PCR METHOD AND PCR KIT FOR INCREASING ALLELIC DISCRIMINATION

Abstract

The present invention relates to a method and a kit for detecting alleles of which the specificity and sensitivity in a DNA polymerase chain reaction (PCR), which is widely used for the detection of minor alleles such as single nucleotide polymorphisms or somatic mutations, are increased. More specifically, the present invention relates to a PCR-based method and kit for single nucleotide polymorphism (SNP) genotyping and somatic mutation detection, the method and kit adding a partially or fully double-stranded oligonucleotide for increasing discrimination to a PCR solution for selective amplification of alleles, so that PCR amplification is not affected when a primer 3′ terminal base is complementary (3′-matched) to a template but PCR amplification is strongly inhibited when a 3′ terminal base is not complementary (3′-mismatched).

Claims

1. A PCR kit for detection of a mutant, the kit comprising: (a) a forward primer and a reverse primer for at least one template including a target DNA sequence having a potential mutation locus; (b) a DNA polymerase for DNA polymerization from the forward primer and the reverse primer which combine with the template; and (c) a discrimination-boosting oligonucleotide which is complementary to none of the template, the forward primer, and the reverse primer, can each reversibly combine with the DNA polymerase, and each form, in part or in entirety, a duplex.

2. The PCR kit of claim 1, further comprising: (d) at least one template including a target DNA sequence having a potential mutation locus.

3. The PCR kit of claim 1, wherein a first base at the 3′ terminus of the forward primer corresponds to the potential mutation locus of the target DNA sequence.

4. The PCR kit of claim 1, wherein the forward primer is an allele-specific (AS) primer or an amplification refractory mutation system (ARMS) primer.

5. The PCR kit of claim 1, wherein the discrimination-boosting oligonucleotide is at least one selected from among a DNA duplex, an RNA/DNA hybrid duplex, a double-stranded oligonucleotide, a partially or entirely complementary oligonucleotide single strand(s) capable of forming a partial or entire DNA duplex, a partially or entirely complementary oligonucleotide single strand(s) capable of forming a partial or entire DNA/RNA hybrid duplex, a partially or entirely complementary oligonucleotide single strand(s) capable of forming a partial or entire double-stranded oligonucleotide, and an oligonucleotide capable of forming a partial or perfect hairpin duplex.

6. The PCR kit of claim 1, wherein the discrimination-boosting oligonucleotide includes any sequence.

7. The PCR kit of claim 1, wherein the mutation in the target DNA sequence is a single nucleotide polymorphism.

8. The PCR kit of claim 1, wherein the DNA polymerase is a thermostable DNA polymerase.

9. The PCR kit of claim 8, wherein the DNA polymerase is a wild-type or a mutant DNA polymerase.

10. The PCR kit of claim 1, wherein the discrimination-boosting oligonucleotide has a length of 10 to 100 bases (both inclusive).

11. The PCR kit of claim 1, wherein the discrimination-boosting oligonucleotide has a length of 15 to 50 bases (both inclusive).

12. The PCR kit of claim 1, wherein the discrimination-boosting oligonucleotide has a Tm identical or greater than an annealing temperature set forth for PCR amplification.

13. The PCR kit of claim 1, wherein the discrimination-boosting oligonucleotide has a Tm of 50-85° C.

14. The PCR kit of claim 1, further comprising a fluorescence resonance energy transfer probe modified with a reporter and a quencher.

15. A method for detection of a genetic mutation, the method comprising the steps of: (a) providing a template including a target DNA sequence having a potential mutation locus, a forward primer and a reverse primer both combining with the template, a DNA polymerase polymerizing DNA from the forward primer and the reverse primer, a discrimination-boosting oligonucleotides that is complementary to none of the template, the forward primer, and the reverse primer, can reversibly combine with the DNA polymerase, and can form a partial or entire a duplex; (b) performing a polymerase chain reaction using the DNA polymerase in the presence of the template, the forward primer, the reverse primer, and the discrimination-boosting oligonucleotide; and (c) acquiring an amplification curve from the reaction of step (b).

16. The method of claim 15, further comprising a step of (d) determining from the amplification curve acquired in step (c) whether the target DNA sequence includes a mutation.

17. The method of claim 15, wherein a first base at the 3′ terminus of the forward primer corresponds to the potential mutation locus of the target DNA sequence.

18. The method of claim 15, wherein the forward primer is an allele-specific (AS) primer or an amplification refractory mutation system (ARMS) primer.

19. The method of claim 15, wherein the discrimination-boosting oligonucleotide is at least one selected from among a DNA duplex, an RNA/DNA hybrid duplex, a double-stranded oligonucleotide, a partially or entirely complementary oligonucleotide single strand(s) capable of forming a partial or entire DNA duplex, a partially or entirely complementary oligonucleotide single strand(s) capable of forming a partial or entire DNA/RNA hybrid duplex, a partially or entirely complementary oligonucleotide single strand(s) capable of forming a partial or entire double-stranded oligonucleotide, and an oligonucleotide capable of forming a partial or perfect hairpin duplex.

20. The method of claim 15, wherein the discrimination-boosting oligonucleotide includes any sequence.

21. The method of claim 15, wherein, the mutation in the target DNA sequence is a single nucleotide polymorphism.

22. The method of claim 15, wherein the DNA polymerase is a thermostable DNA polymerase.

23. The method of claim 15, wherein the DNA polymerase is a wild-type or a mutant DNA polymerase.

24. The method of claim 15, wherein the discrimination-boosting oligonucleotide has a length of 10 to 100 bases (both inclusive).

25. The method of claim 15, wherein the discrimination-boosting oligonucleotide has a length of 15 to 50 bases (both inclusive).

26. The method of claim 15, wherein the discrimination-boosting oligonucleotide has a Tm identical or greater than an annealing temperature set forth for PCR amplification.

27. The method of claim 15, wherein the discrimination-boosting oligonucleotide has a Tm of 50-85° C.

28. The method of claim 15, further comprising employing a fluorescence resonance energy transfer probe modified with a reporter and a quencher in steps (a) or (b).

29. The method of claim 16, further comprising employing a fluorescence resonance energy transfer probe modified with a reporter and a quencher in steps (a) or (b).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0081] FIG. 1 shows a conceptual diagram elucidating the mechanism of PCR according to the presence or absence of a discrimination-boosting oligonucleotide (“dbOligo”) (I) and amplification curves of AS-PCR obtained according to the presence or absence of dbOligo (II).

