Allele-specific amplification using composition of overlapping non-allele-specific primer and allele-specific blocker oligonucleotides

Abstract

To provide a composition of overlapping a non-allele-specific primer oligonucleotide and an allele-specific blocker oligonucleotide for allele-specific amplification.SOLUTION: Provided is an oligonucleotide composition including a blocker and a first primer oligonucleotide. The blocker oligonucleotide includes a first sequence having a target-neutral subsequence and a blocker variable subsequence. The non-target-specific subsequence is flanked on its 3' and 5' ends by the target-neutral subsequence and is continuous with the target-neutral subsequence. The first primer oligonucleotide is sufficient to induce enzymatic extension, where the first primer oligonucleotide includes a second sequence. The second sequence overlaps with the 5' end of the target-neutral subsequence by at least 5 nucleotides, where the second sequence includes an overlapping subsequence and a non-overlapping subsequence. The second sequence does not include the non-target-specific subsequence.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 000,114, filed May 19, 2014, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Sequence Listing The Sequence Listing is submitted with this application in electronic form only and is incorporated herein by reference. The Sequence Listing text file "14-21013-WO(260947.00251)_SL.txt" was created on May 18, 2015 and is 13,144 bytes in size. [Background technology]

[0003] Small differences in DNA and RNA sequences can lead to large differences in the overall physical health and wellness of organisms, including humans. For example, a single base change in a bacterial genome can lead to antibiotic resistance, and a single base change in a human genome can lead to cancer progression. With the maturation of the field of genomics and the concomitant discovery of many nucleic acid biomarker sequences and molecules, there is a strong demand from the biotechnology industry to develop reliable, robust, inexpensive and accurate nucleic acid assays that can distinguish single base changes. In particular, many PCR-based assays have been developed, which are allele-specific.

[0004] PCR-based approaches for selective detection of trace mutations can be broadly classified into two families of approaches: (A) allele-specific primers and (B) non-allele-specific primers with allele-specific blockers.

[0005] Examples of allele-specific primers include the Amplification Refractory Mutation System (ARMS), Allele-Specific Blocker PCR (ASB-PCR), and Competitive Allele-Specific TaqMan PCR (castPCR). (B) Examples include PNA blocker PCR and co-amplification with lower denaturing temperature PCR (COLD-PCR).

[0006] Prior to the present invention, allele-specific primers were generally superior for detecting known single-base mutations (higher mutation sensitivity), whereas non-allele-specific primers with allele-specific blockers were generally applied for detecting closely clustered multiple mutations (hot spots) or unknown mutation sequences.

[0007] Allele-specific PCR primers, such as ARMS, ASB-PCR, castPCR, etc., generally have an allele-specific nucleotide at the 3'-most position of the primer. Allele-specific PCR primers utilize the discriminatory power of DNA polymerase enzymes to specifically extend correctly paired bases, but are limited by the fact that if an incorrect DNA polymerase extension event occurs, traces of the allele nucleotide that was part of the primer will be incorporated into the template, making the subsequent amplification cycles non-specific. Allele-specific PCR primers are only specific up to the first incorrect amplification event. Allele-specific detection with non-allele-specific primers requires precise denaturation temperature control and restriction analysis of smaller sequences. Allele-specific detection with non-allele-specific primers has low mutation sensitivity and is more vulnerable to polymerase-introduced errors.

[0008] Therefore, there is a need for a composition of non-allele-specific primers and allele-specific blocker oligonucleotides with allele-specific amplification and improved mutation sensitivity.Therefore, the development of new approaches suitable for detecting low-abundance targets in a large excess of variants is essential for diagnostic applications.Thus, the present disclosure provides a composition of non-allele-specific primers and allele-specific blocker oligonucleotides to provide more accurate and efficient tools for disease diagnostic applications, such as cancer diagnosis. Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention relates to compositions of overlapping non-allele-specific primer oligonucleotides and allele-specific blocker oligonucleotides for allele-specific amplification. [Means for solving the problem]

[0010] The present invention provides an oligonucleotide composition comprising a blocker oligonucleotide and a first primer oligonucleotide. The blocker oligonucleotide comprises a first sequence having a target-neutral subsequence and a blocker variable subsequence. However, in some instances, the blocker oligonucleotide may be a first sequence having a target-neutral subsequence and a blocker variable subsequence. The blocker oligonucleotide may not include the blocker variable subsequence when the target nucleic acid to be detected is for the detection of an insertion. The blocker variable subsequence is flanked at its 3' and 5' ends by and contiguous with a target neutral subsequence. The first primer oligonucleotide is sufficient to direct enzymatic extension; wherein the first primer oligonucleotide comprises a second sequence. Such that the second sequence comprises overlapping and non-overlapping subsequences. The second sequence overlaps with the 5' end of the target neutral subsequence by at least 5 nucleotides. The second sequence does not contain any sequence homologous to the blocker variable subsequence.

[0011] The present invention further relates to a method for the amplification of a target sequence, the method comprising obtaining a sample containing one or more copies of a first nucleic acid having a variant sequence and at least one copy of a second nucleic acid; wherein the second nucleic acid has a target sequence. The target sequence and the variant sequence each comprise a homologous subsequence and a variable subsequence. The variable subsequence further comprises at least one nucleotide. Furthermore, the variable subsequence of the target sequence is a target specific subsequence and the variable subsequence of the variant sequence is a non-target specific subsequence.

[0012] After obtaining the sample, a blocker oligonucleotide is introduced into the sample. The blocker oligonucleotide comprises a first sequence having a target neutral subsequence and a blocker variable subsequence. The target neutral subsequence is complementary to a portion of a homology subsequence, and the blocker variable subsequence is complementary to a non-target specific subsequence. Further, the blocker variable subsequence is flanked at its 3' and 5' ends by and contiguous with the target neutral subsequence.

[0013] A first primer oligonucleotide is then introduced to the sample. The first primer oligonucleotide is sufficient to induce enzymatic extension. The first primer oligonucleotide comprises a second sequence that is complementary to a second portion of the homologous subsequence. Furthermore, the second sequence overlaps with the 5' end of the target neutral subsequence by at least 5 nucleotides, such that the second sequence comprises an overlapping subsequence and a non-overlapping subsequence. The second sequence does not comprise any sequence that is complementary to a variable subsequence.

[0014] In the next step, a DNA polymerase, nucleoside triphosphates, and one or more reagents necessary for polymerase-based nucleic acid amplification are introduced to the sample; the sample is reacted under conditions sufficient to achieve nucleic acid amplification.

[0015] An advantage of the present invention is the improved mutation sensitivity that matches and / or exceeds allele-specific primer approaches.

[0016] Another advantage of the present invention is the simple two-step thermal cycling protocol with remarkable (8° C.) temperature robustness.

[0017] Yet another advantage of the present invention is that it provides inexpensive DNA primer and blocker reagents without complex backbone or nucleotide modifications.

[0018] Yet another advantage of the present invention is its compatibility with high fidelity enzymes.

[0019] Yet another advantage of the present invention is that allele-specific blocker binds more strongly to variant than to target, so that non-allele-specific primer displaces target-bound blocker more favorably than variant-bound blocker.The advantage of using said non-allele-specific primer is that pseudo-amplified variants result in amplification products (amplicons) that carry variant sequences rather than target sequences.Therefore, specificity increases with each cycle.

