).Attorney Docket No.: 6010-0013WO01 9. The nucleic acid primer set of any one of claims 1 to 8, wherein the spacer sequence comprises an alkylene or a heteroalkylene group.

10. The nucleic acid primer set of any one of claims 1 to 9, wherein the spacer sequence comprises a C10 to C30 alkylene or a heteroalkylene group.

11. The nucleic acid primer set of any one of claims 1 to 10, wherein the hairpin primer sequence is double stranded and allows the outer primer to hybridize with the template.

12. The nucleic acid primer set of any one of claims 1 to 11, wherein the hairpin primer sequence is double stranded and allows the universal flanking primer to hybridize to the amplicon.

13. The nucleic acid primer set of any one of claims 1 to 12, wherein each of the first inner primer sequences a′ and e′ comprises at least one first modified base.

14. The nucleic acid primer set of any one of claims 1 to 13, wherein the at least one first modified base is a stabilizing base.

15. The nucleic acid primer set of any one of claims 1 to 14, wherein the at least one first modified base is a stabilizing base selected from the group consisting of Locked nucleic acids (LNAs), unlocked nucleic acids (UNAs), AP-dC (G-clamp), 2-aminoadenine, 5- methylcytosine, C(5)-propynylcytosine, C(5)-propynyluracil, 2-aminoadenine, 2- thiothymine, deoxyinosine, 7-alkyl-7-deazaguanine, 2′-hypoxanthine, or 7-nitro-7- deazahypoxanthine, 3-(2′-deoxy-beta-D-ribofuranosyl)pyrrolo-[2,3-d]-pyrimidine-2- (3H)-one, N4-alkylcytosine, 2-thiocytosine, and combinations thereof.

16. The nucleic acid primer set of any one of claims 1 to 15, wherein the at least one first modified base is a stabilizing base and provides added stability to the primer, thereby facilitating the primer’s access to a target sequence.Attorney Docket No.: 6010-0013WO01 17. The nucleic acid primer set of any one of claims 1 to 16, wherein the at least one first modified base is a stabilizing base, wherein this added stability gives the primer access to a target sequence and wherein the added stability does not prevent displacement of the hairpin sequence by a DNA polymerase and / or copying of the flanking primer region.

18. The nucleic acid primer set of any one of claims 1 to 17, wherein the at least one second modified base is a stabilizing base.

19. The nucleic acid primer set of any one of claims 1 to 18, wherein the at least one second modified base which is a stabilizing base is selected from the group consisting of Locked nucleic acids (LNAs), unlocked nucleic acids (UNAs), AP-dC (G-clamp), 2- aminoadenine, 5-methylcytosine, C(5)-propynylcytosine, C(5)-propynyluracil, 2- aminoadenine, 2-thiothymine, deoxyinosine, 7-alkyl-7-deazaguanine, 2′-hypoxanthine, or 7-nitro-7-deazahypoxanthine, 3-(2′-deoxy-beta-D-ribofuranosyl)pyrrolo-[2,3-d]- pyrimidine-2-(3H)-one, N4-alkylcytosine, 2-thiocytosine, and combinations thereof.

20. The nucleic acid primer set of any one of claims 1 to 19, wherein the at least one second modified base which is a stabilizing base provides added stability to the primer, thereby facilitating the primer’s access to a target sequence.

21. The nucleic acid primer set of any one of claims 1 to 20, wherein the at least one second modified base which is a stabilizing base, wherein this added stability gives the primer access to a target sequence and wherein the added stability does not prevent displacement of the hairpin sequence by a DNA polymerase and / or copying of the flanking primer region.