[0082] (A) AS-PCR in the absence of dbOligo; (B) AS-PCR in the presence of dbOligo

[0083] K.sub.cat1, K.sub.1, and K.sub.−1: reaction constants of enzyme in the corresponding steps for (A) in the absence of dbOligo when a 3′-matched primer is employed,

[0084] K.sub.cat1d, K.sub.1d, and K.sub.−1d: reaction constant of enzyme in the corresponding steps for (B) in the presence of dbOligo when a 3′-matched primer is employed,

[0085] K.sub.cat2, K.sub.2, and K.sub.−2: reaction constants of enzyme in the corresponding steps for (A) in the absence of dbOligo when a 3′-mismatched primer is employed,

[0086] K.sub.cat2d, K.sub.2d, and K.sub.−2d: reaction constant of enzyme in the corresponding steps for (B) in the presence of dbOligo when a 3′-mismatched primer is employed.

[0087] The dbOligo has the sequence of SEQ ID NO: 14 or 15 and is added in an amount of 20 pmol or not added (Test No. 1).

[0088] Specific amplification ratio is calculated to be 2.sup.ΔCt,

[0089] Specific amplification ratio=amplification of 3′matched DNA/amplification of 3′mismatched DNA.

[0090] FIG. 2 shows amplification curves elucidating discrimination ability of allele-specific PCR according to amounts of dbOligo.

[0091] m, mutated template (matches the 3′ terminus of the primer);

[0092] w, wild-type template (mismatches the 3′ terminus of the primer).

[0093] The dbOligo used has the sequence of SEQ ID NO: 13 and is used in an amount of 0, 10, 20, 40, 60, or 80 pmol.

[0094] FIG. 3 shows amplification curves elucidating the effect of dbOligo on modified Taq DNA polymerase and wild-type Taq DNA polymerase. The dbOligo used has the sequence of SEQ ID NO: 13 and is added in an amount of 40 pmol.

[0095] Wt-Taq, wild-type Taq DNA polymerase; Mut-Taq, modified (R536K) Taq DNA polymerase, m, mutated template (matches the 3′ terminus of the primer);

[0096] w, wild-type template (mismatches the 3′ terminus of the primer).

[0097] FIG. 4 shows amplification curves and electrophoresis photographic images elucidating the effect of dbOligo in PCR without using a hydrolysis probe.

BEST MODE FOR CARRYING OUT THE INVENTION

[0098] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as those commonly understood by those skilled in the art. In general, the nomenclature used in this specification is well-known and commonly used in the art.

[0099] The present disclosure may be understood more readily by reference to the following detailed description taken in connection with the accompanying figures and examples, which form a part of this disclosure. It is to be understood that this disclosure is not limited to the specific devices, methods, applications, conditions or parameters described and/or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed disclosure. As used in the specification including the appended claims, the singular forms “a,” “an,” and “the” include the plural, and reference to a particular numerical value includes at least that particular value, unless the context clearly dictates otherwise.

[0100] Unless otherwise stated, the embodiments of the present disclosure utilize molecular biological methods commonly known in the art to which the present disclosure belongs.

[0101] As used herein, the term “base” refers to a nucleobase and is intended to encompass canonical purine and pyrimidine bases including adenine, guanine, cytosine, uracil, and thymine, whether natural or synthetic, and their analogs and derivatives, but with no limitations thereto.

[0102] As used herein, the term “nucleotide” refers to a basic building block of nucleic acids, which is composed of a sugar, a base, and a phosphate group. The sugar, which is ribose or deoxyribose, has a base bonded to C-1′ thereof and a phosphate group bonded to C-5′ thereof. Here, the term “nucleotide” encompasses nucleotide analogs. The sugar may or may not be substituted by other structural analogs. Examples of nucleic acid analogs composed of such compounds include phosphorothioate DNA, PNA (peptide nucleic acid), phosphoramidate DNA, morpholino, and LNA (locked nucleic acid), but are not limited thereto.

[0103] As used herein, the terms “nucleic acid”, “polynucleotide”, “oligonucleotide”, “oligomer” or other terms equivalent thereto are used to cover polymers of various monomers including polymers of monomers corresponding to nucleobase, i.e., polymers of monomers such as deoxyribonucleic acids, ribonucleic acids, phosphorothioate DNA, LNA (locked nucleic acid), PNA (peptide nucleic acid), etc., and polymers structurally similar thereto (e.g., morpholinos).

[0104] The term “oligonucleotide”, as used herein, means a short polynucleotide. Usually, an oligonucleotide is about 250 nucleotides or less, about 200 nucleotides or less, or 100 nucleotides or less in length.

[0105] In the present disclosure, the “oligonucleotide” may include modified oligonucleotides. Here, the term “modification” indicates any modification on nucleobases (e.g., purine analogs, pyrimidine analogs, inverted base, methylated analogs, fluoro analogs, etc.), linking regions (linkers) of nucleosides (e.g., amino (NH.sub.2) linker, carboxyl linker, thiol (SH) linker, etc.), the phosphate group, 5′-terminal, 3′-terminal, or internal bases of oligonucleotides, and combinations thereof.

[0106] As used herein, the term “template” means “template nucleic acid” which is used to set the genetic sequence of new strands during replication in PCR. All of DNA strands which are naturally present, naturally occur, and are artificially synthesized, fall within the scope of the template.

[0107] As used herein, the term “target” refers to a nucleic acid to be analyzed.

[0108] On the whole, a PCR buffer may contain non-essential components such as DMSO, betaine, an aptamer, or an antibody in addition to essential components such as Mg.sup.++, and dNTP.

[0109] For PCR using a primer that allows for discrimination of two alleles according to the presence or absence of a 3′ mismatch, a PCR buffer may contain a discrimination-boosting oligonucleotide selected from among a DNA duplex, an RNA/DNA hybrid duplex, a double-stranded oligonucleotide, a partially or entirely complementary oligonucleotide single strand(s) capable of forming a partial or entire DNA duplex, a partially or entirely complementary oligonucleotide single strand(s) capable of forming a partial or entire DNA/RNA hybrid duplex, a partially or entirely complementary oligonucleotide single strand(s) capable of forming a partial or entire double-stranded oligonucleotide, and an oligonucleotide capable of forming a partial or perfect hairpin duplex, so as to further increase discrimination of alleles (ΔCt or ΔCp, ΔCq).

