Method for amplifying target nucleic acid

文档序号：1642946 发布日期：2019-12-20 浏览：33次中文

阅读说明：本技术 扩增靶核酸的方法 (Method for amplifying target nucleic acid ) 是由马兆春于 2018-09-19 设计创作，主要内容包括：本公开提供了扩增靶核酸的方法和用于所述方法的试剂盒,其中所述方法包括：(a)提供反应混合物,所述反应混合物包含：(i)包含或怀疑包含所述靶核酸的核酸样品,(ii)多个引物对,其中每种类型引物对的至少一个引物与所述靶核酸的一部分互补,并且每个引物对具有至少一个封闭引物,所述封闭引物包含能够阻断聚合酶延伸的封闭基团,(iii)核酸聚合酶,和(iv)去封闭剂,所述去封闭剂能够使所述靶核酸通过使用所述封闭引物由所述核酸聚合酶进行聚合；和(b)在用于扩增所述靶核酸的条件下孵育所述反应混合物。本公开还提供了对靶核酸进行测序的方法和用于所述方法的试剂盒。(The present disclosure provides methods of amplifying a target nucleic acid and kits for use in the methods, wherein the methods comprise: (a) providing a reaction mixture comprising: (i) a nucleic acid sample comprising or suspected of comprising the target nucleic acid, (ii) a plurality of primer pairs, wherein at least one primer of each type of primer pair is complementary to a portion of the target nucleic acid, and each primer pair has at least one blocking primer comprising a blocking group capable of blocking polymerase extension, (iii) a nucleic acid polymerase, and (iv) a deblocking agent capable of polymerizing the target nucleic acid by the nucleic acid polymerase using the blocking primer; and (b) incubating the reaction mixture under conditions for amplification of the target nucleic acid. The disclosure also provides methods of sequencing a target nucleic acid and kits for use in the methods.)

1. A method of amplifying a target nucleic acid, wherein the method comprises:

(a) providing a reaction mixture comprising: (i) a nucleic acid sample comprising or suspected of comprising the target nucleic acid, (ii) at least 20 different types of primer pairs, wherein at least one primer of each type of primer pair is complementary to a portion of the target nucleic acid and each primer pair has at least one blocking primer comprising a blocking group capable of blocking polymerase extension, (iii) a nucleic acid polymerase, and (iv) a deblocking agent capable of polymerizing the target nucleic acid by the nucleic acid polymerase through the blocking primer; and

(b) incubating the reaction mixture under conditions for amplifying the target nucleic acid.

2. The method of claim 1, wherein the blocking group is located at or near the 3' terminus of each blocking primer.

3. The method of claim 1, wherein the blocking group is a 2',3' -dideoxynucleotide, a ribonucleotide residue, a 2',3' SH nucleotide, or a 2' -O-PO3 nucleotide.

4. The method of claim 1, wherein the blocking primer is further modified to reduce amplification of unwanted nucleic acids.

5. The method of claim 4, wherein the modification is the introduction of at least one mismatched nucleotide in the primer.

6. The method of claim 5, wherein the mismatched nucleotide is 2-18bp from the nucleotide with the blocking group.

7. The method of claim 5, wherein the mismatched nucleotide is 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17bp from the nucleotide having the blocking group.

8. The method of claim 5, wherein the mismatched nucleotide base is located 5' to the nucleotide having the blocking group.

9. The method of claim 4, wherein the modification is a modification of: which serves to raise the Tm between the blocking primer and the target nucleic acid.

10. The method of claim 9, wherein the modification is a modification of: which serves to form an additional bridge connecting the 2 'oxygen and the 4' carbon of at least one nucleotide of the blocking primer.

11. The method of claim 1, wherein there are no more than 20 complementary nucleotide pairings and no more than 50% sequence complementarity between any two primers.

12. The method of claim 11, wherein there are no more than 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7,6, or 5 complementary nucleotide pairings between any two primers of two different types of primer pairs.

13. The method of claim 1, wherein the reaction mixture comprises at least 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 400, 500, 600, 700, 800, 900, or 1000 different types of primer pairs.

14. The method of claim 1, wherein each of the primers is 8 to 100 nucleotides in length.

15. The method of claim 1, wherein the different types of primer pairs can complementarily bind different target nucleic acids or different sequences in the same target nucleic acid.

16. The method of claim 1, wherein the deblocking agent is CS5 DNA polymerase, ampliTaq or KlenTaq polymerase with the F667Y mutation, pyrophosphate, or RNase H2, the CS5 DNA polymerase having a mutation selected from G46E, L329A, Q601R, D640G, I669F, S671F, E678G, or a combination of these mutations.

17. The method of claim 1, wherein the target nucleic acid is single-stranded or double-stranded DNA.

18. The method of claim 1, wherein the target nucleic acid is double-stranded DNA with a single or double adapter tag attached thereto or single-stranded DNA with a single adapter tag attached thereto.

19. The method of claim 18, wherein the reaction mixture further comprises at least one primer that is complementary to all or part of the adapter tag.

20. The method of claim 1, wherein the target nucleic acid is a double-stranded DNA comprising a single or double molecular index tag or a single-stranded DNA comprising a single molecular index tag.

21. The method of claim 20, wherein the molecular index tag comprises a unique identifier nucleic acid sequence and an adapter tag.

22. The method of claim 21, wherein the reaction mixture further comprises at least one primer that is complementary to all or part of the adapter tag.

23. The method of claim 1, wherein the primers have a common tail sequence at or near the 5' end of the primers.

24. The method of claim 23, wherein the common tail sequence is useful as a molecular index tag, a sample index tag, or an adaptor tag, or a combination of three tags.