22. The nucleic acid primer set of any one of claims 1 to 21, wherein each of the outer primer sequence a and the universal flanking primer sequence e comprises at least one second modified base which is stabilizing base.Attorney Docket No.: 6010-0013WO01 23. The nucleic acid primer set of any one of claims 1 to 22, wherein at least one second modified base which is a stabilizing base selected from the group consisting of Locked nucleic acids (LNAs), unlocked nucleic acids (UNAs), AP-dC (G-clamp), 2- aminoadenine, 5-methylcytosine, C(5)-propynylcytosine, C(5)-propynyluracil, 2- aminoadenine, 2-thiothymine, deoxyinosine, 7-alkyl-7-deazaguanine, 2′-hypoxanthine, or 7-nitro-7-deazahypoxanthine, 3-(2′-deoxy-beta-D-ribofuranosyl)pyrrolo-[2,3-d]- pyrimidine-2-(3H)-one, N4-alkylcytosine, 2-thiocytosine, and combinations thereof.

24. The nucleic acid primer set of any one of claims 1 to 23, 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, as compared to pairing between the first and second modified bases.

25. The nucleic acid primer set of any one of claims 1 to 24, wherein the primer set is capable of amplifying the target nucleic acid at the rate of at least 3number of cyclesduring an exponential phase of amplification.

26. The nucleic acid primer set of any one of claims 1 to 25, wherein the primer set permits detection of a single-copy nucleic acid in a biological sample within about 12%-42% fewer amplification cycles than would be required for said detection using only a single forward and a single reverse primer.

27. The nucleic acid primer set of any one of claims 1 to 26, wherein the primer set additionally comprises at least one second primer capable of specifically hybridizing to the second template strand.

28. The nucleic acid primer set of claim 27, wherein the second primer comprises oligonucleotides in the form of, or capable of forming, at least two second primers 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,Attorney Docket No.: 6010-0013WO01 the second outer primer comprising a primer sequence f that specifically hybridizes to second template strand sequence f′, primer sequence f optionally comprising at least one first modified base(s); and the second inner primer comprising a single-stranded primer sequence g that specifically hybridizes to second template strand sequence g′, wherein g′ is adjacent to, and 5′ of, f′, and wherein single-stranded primer sequence g is linked at its 5′ end to a double-stranded hairpin primer sequence comprising: a primer sequence f adjacent to, and 5′ of, single-stranded primer sequence g; and a spacer sequence which is adjacent to, and 5' of primer sequence f; a primer sequence f' which is adjacent to, and 5' of the spacer sequence, wherein primer sequence f' is complementary to primer sequence f, wherein primer sequence f′ comprises at least one second modified base.

29. The nucleic acid primer set of any one of claims 27 to 28, wherein the second primer comprises oligonucleotides in the form of, or capable of forming, at least two second primers 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 comprising an outer primer sequence j that is capable of specifically hybridizing to primer sequence j′, wherein outer primer sequence j comprises a universal flanking primer sequence j, further wherein outer primer sequence j optionally comprises at least one first modified base; and the second inner primer comprising a single-stranded primer sequence g that specifically hybridizes to second template strand sequence g′, wherein g′ is adjacent to, and 5′ of, f′, and wherein single-stranded primer sequence g is linked at its 5′ end to a double-stranded hairpin primer sequence comprising: a primer sequence j adjacent to, and 5′ of, single-stranded primer sequence g; and a spacer sequence which is adjacent to, and 5' of primer sequence j;Attorney Docket No.: 6010-0013WO01 a primer sequence j' which is adjacent to, and 5' of the spacer sequence, wherein primer sequence j' is complementary to primer sequence j, wherein primer sequence j′ comprises at least one second modified base.

30. The nucleic acid primer set of any one of claims 27 to 29, wherein the universal flanking primer sequence e and the universal flanking primer sequence j are the same.

31. The nucleic acid primer set of any one of claims 27 to 30, wherein universal flanking primer sequence e is from about 12 to about 50 bases in length, and universal flanking primer sequence j is from about 12 to about 50 bases in length.

32. The nucleic acid primer set of any one of claims 27 to 31, wherein the universal flanking primer sequence has no significant homology to any segment in the template strand.

33. The nucleic acid primer set of any one of claims 27 to 32, wherein the universal flanking primer sequence lacks secondary structure.

34. The nucleic acid primer set of any one of claims 27 to 33, wherein the universal flanking primer sequence is optimized for Tm.