[0110] In addition, the discrimination-boosting oligonucleotide used for PCR in the present disclosure may include oligonucleotides which are rendered to form a duplex by, for example, chemically coupling two or more single strands having partially or entirely complementary sequences through psoralen or a structurally similar compound thereto, or a chemical linker (e.g., disulfide linker, bismalemide linker, etc.), or by linking termini of the oligonucleotides or extending the oligonucleotide to form a complementary sequence, such as a hairpin loop structure, within single strands. Preferable among them is the formation of a complementary sequence within single strands of oligonucleotides whereby a duplex can be easily formed. This is because a duplex is more easily formed with a single strand, which is not dispersed in a buffer, has a partially or entirely self-complementary sequence, compared to two separate strands which have partially or entirely complementary sequences to each other. Therefore, it is advantageous for accomplishing the goal of the present disclosure to form a duplex by using a chemical method for preventing physical separation of two complementary sequences or a method for making a complementary sequence within a single strand.

[0111] The discrimination-boosting oligonucleotide of the present disclosure for a DNA polymerase may vary in binding affinity for DNA polymerase, depending on various factors including the base sequence thereof, kinds of specific nucleic acids, or oligonucleotide length. In the present disclosure, however, the binding affinity is not limited by specific base sequences, specific kinds of nucleic acids, or a specific range of oligonucleotide length.

[0112] When the complementary sequence accounting for the formation of a duplex is too long or when there are many kinds of complementary oligonucleotide pairs in a PCR solution, the formation of a duplex is reciprocally impeded or the formation of a non-specific duplex is caused during PCR. For instance, when a genomic DNA is denatured during PCR, the resulting single strands are too long to form an accurate duplex again, which makes it difficult to attain the goal of the present disclosure. Thus, the complementary sequence suitable for forming a duplex is preferably 10 bases in length and more preferably 15 to 50 bases in length, with no specific limitations imparted to the complementary sequence and kinds of nucleic acids.

[0113] In the present disclosure, a PCR solution may contain discrimination-boosting oligonucleotide in an amount of 0.01-1,000 pmol, 0.1-500 pmol, 0.1-400 pmol, 0.1-300 pmol, 0.1-200 pmol, 0.1-100 pmol, or 1-80 pmol per 20 μL of the PCR solution. However, the amount of the discrimination-boosting oligonucleotide may vary depending on the sequence of a target gene to be detected, a sample, and PCR conditions, and is not limited to specific concentration.

[0114] The present disclosure is applicable to PCR using a DNA polymerase, especially polymerases of family A (E. coli Pol I lineage). This polymerase may be selected from DNA polymerases derived from thermophilic bacteria, particularly thermophilic eubacteria, and more particularly Thermus spp., Thermotoga spp. Thermococcus spp., Deinococcus spp., and Bacillus spp.

[0115] In accordance with the present disclosure, a duplex of the discrimination-boosting oligonucleotide has a melting temperature (Tm) higher than an annealing temperature of general PCR. For instance, when a duplex of the discrimination-boosting oligonucleotide has a Tm lower than an annealing temperature in PCR, the discrimination-boosting oligonucleotide has difficulty in forming a duplex, leading to a decrease in binding affinity for DNA polymerase. Thus, the discrimination-boosting oligonucleotide used in the present disclosure is particularly configured to have a base sequence such that its duplex region has a Tm higher than an annealing temperature of general PCR.

[0116] The advantages of the present disclosure can be explained through the mechanism depicted in FIG. 1. This mechanism is provided to more accurately understand the present disclosure, but is not to be construed to perfectly describe the entirety of the present disclosure. However, even if the description of the mechanism of the present disclosure is not complete, the effect of the present disclosure should not be denied for this reason.

[0117] FIG. 1 elucidates the mechanism of PCR in terms of kinetic parameters when a primer's 3′ terminus makes a match (left panel, “3′-matched”) or a mismatch (right panel, “3′-mismatched”) with a template. The 3′-matched and the 3′-mismatched are each subjected to two conditions: (A) PCR in the absence of a discrimination-boosting oligonucleotide (dbOligo); and (B) PCR in the presence of dbOligo. In the drawings, “DNAP” stands for DNA polymerase; “dbOligo” for an oligonucleotide that has any sequence and is added in a double-stranded form or forms a duplex in a reaction solution; “K.sub.1” and “K.sub.−1” for kinetic parameters in a forward and a reverse direction, respectively, under the condition of making a match between a primer's 3′ terminus and a template upon PCR in the absence of dbOligo; “K.sub.1d” and “K.sub.−1d” for kinetic parameters in a forward and a reverse diction, respectively, under the condition of making a match between a primer's 3′ terminus and a template upon PCR in the presence of dbOligo; “K.sub.2” and “K.sub.−2” for kinetic parameters in a forward and a reverse direction, respectively, under the condition of making a mismatch between a primer's 3′ terminus and a template upon PCR in the absence of dbOligo; and “K.sub.2d” and “K.sub.−2d” for kinetic parameters in a forward and a reverse diction, respectively, under the condition of making a mismatch between a primer's 3′ terminus and a template upon PCR in the presence of dbOligo.

[0118] According to the match or mismatch between a template and a primer's 3′ terminus, the DNA synthesis process of a DNA polymerase (hereinafter, referred to as “DNAP”) is very complicated. Various factors implicated in enzyme kinetics include a primer (hereinafter referred to as “P”) serving as a substrate, a template DNA (hereinafter, 3′-matched template DNA is abbreviated to “T.sub.1”, 3′-mismatched template DNA is abbreviated to “T.sub.2” for convenience), a hybrid thereof (P/T.sub.1 or P/T.sub.2), DNA polymerase (DNAP), dNTPs, Mg.sup.++, and PPi.

[0119] Upon the formation of DNAP.Math.P/T complex, K.sub.cat is greatly different according to the 3′ match (DNAP.Math.P/T.sub.1) or 3′ mismatch (DNAP.Math.P/T.sub.2). Reportedly, PCR very quickly starts when a primer's 3 terminus matches a template, compared to when a primer's 3′ terminus mismatches a template {Clin Chem. 64(5):801-809 (2018)}. According to the report, K.sub.cat/Km is approximately 100 to 1000-fold higher for 3′-match than 3′-mismatch. This is because K.sub.cat is substantially lowered (approximately 10- to 600-fold) and Km is slightly increased (up to 3-fold) for 3′-mismatch.