25. The method of claim 1, wherein the reaction mixture further comprises a high fidelity polymerase.

26. The method of claim 25, wherein the high fidelity polymerase is a PFU DNA polymerase.

27. The method of claim 1, wherein the step (b) of incubating the reaction mixture under conditions for amplification of the target nucleic acid comprises the steps of: denaturing the target nucleic acid; annealing the primer to the target nucleic acid to allow formation of a nucleic acid-primer hybrid; and incubating the nucleic acid-primer hybrid to allow the nucleic acid polymerase to amplify the target nucleic acid.

28. The method of claim 1, wherein the following steps are repeated at least 1, 5, 10, 15, 20, 25, 30, 35, 40, or 50 times: denaturing the target nucleic acid; annealing the primer to the target nucleic acid to allow formation of a nucleic acid-primer hybrid; and incubating the nucleic acid-primer hybrid to allow the nucleic acid polymerase to amplify the target nucleic acid.

29. The method of claim 1, wherein said step (b) is repeated from about 20 to about 50 times.

30. The method of claim 1, wherein the target nucleic acid in the nucleic acid sample is no more than 1 copy, 2 copies, 5 copies, 8 copies, 10 copies, 20 copies, 30 copies, 50 copies, 80 copies, or 100 copies.

31. The method of claim 1, wherein the molar percentage of target nucleic acid in the nucleic acid sample is less than 50%, 20%, 10%, 5%, 3%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.005%, or 0.001%.

32. The method of claim 1, wherein nucleic acids other than the target nucleic acid are not substantially amplified in step (b).

33. The method of claim 1, wherein the molar percentage of undesired nucleic acids in the reaction product obtained from step (b) is less than 20%, 15%, 10%, 5%, 3%, 2%, or 1%.

34. The method of claim 1, wherein the method is for selectively enriching for mutant nucleic acids in a sample comprising wild-type nucleic acids.

35. The method of claim 34, wherein at least one blocking primer is complementary to the mutant nucleic acid at the mutant residue, and the nucleotide of the blocking primer corresponding to the mutant residue has the blocking group.

36. A method of sequencing a target nucleic acid, wherein the method comprises:

(b) incubating the reaction mixture under conditions for amplification of the target nucleic acid; and

37. The method of claim 36, wherein the method is for sequencing by capillary electrophoresis, PCR, or high throughput sequencing.

38. The method of claim 36, wherein the blocking primer is further modified to reduce amplification of unwanted nucleic acids.

39. The method of claim 36, wherein the reaction mixture further comprises a high fidelity polymerase.

40. A method of sequencing a target nucleic acid, wherein the method comprises:

(b) incubating the reaction mixture under conditions for amplification of the target nucleic acid;

(c) adding an adapter tag, a molecular index tag and/or a sample index tag to the reaction product obtained from step (b); and

(d) determining the sequence of the reaction product obtained from step (c).

41. The method of claim 40, wherein the method is for sequencing by capillary electrophoresis, PCR, or high throughput sequencing.

42. The method of claim 40, wherein the blocking primer is modified to reduce amplification of unwanted nucleic acids.

43. The method of claim 40, wherein the reaction mixture further comprises a high fidelity polymerase.

44. A kit for amplifying a target nucleic acid, wherein the kit comprises: (i) at least 20 different types of primer pairs, wherein at least one primer of each type of primer pair is complementary to a portion of the target nucleic acid, and each primer pair has at least one blocking primer comprising a blocking group capable of blocking polymerase extension, (ii) a nucleic acid polymerase, and (iii) a deblocking agent capable of polymerizing the target nucleic acid by the nucleic acid polymerase through the blocking primer.

45. The kit of claim 44, wherein the blocking primer is modified to reduce amplification of unwanted nucleic acids.

46. The kit of claim 44, wherein the reaction mixture further comprises a high fidelity polymerase.

47. A method of amplifying a target nucleic acid, wherein the method comprises:

(a) providing a reaction mixture comprising: (i) a nucleic acid sample comprising or suspected of comprising the target nucleic acid, (ii) at least one type of primer complementary to a portion of the target nucleic acid, and each type of primer having at least one blocking primer comprising a blocking group capable of blocking polymerase extension, wherein the blocking primer is modified to reduce amplification of unwanted nucleic acids, (iii) a nucleic acid polymerase, and (iv) a deblocking agent capable of polymerizing the target nucleic acid by the nucleic acid polymerase through the blocking primer; and

(b) incubating the reaction mixture under conditions for amplifying the target nucleic acid.

48. The method of claim 47, wherein the modification is the introduction of at least one mismatched nucleotide in the primer.

49. The method of claim 48, wherein the mismatched nucleotide is 2-18bp from the nucleotide with the blocking group.

50. The method of claim 48, wherein the mismatched nucleotide is 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17bp from the nucleotide having the blocking group.

51. The method of claim 48, wherein the mismatched nucleotide is located 5' to the blocking group.

52. The method of claim 48, wherein the modification is a modification of: which serves to reduce the affinity between the blocking primer and the target nucleic acid.

53. The method of claim 52, wherein the modification is a modification of: which serves to form an additional bridge connecting the 2 'oxygen and the 4' carbon of at least one nucleotide of the blocking primer.

54. The method of claim 47, wherein the method is used to selectively enrich for mutant nucleic acids in a sample comprising wild-type nucleic acids.

55. The method of claim 54, wherein a blocking primer is complementary to the mutant nucleic acid at the mutant residue, and the nucleotide of the blocking primer corresponding to the mutant residue has the blocking group.

56. A kit for amplifying a target nucleic acid, wherein the kit comprises: (i) at least one type of primer complementary to a portion of the target nucleic acid, and each type of primer having at least one blocking primer comprising a blocking group capable of blocking polymerase extension, wherein the blocking primer is modified to reduce amplification of an undesired nucleic acid, (ii) a nucleic acid polymerase, and (iii) a deblocking agent capable of polymerizing the target nucleic acid by the nucleic acid polymerase through the blocking primer.