35. The nucleic acid primer set of any one of claims 27 to 34, wherein the universal flanking primer sequence is optimized for GC content.

36. The nucleic acid primer set of any one of claims 27 to 3528, wherein the universal flanking primer sequence e comprises a sequence that is at least 90% identical or complementary to at least 12 contiguous nucleotides of ATGTCCGCCTACTTTA (SEQ ID NO: 1).

37. The nucleic acid primer set of any one of claims 27 to 36, wherein the universal flanking primer sequence j comprises a sequence that is at least 90% identical or complementary to at least 12 contiguous nucleotides of ATGTCCGCCTACTTTA (SEQ ID NO: 1).Attorney Docket No.: 6010-0013WO01 38. The nucleic acid primer set of any one of claims 27 to 37, wherein the spacer sequence comprises an alkylene or a heteroalkylene group.

39. The nucleic acid primer set of any one of claims 27 to 38, wherein the spacer sequence comprises a C10to C30alkylene or a heteroalkylene group.

40. The nucleic acid primer set of any one of claims 27 to 39, wherein the hairpin primer sequence is double stranded and allows the outer primer to hybridize with the template.

41. The nucleic acid primer set of any one of claims 27 to 40, wherein the hairpin primer sequence is double stranded and allows the universal flanking primer to hybridize to the amplicon.

42. The nucleic acid primer set of any one of claims 27 to 41, wherein each of the first inner primer sequences a′ and e′ comprises at least one first modified base.

43. The nucleic acid primer set of any one of claims 27 to 42, wherein the at least one first modified base is a stabilizing base.

44. The nucleic acid primer set of any one of claims 27 to 43, wherein the at least one modified base which is a stabilizing base selected from the group consisting of Locked nucleic acids (LNAs), unlocked nucleic acids (UNAs), AP-dC (G-clamp), 2- aminoadenine, 5-methylcytosine, C(5)-propynylcytosine, C(5)-propynyluracil, 2- aminoadenine, 2-thiothymine, deoxyinosine, 7-alkyl-7-deazaguanine, 2′-hypoxanthine, or 7-nitro-7-deazahypoxanthine, 3-(2′-deoxy-beta-D-ribofuranosyl)pyrrolo-[2,3-d]- pyrimidine-2-(3H)-one, N4-alkylcytosine, 2-thiocytosine, and combinations thereof.

45. The nucleic acid primer set of any one of claims 27 to 44, wherein the at least one first modified base which is a stabilizing base provides added stability to the primer, thereby facilitating the primer’s access to a target sequence.Attorney Docket No.: 6010-0013WO01 46. The nucleic acid primer set of any one of claims 27 to 45, wherein the at least one first modified base which is a stabilizing base, wherein this added stability gives the primer access to a target sequence and wherein the added stability does not prevent displacement of the hairpin sequence by a DNA polymerase and / or copying of the flanking primer region.

47. The nucleic acid primer set of any one of claims 27 to 46, wherein each of the first inner primer sequences f′ and j′ comprises at least one first modified base.

48. The nucleic acid primer set of any one of claims 27 to 47, wherein the at least one second modified base is a stabilizing base.

49. The nucleic acid primer set of any one of claims 27 to 48, wherein the at least one second modified base which is a stabilizing base selected from the group consisting of Locked nucleic acids (LNAs), unlocked nucleic acids (UNAs), AP-dC (G-clamp), 2- aminoadenine, 5-methylcytosine, C(5)-propynylcytosine, C(5)-propynyluracil, 2- aminoadenine, 2-thiothymine, deoxyinosine, 7-alkyl-7-deazaguanine, 2′-hypoxanthine, or 7-nitro-7-deazahypoxanthine, 3-(2′-deoxy-beta-D-ribofuranosyl)pyrrolo-[2,3-d]- pyrimidine-2-(3H)-one, N4-alkylcytosine, 2-thiocytosine, and combinations thereof.