[0120] From the documents, it could be inferred that when a primer's 3′ terminus matches a template, the DNAP.Math.P/T.sub.1 complex, once formed, continually perform repeated cycles of polymerization with dNTPs without detachment of the enzyme on the basis of high K.sub.cat1 until completion of the polymerization whereas the DNAP.Math.P/T.sub.2 complex formed for a mismatch between a primer's 3′ terminus and a template frequently undergoes dissociation and association of the DNA polymerase due to low K.sub.cat2 (K.sub.cat1>>>K.sub.cat2). In the presence of a double-stranded oligonucleotide according to the present disclosure, there is a significant difference in real-time PCR between 3′ match and 3′-mismatch (amplification curves in II of FIG. 1).

[0121] For a 3′ match, similar Ct values were obtained irrespective of the presence or absence of a dbOligo. In light of this fact, similar values are obtained in the reaction speed (K.sub.cat1≈K.sub.cat1d) and the dissociation and association ratio of DNA polymerase for template DNA (K.sub.−1/K.sub.1≈K.sub.−1d/K.sub.1d) between the presence and the absence of dbOligo, leading to the inference that the dbOligo has almost no or little influences on the activity of the DNA polymerase.

[0122] The present inventors predicted that when a primer's 3′ terminus mismatches a template, K.sub.cat2 and K.sub.cat2d, although lower than K.sub.cat1 or K.sub.cat1d, might be similar to each other irrespective of the presence or absence of a dbOligo (K.sub.cat2≈K.sub.cat2d<<<K.sub.cat1≈K.sub.cat1d). Contrary to the prediction, the presence of an dbOligo remarkably decreased the polymerization for 3′ mismatch (3′-mismatched in FIG. 1(B)). The reason is that the DNA polymerase is inferred to decrease in association constant (K.sub.2d) or increase in dissociation constant (K.sub.−2d) for formation of DNAP.Math.P/T.sub.2 in the presence of a dbOligo, compared to the absence of a dbOligo. That is to say, it is inferred that the addition of a dbOligo to a PCR solution might make a great change in the dissociation and association ratio of DNA polymerase for template DNA or product DNA (K.sub.−2/K.sub.2<<<K.sub.−2d/K.sub.2d). This difference of kinetic parameters upon the presence of a dbOligo increases discrimination of PCR between 3′ match and 3′ mismatch.

[0123] As a result, it is possible to explicitly discriminate real-time PCR amplification curves, depending on the 3′ match or mismatch with alleles or mutant genes, so that the goal of the present disclosure can be attained.

[0124] In addition, when exponential amplification proceeds with the effective progression of real-time PCR on the 3′-matched template DNA, the synthesized DNA amplicon serves as a new template and as such, the amount of the enzyme substrate drastically increases. In an early stage of PCR, [P/T.sub.1]≈[P/T.sub.2]<<<[dbOligo]. During PCR amplification, [P/T.sub.1] increases in an exponential manner, with the resultant disappearance of the predominance of [dbOligo] over [P/T.sub.1]. Thus, PCR will be further smoothly conducted. In contrast, the predominance of [dbOligo] over [P/T.sub.2] is kept going for 3′ mismatch between a primer's 3 terminus and a DNA template, repressing PCR progression. Thus, discrimination on the amplification curves can be further increased.

MODE FOR CARRYING OUT THE INVENTION

[0125] A better understanding of the present disclosure may be obtained through the following examples that are set forth to illustrate, but are not to be construed to limit the present disclosure.

[0126] The PCR using a discrimination boosting oligonucleotide (dbOligo) to increase discrimination of alleles or mutated genes was designated “STexS (SNP Typing with excellent specificity)” PCR.

[0127] Below, a detailed description will be given of “STexS” PCR in conjunction with concrete examples.

[0128] <PCR Condition>

[0129] Unless stated otherwise, PCR solutions and conditions used in Examples of the present disclosure are as follows.

[0130] Wild-type target template DNA and mutant target template DNA, forward primers, reverse primers, and hydrolysis probes for signal detection used in PCR are summarized in Table 1, below.

[0131] For use in detecting the variation of the target genes EGFR c.2369 C>T (p.T790M); EGFR c.2573 T>G (p.L858R) and BRAF c.1799 rc. A>T (p.V600E), the forward primers, called AS primers, are designed to match the mutated genes and mismatch 1 base pair of the wild-type genes at the 3′ terminus of each of the primers.

[0132] Enzymes for each PCR were 2 units (0.05-0.08 μM) of Taq DNA polymerase (GenoTech). PCR buffer (10 mM Tris, pH 9.0, 1.5 mM MgCl.sub.2, 60 mM KCl, 10 mM (NH.sub.4).sub.2SO.sub.4) amounted to a total of 20 μl. PCR was conducted using ABI 7500 Real-Time PCR System, starting at 95° C. for 5 min followed by 45 cycles of 95° C. for 30 seconds, 55° C. for 40 seconds. Results are expressed as mean values of measurements for all trials that were tested in triplicate.

[0133] To confirm an improvement in discrimination upon PCR, various types of dbOligo including single-stranded DNA (SD), complementary double-stranded DNA (DD), and single-stranded DNA with an internal complementary sequence (hairpin DNA; HD) were added in an amount of 1 to 80 pmol according to test (Tables 2, 3, and 4).

[0134] Template DNAs were prepared by artificially synthesizing the sequences of SEQ ID NOS: 44, 45, 46, 47, 48, and 49, inserting the sequences into pTOP Blunt V2 (Enzynomics, Korea), transforming the plasmids into E. coli, and digesting the amplified plasmids with proper restriction enzymes. Before use, the purified plasmid DNAs were quantitated.