[0020] This Summary is presented to introduce in a simplified form the disclosure, certain aspects, advantages and novel features of the invention, which are further described in the Detailed Description below. This Summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. [Brief description of the drawings]

[0021] The foregoing summary, as well as the following detailed description of exemplary embodiments, will be better understood when read in conjunction with the accompanying drawings, in which: For the purpose of illustrating the disclosure, there are shown in the drawings exemplary configurations of the disclosure; however, the disclosure is not limited to the specific methods and instrumentalities disclosed therein. [Figure 1] FIG. 1 illustrates a schematic diagram of non-allele-specific primers and allele-specific blockers for allele-specific amplification according to the present invention. [Diagram 2] FIG. 1 illustrates a schematic diagram of the thermodynamic design of primers and blockers according to the present invention. [Diagram 3] Figure 3 illustrates a schematic diagram of the calculation of ΔG° values ​​from sequences according to the present invention. Figure 3 discloses SEQ ID NOs: 1, 33, 1, 34, 2, 33, 2, and 34, respectively, in order of appearance. [Figure 4] FIG. 4 is a schematic diagram illustrating detection of trace alleles in human genomic DNA according to Example 1. FIG. 4 discloses SEQ ID NOs: 3-4, 35, 32, 36, and 32, respectively, in the order of appearance. [Diagram 5] FIG. 5 illustrates a schematic diagram of trace temperature robustness according to Example 2. FIG. 5 discloses, in order of appearance, SEQ ID NOs: 5-6 and 37-38, respectively. [Figure 6A] FIG. 1 illustrates a schematic of increased primer design flexibility according to Example 3. [Figure 6B] FIG. 6A illustrates a schematic diagram of the nucleic acid sequences of four sets of primer and blocker pairs for amplifying human SNP rs3789806 according to Example 3. FIG. 6B discloses SEQ ID NOs: 5-12 and 39-40, respectively, in order of appearance. [Figure 7A] FIG. 14 illustrates a schematic of a simulation of the effect of ΔG°rxn1 at fixed ΔΔG°=2 kcal / mol according to Example 4. [Figure 7B(I)]FIG. 7B illustrates a schematic diagram of the experimental validation of SMAD7 rs4939827 according to Example 4. FIG. 7B(I) discloses SEQ ID NOs: 3, 13-16, 4, 17, and 35-36, respectively, in order of appearance. [Figure 7B(II)] FIG. 7B illustrates a schematic diagram of the experimental validation of SMAD7 rs4939827 according to Example 4. FIG. 7B(II) discloses SEQ ID NOs: 3, 18-23, 36, and 35, respectively, in order of appearance. [Figure 8] FIG. 8 illustrates a schematic diagram of blockers with 3' non-homologous regions according to Example 5. FIG. 8 discloses SEQ ID NOs: 5, 24, and 37-38, respectively, in order of appearance. [Figure 9A] FIG. 1 illustrates a schematic of multiplexing using TaqMan® probes according to Example 6. [Figure 9B] FIG. 1 illustrates a schematic of multiplexing using TaqMan® probes according to Example 6. [Figure 10] FIG. 1 illustrates a schematic diagram of a protector for primers and blockers according to Example 7. [Figure 11A] FIG. 11A illustrates a schematic diagram of detection of the deletion (STAG2 rs200841330) according to Example 8. FIG. 11A discloses, in order of appearance, SEQ ID NOs: 25-26 and 41-42, respectively. [Figure 11B] FIG. 11B illustrates a schematic of the detection of an insertion (STAG2 rs200841330) according to Example 8. FIG. 11B discloses SEQ ID NOs: 25, 43, 42, and 41, respectively, in order of appearance. [Figure 12] FIG. 12 illustrates a schematic diagram of upstream blockers according to Example 9. FIG. 12 discloses, in order of appearance, SEQ ID NOs: 27-28 and 44-45, respectively. [Figure 13] FIG. 1 illustrates a schematic diagram of specific amplification of fungal ribosomal RNA-encoding DNA according to Example 10. [Figure 14]FIG. 14 is a schematic diagram illustrating Dual NASP according to Example 11. FIG. 14 discloses, in order of appearance, SEQ ID NOs: 29, 10, 46-49, and 30-31, respectively. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. The drawings described are schematic only and are non-limiting. In the drawings, the size of some of the elements may be exaggerated or distorted and are not drawn to scale for illustrative purposes. Unless something else is specifically specified, if an element of the invention is designated as "a" or "an" in the first occurrence, it will be designated as "the" or "said" on the second or subsequent occurrence.

[0023] The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all, embodiments of the invention are shown. Indeed, the invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy all legal requirements.

[0024] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0025] As used herein, the term "blocker oligonucleotide" refers to at least one continuous strand of about 12 to about 100 nucleotides in length, and, when so designated herein, may further comprise a functional group or nucleotide sequence at its 3' end, which prevents enzymatic extension during an amplification process such as the polymerase chain reaction.

[0026] As used herein, the term "primer oligonucleotide" refers to a molecule that contains at least one contiguous strand of about 12 to about 100 nucleotides in length and is sufficient to permit enzymatic extension during an amplification process such as the polymerase chain reaction.

[0027] As used herein, the term "target neutral subsequence" refers to a sequence of nucleotides that is complementary to a sequence in both the target nucleic acid and the variant nucleic acid. For example, a desired nucleic acid sequence (target nucleic acid) targeted for amplification may be present in a sample that contains nucleic acid molecules that have a sequence that is largely homologous to the target nucleic acid, with the exception of a variable region (variant nucleic acid), and in some instances, such a variable region differs from the target nucleic acid by only a single nucleotide. In this example, the target neutral subsequence is complementary to at least a portion of the homologous sequence shared between the two nucleic acids that is not a variable region. Thus, as used herein, the term "blocker variable subsequence" refers to a nucleotide sequence of a blocker oligonucleotide that is complementary to a variable region of a variant nucleic acid.

[0028] As used herein, the term "overlapping subsequence" refers to a nucleotide sequence of at least 5 nucleotides of a primer oligonucleotide that is homologous to a portion of a blocker oligonucleotide sequence used in the compositions described herein. The overlapping subsequence of the primer oligonucleotide may be homologous to any portion of the target neutral subsequence of the blocker oligonucleotide, whether 5' or 3' of the blocker variable subsequence. Thus, the term "non-overlapping subsequence" refers to a sequence of a primer oligonucleotide that is not an overlapping subsequence.

[0029] As used herein, the term "target sequence" refers to the nucleotide sequence of a nucleic acid that has a desired allele, such as a single nucleotide polymorphism, to be amplified, identified, or otherwise isolated. As used herein, the term "variant sequence" refers to the nucleotide sequence of a nucleic acid that does not have a desired allele. For example, in some instances, the variant sequence has a wild-type allele, while the target sequence has a mutant allele. Thus, in some instances, the variant sequence and the target sequence are derived from a common locus in a genome, and the respective sequences may be substantially homologous except for the region that has the desired allele, nucleotide, or group of nucleotides that varies between the two.

[0030] The present invention relates to an oligonucleotide composition. In an embodiment of the invention, the oligonucleotide composition comprises a non-allele-specific primer oligonucleotide and an allele-specific blocker oligonucleotide for allele-specific amplification.

[0031] In another embodiment of the invention, the oligonucleotide composition comprises a blocker oligonucleotide and a first primer oligonucleotide. The blocker oligonucleotide comprises a first sequence having a target neutral subsequence and a blocker variable subsequence. The blocker variable subsequence is adjacent to and contiguous with the target neutral subsequence at its 3' and 5' ends. In other words, the blocker variable subsequence may divide the target neutral subsequence into two portions, a portion to the 3' and a portion to the 5' of the blocker variable subsequence. The first primer oligonucleotide is sufficient to induce enzymatic extension; wherein the first primer oligonucleotide comprises a second sequence. The second sequence comprises overlapping and non-overlapping subsequences. The sequence of overlaps with the target neutral subsequence by at least 5 nucleotides. The second sequence does not include a blocker variable subsequence. Thus, the primer oligonucleotide may be characterized as a non-allele specific primer. The overlap region may be homologous to a portion of the target neutral subsequence of the blocker oligonucleotide either 5' to the blocker variable subsequence or 3' to the blocker variable subsequence. As shown in FIG. 1, the primer oligonucleotide overlap subsequence is homologous to the target neutral subsequence of the blocker 5' to the blocker variable subsequence (denoted as "C" in blocker), where the target neutral subsequence is the portion of the blocker on either side of the blocker variable subsequence C. FIG. 12 depicts an example where the primer overlap subsequence is homologous to a portion of the target neutral subsequence of the blocker that is 3' to the blocker variable subsequence C.