50. The nucleic acid primer set of any one of claims 27 to 49, wherein the at least one second modified base which is a stabilizing base provides added stability to the primer, thereby facilitating the primer’s access to a target sequence.

51. The nucleic acid primer set of any one of claims 27 to 50, wherein the at least one second modified base which is a stabilizing base, wherein this added stability gives the primer access to a target sequence and wherein the added stability does not prevent displacement of the hairpin sequence by a DNA polymerase and / or copying of the flanking primer region.Attorney Docket No.: 6010-0013WO01 52. The nucleic acid primer set of any one of claims 27 to 51, 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, as compared to pairing between the first and second modified bases.

53. The nucleic acid primer set of any one of claims 27 to 52, wherein the primer set is capable of amplifying the target nucleic acid at the rate of at least 6number of cyclesduring an exponential phase of amplification.

54. The nucleic acid primer set of any one of claims 27 to 53, wherein the primer set permits detection of a single-copy nucleic acid in a biological sample within about 36%-66% fewer amplification cycles than would be required for said detection using only a single forward and a single reverse primer.

55. The nucleic acid primer set of any one of claims 27 to 54, wherein the primer set is free of GC-rich regions.

56. A kit comprising the nucleic acid primer set of any one of claims 27 to 55, an amplification buffer comprising a DNA polymerase lacking 5′-3′ exonuclease activity, a mixture of nucleotides, and optionally polyethylene glycol (PEG) 8000.

57. 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) adding the sample to a polymerase chain reaction (PCR) reaction mixture comprising: (i) a primer set comprising oligonucleotides in the form of, or capable of forming, 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,Attorney Docket No.: 6010-0013WO01 the first outer primer comprising outer primer sequence a that specifically hybridizes to first template strand sequence a′, outer primer sequence a optionally comprising at least one first modified base; and the first inner primer comprising a single-stranded primer sequence b that specifically hybridizes to first template strand sequence b′, wherein b′ is adjacent to, and 5′ of, a′, and wherein single-stranded primer sequence b is linked at its 5′ end to a double- stranded hairpin primer sequence comprising: primer sequence a adjacent to, and 5′ of, single-stranded primer sequence b; and a spacer sequence which is adjacent to, and 5' of primer sequence a; a primer sequence a', which is adjacent to, and 5' of the spacer sequence, wherein primer sequence a' is complementary to primer sequence a, wherein primer sequence a′ comprises at least one second modified base, wherein the hybridization is carried out under conditions wherein the primers anneal to their template strands, if present; and (ii) an amplification buffer comprising a DNA polymerase lacking 5′-3′ exonuclease activity, and a mixture of nucleotides; (b) amplifying the target nucleic acid, if present, under conditions where strand displacement occurs, thereby producing amplicons that include the sequences of all primers employed in the amplification reaction.

58. The method of claim 57 wherein said PCR reaction mixture comprises polyethylene glycol (PEG).

59. 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) adding the sample to a polymerase chain reaction (PCR) reaction mixture comprising:Attorney Docket No.: 6010-0013WO01 (i) a primer set comprising oligonucleotides in the form of, or capable of forming, 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 comprising outer primer sequence e that is capable of specifically hybridizing to the first inner primer sequence e′, wherein outer primer sequence e comprises a universal flanking primer sequence e, further wherein outer primer sequence e optionally comprises at least one first modified base; the first inner primer comprising a single-stranded primer sequence b that specifically hybridizes to first template strand sequence b′, wherein b′ is adjacent to, and 5′ of, a′, and wherein single-stranded primer sequence b is linked at its 5′ end to a double- stranded hairpin primer sequence comprising: primer sequence e adjacent to, and 5′ of, single-stranded primer sequence b; and a spacer sequence which is adjacent to, and 5' of primer sequence e; a primer sequence e', which is adjacent to, and 5' of the spacer sequence, wherein primer sequence e' is complementary to primer sequence e, wherein primer sequence e′ comprises at least one second modified base; and (ii) an amplification buffer comprising a DNA polymerase lacking 5′-3′ exonuclease activity, and a mixture of nucleotides, (b) amplifying the target nucleic acid, if present, under conditions where strand displacement occurs, thereby producing amplicons that include the sequences of all primers employed in the amplification reaction.