57. The kit of claim 56, wherein the blocking primer is modified to reduce the affinity between the blocking primer and the target nucleic acid.

Background

Whether nascent human genetic mutations or somatic human genetic mutations, they are important information for understanding human genetic diseases (Ku, c.s.et al, a new era in the discovery of de novo mutations understinghuman genetic diseases, Hum Genomics 6,27,2012), cancer biology (Helleday, t.et al, mechanismis understinguishing biological signals in human cancers, Nat Rev genet15,585-598,2014) and potential anticancer therapies. It has long been known that a nascent mutation causes genetic disease, and it also plays an important role in rare and common forms of neurodevelopmental disease, including intellectual disability, autism, and schizophrenia (Veltman, j.a. et al, De novo mutations in human genetic disease, NatRev Genet 13, 565-. Somatic mutations in the cancer genome have been extensively studied and are believed to have key roles in understanding the origin, risk of cancer and finding potential biomarkers for therapeutic use. Detection of these gene mutations is important for the diagnosis of disease and treatment of patients.

Due to the limitations of genome sequencing technology, the past studies of new mutations or somatic mutations in human genomes have been very difficult. However, the development of high throughput Next Generation Sequencing (NGS) technology has greatly facilitated the study of such mutations. Whole Genome Sequencing (WGS) and Whole Exome Sequencing (WES) can now be performed on both parental and progeny core families to identify nascent point mutations in the entire genome or within protein-coding regions, respectively.

WGS and WES are good tools for genetic mutation research, but they are still cost prohibitive for routine clinical use. In some cases, genetic analysis of these genes can be more efficient and cost effective if only a single set of genetic mutations is known to be associated with certain diseases or specific drug responses. To perform limited re-sequencing of a set of genes (panels) it is necessary to capture these genes prior to performing NGS. The capture process can be accomplished using hybridization or amplicon methods. For the hybrid capture method, gDNA is first physically fragmented or enzymatically digested, and then synthetic oligonucleotides are hybridized to the target region in solution to capture the desired sequence. The amplicon-based method is direct capture of the desired region by PCR primer amplification. Hybrid capture methods can be scaled to large numbers of genes, but the hybridization step typically takes place overnight, requiring many days for the entire process. It also requires a gDNA material input of at least 1 to 2. mu.g. The amplicon-based method takes less time and requires only 10 to 50ng of gDNA input, so it is suitable for performing with limited DNA input from clinical samples. However, multiplex PCR primers also produce non-specific amplification products, especially when the number of PCR primers is increased. In fact, most PCR products are non-specific amplicons when the number of primers is close to hundreds. Thus, amplicon-based methods typically use an enzymatic digestion step to reduce non-specific amplification products, followed by an additional ligation step or use of multiple cleaning steps to reduce those non-specific products. Those non-specific amplification products not only require multiple steps during sequencing library generation, but also introduce sequencing data errors.

Recently, the detection of low frequency mutations has important applications in basic and clinical research, and thus it has become a rapidly developing field of interest. A rare mutation, circulating cell-free DNA (cfDNA) from human plasma, was used for prenatal screening (Chiu, R.W.et al, Noninversive predictive binary amplified by-throughput screening, Prenat Diagn 32,401-406,2012), while circulating tumor DNA (ctDNA) has been shown to contain a marker mutation for cancer cells. ctDNA is likely to be a novel non-invasive biomarker that facilitates early cancer detection at the surgical curable stage, reduces the necessity of repeated tissue biopsies, and detects early recurrence of disease, thereby improving the efficacy of targeted therapies. For cancers that are usually detected at late stages (including lung, pancreatic and ovarian cancer, etc.), ctDNA detection with high sensitivity can be used as an important screening test for detecting typical (typically) terminal metastatic cancers at an earlier, potentially curable stage. By continuous ctDNA monitoring of the patient's blood, changes in the composition and amount of ctDNA can be used to monitor the progression of cancer in real time, improving patient safety and avoiding the costs associated with repeated tissue biopsies.

Unfortunately, the detection of ctDNA remains difficult because its presence (especially in early cancer patients) is relatively low. To date, several available techniques have been developed for detecting ctDNA, including BEAMing, digital PCR, and next generation sequencing. All of these methods can detect low frequency mutations by evaluating individual molecules one by one. NGS is advantageous over traditional methods in that a large amount of sequencing information can be readily obtained in an automated manner. However, NGS cannot generally be used to detect rare mutations because of their high error rates associated with the production and sequencing processes of NGS libraries. Some of these errors may be caused by introduced mutations, which may occur during template preparation, during pre-amplification steps required for library preparation, and further during solid phase amplification performed on the instrument itself. Other errors can be attributed to base misincorporation and base-recognition (base-trapping) errors during sequencing.

Thus, there is a continuing need for a new method that eliminates non-specific amplification products during multiplex PCR reactions, thereby directly generating sequencing libraries without additional digestion and ligation steps, and a new method that reduces error rates so that rare mutations can be reliably detected by using current NGS instruments.

Disclosure of Invention

One aspect of the present invention provides a method of amplifying a target nucleic acid, wherein the method comprises: (a) providing a reaction mixture comprising: (i) a nucleic acid sample comprising or suspected of comprising the target nucleic acid, (ii) at least 20 different types of primer pairs, wherein at least one primer of each type of primer pair is complementary to a portion of the target nucleic acid and each primer pair has at least one blocking primer comprising a blocking group capable of blocking polymerase extension, (iii) a nucleic acid polymerase, and (iv) a deblocking agent capable of polymerizing the target nucleic acid by the nucleic acid polymerase through the blocking primer; and (b) incubating the reaction mixture under conditions for amplification of the target nucleic acid.

In some embodiments, the blocking group is located at or near the 3' terminus of each blocking primer. In some embodiments, the blocking group is a 2',3' -dideoxynucleotide, a ribonucleotide residue, a 2',3' SH nucleotide, or a 2' -O-PO₃A nucleotide.