[0032] In yet another embodiment of the invention, the blocker oligonucleotide further comprises a functional group or a region of non-complementary sequence at or near the 3' end that prevents enzymatic extension. In one example, the functional group of the blocker oligonucleotide is selected from the group consisting of, but not limited to, a 3-carbon spacer or a dideoxynucleotide.

[0033] The second sequence has a standard free energy of hybridization (ΔG° PT ) and the first sequence gives rise to a standard free energy of hybridization (ΔG° BT ), which results in the following condition: +2kcal / mol≧ΔG° PT -ΔG° BT ≧-8kcal / mol Meet the following.

[0034] Non-overlapping subsequences have a standard free energy of hybridization (ΔG° 3 ), which results in the following condition: -4kcal / mol≧ΔG° 3≧-12kcal / mol Meet the following.

[0035] In one example, the second sequence overlaps with the 5' end of the target neutral subsequence by about 5 nucleotides to about 40 nucleotides, hi another example, the second sequence overlaps with the 5' end of the target neutral subsequence by about 7 nucleotides to about 30 nucleotides.

[0036] In one example, the concentration of the blocker oligonucleotide is about 2 to about 10,000 times higher than the concentration of the first primer oligonucleotide, and in another example, the concentration of the blocker oligonucleotide is about 5 to about 1,000 times higher than the concentration of the first primer oligonucleotide.

[0037] In yet another embodiment of the invention, the oligonucleotide composition further comprises a second primer oligonucleotide sufficient to direct enzymatic extension, the second primer oligonucleotide comprising a third sequence that does not overlap with the first sequence or the second sequence, is target-neutral, and is sufficient for use with the first primer oligonucleotide to amplify a region of a nucleic acid using polymerase chain reaction.

[0038] In yet another embodiment of the invention, the oligonucleotide composition further comprises a second blocker oligonucleotide. The second blocker oligonucleotide comprises a fourth sequence having a second target neutral subsequence and a second blocker variable subsequence. The second blocker variable subsequence is generally designed to bind to an antisense sequence of the target and therefore has the same sequence as a non-target specific subsequence of the target nucleic acid. The second blocker variable subsequence is flanked at its 3' and 5' ends by a second target neutral subsequence and is contiguous with the second target neutral subsequence. The third sequence comprises a second overlapping subsequence. The third sequence overlaps with the 5' end of the second target neutral subsequence by at least 5 nucleotides such that the third sequence comprises a second non-overlapping subsequence and a second non-overlapping subsequence.

[0039] In yet another embodiment of the invention, the second blocker oligonucleotide further comprises a second functional group or a second non-complementary sequence region at or near the 3' end that prevents enzymatic extension. In one example, the second functional group of the second blocker oligonucleotide is selected from the group consisting of, but not limited to, a 3-carbon spacer or a dideoxynucleotide.

[0040] The third sequence has a standard free energy of hybridization (ΔG° PT2 ), and the fourth sequence gives rise to the standard free energy of hybridization (ΔG° BT2 ), which results in the following condition: +2kcal / mol≧ΔG° PT2 -ΔG° BT2 ≧-8kcal / mol Meet the following.

[0041] The second non-overlapping subsequence has a standard free energy of hybridization (ΔG° 6 ), which results in the following condition: -4kcal / mol≧ΔG° 6 ≧-12kcal / mol Meet the following.

[0042] In one example, the third sequence overlaps with the 5' end of the second target neutral subsequence by about 5 nucleotides to about 40 nucleotides. In another example, the third sequence overlaps with the 5' end of the second target neutral subsequence by about 7 nucleotides to about 30 nucleotides.

[0043] In any of the above embodiments, the primer oligonucleotide and the blocker oligonucleotide may each be about 12 to about 100 nucleotides in length. In any of the above embodiments, the primer oligonucleotide and the blocker oligonucleotide may each be about 15 to about 90 nucleotides in length. In any of the above embodiments, the primer oligonucleotide and the blocker oligonucleotide may each be about 20 to about 80 nucleotides in length. In any of the above embodiments, the primer oligonucleotide and the blocker oligonucleotide may each be about 20 to about 70 nucleotides in length. In any of the above embodiments, the primer oligonucleotide and the blocker oligonucleotide may each be about 20 to about 60 nucleotides in length. In any of the above embodiments, the primer oligonucleotide and the blocker oligonucleotide may be 21, 22, 23, 24, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length, respectively.

[0044] In one example, the concentration of the second blocker oligonucleotide is about 2 to about 10,000 times higher than the concentration of the second primer oligonucleotide. In another example, the concentration of the second blocker oligonucleotide is about 5 to about 1,000 times higher than the concentration of the second primer oligonucleotide. In another example, the concentration of the second blocker oligonucleotide is about 10 to about 900 times higher than the concentration of the second primer oligonucleotide. In another example, the concentration of the second blocker oligonucleotide is about 20 to about 800 times higher than the concentration of the second primer oligonucleotide. In another example, the concentration of the second blocker oligonucleotide is about 30 to about 700 times higher than the concentration of the second primer oligonucleotide. In another example, the concentration of the second blocker oligonucleotide is about 40 to about 600 times higher than the concentration of the second primer oligonucleotide. In another example, the concentration of the second blocker oligonucleotide is about 10 to about 900 times higher than the concentration of the second primer oligonucleotide. The concentration of the second blocker oligonucleotide is about 50 to about 500 times higher than the concentration of the second primer oligonucleotide. In another example, the concentration of the second blocker oligonucleotide is about 60 to about 400 times higher than the concentration of the second primer oligonucleotide. In another example, the concentration of the second blocker oligonucleotide is about 70 to about 300 times higher than the concentration of the second primer oligonucleotide. In another example, the concentration of the second blocker oligonucleotide is about 80 to about 200 times higher than the concentration of the second primer oligonucleotide. In another example, the concentration of the second blocker oligonucleotide is about 90 to about 100 times higher than the concentration of the second primer oligonucleotide.

[0045] In yet another embodiment of the invention, the oligonucleotide composition further comprises reagents necessary for the polymerase chain reaction.

[0046] In yet another embodiment of the invention, the oligonucleotide composition further comprises a plurality of nucleoside triphosphates.

[0047] In yet another embodiment of the invention, the oligonucleotide composition further comprises a DNA polymerase or an RNA polymerase.

[0048] In yet another embodiment of the invention, the oligonucleotide composition further comprises reagents necessary for the polymerase chain reaction, a plurality of nucleoside triphosphates, and a DNA polymerase.

[0049] In yet another embodiment of the invention, the blocker variable subsequence is a single nucleotide.

[0050] The present invention further relates to a method for the amplification of a target sequence, the method comprising the steps of: (a) obtaining a sample containing one or more copies of a first nucleic acid having a variant sequence and at least one copy of a second nucleic acid; wherein said second nucleic acid has a target sequence. The target sequence and the variant sequence each have a homologous subsequence and a variable subsequence. The variable subsequence further comprises at least one nucleotide, but may comprise from 2 to 5 nucleotides. Furthermore, the variable subsequence of the target sequence is a target specific subsequence and the variable subsequence of the variant sequence is a non-target specific subsequence.