60. The method of claim 59 wherein said PCR reaction mixture comprises polyethylene glycol (PEG).

61. The method of any one of claims 59 to 60, wherein the universal flanking primer sequence is from about 12 to about 50 bases in length.Attorney Docket No.: 6010-0013WO01 62. The method of any one of claims 59 to 61, wherein the universal flanking primer sequence has no significant homology to any segment in the template strand.

63. The method of any one of claims 59 to 62, wherein the universal flanking primer sequence lacks secondary structure.

64. The method of any one of claims 59 to 63, wherein the universal flanking primer sequence is optimized for Tm.

65. The method of any one of claims 59 to 64, wherein the universal flanking primer sequence is optimized for GC content.

66. The method of any one of claims 59 to 65, wherein the universal flanking primer sequence has no significant homology to any segment in the template strand.

67. The method of any one of claims 59 to 66, wherein the universal flanking primer sequence comprises a sequence that is at least 90% identical or complementary to at least 12 contiguous nucleotides of ATGTCCGCCTACTTTA (SEQ ID NO: 1).

68. The method of any one of claims 57 to 67, wherein the spacer sequence comprises an alkylene or a heteroalkylene group.

69. The method of any one of claims 57 to 68, wherein the spacer sequence comprises a C10 to C30 alkylene or a heteroalkylene group.

70. The method of any one of claims 57 to 69, wherein the hairpin primer sequence is double stranded and allows the outer primer to hybridize with the template.

71. The method of any one of claims 57 to 70, wherein the hairpin primer sequence is double stranded and allows the universal flanking primer to hybridize to the amplicon.Attorney Docket No.: 6010-0013WO01 72. The method of any one of claims 57 to 71, wherein each of the first inner primer sequences a′ and e′ comprises at least one first modified base.

73. The method of any one of claims 57 to 72, wherein the at least one first modified base is a stabilizing base.

74. The method of any one of claims 57 to 73, wherein the at least one first modified base which is a stabilizing base is selected from the group consisting of Locked nucleic acids (LNAs), unlocked nucleic acids (UNAs), AP-dC (G-clamp), 2-aminoadenine, 5- methylcytosine, C(5)-propynylcytosine, C(5)-propynyluracil, 2-aminoadenine, 2- thiothymine, deoxyinosine, 7-alkyl-7-deazaguanine, 2′-hypoxanthine, or 7-nitro-7- deazahypoxanthine, 3-(2′-deoxy-beta-D-ribofuranosyl)pyrrolo-[2,3-d]-pyrimidine-2- (3H)-one, N4-alkylcytosine, 2-thiocytosine, and combinations thereof.

75. The method of any one of claims 57 to 74, wherein the at least one first modified base which is a stabilizing base provides added stability to the primer, thereby facilitating the primer’s access to a target sequence.

76. The method of any one of claims 57 to 75, wherein the at least one first modified base which is a stabilizing base, wherein this added stability gives the primer access to a target sequence and wherein the added stability does not prevent displacement of the hairpin sequence by a DNA polymerase and / or copying of the flanking primer region.

77. The method of any one of claims 57 to 76, wherein the at least one second modified base is a stabilizing base.

78. The method of any one of claims 57 to 77, wherein the at least one second modified base which is a stabilizing base is selected from the group consisting of Locked nucleic acids (LNAs), unlocked nucleic acids (UNAs), AP-dC (G-clamp), 2-aminoadenine, 5- methylcytosine, C(5)-propynylcytosine, C(5)-propynyluracil, 2-aminoadenine, 2- thiothymine, deoxyinosine, 7-alkyl-7-deazaguanine, 2′-hypoxanthine, or 7-nitro-7-Attorney Docket No.: 6010-0013WO01 deazahypoxanthine, 3-(2′-deoxy-beta-D-ribofuranosyl)pyrrolo-[2,3-d]-pyrimidine-2- (3H)-one, N4-alkylcytosine, 2-thiocytosine, and combinations thereof.