In some embodiments, the blocking primer is complementary to a portion of the target nucleic acid. In some embodiments, the blocking primer is further modified to reduce amplification of unwanted nucleic acids. In some embodiments, the modification is the introduction of at least one mismatched nucleotide in the primer. In some embodiments, the mismatched nucleotide is 2-18bp from the nucleotide with the blocking group. In some embodiments, wherein the mismatched nucleotide is 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17bp from the nucleotide having the blocking group. In some embodiments, the mismatched nucleotide base is located 5' to the nucleotide having the blocking group. In some embodiments, the modification is a modification of: which serves to reduce the Tm between the blocking primer and the unwanted nucleic acid. In some embodiments, the modification is a modification of: which serves to raise the Tm between the blocking primer and the target nucleic acid. In some embodiments, wherein the modification is a modification of: which serves to form an additional bridge connecting the 2 'oxygen and the 4' carbon of at least one nucleotide of the blocking primer.

In some embodiments, the reaction mixture comprises at least 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 400, 500, 600, 700, 800, 900, or 1000 different types of primer pairs.

In some embodiments, each of the primers is 8 to 100 nucleotides in length.

In some embodiments, the different types of primer pairs can complementarily bind different target nucleic acids or different sequences in the same target nucleic acid.

In some embodiments, wherein the deblocking agent is CS5 DNA polymerase, ampliTaq or KlenTaq polymerase with the F667Y mutation, pyrophosphate, or RNase H2, the CS5 DNA polymerase having a mutation selected from G46E, L329A, Q601R, D640G, I669F, S671F, E678G, or a combination of these mutations.

In some embodiments, the target nucleic acid is single-stranded or double-stranded DNA.

In some embodiments, the target nucleic acid is double-stranded DNA with a single or double adapter tag attached thereto or single-stranded DNA with a single adapter tag attached thereto.

In some embodiments, the reaction mixture further comprises at least one primer that is complementary, in whole or in part, to the adapter tag.

In some embodiments, the target nucleic acid is double-stranded DNA comprising a single or double molecular index tag or single-stranded DNA comprising a single molecular index tag. In some embodiments, the molecular index tag comprises a unique identifier nucleic acid sequence and an adapter tag.

In some embodiments, the primers have a common tail sequence at or near the 5' end of the primers. In some embodiments, the common tail sequence can be used as a molecular index tag, a sample index tag, or an adapter tag, or a combination of all three tags.

In some embodiments, the reaction mixture further comprises a high fidelity polymerase. In some embodiments, the high fidelity polymerase is a PFU DNA polymerase.

In some embodiments, the step (b) of "incubating the reaction mixture under conditions for amplifying the target nucleic acid" comprises the steps of: denaturing the target nucleic acid; annealing the primer to the target nucleic acid to allow formation of a nucleic acid-primer hybrid; and incubating the nucleic acid-primer hybrid to allow the nucleic acid polymerase to amplify the target nucleic acid.

In some embodiments, formation of the nucleic acid-primer hybrid results in deblocking of the blocking group in the primer by a deblocking agent.

In some embodiments, the following steps are repeated at least 1, 5, 10, 15, 20, 25, 30, 35, 40 or 50 times: denaturing the target nucleic acid; annealing the primer to the target nucleic acid to allow formation of a nucleic acid-primer hybrid; and incubating the nucleic acid-primer hybrid to allow the nucleic acid polymerase to amplify the target nucleic acid. In some embodiments, the step (b) is repeated from about 20 times to about 50 times.

In some embodiments, the nucleic acid sample comprises the target nucleic acid. In some embodiments, the target nucleic acid in the nucleic acid sample is no more than 1 copy, 2 copies, 5 copies, 8 copies, 10 copies, 20 copies, 30 copies, 50 copies, 80 copies, or 100 copies. In some embodiments, the molar percentage of target nucleic acid in the nucleic acid sample is less than 50%, 20%, 10%, 5%, 3%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.005%, or 0.001%.

In some embodiments, nucleic acids other than the target nucleic acid are not substantially amplified in step (b). In some embodiments, the molar percentage of undesired nucleic acids in the reaction product obtained from step (b) is less than 20%, 15%, 10%, 5%, 3%, 2%, or 1%.

In some embodiments, the method is for selectively enriching a mutant nucleic acid in a sample comprising a wild-type nucleic acid. In some embodiments, wherein at least one blocking primer is complementary to the mutant nucleic acid at the mutant residue, and the nucleotide of the blocking primer corresponding to the mutant residue has the blocking group.

Another aspect of the disclosure provides a method of sequencing a target nucleic acid, wherein the method comprises: (a) providing a reaction mixture comprising: (i) a nucleic acid sample comprising or suspected of comprising the target nucleic acid, (ii) at least 20 different types of primer pairs, wherein at least one primer of each type of primer pair is complementary to a portion of the target nucleic acid and each primer pair has at least one blocking primer comprising a blocking group capable of blocking polymerase extension, (iii) a nucleic acid polymerase, and (iv) a deblocking agent capable of polymerizing the target nucleic acid by the nucleic acid polymerase through the blocking primer; (b) incubating the reaction mixture under conditions for amplification of the target nucleic acid; and (c) determining the sequence of the reaction product obtained from step (b).

In some embodiments, the method is for sequencing by capillary electrophoresis, PCR, or high throughput sequencing. In some embodiments, the blocking primer is further modified to reduce amplification of unwanted nucleic acids.

In some embodiments, the reaction mixture further comprises a high fidelity polymerase.