[0051] The method further comprises (b) introducing a blocker oligonucleotide to the sample, wherein the blocker oligonucleotide comprises a first sequence having a target neutral subsequence and a blocker variable subsequence, wherein the target neutral subsequence is complementary to a portion of the homology subsequence and the blocker variable subsequence is complementary to a non-target specific subsequence, and wherein the blocker variable subsequence is flanked at its 3' and 5' ends by and contiguous with the target neutral subsequence.

[0052] The method further comprises (c) introducing a first primer oligonucleotide to the sample, the first primer oligonucleotide being sufficient to induce enzymatic extension. The first primer oligonucleotide comprises a second sequence. Further, the second sequence is complementary to a second portion of the homologous subsequence, and the second sequence may overlap with the 5' end of the target neutral subsequence by at least 5 nucleotides, such that the second sequence comprises an overlapping subsequence and a non-overlapping subsequence. The second sequence does not comprise any sequence complementary to a variable subsequence.

[0053] The method includes the steps of: (d) introducing to the sample a DNA polymerase, nucleoside triphosphates, and one or more reagents necessary for polymerase-based nucleic acid amplification; and then (e) The method further includes reacting the sample under conditions sufficient to achieve nucleic acid amplification.

[0054] In an embodiment of the invention, the blocker oligonucleotide further comprises a functional group or a region of non-complementary sequence at or near the 3' end that prevents enzymatic extension. In one example, the functional group is selected from the group consisting of, but not limited to, a 3-carbon spacer or a dideoxynucleotide.

[0055] The second sequence has a standard free energy of hybridization (ΔG° PT ) and the first sequence gives rise to a standard free energy of hybridization (ΔG° BT ), which results in the following condition: +2kcal / mol≧ΔG° PT -ΔG° BT ≧-8kcal / mol Meet the following.

[0056] Non-overlapping subsequences have a standard free energy of hybridization (ΔG° 3 ), which results in the following condition: -4kcal / mol≧ΔG° 3 ≧-12kcal / mol Meet the following.

[0057] In one example, the second sequence overlaps with the 5' end of the target neutral subsequence of the blocker by about 5 nucleotides to about 40 nucleotides (i.e., an "overlapping subsequence"). In other words, the second sequence includes an overlapping subsequence that is homologous to a portion of the target neutral subsequence of the blocker that is 5' to the blocker variable subsequence of the blocker. In another example, the second sequence overlaps with the 5' end of the target neutral subsequence by about 7 nucleotides to about 30 nucleotides. In yet another example, the second sequence includes an overlapping subsequence that is homologous to a portion of the target neutral subsequence of the blocker that is 3' to the blocker variable subsequence of the blocker.

[0058] In one example, the concentration of the blocker oligonucleotide introduced into the sample is about 2 to about 10,000 times higher than the concentration of the first primer oligonucleotide introduced into the sample. In another example, the concentration of the blocker oligonucleotide introduced into the sample is about 5 to about 1,000 times higher than the concentration of the first primer oligonucleotide introduced into the sample. In another example, the concentration of the blocker oligonucleotide introduced into the sample is about 10 to about 500 times higher than the concentration of the first primer oligonucleotide introduced into the sample. In another example, the concentration of the blocker oligonucleotide introduced into the sample is about 20 to about 250 times higher than the concentration of the first primer oligonucleotide introduced into the sample. In another example, the concentration of the blocker oligonucleotide introduced into the sample is about 40 to about 125 times higher than the concentration of the first primer oligonucleotide introduced into the sample. In another example, the concentration of the blocker oligonucleotide introduced into the sample is about 50 to about 100 times higher than the concentration of the first primer oligonucleotide introduced into the sample. In another example, the concentration of the blocker oligonucleotide introduced to the sample is about 300 to about 400 times higher than the concentration of the first primer oligonucleotide introduced to the sample. In another example, the concentration of the blocker oligonucleotide introduced to the sample is about 500 to about 600 times higher than the concentration of the first primer oligonucleotide introduced to the sample.

[0059] In another embodiment of the invention, the DNA polymerase is a thermostable DNA polymerase.

[0060] In yet another embodiment of the invention, the method further comprises exposing the sample to at least 10 cycles of conditions sufficient to achieve nucleic acid amplification, each cycle having at least two different temperature exposures, one temperature exposure being 10 cycles. is at least 85°C and one temperature exposure is no greater than 75°C.

[0061] In yet another embodiment of the invention, the method further comprises the step of introducing to the sample an enzyme selected from the group consisting of a nicking enzyme, a recombinase, a helicase, an RNAse, a reverse transcriptase, or any combination thereof.

[0062] In yet another embodiment of the present invention, the method further comprises introducing a second primer oligonucleotide into the sample. The second primer oligonucleotide comprises a third sequence. Furthermore, the third sequence does not overlap with the variable subsequence. Furthermore, the third sequence is target-neutral and sufficient to be used with the first primer oligonucleotide to amplify a region of the nucleic acid having the target sequence.

[0063] In yet another embodiment of the present invention, the method further comprises introducing a second blocker oligonucleotide into the sample. The second blocker oligonucleotide comprises a fourth sequence; wherein the fourth sequence comprises a second target neutral subsequence and a second blocker variable subsequence. Furthermore, the second blocker variable subsequence is a sequence complementary to the blocker variable subsequence. Furthermore, the second blocker variable subsequence is adjacent to and contiguous with the second target neutral subsequence at its 3' and 5' ends. Additionally, the third sequence overlaps with the 5' end of the second target neutral subsequence by at least 5 nucleotides, such that the third sequence comprises a second overlapping subsequence and a second non-overlapping subsequence.

[0064] In yet another embodiment of the invention, the second blocker oligonucleotide further comprises a second functional group or a second region of non-complementary sequence at or near the 3' end that prevents enzymatic extension. In one example, the second functional group is selected from the group consisting of, but not limited to, a 3-carbon spacer or a dideoxynucleotide.

[0065] The third sequence has a standard free energy of hybridization (ΔG° PT2 ), and the fourth sequence gives rise to the standard free energy of hybridization (ΔG° BT2 ), which results in the following condition: +2kcal / mol≧ΔG° PT2 -ΔG° BT2 ≧-8kcal / mol Meet the following.

[0066] The second non-overlapping subsequence has a standard free energy of hybridization (ΔG° 6 ), which results in the following condition: -4kcal / mol≧ΔG° 6 ≧-12kcal / mol Meet the following.

[0067] In one example, the third sequence overlaps with the 5' end of the second target neutral subsequence by about 5 nucleotides to about 40 nucleotides, hi another example, the third sequence overlaps with the 5' end of the second target neutral subsequence by about 7 nucleotides to about 30 nucleotides.

[0068] In one example, the concentration of the second blocker oligonucleotide introduced to the sample is about 2 to about 10,000 times higher than the concentration of the second primer oligonucleotide introduced to the sample. In another example, the concentration of the second blocker oligonucleotide introduced to the sample is about 5 to about 1,000 times higher than the concentration of the second primer oligonucleotide introduced to the sample.

[0069] In yet another embodiment of the present invention, the method for amplification of a target sequence comprises the steps of removing an aliquot from a sample; and steps (b) to (e) as defined above. The method further includes the step of repeating the steps.