TABLE-US-00001 TABLE 1 Seq. ID Amount Note Target Sequence: EGFR c.2369 C > T (p.T790M) Wild Template 44 1 × 10.sup.7 copies Mutant Template 45 1 × 10.sup.7 copies Forward Primer 1 20 pmol Reverse Primer 7 20 pmol Hydrolysate 10 10 pmol dual labelled Probe Target Sequence: EGFR c.2573 T > G (p.L858R) Wild Template 46 1 × 10.sup.7 copies Mutant Template 47 1 × 10.sup.7 copies Forward Primer 5 20 pmol Reverse Primer 8 20 pmol Hydrolysate 11 10 pmol dual labelled Probe Target Sequence: BRAF c.1799 rc. A > T (p.V600E) Wild Template 48 1 × 10.sup.7 copies Mutant Template 49 1 × 10.sup.7 copies Forward Primer 6 20 pmol Reverse Primer 9 20 pmol Hydrolysate 12 10 pmol dual labelled Probe

<Example 1> Improvement of Discrimination Ability for 3′-Mismatch in Presence of dbOligo

[0135] After PCR using AS primers (forward primer: SEQ ID NO: 1, reverse primer: SEQ ID NO: 7) for targeting EGFR T790M, the discrimination ability ΔCt1 (ΔCt1=Ct of mutated gene—Ct of wild-type gene) was given very low values 1.48-2.16 (Test Nos. 1-6 in Table 2).

[0136] When the complementary double-stranded DNA (DD) was added as an dbOligo in an amount of 20 pmol for PCR (Test No. 1 (SEQ ID NOS: 14/15) and Test No. 2 (SEQ ID NOS: 17/18, 19/20, 14/15, 21/22, 23/24, 25/26)), ΔCt2 (ΔCt2=Ct of mutated gene in the presence of dbOligo—Ct of wild-type gene in the presence of dbOligo) was measured to 3.04-6.79, with ΔΔCt (ΔΔCt=ΔCt2-ΔCt1) amounting to 1.56-4.56, demonstrating a great improvement in discrimination ability (Test Nos. 1 and 2 in Table 2, panel II in FIG. 1 II; Test No. 1).

[0137] For test groups in which the single-stranded DNA (SD) having the same sequence as one strand of DD was added in an amount of 20-40 pmol (Test No. 4 (SEQ ID NO: 25 and SEQ ID NO: 26)), ΔCt2 was measured to be 2.28-2.82, with ΔΔCt amounting to 0.20-0.74, which was insignificant in discrimination ability, compared to the complementary double-stranded DNA (DD) groups [Test No. 4 (SEQ ID NOS: 25/26)], with ΔCt2=5.66 and ΔΔCt=3.58 (Table 2). This result shows that the discrimination-boosting oligonucleotide (dbOligo) is very important in enhancing the discrimination ability.

[0138] In addition, all the tests with the hairpin DNA (HD), which forms a duplex due to its complementary base sequence within a single strand, were measured to have ΔCt2 of 2.46-5.99 and a ΔΔCt of 0.30-3.51, exhibiting a great improvement in discrimination ability (Test No. 1 (SEQ ID NO: 16) and Test No. 5 (SEQ ID NOs: 16, 27, 13, 28, 29, 30, 31)) (Table 2), like DD.

TABLE-US-00002 TABLE 2 dbOligo Type,* ΔCt Duplex (Mutant Ct- ΔΔCt Test Seq. No, ** Amount Wild type Ct)*** (ΔCt2- No. ID Tm*** (pmol) ΔCt1# ΔCt2## ΔCt1) 1 14/15 DD, 24, 68 20 2.23  6.79  4.56 16 HD, 24, 68  5.74  3.51 2 17/18 DD, 20, 64 20 1.48  3.04  1.56 19/20 DD, 22, 66  5.31  3.83 14/15 DD, 24, 68  4.57  3.09 21/22 DD, 26, 71  4.02  2.54 23/24 DD, 28, 72  5.66  4.18 25/26 DD, 30, 74  5.70  4.22 3 25/26 DD, 30, 74 10 1.57  4.58  3.01 20  5.67  4.10 40  7.26  5.69 60  8.26  6.69 80 15.07 13.5  4 25/26 DD, 30, 74 20 2.08  5.66  3.58 25 SD, 30, —  2.82  0.74 26 SD, 30, —  2.46  0.38 25 SD, 30, — 40  2.28  0.20 26 SD, 30, —  2.37  0.29 5 16 HD, 24, 68 20 2.16  5.52  3.36 27 HD, 22, 66  5.52  3.36 13 HD, 20, 64  5.99  3.83 28 HD, 18, 60  5.68  3.52 29 HD, 16, 58  3.70  1.54 30 HD, 14, 54  3.15  0.99 31 HD, 12, 47  2.46  0.30 6 13 HD, 20, 64 10 1.23  3.69  2.46 20  4.84  3.61 40  6.72  5.49 60  7.34  6.11 80  9.05  7.82 * dbOligo type: SD, single-stranded DNA; DD, double-stranded DNA; HD, hairpin structure DNA, ** Duplex no: Number of bases in duplex, ***Tm: melting temperature of double-stranded oligonucleotide, #ΔCt1: ACt in the absence of dbOligo, ##ΔCt2: ACt in the presence of dbOligo.

<Example 2> Discrimination Ability for 3′-Mismatch According to Concentration of dbOligo

[0139] Discrimination ability was tested according to amounts of the discrimination-boosting oligonucleotide (dbOligo) (Table 2 (Test Nos. 3 and 6)). Compared to DD-type dbOligo, HD-type dbOligo allowed a low deviation over repeated tests and thus was identified to be more consistent in PCR discrimination. For both cases, the discrimination ability was improved in a dose-dependent manner over the range of 10-80 pmol in a PCR solution. ΔCt2 was measured to be up to 15.07 and maximum ΔΔCt was 13.5 (Table 2; Test No. 3 (SEQ ID NOs: 25/26) and Test No. 6 (SEQ ID NO: 13)) (FIG. 2).

<Example 3> Discrimination Ability for 3′-Mismatch According to Duplex Length or Tm of dbOligo

[0140] Examination was made of relationship between the duplex length or melting temperature (Tm) of duplex-forming dbOligo and 3′-mismatch discrimination was examined (Table 2 (Test Nos. 2 and 5)).

[0141] When the DD-type dbOligo was made to increase in duplex length from 20 base pairs to 30 base pairs and in Tm from 64° C. to 74° C., the discrimination ability was improved from ΔCt2=3.04 (ΔΔCt=1.56) to ΔCt2=5.70 (ΔΔCt=4.22) (Table 2; Test No. 2 (SEQ ID NOs: 17/18, 19/20, 14/15, 21/22, 23/24, 25/26)).