Yet another aspect of the disclosure provides a method of sequencing a target nucleic acid, wherein the method comprises: (a) providing a reaction mixture comprising: (i) a nucleic acid sample comprising or suspected of comprising the target nucleic acid, (ii) at least 20 different types of primer pairs, wherein at least one primer of each type of primer pair is complementary to a portion of the target nucleic acid and each primer pair has at least one blocking primer comprising a blocking group capable of blocking polymerase extension, (iii) a nucleic acid polymerase, and (iv) a deblocking agent capable of polymerizing the target nucleic acid by the nucleic acid polymerase through the blocking primer; (b) incubating the reaction mixture under conditions for amplification of the target nucleic acid; (c) adding an adapter tag, a molecular index tag and/or a sample index tag to the reaction product obtained from step (b); and (d) determining the sequence of the reaction product obtained from step (c).

In some embodiments, the method is for sequencing by capillary electrophoresis, PCR, or high throughput sequencing.

In some embodiments, wherein the blocking primer is modified to reduce amplification of the unwanted nucleic acid.

In some embodiments, wherein the reaction mixture further comprises a high fidelity polymerase.

Yet another aspect of the present disclosure provides a kit for amplifying a target nucleic acid, wherein the kit comprises: (i) at least 20 different types of primer pairs, wherein at least one primer of each type of primer pair is complementary to a portion of the target nucleic acid, and each primer pair has at least one blocking primer comprising a blocking group capable of blocking polymerase extension, (ii) a nucleic acid polymerase, and (iii) a deblocking agent capable of polymerizing the target nucleic acid by the nucleic acid polymerase through the blocking primer.

In some embodiments, the blocking primer is modified to reduce amplification of the unwanted nucleic acid.

In some embodiments, the reaction mixture further comprises a high fidelity polymerase.

Yet another aspect of the present disclosure provides a method of amplifying a target nucleic acid, wherein the method comprises: (a) providing a reaction mixture comprising: (i) a nucleic acid sample comprising or suspected of comprising the target nucleic acid, (ii) at least one type of primer complementary to a portion of the target nucleic acid, and each type of primer having at least one blocking primer comprising a blocking group capable of blocking polymerase extension, wherein the blocking primer is modified to reduce amplification of unwanted nucleic acids, (iii) a nucleic acid polymerase, and (iv) a deblocking agent capable of polymerizing the target nucleic acid by the nucleic acid polymerase through the blocking primer; and (b) incubating the reaction mixture under conditions for amplification of the target nucleic acid.

In some embodiments, the modification is the introduction of at least one mismatched nucleotide in the primer.

In some embodiments, the mismatched nucleotide is 2-18bp from the nucleotide with the blocking group.

In some embodiments, the mismatched nucleotide is 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17bp from the nucleotide having the blocking group.

In some embodiments, the mismatched nucleotide is located 5' to the blocking group.

In some embodiments, the modification is a modification of: which serves to reduce the affinity between the blocking primer and the target nucleic acid.

In some embodiments, the modification is a modification of: which serves to form an additional bridge connecting the 2 'oxygen and the 4' carbon of at least one nucleotide of the blocking primer.

In some embodiments, the method is for selectively enriching a mutant nucleic acid in a sample comprising a wild-type nucleic acid.

In some embodiments, a blocking primer is complementary to a portion of the target nucleic acid. In some embodiments, the blocking primer is complementary to the mutant nucleic acid at the mutant residue, and the nucleotide of the blocking primer corresponding to the mutant residue has the blocking group.

Yet another aspect of the present disclosure provides a kit for amplifying a target nucleic acid, wherein the kit comprises: (i) at least one type of primer complementary to a portion of the target nucleic acid, and each type of primer having at least one blocking primer comprising a blocking group capable of blocking polymerase extension, wherein the blocking primer is modified to reduce amplification of an undesired nucleic acid, (ii) a nucleic acid polymerase, and (iii) a deblocking agent capable of polymerizing the target nucleic acid by the nucleic acid polymerase through the blocking primer

In some embodiments, the blocking primer is modified to reduce the affinity between the blocking primer and the target nucleic acid.

Drawings

FIG. 1: NGS library construction was performed on genomic DNA by multiplex PCR.

FIG. 2: NGS library construction was performed on fragmented DNA by multiplex PCR.

FIG. 3: NGS library construction was performed by multiplex PCR on fragmented DNA with single-stranded molecular index tags.

FIG. 4: NGS library construction was performed by multiplex PCR on fragmented DNA with double-stranded molecular index tags.

FIG. 5: the mutant sequences in genomic DNA were selectively amplified by multiplex PCR.

FIG. 6: mutant enriched NGS library construction of fragmented DNA by multiplex PCR.

FIG. 7: mutant enriched NGS library construction by multiplex PCR on fragmented DNA with single-stranded molecular index tags.

FIG. 8: NGS library construction was performed by multiplex PCR on fragmented DNA with double-stranded molecular index tags.

FIG. 9. example 1, 196 reactions were performed on genomic DNA samples for a total of six separate reactions, followed by sequencing on a MiSeq sequencer, and the resulting normalized reading for each amplicon.

FIG. 10. example 1, 196 reactions were performed on genomic DNA samples for a total of six separate reactions, followed by sequencing on a MiSeq sequencer, and the normalized reading for each amplicon was obtained as a function of the percentage of amplicon GC.

FIG. 11. general workflow for assay design and NGS data analysis for multiplex PCR reactions.

FIG. 12 is an electrophoretogram of different mutant nucleic acids selectively enriched after multiplex PCR reactions in example 2.

FIG. 13 electrophoretic picture of selectively enriched mutant nucleic acids after multiplex PCR reaction in example 3.

FIG. 14 electrophoretic picture of selectively enriched mutant nucleic acids after multiplex PCR reaction in example 4.

FIG. 15 schematic representation of multiplex PCR and library construction in example 1.

Detailed Description

One aspect of the present invention provides a method of amplifying a target nucleic acid, wherein the method comprises: (a) providing a reaction mixture; and (b) incubating the reaction mixture under conditions for amplification of the target nucleic acid.