79. The method of any one of claims 57 to 78, wherein the at least one second modified base which is a stabilizing base provides added stability to the primer, thereby facilitating the primer’s access to a target sequence.

80. The method of any one of claims 57 to 79, wherein the at least one second modified base which is a stabilizing base, wherein this added stability gives the primer access to a target sequence and wherein the added stability does not prevent displacement of the hairpin sequence by a DNA polymerase and / or copying of the flanking primer region.

81. The method of any one of claims 57 to 80, wherein the universal flanking primer sequence e is optimized for primer-artifact reduction.

82. method of any one of claims 57 to 81, wherein each of the outer primer sequence a and the universal flanking primer sequence e comprises at least one second modified base which is stabilizing base.

83. method of any one of claims 57 to 82, wherein at least one second modified base which is a stabilizing base selected from the group consisting of Locked nucleic acids (LNAs), unlocked nucleic acids (UNAs), AP-dC (G-clamp), 2-aminoadenine, 5-methylcytosine, C(5)-propynylcytosine, C(5)-propynyluracil, 2-aminoadenine, 2-thiothymine, deoxyinosine, 7-alkyl-7-deazaguanine, 2′-hypoxanthine, or 7-nitro-7-deazahypoxanthine, 3-(2′-deoxy-beta-D-ribofuranosyl)pyrrolo-[2,3-d]-pyrimidine-2-(3H)-one, N4- alkylcytosine, 2-thiocytosine, and combinations thereof.

84. The method of any one of claims 57 to 83, 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, as compared to pairing between the first and second modified bases.Attorney Docket No.: 6010-0013WO01 85. The method of any one of claims 57 to 84, wherein the primer set is capable of amplifying the target nucleic acid at the rate of at least 3number of cyclesduring an exponential phase of amplification.

86. The method of any one of claims 57 to 85, wherein the primer set permits detection of a single-copy nucleic acid in a biological sample within about 12%-42% fewer amplification cycles than would be required for said detection using only a single forward and a single reverse primer.

87. The method of any one of claims 57 to 86, wherein the primer set additionally comprises at least one second primer capable of specifically hybridizing to the second template strand.

88. The method of claim 87 wherein the second primer comprises oligonucleotides in the form of, or capable of forming, at least two second primers 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 comprising a primer sequence f that specifically hybridizes to second template strand sequence f′, primer sequence f optionally comprising at least one first modified base; and the second inner primer comprising a single-stranded primer sequence g that specifically hybridizes to second template strand sequence g′, wherein g′ is adjacent to, and 5′ of, f′, and wherein single-stranded primer sequence g is linked at its 5′ end to a double-stranded hairpin primer sequence comprising: a primer sequence f adjacent to, and 5′ of, single-stranded primer sequence g; and a spacer sequence which is adjacent to, and 5' of primer sequence f; a primer sequence f' which is adjacent to, and 5' of the spacer sequence, wherein primer sequence f' is complementary to primer sequence f, wherein primer sequence f′ comprises at least one second modified base,Attorney Docket No.: 6010-0013WO01 wherein the hybridization is carried out under conditions wherein the primers anneal to their template strands, if present; and (ii) an amplification buffer comprising a DNA polymerase lacking 5′-3′ exonuclease activity, and a mixture of nucleotides; (b) amplifying the target nucleic acid, if present, under conditions where strand displacement occurs, thereby producing amplicons that include the sequences of all primers employed in the amplification reaction.