[0070] FIG. 1 illustrates a schematic diagram of non-allele-specific primers and allele-specific blockers for allele-specific amplification. The nucleic acid reagent mixture includes two oligonucleotide species, a primer (primer oligonucleotide) and a variant-specific blocker (blocker oligonucleotide), which work together to enable reliable amplification of rare targets in conjunction with polymerase-based amplification methods. The blocker may be modified at the 3' end with a non-extendible modification such as a dideoxynucleotide or a 3-carbon linker. The sequences of the primer and blocker are rationally designed based on the thermodynamics of their hybridization to the target and variant nucleic acid sequences. In some embodiments, the blocker is present in a significantly higher concentration than the primer, such that a preponderance of the target and variant nucleic acid sequences bind to the blocker before binding to the primer. The primer binds transiently to the blocker-target or blocker-variant molecule and has the potential to displace the blocker upon binding to the target or variant. Because the blocker sequence is specific to the variant target, displacement from the variant is less thermodynamically favored than displacement from the target. Thus, non-allele specific primers amplify target sequences with higher yield / efficiency than they do variant sequences, and the non-allele specific nature of the primers means that spuriously amplified variant sequences will carry the variant allele rather than the target allele, and subsequent amplification cycles will also show amplification bias in favor of the target.

[0071] A high concentration of variant-specific blocker provides a high probability that the blocker will initially bind to both the target and the variant before the primer has a chance to bind. The primer may initiate binding to both the target-blocker or variant-blocker complex via a unique region that does not overlap with the blocker, and then displace the blocker via an enzyme-free strand displacement process.

[0072] FIG. 2 illustrates a schematic of the thermodynamic design of primers and blockers. Primer and blocker sequences are rationally designed to achieve desirable reaction thermodynamics, where the primer, blocker, target, and variant nucleic acid sequences are subdivided into regions containing consecutive nucleotides, numbered 1 through 8 in FIG. 2. The standard free energy of hybridization between the primer and target (ΔG° PT ) and the standard free energy of hybridization between the blocker and the target (ΔG° BT ) satisfies: +2kcal / mol≧ΔG° PT -ΔG° BT ≧-8kcal / mol

[0073] A primer and blocker system that satisfies the above will disproportionately produce a large difference in hybridization yield in each cycle between primer binding to the target and primer binding to the variant, resulting in target-specific amplification.

[0074] The enzymatic free strand displacement process is driven by the relative thermodynamics of primer binding versus blocker binding. Blockers bind to targets (where a more negative ΔG° indicates a strong favorability), so the standard free energy of binding, ΔG°, is BT , ΔG° BT >ΔG° BV ) for variants (standard free energy of binding ΔG° BV In another embodiment of the invention, the primer hybridizes more favorably to the target (standard free energy of binding ΔG° PT , ΔG° PV = ΔG° PT ) for the variant (standard free energy of binding ΔG° PV ) to hybridize.

[0075] The reaction of a primer displacing a blocker bound to a target, BT+P<=>PT+B, has a standard free energy of ΔG° rxn1 = ΔG° PT -ΔG°BT This has a standard free energy ΔG° rxn2 = ΔG° PV -ΔG° BV Binding to variants with For good performance of the present invention, primers and blockers are usually rxn1 ≦0 and ΔG° rxn2 0. However, the relative concentrations of blocker and primer can affect the equilibrium distribution and effective thermodynamics of the reaction, so ΔG° rxn1 and ΔG° rxn2 The guidelines are not absolute.

[0076] FIG. 3 illustrates a schematic diagram of the calculation of ΔG° values ​​from sequences. There are different conventions for calculating ΔG° for different domain interactions; an exemplary energy calculation based on the nearest neighbor model is shown in FIG. 1-5 , ΔG° 4-7 , and ΔG° 4-8 The term is the standard free energy of hybridization initiation (ΔG° init ) and an extra base stack adjacent to the sixth region. The standard free energy ΔG° calculated using other methods has the same ΔG° PT , ΔG° PV , ΔG° BT , and ΔG° BV values, but individual regions (e.g., ΔG° 3-6 The thermodynamics of ΔG° may be different. The illustrative examples are intended to show the method of ΔG° calculation used in the present invention and are not intended to suggest sequences for the assay; the sequences listed may be too short for stable binding. ΔG° from primer sequences, blocker sequences, target sequences, variant sequences, operating temperature, and operating buffer conditions. PT , ΔG° PV , ΔG° BT , and ΔG° BV The calculation of is known to those skilled in the art.

[0077] Figure 4 shows the probe and blocker design of the present invention and the subsequences around the SMAD7 locus of two human repository samples, NA18537 and NA18562. The primer / blocker set design was designed to specifically amplify the A variant of the SMAD7 single nucleotide polymorphism (SNP). The blocker amplifies the G-barrier variant of the SMAD7 SNP. The Taq DNA was designed to be perfectly complementary to the variant template carrying the Taq DNA fragment. It is functionalized at the 3' end with a 3-carbon linker (C3) that prevents enzymatic extension by DNA polymerase. The reverse primer is attached 3' to the forward primer and is not specific for either allele. To illustrate the terminology used to describe the various subsequences of the primer and blocker, the oligonucleotides can be described as follows: The blocker oligonucleotide comprises a target neutral subsequence that includes sequences to the 5' and 3' of a cytosine nucleotide that is shown outside the alignment of the rest of the sequence, in this example the cytosine nucleotide represents the blocker variable subsequence of the blocker. The overlapping subsequence of the primer oligonucleotide comprises a sequence homologous to the blocker sequence on the 5' side of the blocker variable subsequence (cytosine).

[0078] FIG. 5 shows the results of human SNP rs at annealing / extension temperatures ranging from 56°C to 66°C. 1 shows the performance of a primer blocker pair targeting the 3789806(C / G)C allele.

[0079] 6A and 6B show primer designs with more design space to avoid unexpected interactions.

[0080] 7A and 7B show ΔG° rxn1 We show that it is possible to control the behavior of allele-specific PCR through the design of ΔG° rxn1The simulation in FIG. 7A suggests that if primers and blockers are designed such that G is more positive, the ΔCq value will be larger. rxn1 At more positive values ​​of , the amplification of the target is slower, resulting in larger values ​​of Cq. The simulations assume a fixed ΔΔG° of 2 kcal / mol. The top subgraph shows the effect of different values ​​of ΔG° on the hybridization yield. rxn1 (star) and ΔG° rxn2 The bottom subgraph shows the predicted effect of (dots) on the amplification of the target and variants for each primer and blocker pair. The curves are shown. In all cases shown, the concentration ratio of blocker to primer is 20:1. Figure 7B(I) and Figure 7B(II) show the effect of ΔG° on amplification selectivity. rxn1 The experimental results are shown below. rxn1 Values ​​of ΔG° were achieved through lengthening or shortening the 3' end of the blocker. As expected, more positive ΔG° rxn1 This results in a larger Cq for both the target and the variant. We have determined that the optimal intermediate ΔG° results in a larger ΔCq for the target and a relatively small Cq delay (Cq<28). rxn1 Unlike the simulation, ΔCq is ΔG° rxn1 The Cq concentration did not increase monotonically with Cq. Primer dimer formation and non-specific amplification result in Cq ceiling.

[0081] A kinetic simulation of the PCR process is expressed as follows: Tfn+1=Tfn+Pn·[Pn / (Pn+Bn)+Y(ΔG° rxn1 )·Bn / (Pn+Bn)]·Trn Trn+1=Trn+Rn·Tfn Vfn+1=Vfn+Pn·[Pn / (Pn+Bn)+Y(ΔG° rxn2 )·Bn / (Pn+Bn)]·Vrn Vrn+1=Vrn+Rn Vfn where Tfn is the normalized concentration of the target forward strand at cycle n, Trn is the normalized concentration of the target reverse strand at cycle n, Vfn is the normalized concentration of the variant forward strand at cycle n, Vrn is the normalized concentration of the variant forward strand at cycle n, Pn is the normalized concentration of the forward primer at cycle n, Bn is the normalized concentration of the blocker at cycle n, Rn is the normalized concentration of the reverse primer at cycle n, and Y(ΔG°) is the hybridization yield of the displacement reaction at a given ΔG° reaction standard free energy. The simulation results are shown in Figure 7A.