Providing a reaction mixture

In some embodiments, a reaction mixture for detecting a target nucleic acid of the present disclosure comprises: (i) a nucleic acid sample comprising or suspected of comprising the target nucleic acid, (ii) at least 20 different types of primer pairs, wherein at least one primer of each type of primer pair is complementary to a portion of the target nucleic acid, and each primer pair has at least one blocking primer comprising a blocking group capable of blocking polymerase extension, (iii) a nucleic acid polymerase, and (iv) a deblocking agent capable of polymerizing the target nucleic acid by the nucleic acid polymerase through the blocking primer

Nucleic acid sample

The term "nucleic acid" as used in this disclosure refers to a biopolymer of nucleotide bases and may include, but is not limited to, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), microrna (mirna), and Peptide Nucleic Acid (PNA), modified oligonucleotides (e.g., oligonucleotides comprising nucleotides unconventional to biological RNA or DNA, such as 2' -O-methylated oligonucleotides), and the like. The nucleotides may be natural or unnatural, substituted or unsubstituted, modified or unmodified. The nucleotides may be linked by phosphodiester linkages, or by phosphorothioate linkages, methylphosphonate linkages, boranophosphate linkages, and the like. The polynucleotide may additionally comprise non-nucleotide elements such as tags, quenchers, blocking groups, etc. The nucleic acid may be, for example, single-stranded or double-stranded.

The term "DNA" as used in this disclosure refers to deoxyribonucleic acid, which is a long-chain polymer biomacromolecule that forms the genetic instruction. Subunits of DNA are nucleotides. Each nucleotide in DNA consists of a nitrogenous base, a five-carbon sugar (2-deoxyribose), and a phosphate group. Adjacent nucleotides are linked by a diester formed by deoxyribose and phosphate, forming a long chain framework. In general, there are four types of nitrogenous bases in a DNA nucleotide, namely adenine (A), guanine (G), cytosine (C), and thymine (T). Bases on two long strands of DNA are paired via hydrogen bonds, with adenine (a) paired with thymine (T) and guanine (G) paired with cytosine (C).

The term "nucleic acid sample" as used in the present disclosure refers to any sample containing nucleic acids, including but not limited to cells, tissues, and body fluids, among others. In some embodiments, the nucleic acid sample is a tissue, e.g., a biopsy or a paraffin-embedded tissue. In some embodiments, the nucleic acid sample is a bacterium or a cell of an animal or plant. In some other embodiments, the nucleic acid sample is a bodily fluid, e.g., blood, plasma, serum, saliva, amniocentesis fluid, pleural effusion, peritoneal effusion, and the like. In some embodiments, the nucleic acid sample is blood, serum, or plasma.

In some embodiments, the nucleic acid sample comprises or is suspected of comprising the target nucleic acid.

The term "target nucleic acid" or "target region" as used in this disclosure refers to any region or sequence of a nucleic acid that is intentionally amplified.

In some embodiments, the target nucleic acid is DNA, RNA, or a hybrid or a mixture thereof. In some embodiments, the target nucleic acid is genomic DNA. In some embodiments, the target nucleic acid is cell-free dna (cfdna). In some embodiments, the target nucleic acid is circulating tumor dna (ctdna).

As used in this disclosure, "cell-free DNA" refers to DNA released from cells and present in the circulatory system (e.g., blood), the source of which is generally considered genomic DNA released during apoptosis.

As used in this disclosure, "circulating tumor DNA" refers to cell-free DNA derived from tumor cells. In humans, tumor cells release their genomic DNA into the blood due to factors such as apoptosis and immune response. Circulating tumor DNA usually occupies only a small fraction of cell-free DNA, since normal cells can also release their genomic DNA into the blood.

In some embodiments, the target nucleic acid is single-stranded or double-stranded DNA. In some embodiments, the target nucleic acid is all or part of one or more genes selected from the group consisting of: ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CDKN2A, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, EZH2, FBXW7, FGFR1, FGFR2, FGFR3, FLT3, GNA11, GNAQ, GNAs, HNF1A, HRAS, IDH1, IDH2, JAK2, JAK3, KDR, KIT, KRAS, MET, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, smarcarcb 1, SMO, SRC, STK11, TP53, and VHL.

In some embodiments, the amount of target nucleic acid in the nucleic acid sample is no more than 1 copy, 2 copies, 3 copies, 4 copies, 5 copies, 6 copies, 7 copies, 8 copies, 9 copies, 10 copies, 12 copies, 15 copies, 18 copies, 20 copies, 30 copies, 50 copies, 80 copies, or 100 copies. In some embodiments, the molar percentage (moles/mole) of target nucleic acid in the nucleic acid sample is less than 50%, 20%, 10%, 8%, 6%, 5%, 3%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.005%, or 0.001%. In some embodiments, the ratio of moles of target nucleic acid to moles of non-target nucleic acid in the nucleic acid sample is less than 50%, 20%, 10%, 8%, 6%, 5%, 3%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.005%, or 0.001%.

In some embodiments, the target nucleic acid is a DNA fragment. In some embodiments, the target nucleic acid is 0.01-5kb, 0.1-1kb, 1-2kb, 2-3kb, 3-4kb, 4-5kb, 0.2-0.4kb, 0.5-1kb, 0.1-0.5kb, 0.01-0.4kb, 0.01-0.3kb, 0.01-0.25kb, 0.02-0.25kb, 0.05-0.3kb, or 0.05-0.25kb in size. The DNA fragments can be obtained by conventional techniques in the art (e.g., physical disruption, cleavage with a specific restriction endonuclease, etc.).

In some embodiments, the target nucleic acid is double-stranded DNA linked to a single-linker tag or a double-linker tag or single-stranded DNA linked to a single-linker tag.