[0142] Likewise, as the HD type decreased in duplex length from 24 based to 12 bases (with the resultant decrease of Tm from 68° C. to 47° C.) (Table 2; Test No. 5 (SEQ ID NOs: 16, 27, 13, 28, 29, 30, and 31)), the discrimination ability was reduced to a ΔCt2 of 2.46 and a ΔΔCt of 0.30. When a double-stranded DNA (SEQ ID NO: 31) was endowed with a Tm (47° C.) significantly lower than a PCR annealing temperature 55° C., the discrimination ability drastically decreased.

[0143] Both results indicate that the presence of a double-stranded oligonucleotide with a high Tm or with a large number of base pairs accounting for the duplex as a dbOligo improves the discrimination ability and low discrimination ability is detected in the absence of the dbOligo.

<Example 4> Discrimination Ability According to Sequence of Hairpin Structure dbOligo

[0144] When the non-complementary region in HD-type dbOligo, that is, the intermediate sequence that does not participate in duplex form, was arbitrarily changed, the influence attributed to kinds of nucleotides in dbOligo was not significant in the light of ΔCt2=6.12 (ΔΔCt=3.85) to ΔCt2=7.42 (ΔΔCt=5.15) (Table 3; Test No. 7 (SEQ ID Nos: 13, 32, 33, 34, and 35)).

[0145] In addition, when the non-complementary region in HD-type dbOligo was extended by increasing, for example, the number of adenine base from 3 to 10, the discrimination was slightly reduced with the increase of the non-complementary sequence length, but still remained higher than that of the control in the absence of dbOligo (Table 3; Test No. 8 (SEQ ID NOs: 36, 37, 38, and 39)).

[0146] In addition, a change in the kind of the complementary sequence of dbOligo resulted in a difference from ΔCt2=4.00 (ΔΔCt=1.60) to ΔCt2=6.04 (ΔΔCt=3.64). Even an extremely replicated sequence such as poly A/T or poly G/C brought about an improvement in discrimination although it was not significant. The data imply that the efficiency of discrimination may differ from one complementary sequence of dbOligo to another (Table 3; Test No. 9 (SEQ ID NOs: 13, 40, 41, 42, and 43)).

TABLE-US-00003 TABLE 3 dbOligo Type, * ΔCt Duplex (Mutant Ct- ΔΔCt Test Seq. No., ** Wild type Ct) (ΔCt2- No. Test Content ID Tm*** Amount ΔCt1# ΔCt2## ΔCt1) 7 HD (A) 5  13 HD, 20, 64 20 2.27 6.12 3.85 mismatched (T) 5  32 6.92 4.65 Seq.- (C) 5  33 6.91 4.64 Species (G) 5  34 8.97 6.70 AGAGAC 35 7.42 5.15 8 HD (A) 3  36 HD, 20, 64 20 1.76 6.16 4.40 mismatched (A) 5  13 6.66 4.90 Seq.- (A) 7  37 5.72 3.96 Numbers (A) 9  38 4.80 3.04 (A) 10 39 4.43 2.67 9 HD random 13 HD, 20, 64 20 2.40 6.04 3.64 matched random 40 HD, 20, 57 5.89 3.49 Seq. random 41 HD, 20, 55 5.13 2.73 polyA/T 42 HD, 22, 38 4.00 1.60 polyG/C 43 HD, 20, 85 4.76 2.36 * dbOligo type: SD, single-stranded DNA; DD, double-stranded DNA; HD, hairpin structure DNA, ** Duplex no: Number of bases in duplex, ***Tm: melting temperature of double-stranded oligonucleotide, #ΔCt1: ACt in the absence of dbOligo, ##ΔCt2: ACt in the presence of dbOligo.

<Example 5> Discrimination Ability for 3′-Mismatch According to Template Concentration in Presence of dbOligo

[0147] When the template DNA was used in various amounts of 1×10.sup.4 copies to 1×10.sup.7 copies for targeting EGFR T790M (Table 4; Test No. 10), ΔCt1 of the control without dbOligo was insignificant (1.15-1.50), but ΔCt2 of the test group with dbOligo was as large as 7.53-8.83 (ΔΔCt 6.38-7.33), with no significant difference in discrimination according to amounts of template DNA. The data obtained suggest that discrimination ability for 3′-mismatch can be easily improved even in test groups using various concentrations of template DNA.

<Example 6> Discrimination Ability for 3′-Mismatch in ARMS PCR in Presence of dbOligo

[0148] ARMS PCR was also designed to increase discrimination for a primer with 3′-mismatch. Discrimination was examined for PCR using ARMS primers in the presence of a double-stranded oligomer (Table 4, Test No. 11). All the three types of ARMS primers (SEQ ID NOS: 2, 3, and 4) exhibited higher ΔCt1 (6.50-7.67), compared to AS primer (SEQ ID NO: 1), but ΔCt2 was measured to be 10.06-10.57 in the presence of dbOligo (SEQ ID NO: 13), implying that the double-stranded oligonucleotide, when used in the PCR, additionally improves the discrimination (ΔΔCt of 2.39 to 3.24). Taken together, the results indicate that the double-strand oligonucleotides can improve discrimination for 3′-mismatch in ARMS PCR as well as in AS PCR.

<Example 7> Discrimination Ability for 3′-Mismatch According to Kind of Mutant Base of Target Gene in Presence of dbOligo

[0149] There are various base mismatches of SNP in living organisms. In Examples 1-6, discrimination ability for 3′-mismatch in the presence of dbOligo was examined by tests for targeting T790M, that is, for discriminating C and T bases (template DNA SEQ ID NOS: 44 and 45). In this Example, discrimination ability for T and G bases in EGFR L858R (temple DNA SEQ ID NOS: 46 and 47) and A and T bases in BRAF V600E (rc) (template DNA SEQ ID NO: 48, 49) were examined by real-time PCR using suitable AS primers in the presence of a double-stranded oligonucleotide. Although there was a difference in ΔCt1 according to the sequence or 3′ terminal base of template DNA (T790M, 2.20; L858R, 8.14; V600E, 6.75), ΔCt2 was measured to be, 4.48 for T790M; 10.45 for L858R; and 11.43 for V600E and ΔΔCt was measured to be 2.28 for T790M; 2.31 for L858R; and 4.68 for V600E when the double-stranded oligonucleotide was added. That is, the presence of the double-stranded oligonucleotide was identified to increase the discrimination ability in all of the test groups although there are partial differences in discrimination ability according to the sequence of target genes or kind of 3′-mismatch bases (Table 4, Test No. 12).