[0082] Figure 8 shows a blocker with a non-homologous sequence at the 3' end due to non-extendable chemical functionalization. The non-homologous sequence does not bind to downstream regions of either the target or variant, so the non-homologous region effectively prevents enzymatic extension. The target Cq and ΔCq values ​​are similar to those shown in Example 1, indicating that similar functional groups at the 3' end, such as a 3-carbon spacer or dideoxynucleotide, a non-homologous sequence, can effectively prevent enzymatic extension, respectively, where fP, rP, and B represent the forward primer, reverse primer, and blocker (relative to the forward primer).

[0083] In standard blocking PCR, the forward primer only biases the amplification of the target sequence; the reverse primer approximately equally amplifies both the target and the variant. If both the forward and reverse primers bias the amplification of only the target sequence, the amplification specificity can be further improved (with approximately quadratic improvement in mutation sensitivity). To achieve dual target-specific amplification, the forward and reverse primers are designed to be separated by a short distance, and they overlap with the variant-specific blocker. In that limit, the primer binding regions are separated by a single nucleotide whose species varies between the target and the variant. The forward and reverse variant-specific blockers are partially complementary to each other, as they bind to the complementary strand of the target and both span the mutation region. The blockers are designed such that, although partially complementary under the operating conditions, the majority of the blocker molecules do not hybridize to each other.

[0084] 9A and 9B show the primer-blocker systems designed for human BRAF rs3789806 and IL23R rs1884444 SNPs, respectively. These were multiplexed in the same reaction. TaqMan® probes targeting the corresponding downstream of the blocker binding regions were designed and used as the readout mechanism.

[0085] Figure 10 shows that non-specific binding of primers or blockers to other primers, blockers, or templates results in non-specific amplification. The bottom section provides a protector design that suppresses non-specific binding of primers and blockers. The protector oligonucleotides are partially complementary to the primer and blocker sequences. The presence of a stoichiometric excess of protector results in both primers and blockers being partially double-stranded. The blocker has a new region 11 whose sequence is non-complementary to region 5 in the nucleic acid sequence, and the protector has a new region 10 whose sequence is complementary to region 11.

[0086] Figures 11A and 11B show primers and blockers designed to detect deletions and insertions, respectively. The primers are non-target specific, and the method can also be used to detect deletions or insertions of an uncertain number of bases or uncertain positions.

[0087] Figure 12 shows a subembodiment of the invention by placing a primer 3' to the SNP position and a blocker 5' to the primer. This implementation produces hybridization specificity only in the first cycle, and the ΔCq is smaller than the preferred design.

[0088] FIG. 13 shows the sequence of fungal 18S DNA fragments from three pathogenic fungal species in a large excess of homologous human DNA. Because the fungal 18S subsequence and its homologous human subsequence differ in multiple regions, we designed corresponding blocker species for both the forward and reverse primers to maximize amplification specificity.

[0089] Figure 14 describes the use of target-specific amplification for both forward and reverse primers by using two sets of variant allele blockers, one for each primer. The blockers are partially complementary to each other, but many of them do not hybridize to each other under operating conditions. The blockers and primers are designed so that the forward primer cannot extend off the reverse blocker and the reverse primer cannot extend off the forward blocker. Figure 14 shows preliminary experimental results.

[0090] The primer and blocker combination may be applied to other enzymatic amplification assays for nucleic acids, including, but not limited to, nicking enzymatic amplification reaction (NEAR), loop-mediated isothermal amplification (LAMP), and rolling circle amplification (RCA).

[0091] The present invention is particularly suitable for detecting low abundance target with large excess of variant.For cancer diagnosis application of the present invention, variant can refer to wild type DNA sequence and target can refer to cancer DNA sequence.For infectious disease diagnosis application of the present invention, variant can refer to pathogen DNA sequence and target can refer to homologous human DNA sequence. [Example] EXAMPLES

[0092] FIG. 4 shows the probe and blocker design and the subsequences around the SMAD7 locus of two human repository samples, NA18537 and NA18562, in accordance with the present invention. A healthy human single nucleotide polymorphism (SNP) rs4939827 (C / T) in the SMAD7 locus was selected for proof-of-concept validation. The T allele was arbitrarily designated as the target and the C allele as the variant. Human repository genomic DNA samples NA18562 (homozygous for the C allele) and NA18537 (homozygous for the T allele) were purchased from Coriell® and the corresponding nucleotide sequences were analyzed using 1000Genomes. Retrieved from the website. To prevent unintended polymerization, the 3' end of the blocker was modified with a C3 spacer. qPCR experimental results distinguish 0.1% target (99.9% variant) from 0% target (100% variant). 0.1% target samples were prepared by mixing 0.1% NA18537 with 99.9% NA18562. The average difference in quantification cycles (ΔCq) between 100% variant and 100% target was 14.8, and the average difference between 100% variant (denoted as "WT" in the graph) and 0.1% target was 4.2. All experiments were performed on a 96-well plate in a Bio-Rad CFX96™ qPCR instrument. The experiments were carried out in a 30 well plate. In each well, 200 nM primer, 2 μM blocker, and 20 ng human genomic DNA were mixed in Bio-rad iTaq™ SYBR® Green Supermix. After a 3 min initiation process at 95°C, 65 cycles were performed with a 10 s denaturation step at 95°C and a 30 s annealing / extension step at 60°C. The example experiments shown in Figures 5-12 used a similar protocol.

[0093] [Table 1]

[0094] Table 1 shows examples of allele-specific amplification results for four sets of SNP pairs in human genomic DNA samples NA18562 and NA18537 from the 1000Genomes database. For each SNP pair, primer and blocker designs for each allele variant were designed and tested. The base type shown in the "Blocker" column represents the suppressed allele (variant), and "-" means no blocker was added. In all cases, large ΔCq values ​​between the two alleles were observed, ranging from 7.1 to 14.6. All experiments were performed in triplicate with 400 nM of each primer and 4 μM of blocker in Bio-rad iTaq™ SYBR® Green Supermix. The results showed the performance of the thermodynamically determined designs without any empirical optimization of primer and blocker sequences or experimental conditions. EXAMPLES

[0095] FIG. 5 shows the results of human SNP rs37 at annealing / extension temperatures ranging from 56°C to 66°C. Example 2 of the present invention shows the performance of a primer-blocker pair targeting the 89806(C / G)C allele. Example 2 shows good allele-specific amplification from 56° C. to 64° C., demonstrating a temperature robustness of at least 8° C. 400 nM of each primer and 4 μM blocker was used and other experimental conditions were the same as those described in Example 1, except for the annealing / extension temperature. EXAMPLES

[0096] 6A and 6B show Example 3 of the present invention. The primer design shown in the present invention provided more design space to avoid unexpected interactions. Four sets of primers were designed to specifically amplify the human SNPrs3789806C allele. The nucleic acid sequences of the primer and blocker pair show a ΔCq > 11. 400 nM of each primer and 4 μM of blocker were used, and other experimental conditions were the same as those described in Example 1. EXAMPLES