The term "adaptor tag" as used in the present disclosure refers to a specific DNA sequence attached to one or both ends of a nucleic acid (single-stranded or double-stranded nucleic acid) as desired, and the length of the adaptor is typically within 5-50 bp. The adapter tags can be used to facilitate amplification of the target nucleic acid and/or to sequence the amplified target nucleic acid. In some embodiments, the linker tag is used to facilitate ligation of tags for sequencing (e.g., ligation of P5 and P7 tags for Illumina MiSeq sequencers). In some embodiments, the adapter tag is attached to only one of the 3 'end or the 5' end of the single stranded nucleic acid. In some embodiments, the adapter tags are attached to both ends of a single-stranded nucleic acid. In some embodiments, one adaptor tag is attached to the 3 'end or the 5' end of each strand in the double stranded nucleic acid. For example, one adaptor tag is attached to the 3 'end of one strand of a double stranded nucleic acid and one adaptor tag is attached to the 5' end of the other strand of the double stranded nucleic acid, and the two adaptor tags are identical or complementary to each other. In some embodiments, two adaptor tags are attached to both ends of each strand in a double-stranded nucleic acid.

The linker tag may be attached to the nucleic acid by conventional techniques in the art. In some embodiments, when the target nucleic acid is double-stranded DNA, the adaptor tag can be attached to the nucleic acid by: (a) providing a linker-ligated nucleic acid that is designed to contain a sequence to be ligated to the end of one strand of DNA (e.g., the linker-ligated nucleic acid contains a hybridizing complementary region, or a short sequence such as poly-T that hybridizes randomly); (b) the linker-connecting nucleic acid hybridizes to the strand of the DNA; and (c) adding a polymerase (e.g., reverse transcriptase) to extend the adaptor-ligated nucleic acid after the hybridizing, thereby ligating the adaptor tag to the end of the target DNA fragment. To attach another linker to the other end of the same strand or to another strand of the DNA, a linker-connecting nucleic acid can be designed and steps (b) - (c) can be repeated as desired. In some other embodiments, when the DNA fragment is double stranded and the end of the DNA fragment is a sticky end, the adaptor tag may be attached to the nucleic acid by: (a) designing the adaptor-ligated nucleic acid to include a sequence to be ligated to the sticky end; (b) complementarily annealing the adaptor-ligated nucleic acid to the sticky end; (c) ligating said adaptor-ligated nucleic acid to the double strand of said target DNA with a ligase, thereby attaching said adaptor to said ends of said DNA fragments.

The term "molecular index tag" as used in the present disclosure refers to a nucleic acid sequence used as a tag, which may be linked to or present at the 5 'end, 3' end, or both ends of a DNA fragment. In DNA sequencing, particularly in high throughput sequencing technologies, molecular index tags are used to label specific DNA molecules. After amplification and sequencing, the counting of the molecular index sequence is used to label specific DNA molecules and can be used as a basis for determining the expression level of the labeled genes, or to track the information of DNA molecules amplified from the same original molecules, thereby correcting random errors of DNA sequences during amplification and sequencing.

In some embodiments, the molecular index tag is exogenous and is attached to the target nucleic acid by PCR (e.g., as described in MoCloskey M. L. et al, Encoding PCR products with a batch-templates and databases. biochem Genet 45:761-767,2014 or Paraswap P, et al, A pyrosequencing-related nucleic acid sequences attached to large-scale sample multiplexing. nucleic Acids Res. 35: E130,2017) or ligation (e.g., as described in Craig D W, et al, Identification of genetic variants using bar-coded multiplexed sequences Nat. 5:887, or Miner B893, sample 135,2004). In some embodiments, the molecular index tag or the unique identifier nucleic acid sequence therein may be a random sequence (i.e., formed of randomly arranged a/T/C/G).

In some embodiments, the molecular index tag or the unique identifier nucleic acid sequence therein is endogenous, being the sequence at both ends of a randomly spliced fragment.

More information about molecular index tags can be found in U.S. Pat. No. 20140227705 and U.S. Pat. No. 20150044687.

Primer and method for producing the same

The term "primer" as used in the present disclosure refers to an oligonucleotide that is capable of acting as a point of initiation of synthesis when placed under conditions that initiate primer extension. The primer may comprise a native ribonucleic acid, deoxyribonucleic acid, or other form of native nucleic acid. The primers may also comprise non-natural nucleic acids (e.g., LNA, ZNA, etc.).

Primers can be made by using any suitable method, such as the phosphotriester and phosphodiester methods or automated embodiments thereof. In one such automated embodiment, diethylphosphoramidite is used as the starting material and can be synthesized as described in Beaucage et al, Tetrahedron Letters,22: 1859-. One method of synthesizing primers on a modified solid support is described in U.S. Pat. No. 4,458,006. Primers that have been isolated from biological sources, such as restriction endonuclease digests, can also be used. In some embodiments, a primer with a blocking nucleotide at the 3' end (Nuc Aci Res 2002,30(2)) can be synthesized using terminal transferase (Gibco BRL).

The term "primer pair" as used in the present disclosure refers to a pair of primers consisting of a forward primer and a reverse primer, which are respectively complementary to a portion of a sequence to be amplified, wherein the forward primer defines a starting point of the amplification sequence and the reverse primer defines a termination point of the amplification sequence. The term "complementary" when used to describe the relationship between a primer and a sequence to be amplified means that the primer is complementary to the sequence to be amplified or complementary to the sequence to be amplified.

Primer pairs can be designed based on the sequence of the target nucleic acid. In some embodiments, at least one primer of each type of primer pair is complementary to a portion of the target nucleic acid. In some embodiments, when the target sequence (assuming it is double-stranded DNA) has an adaptor tag, one primer of a primer pair may be complementary to a portion of the target sequence (a portion on one strand) and the other primer may be complementary to the adaptor tag (an adaptor tag on the other strand).