<Example 8> Discrimination Ability for 3′-Mismatch According to Polymerase in Presence of dbOligo

[0150] Modified polymerases may be used to increase the discrimination of PCR for 3′-mismatch. In this Example, a test for discrimination ability for 3′-mismatch was conducted using mutant (R536K) Taq DNA polymerase known to increase discrimination for 3′-mismatch (Table 4, Test No. 13; and FIG. 3). The mutant Taq DNA polymerase (Mut Taq (R536K)) slightly increased ΔCt1(=3.53), compared to wild-type Taq DNA polymerase (Wt-Taq) (ΔCt1=2.78). In addition, ΔCt2=8.21 and ΔΔCt=4.68 were detected in the presence of dbOligo. From this result, it was understood that although the mutant Taq DNA polymerase increased in part discrimination for 3′-mismatch, the presence of dbOligo further improved discrimination for 3′-mismatch. Particularly, dbOligo was observed to increase discrimination even for the unmodified, wild-type polymerase as measured by ΔCt1=2.78 for control without dbOligo and ΔCt2=7.56 (ΔΔCt=4.78) for a test group with dbOligo. A high level of discrimination for 3′-mismatch was obtained only by adding dbOligo without modifying the enzyme (FIG. 3). In addition, the results indicate that the discrimination can be improved by using the double-stranded oligonucleotide for real-time PCR irrespective of wild-type polymerase or modified polymerase.

TABLE-US-00004 TABLE 4 ΔCt (Mutant Ct- dbOligo Wild type Ct) Type, * ΔCt of ΔCt of Duplex dbOligo dbOligo ΔΔCt Test Seq. No, ** (−) (+) (ΔCt2- No. Test Content ID Tm*** Amount (ΔCt1) (ΔCt2) ΔCt1) 10 Template 1 × 10{circumflex over ( )}7 13 HD, 20, 64 80 1.47  7.98 6.51 DNA 1 × 10{circumflex over ( )}6 1.15  7.53 6.38 (copies) 1 × 10{circumflex over ( )}5 1.31  8.46 7.15 1 × 10{circumflex over ( )}4 1.50  8.83 7.33 11 ARMS 2 13 HD, 20, 64 20 7.67 10.06 2.39 primer 3 6.50 10.47 3.97 (Seq. ID) 4 7.33 10.57 3.24 12 Detected T790M C > T 13 HD, 20, 64 20 2.20  4.48 2.28 Mutants L858R T > G 8.14 10.45 2.31 bases V600E (rC) 6.75 11.43 4.68 (Mutant A > T base, Wild > Mutant base) 13 Polymerase Taq-WT 13 HD, 20, 64 40 2.78 7.56 4.78 Mut-Taq 3.53 8.21 4.68 (R536K) * dbOligo type: SD, single-stranded DNA; DD, double-stranded DNA; HD, hairpin structure DNA, ** Duplex no: Number of bases in duplex, ***Tm: melting temperature of double-stranded oligonucleotide, #ΔCt1: ACt in the absence of dbOligo, ##ΔCt2: ACt in the presence of dbOligo.

<Example 9> PCR in Presence of dbOligo without Hydrolysis Probe

[0151] Examination was made of an improvement in discrimination for 3′-mismatch in the presence of the double-stranded oligonucleotide under the condition of using no hydrolysis probes. Using CFX96™ Real-Time PCR Detection System, PCR was performed in the presence of 20 pmol of the double-stranded oligonucleotide (SEQ ID NO: 13) and SYBR Green I without employing a hydrolysis probe, with the DNA of BRAF V600E (rc) serving as a template. As a result, the addition of dbOligo improved discrimination for 3′-mismatch even in the condition of using no hydrolysis probes (amplification curves in FIG. 4). In addition, the PCR product obtained by PCR without SYBR Green I was identified by agarose gel electrophoresis. The electrophoresis results showed explicit discrimination in the presence of dbOligo (electrophoresis images of FIG. 4).