[0097] 7A, 7B(I) and 7B(II) show ΔG° rxn1 Example 4 of the present invention shows the control of allele-specific PCR behavior through the design of ΔG° rxn1 The simulation in FIG. 7A suggested that if primers and blockers are designed such that G is more positive, the ΔCq value would be larger. However, ΔG rxn1 At more positive values ​​of , the amplification of the target is slower, resulting in larger values ​​of Cq. The simulations assumed a fixed ΔΔG° of 2 kcal / mol. The top subgraph shows the effect of different values ​​of ΔG° on the hybridization yield. rxn1 (star) and ΔG° rxn2 The effect of ΔG° (dots) on amplification selectivity was predicted. The lower subgraph predicted simulated amplification curves of the target and variants for each primer and blocker pair. In all cases shown, the concentration ratio of blocker to primer was 20:1. Figure 7B(I) and Figure 7B(II) show the effect of ΔG° on amplification selectivity.rxn1 The experimental results are shown below. rxn1 Values ​​of ΔG° were achieved through extension or shortening of the 3' end of the blocker. As expected, more positive ΔG° rxn1 This resulted in a larger Cq for both the target and the variant. rxn1 resulted in the largest ΔCq and a relatively small Cq delay (Cq<28) for the target. Unlike the simulation, ΔCq was smaller than ΔG° because primer dimer formation and nonspecific amplification occurred due to Cq sealing. rxn1 The concentration of each primer was 200 nM and the blocker was 2 μM. The other experimental conditions were the same as those described in Example 1. EXAMPLES

[0098] FIG. 8 shows Example 5 of the present invention for a blocker with a non-homologous sequence at the 3' end rather than non-extendable chemical functionalization. The non-homologous sequence does not bind to downstream regions of either the target or the variant, so the non-homologous region effectively prevents enzymatic extension. 400 nM of each primer and 4 μM of blocker were used, and other experimental conditions were the same as those described in Example 1. The target Cq and ΔCq values ​​are similar to those shown in FIG. 4, indicating that similar functional groups at the 3' end, such as 3-carbon spacers or dideoxynucleotides, non-homologous sequences, can effectively prevent enzymatic extension. In FIG. 8, fP, rP, and B represent the forward primer, reverse primer, and blocker (for the forward primer), respectively. EXAMPLES

[0099] Figures 9A and 9B show Example 6 of the present invention. To perform real-time PCR of our non-allele-specific primers and allele-specific blockers, a reverse primer was also required, which was not allele-specific. SYBR® Green Supermix and TaqMan® were used in the experiment, and the results showed that there was no significant difference between the two. Human BRAF rs3789806 and IL23Rrs18 The primer-blocker system designed for the 84444 SNPs is the same The TaqM targeting the corresponding downstream of the blocker binding region was multiplexed in the reaction. An® probe was designed and used as a readout mechanism. The TaqMan® probe for the rs3789806 amplicon was modified with a FAM fluorophore, an Iowa Black FQ quencher, and an internal ZEN quencher to obtain the IL23R rs18 The TaqMan® probe for the 84444 amplicon was modified with a Cy5 fluorophore and an Iowa Black RQ quencher. Figure 9B shows the results of multiplex detection of the BRAF rs3789806C allele and the IL23R rs1884444 T allele. The base type indicates the allele (variant) that was suppressed by the blocker. Multiplex detection of the 444 G allele. 400 nM of each primer, 4 μM of each blocker, and 200 nM of each TaqMan® probe were mixed in Bio-rad iQ Supermix. The qPCR protocol was as described in Example 1. was the same as that shown in EXAMPLES

[0100] Figure 10 shows Example 7 of the present invention for non-specific binding of primers or blockers to other primers, blockers, or templates. Example 7 resulted in non-specific amplification. The lower section provided a protector design to suppress non-specific binding of primers and blockers. The blocker had a new region 11 whose sequence was non-complementary to region 5 in the nucleic acid sequence, and the protector had a new region 10 whose sequence was complementary to region 11. EXAMPLES

[0101] FIG. 11A and FIG. 11B show Example 8 of the present invention for primers and blockers designed to detect deletions and insertions. The primers were used to demonstrate the results of the validation study. 400 nM of each primer and 4 μM of blocker were used, and other experimental conditions were the same as those shown in Example 1. Because the primers are non-allele specific, this method can also be used to detect deletions or insertions of uncertain numbers of bases or uncertain positions. EXAMPLES

[0102] Figure 12 shows Example 9 of the present invention by placing a primer 3' to the SNP position and a blocker 5' to the primer. Example 9 produced hybridization specificity only in the first cycle, and ΔCq was smaller than the preferred design. The primer concentration was 400 nM and the blocker concentration was 4 μM. The experimental conditions were the same as those shown in Example 1. EXAMPLES

[0103] FIG. 13 shows the sequence of fungal 18S DNA fragments from three pathogenic fungal species in a large excess of homologous human DNA. Example 10 of the present invention shows the selective amplification of a gBlock sequence. Since the fungal 18S subsequence and its homologous human subsequence differ in multiple regions, corresponding blocker species were designed for both the forward and reverse primers to maximize amplification specificity. gBlock fragments (sequence-verified 400 bp double-stranded DNA, IDT) of 18S subsequences from three fungal species and the consensus sequence in humans were used in the experiment. 60,000 copies (0.1%) of the DNA gBlock fragment of each fungus, 60 copies 0 copies (100% human) and 0 copies (0.0001%) were mixed separately with 60,000,000 copies of human DNA. In all cases, the results showed that sequences were detected at less than one part per million. 200 nM of each primer and 1 μM of each blocker were used, and other experimental conditions were the same as those shown in Example 1. EXAMPLES

[0104] Figure 14 shows Example 11 of the present invention. Figure 14 describes the use of allele-specific amplification with both forward and reverse primers; one using two sets of variant allele blockers for each primer. Although the blockers are partially complementary to each other, the operating conditions did not support much hybridization with each other. The blockers and primers were designed so that the forward primer could not extend off the reverse blocker and the reverse primer could not extend off the forward blocker. Figure 14 presents preliminary experimental results.

[0105] The foregoing descriptions of specific embodiments of the present disclosure have been presented for purposes of illustration and description. The exemplary embodiments have been chosen and described in order to best explain the principles of the invention and its practical application, thereby enabling others skilled in the art to best utilize the invention, and various embodiments with various modifications are suitable for the particular uses intended.

Claims

1. A method for amplifying a target sequence, comprising the steps of: a. obtaining a sample containing one or more copies of a first nucleic acid comprising a variant sequence, and potentially containing at least one copy of a second nucleic acid comprising the target sequence, wherein the target sequence and the variant sequence each comprise a homologous subsequence and a variable subsequence, the variable subsequence comprising an insertion or deletion present in the target sequence but not present in the variant sequence, the variable subsequence of the target sequence being a target specific subsequence, and the variable subsequence of the variant sequence being a non-target specific subsequence; b. introducing a blocker oligonucleotide into the sample comprising a first sequence comprising a target neutral subsequence and a blocker variable subsequence, the blocker variable subsequence being flanked at its 3' and 5' ends by and contiguous with the target neutral subsequence, the blocker variable subsequence dividing the target neutral subsequence into two portions, a portion to the 3' of the blocker variable subsequence and a portion to the 5' of the blocker variable subsequence, the target neutral subsequence being complementary to portions of the homology subsequence of both the first target nucleic acid and a first variant nucleic acid, the blocker variable subsequence being complementary to a first variant subsequence of the first variant nucleic acid, and the blocker oligonucleotide comprising a non-complementary sequence region at its 3' end that prevents enzymatic extension and that is not complementary to sequences contiguous with the target neutral subsequence; c. introducing a first primer oligonucleotide into the sample, said first primer oligonucleotide sufficient to induce enzymatic extension, said first primer oligonucleotide comprising a second sequence, said second sequence being complementary to a second portion of said homologous subsequence, said second sequence overlapping with said target neutral subsequence of said blocker oligonucleotide by at least 5 nucleotides such that said second sequence comprises overlapping and non-overlapping subsequences, and said second sequence does not comprise any sequence complementary to said variable subsequence; the second sequence of the blocker oligonucleotide gives rise to a standard free energy of hybridization (ΔG°PT) to the target sequence, the first sequence of the blocker oligonucleotide gives rise to a standard free energy of hybridization (ΔG°BT) to the target sequence that satisfies the following condition: +2 kcal / mol≧ΔG°PT-ΔG°BT≧−8 kcal / mol; and the non-overlapping subsequences give rise to a standard free energy of hybridization (ΔG°3) to the target sequence that satisfies the following condition: −4 kcal / mol≧ΔG°3≧−12 kcal / mol; d. introducing to the sample one or more reagents necessary for DNA polymerase, nucleoside triphosphates, and polymerase-based nucleic acid amplification; and e. reacting the sample under conditions sufficient to achieve nucleic acid amplification;

2. The method of claim 1, wherein a variable subsequence is an insertion that is present in the target sequence but not in the variant sequence, the variable subsequence of the variable sequence is absent, and a blocker variable subsequence is absent.