89. The method of any one of claims 59 to 88 wherein the second primer comprises oligonucleotides in the form of, or capable of forming, at least two second primers 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 comprising an outer primer sequence j that is capable of specifically hybridizing to primer sequence j′, wherein outer primer sequence j comprises a universal flanking primer sequence j, further wherein outer primer sequence j optionally comprises at least one first modified base, the second inner primer comprising a single-stranded primer sequence g that specifically hybridizes to second template strand sequence g′, wherein g′ is adjacent to, and 5′ of, f′, and wherein single-stranded primer sequence g is linked at its 5′ end to a double-stranded hairpin primer sequence comprising: a primer sequence j adjacent to, and 5′ of, single-stranded primer sequence g; and a spacer sequence which is adjacent to, and 5' of primer sequence j; a primer sequence j' which is adjacent to, and 5' of the spacer sequence, wherein primer sequence j' is complementary to primer sequence j, wherein primer sequence j′ comprises at least one modified base, wherein the hybridization is carried out under conditions wherein the primers anneal to their template strands, if present; andAttorney Docket No.: 6010-0013WO01 (ii) an amplification buffer comprising a DNA polymerase lacking 5′-3′ exonuclease activity, and a mixture of nucleotides; (b) amplifying the target nucleic acid, if present, under conditions where strand displacement occurs, thereby producing amplicons that include the sequences of all primers employed in the amplification reaction.

90. The method of any one of claims 59 to 89, wherein the spacer universal flanking primer sequence e and the universal flanking primer sequence j are the same.

91. The method of any one of claims 59 to 90, wherein universal flanking primer sequence e is from about 12 to about 50 bases in length, and universal flanking primer sequence j is from about 12 to about 50 bases in length.

92. The method of any one of claims 59 to 91, wherein the universal flanking primer sequence has no significant homology to any segment in the template strand.

93. The method of any one of claims 59 to, wherein the universal flanking primer sequence lacks secondary structure.

94. The method of any one of claims 59 to 93, wherein the universal flanking primer sequence is optimized for Tm.

95. The method of any one of claims 59 to 94, wherein the universal flanking primer sequence is optimized for GC content.

96. The method of any one of claims 59 to 95, wherein the universal flanking primer sequence e comprises a sequence that is at least 90% identical or complementary to at least 12 contiguous nucleotides of ATGTCCGCCTACTTTA (SEQ ID NO: 1).

97. The method of any one of claims 59 to 96, wherein the universal flanking primer sequence j comprises a sequence that is at least 90% identical or complementary to at least 12 contiguous nucleotides of ATGTCCGCCTACTTTA (SEQ ID NO: 1).Attorney Docket No.: 6010-0013WO01 98. The method of any one of claims 59 to 97, wherein the spacer sequence comprises an alkylene or a heteroalkylene group.

99. The method of any one of claims 59 to 98, wherein the spacer sequence comprises a C10to C30 alkylene or a heteroalkylene group.

100. The method of any one of claims 59 to 991 to 6, wherein the hairpin primer sequence is double stranded and allows the outer primer to hybridize with the template.

101. The method of any one of claims 59 to 100, wherein the hairpin primer sequence is double stranded and allows the universal flanking primer to hybridize to the amplicon.

102. The method of any one of claims 59 to 101, wherein each of the first inner primer sequences a′ and e′ comprises at least one first modified base.

103. The method of any one of claims 59 to 102, wherein the at least one first modified base is a stabilizing base.

104. The method of any one of claims 59 to 103, wherein the at least one modified base which is a stabilizing base selected from the group consisting of Locked nucleic acids (LNAs), unlocked nucleic acids (UNAs), AP-dC (G-clamp), 2-aminoadenine, 5- methylcytosine, C(5)-propynylcytosine, C(5)-propynyluracil, 2-aminoadenine, 2- thiothymine, deoxyinosine, 7-alkyl-7-deazaguanine, 2′-hypoxanthine, or 7-nitro-7- deazahypoxanthine, 3-(2′-deoxy-beta-D-ribofuranosyl)pyrrolo-[2,3-d]-pyrimidine-2- (3H)-one, N4-alkylcytosine, 2-thiocytosine, and combinations thereof.