TABLE-US-00005 TABLE 5 No. sequence remarks 1 agccgaaggg catgagctgc a f. primer 2 agccgaaggg catgagctac a f. primer 3 agccgaaggg catgagctgt a f. primer 4 agccgaaggg catgagcatc a f. primer 5 gcatgtcaag atcacagatt ttgggcg f. primer 6 ggacccactc catcgagatt tct f. primer 7 agtgtggaca acccccacgt gtgc r. primer 8 ctggctgacc taaagccacc tc r. primer 9 cacctcagat atatttcttc atgaagac probe 10 cggtggaggt gaggcagatg probe 11 taccatgcag aaggaggc probe 12 tagaccaaaa tcacctattt ttactg probe 13 gggacagtcg gaggactcgt aaaaaacgag tcctccgact gtccc dbOligo 14 gggacagtcg gaggactcgt ctgg dbOligo 15 ccagacgagt cctccgactg tccc dbOligo 16 gggacagtcg gaggactcgt ctggaaaaac cagacgagtc ctccgactgt ccc dbOligo 17 gggacagtcg gaggactcgt dbOligo 18 acgagtcctc cgactgtccc dbOligo 19 gggacagtcg gaggactcgt ct dbOligo 20 agacgagtcc tccgactgtc cc dbOligo 21 gggacagtcg gaggactcgt ctggca dbOligo 22 tgccagacga gtcctccgac tgtccc dbOligo 23 gggacagtcg gaggactcgt ctggcaca dbOligo 24 tgtgccagac gagtcctccg actgtccc dbOligo 25 gggacagtcg gaggactcgt ctggcacagg dbOligo 26 cctgtgccag acgagtcctc cgactgtccc dbOligo 27 gggacagtcg gaggactcgt ctaaaaaaga cgagtcctcc gactgtccc dbOligo 28 gggacagtcg gaggactcaa aaagagtcct ccgactgtcc c dbOligo 29 gggacagtcg gaggacaaaa agtcctccga ctgtccc dbOligo 30 gggacagtcg gaggaaaaac ctccgactgt ccc dbOligo 31 gggacagtcg gaaaaaatcc gactgtccc dbOligo 32 gggacagtcg gaggactcgt tttttacgag tcctccgact gtccc dbOligo 33 gggacagtcg gaggactcgt cccccacgag tcctccgact gtccc dbOligo 34 gggacagtcg gaggactcgt gggggacgag tcctccgact gtccc dbOligo 35 gggacagtcg gaggactcgt agagcacgag tcctccgact gtccc dbOligo 36 gggacagtcg gaggactcgt aaaacgagtc ctccgactgt ccc dbOligo 37 gggacagtcg gaggactcgt aaaaaaaacg agtcctccga ctgtccc dbOligo 38 gggacagtcg gaggactcgt aaaaaaaaaa cgagtcctcc gactgtccc dbOligo 39 gggacagtcg gaggactcgt aaaaaaaaaa acgagtcctc cgactgtccc dbOligo 40 ggagatacgt gacaggactc aaaaagagtc ctgtcacgta tctcc dbOligo 41 gaaccctcgg taaacagaag aaaaaacttc tgtttaccga gggttc dbOligo 42 aaaaaaaaaa aaaaaaaaaa aacccccctt tttttttttt tttttttttt dbOligo 43 gggggggggg gggggggggg aaaaaccccc cccccccccc ccccc dbOligo 44 cacgcacaca catatcccca tggcaaactc ttgctatccc aggagcgcag accgcatgtg  60 template aggatcctgg ctccttatct cccctccccg tatctccctt ccctgattac ctttgcgatc 120 tgcacacacc agttgagcag gtactgggag ccaatattgt ctttgtgttc ccggacatag 180 tccaggaggc agccgaaggg catgagctgc gtgatgagct gcacggtgga ggtgaggcag 240 atgcccagca ggcggcacac gtgggggttg tccacgctgg ccatcacgta ggcttcctgg 300 agggagggag aggcacgtca gtgtggcttc gcatggtggc cagaaggagg ggcacatgga 360 ccccttccag gtgaagacgc atgaatgcga tcttgagttt caaaatacgt actcatggag 420 gaaaagctgt gcctgcaaaa gacctagc 45 cacgcacaca catatcccca tggcaaactc ttgctatccc aggagcgcag accgcatgtg  60 template aggatcctgg ctccttatct cccctccccg tatctccctt ccctgattac ctttgcgatc 120 tgcacacacc agttgagcag gtactgggag ccaatattgt ctttgtgttc ccggacatag 180 tccaggaggc agccgaaggg catgagctgc atgatgagct gcacggtgga ggtgaggcag 240 atgcccagca ggcggcacac gtgggggttg tccacgctgg ccatcacgta ggcttcctgg 300 agggagggag aggcacgtca gtgtggcttc gcatggtggc cagaaggagg ggcacatgga 360 ccccttccag gtgaagacgc atgaatgcga tcttgagttt caaaatacgt actcatggag 420 gaaaagctgt gcctgcaaaa gacctagc 46 cgccagccat aagtcctcga cgtggagagg ctcagagcct ggcatgaaca tgaccctgaa  60 template ttcggatgca gagcttcttc ccatgatgat ctgtccctca cagcagggtc ttctctgttt 120 cagggcatga actacttgga ggaccgtcgc ttggtgcacc gcgacctggc agccaggaac 180 gtactggtga aaacaccgca gcatgtcaag atcacagatt ttgggctggc caaactgctg 240 ggtgcggaag agaaagaata ccatgcagaa ggaggcaaag taaggaggtg gctttaggtc 300 agccagcatt ttcctgacac cagggaccag gctgccttcc cactagctgt attgtttaac 47 cgccagccat aagtcctcga cgtggagagg ctcagagcct ggcatgaaca tgaccctgaa  60 template ttcggatgca gagcttcttc ccatgatgat ctgtccctca cagcagggtc ttctctgttt 120 cagggcatga actacttgga ggaccgtcgc ttggtgcacc gcgacctggc agccaggaac 180 gtactggtga aaacaccgca gcatgtcaag atcacagatt ttgggcgggc caaactgctg 240 ggtgcggaag agaaagaata ccatgcagaa ggaggcaaag taaggaggtg gctttaggtc 300 agccagcatt ttcctgacac cagggaccag gctgccttcc cactagctgt attgtttaac 360 48 aaaatattcg ttttaagggt aaagaaaaaa gttaaaaaat ctatttacat aaaaaataag  60 template aacactgatt tttgtgaata ctgggaacta tgaaaatact atagttgaga ccttcaatga 120 ctttctagta actcagcagc atctcagggc caaaaattta atcagtggaa aaatagcctc 180 aattcttacc atccacaaaa tggatccaga caactgttca aactgatggg acccactcca 240 tcgagatttc actgtagcta gaccaaaatc acctattttt actgtgaggt cttcatgaag 300 aaatatatct gaggtgtagt aagtaaagga aaacagtaga tctcattttc ctatcagagc 360 aagcattatg aagagtttag gtaagagatc taatttctat aattctgtaa tataatattc 420 tttaaaacat agtacttcat ctttcctctt agagtcaata agtatgtcta aaacaatgat 480 tagttctatt tagcctatat a 49 aaaatattcg ttttaagggt aaagaaaaaa gttaaaaaat ctatttacat aaaaaataag  60 template aacactgatt tttgtgaata ctgggaacta tgaaaatact atagttgaga ccttcaatga 120 ctttctagta actcagcagc atctcagggc caaaaattta atcagtggaa aaatagcctc 180 aattcttacc atccacaaaa tggatccaga caactgttca aactgatggg acccactcca 240 tcgagatttc tctgtagcta gaccaaaatc acctattttt actgtgaggt cttcatgaag 300 aaatatatct gaggtgtagt aagtaaagga aaacagtaga tctcattttc ctatcagagc 360 aagcattatg aagagtttag gtaagagatc taatttctat aattctgtaa tataatattc 420 tttaaaacat agtacttcat ctttcctctt agagtcaata agtatgtcta aaacaatgat 480 tagttctatt tagcctatat a

INDUSTRIAL APPLICABILITY

[0152] The PCR kit or method employing a discrimination-boosting oligonucleotide according to the present disclosure remarkably enhances specificity and sensitivity in general PCR as well as real-time PCR, thus making it possible to easily identify the position and amplification of complementary or non-complementary mutation loci. Therefore, the PCR kit or method of the present disclosure is advantageous for detecting alleles from a sample containing a mixture of trace amounts of various species and can easily detect a mutant gene even if it is present at a trace amount in a sample, finding a wide spectrum of applications in the gene examination of agricultural, aquatic, and livestock products and in the diagnostic medical field.

SEQUENCE LISTING

[0153] The electronic file attached