3. The method of claim 1, wherein the variable subsequence is a deletion that is present in the target sequence but not in the variant sequence.

4. The method of claim 1, wherein the DNA polymerase is a thermostable DNA polymerase and the conditions sufficient to achieve nucleic acid amplification include subjecting the sample to at least 10 cycles, each cycle including at least two different temperature exposures, one temperature exposure being at least 85°C and one temperature exposure being no greater than 75°C.

5. The method of claim 1, wherein the overlapping subsequence comprises a portion of the 5' end of the target neutral subsequence, said portion being from about 5 nucleotides to about 40 nucleotides, or optionally from about 7 nucleotides to about 30 nucleotides.

6. The method of claim 1, wherein the concentration of the blocker oligonucleotide introduced to the sample is about 2 to about 10,000 times higher than the concentration of the first primer oligonucleotide introduced to the sample.

7. The method of claim 1, wherein the concentration of the blocker oligonucleotide introduced to the sample is about 5 to about 1,000 times higher than the concentration of the first primer oligonucleotide introduced to the sample.

8. An oligonucleotide composition for carrying out any of the methods of claims 1 to 7, comprising: (a) a first blocker oligonucleotide comprising a first sequence comprising a first target neutral subsequence and a first blocker variable subsequence, the first blocker variable subsequence is flanked at its 3' and 5' ends by and contiguous with the first target neutral subsequence, the first blocker variable subsequence divides the first target neutral subsequence into two portions, a portion to the 3' of the first blocker variable subsequence and a portion to the 5' of the first blocker variable subsequence, the first target neutral subsequence is complementary to a first portion of a homology subsequence of both the first target nucleic acid and the first variant nucleic acid, the first blocker variable subsequence is complementary to a first variant subsequence of the first variant nucleic acid, and the first blocker oligonucleotide includes a non-complementary sequence region at its 3' end that prevents enzymatic extension and is not complementary to sequences contiguous with the first target neutral subsequence. the first blocker oligonucleotide; (b) a first primer oligonucleotide sufficient to direct enzymatic extension for amplification of a desired first target sequence, said first primer oligonucleotide comprising a second sequence complementary to a second portion of a homology subsequence of both said first target nucleic acid and said first variant nucleic acid, said second sequence overlapping with said target neutral subsequence by at least 5 nucleotides such that said second sequence comprises a first overlapping subsequence and a first non-overlapping subsequence, and said second sequence does not comprise said first blocker variable subsequence; The second sequence gives rise to a standard free energy of hybridization (ΔG°PT) for the target nucleic acid, and the first sequence gives rise to a standard free energy of hybridization (ΔG°BT) for the target nucleic acid, which satisfies the following condition: +2 kcal / mol≧ΔG°PT-ΔG°BT≧−8 kcal / mol; the first non-overlapping subsequence of the first primer oligonucleotide generates a standard free energy of hybridization (ΔG°3) to the target nucleic acid that satisfies the following condition: -4 kcal / mol≧ΔG°3≧-12 kcal / mol; the first primer oligonucleotide; (c) a second blocker oligonucleotide comprising a fourth sequence comprising a second target neutral subsequence and a second blocker variable subsequence, the second blocker variable subsequence is flanked at its 3' and 5' ends by and contiguous with the second target neutral subsequence, the second blocker variable subsequence divides the second target neutral subsequence into two portions, a portion to the 3' of the second blocker variable subsequence and a portion to the 5' of the second blocker variable subsequence, the second target neutral subsequence is complementary to a first portion of a homology subsequence of both the second target nucleic acid and the second variant nucleic acid, the second blocker variable subsequence is complementary to a second variant subsequence of the second variant nucleic acid, and the second blocker oligonucleotide includes a non-complementary sequence region at its 3' end that prevents enzymatic extension and is not complementary to sequences contiguous with the second target neutral subsequence. the second blocker oligonucleotide; (d) a second primer oligonucleotide sufficient to direct enzymatic extension for amplification of a desired second target sequence, said second primer oligonucleotide comprising a third sequence complementary to a second portion of a homology subsequence of both said second target nucleic acid and said second variant nucleic acid, said third sequence overlapping with said second target neutral subsequence by at least 5 nucleotides such that said third sequence comprises a second overlapping subsequence and a second non-overlapping subsequence, and said third sequence does not comprise said second blocker variable subsequence; The third sequence generates a standard free energy of hybridization (ΔG°PT2) to the second target nucleic acid, and the fourth sequence generates a standard free energy of hybridization (ΔG°BT2) to the target nucleic acid, satisfying the following condition: +2 kcal / mol≧ΔG°PT2−ΔG°BT2≧−8 kcal / mol; the second non-overlapping subsequence of the second primer oligonucleotide generates a standard free energy of hybridization (ΔG°3) to the second target nucleic acid that satisfies the following condition: -4 kcal / mol≧ΔG°3≧-12 kcal / mol; the second primer oligonucleotide; 9. (a) a first overlapping subsequence comprises a portion of a 5' end of a first target neutral subsequence, said portion being from about 5 nucleotides to about 40 nucleotides; (b) the second overlapping subsequence comprises a portion of the 5' end of the second target neutral subsequence, said portion being from about 5 nucleotides to about 40 nucleotides; or (c) both (a) and (b); The oligonucleotide composition of claim 8.

10. (a) a first overlapping subsequence comprises a portion of a 5' end of a first target neutral subsequence, said portion being from about 7 nucleotides to about 30 nucleotides; (b) the second overlapping subsequence comprises a portion of the 5' end of the second target neutral subsequence, said portion being from about 7 nucleotides to about 30 nucleotides; or (c) both (a) and (b); The oligonucleotide composition of claim 8. (a) the concentration of the first blocker oligonucleotide is about 2 to about 10,000 times higher than the concentration of the first primer oligonucleotide; (b) the concentration of the second blocker oligonucleotide is about 2 to about 10,000 times greater than the concentration of the second primer oligonucleotide; or (c) both (a) and (b); The oligonucleotide composition of claim 8. (a) the concentration of the first blocker oligonucleotide is about 5 to about 1,000 times higher than the concentration of the first primer oligonucleotide; (b) the concentration of the second blocker oligonucleotide is about 5 to about 1,000 times greater than the concentration of the second primer oligonucleotide; or (c) both (a) and (b); The oligonucleotide composition of claim 8. (a) a reagent necessary for a polymerase chain reaction method; (b) a plurality of nucleoside triphosphates; or (c) a DNA polymerase; The oligonucleotide composition of claim 8, further comprising:

14. (a) the first blocker variable subsequence is absent; (b) the second blocker variable subsequence is absent; or (c) both (a) and (b); The oligonucleotide composition of claim 8. (a) the concentration of the first blocker oligonucleotide is about 10 to about 500 times higher than the concentration of the first primer oligonucleotide; and / or (b) the concentration of the second blocker oligonucleotide is about 10 to about 500 times greater than the concentration of the second primer oligonucleotide; The oligonucleotide composition of claim 8.

16. The oligonucleotide composition of claim 8, further comprising a target nucleic acid.

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