105. The method of any one of claims 59 to 104, wherein the at least one first modified base which is a stabilizing base provides added stability to the primer, thereby facilitating the primer’s access to a target sequence.Attorney Docket No.: 6010-0013WO01 106. The method of any one of claims 59 to 105, wherein the at least one first modified base which is a stabilizing base, wherein this added stability gives the primer access to a target sequence and wherein the added stability does not prevent displacement of the hairpin sequence by a DNA polymerase and / or copying of the flanking primer region.

107. The method of any one of claims 59 to 106, wherein each of the first inner primer sequences a′ and e′ comprises at least one first modified base.

108. The method of any one of claims 59 to 107, wherein the at least one second modified base is a stabilizing base.

109. The method of any one of claims 59 to 108, wherein the at least one second modified base which is a stabilizing base selected from the group consisting of Locked nucleic acids (LNAs), unlocked nucleic acids (UNAs), AP-dC (G-clamp), 2- aminoadenine, 5-methylcytosine, C(5)-propynylcytosine, C(5)-propynyluracil, 2- aminoadenine, 2-thiothymine, deoxyinosine, 7-alkyl-7-deazaguanine, 2′-hypoxanthine, or 7-nitro-7-deazahypoxanthine, 3-(2′-deoxy-beta-D-ribofuranosyl)pyrrolo-[2,3-d]- pyrimidine-2-(3H)-one, N4-alkylcytosine, 2-thiocytosine, and combinations thereof.

110. The method of any one of claims 59 to 109, wherein the at least one second modified base which is a stabilizing base provides added stability to the primer, thereby facilitating the primer’s access to a target sequence.

111. The method of any one of claims 59 to 110, wherein the at least one second modified base which is a stabilizing base, wherein this added stability gives the primer access to a target sequence and wherein the added stability does not prevent displacement of the hairpin sequence by a DNA polymerase and / or copying of the flanking primer region.

112. The method of any one of claims 59 to 111, wherein the universal flanking primer sequence e is optimized for primer-artifact reduction.Attorney Docket No.: 6010-0013WO01 113. The method of any one of claims 59 to 113, wherein the universal flanking primer sequence has no significant homology to any segment in the template strand.

114. The method of any one of claims 59 to 113, wherein the universal flanking primer sequence lacks secondary structure.

115. The method of any one of claims 59 to 114, wherein the universal flanking primer sequence is optimized for Tm.

116. The method of any one of claims 59 to 115, wherein the universal flanking primer sequence is optimized for GC content.

117. The method of any one of claims 59 to 116, wherein the universal flanking primer sequence comprises a sequence that is at least 90% identical or complementary to at least 12 contiguous nucleotides of ATGTCCGCCTACTTTA (SEQ ID NO: 1).

118. The method of any one of claims 59 to 117, 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, as compared to pairing between the first and second modified bases.

119. The method of any one of claims 59 to 118, wherein the primer set is capable of amplifying the target nucleic acid at the rate of at least 6number of cycles during an exponential phase of amplification.

120. The method of any one of claims 59 to 119, wherein the primer set permits detection of a single-copy nucleic acid in a biological sample within about 36%-66% fewer amplification cycles than would be required for said detection using only a single forward and a single reverse primer.Attorney Docket No.: 6010-0013WO01 121. The method of any one of claims 59 to 120, wherein the primer set is free of GC- rich regions.

122. The method of any one of claims 59 to 121, wherein the primer set is capable of amplifying the target nucleic acid at the rate of at least 6number of cyclesduring an exponential phase of amplification.

123. The method of any one of claims 59 to 122wherein said PCR reaction mixture comprises polyethylene glycol (PEG).

124. The method of any one of claims 59 to 123, wherein said PCR reaction mixture comprises PEG at a concentration of at least about 2 percent.

125. The method of any one of claims 59 to 124, wherein said PCR reaction mixture comprises PEG 8000 at a concentration of at least about 2 percent.

126. The method of any one of claims 59 to 125, wherein the primer set permits detection of a single-copy nucleic acid in a biological sample within about 36%-66% fewer amplification cycles than would be required for said detection using only a single forward and a single reverse primer.

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