In some embodiments, each primer pair has at least one blocking primer comprising a blocking group capable of blocking polymerase extension. In some embodiments, both primers in each primer pair are blocked primers comprising a blocking group capable of blocking polymerase extension.

The term "blocked primer" as used in this disclosure refers to a primer having a blocking group.

The term "blocking group" as used in this disclosure refers to any chemical group covalently linked in a nucleic acid strand and capable of blocking polymerase extension. In some embodiments, the nucleotide having a blocking group is a modified nucleotide at or near the 3' terminus of each blocking primer. In some embodiments, the nucleotide having a blocking group is no more than 6bp, 5bp, 4bp, 3bp, 2bp, or 1bp from the 3' end of each blocking primer. In some embodiments, when the methods of the present disclosure are used to selectively enrich for a mutant nucleic acid in a sample comprising a wild-type nucleic acid, the blocking group is located at a nucleotide that is complementary to a corresponding mutant nucleotide of the mutant nucleic acid, but is not complementary to a corresponding nucleotide of the wild-type nucleic acid.

In some embodiments, the blocking group is a 2',3' -dideoxynucleotide, a ribonucleotide residue, a 2',3' SH nucleotide, or a 2' -O-PO₃A nucleotide. When the blocking group is a ribonucleotide residue, the blocking primer is a primer that: which is provided with oneRibonucleotide residues and the other residues are all deoxyribonucleotide residues.

More information on blocking groups and blocking primers can be found in U.S. Pat. No. 9,133,491, U.S. Pat. No. 6,534,269 and Joseph R.D.et al, RNase H-dependent PCR (rhPCR) improved detection and single nucleotide polymorphism detection using blocked primers, BMC Biotechnology 11:80,2011.

In some embodiments, the blocking primer is complementary to a portion of the target nucleic acid.

In some embodiments, the primer is 5 to 100 nucleotides in length. In some embodiments, the primer is at least 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, or 40 nucleotides in length. In some embodiments, the primer is no more than 100, 90, 80, 70, 60, 50, 40, 35, 30, 25, or 20 nucleotides in length.

In some embodiments, a primer comprises a complementary region complementary to the target sequence and a common tail sequence located at or near the 5' end of the primer. In some embodiments, the common tail sequence can be used as a molecular index tag, an adapter tag, or a sample index tag, or a combination of all three tags.

The term "sample index tag" as used in this disclosure refers to a series of unique nucleotides (i.e., each sample index tag is unique) and can be used to achieve sample multiplexing such that each sample can be validated based on its sample index tag. In some embodiments, for each sample in a set of samples, there is a unique sample index tag, and the samples are pooled during sequencing. For example, if 12 samples are combined into a single sequencing reaction, there are at least 12 unique sample index tags, such that each sample is labeled in a unique manner.

In some embodiments, the blocking primer is modified to further reduce amplification of the unwanted nucleic acid.

In some embodiments, the modification is the introduction of at least one mismatched nucleotide in the primer. In some embodiments, the mismatched nucleotide base is located 5' to the nucleotide having the blocking group.

The term "mismatched nucleotide" as used in this disclosure refers to a nucleotide of a first nucleic acid (e.g., a primer) that is not capable of pairing with a nucleotide at a corresponding position of a second nucleic acid (e.g., a target nucleic acid) when the first nucleic acid and the second nucleic acid are aligned.

The preferred or acceptable position of the mismatch nucleotide can be determined by conventional techniques. For example, the mismatched nucleotides are introduced at different positions in the blocking primers and those blocking primers are used to amplify the target nucleic acid, respectively, and then a preferred or acceptable position for the mismatched nucleotides of the target nucleic acid can be determined based on the amplification results (e.g., a position that reduces amplification of unwanted nucleic acids or false positive results is a preferred or acceptable position). The position of the mismatch nucleotide may vary depending on the target nucleic acid or the structure of the blocking primer. In some embodiments, the mismatched nucleotide is 2-18bp from the nucleotide with the blocking group. In some embodiments, the mismatched nucleotide is 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17bp from the nucleotide having a blocking group. In some embodiments, the mismatched nucleotide is no less than 2,3, 4, 5, 6, 7, 8, 9, or 10bp from the nucleotide having a blocking group. In some embodiments, the mismatched nucleotide is no more than 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7,6, 5,4, 3, or 2bp from the nucleotide containing the blocking group.

In some embodiments, the modification is a modification to increase the melting temperature (Tm) between the blocking primer and the target nucleic acid. In some embodiments, the modification is a modification to reduce the melting temperature (Tm) between the blocking primer and the unwanted nucleic acid, which can be a wild-type nucleic acid in a method of selectively enriching for a mutant nucleic acid in a sample. In some embodiments, wherein the modification is a modification (e.g., Locked Nucleic Acid (LNA)) to form an additional bridge connecting the 2 'oxygen and 4' carbon of at least one nucleotide of the blocking primer, see, e.g., Karkare S et al, formulating nucleic acid analogs and chemicals, chromatography diagnostic and applications of PNA, LNA, and morpholino. applied Microbiol Biotechnol71(5):575-, 586,2006 and Vester B et al, LNA (locked nucleic acid) high-affinity synthesis of complementary RNA and biochemistry 43(42):13233-, 13241, 2004.

In some embodiments, the reaction mixture comprises at least 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 400, 500, 600, 700, 800, 900, or 1000 different types of primer pairs. In some embodiments, the different types of primer pairs are complementary to different target nucleic acid fragments or to different sequences in the same target nucleic acid fragment.

The inventors of the present disclosure performed simulation experiments to evaluate the probability of forming primer pairs between randomly generated primers having a certain length. The inventors randomly generated 10 to 490 primer pairs of 20bp in length to form different primer pools, and examined the formation of primer dimers between any one primer and the other primers in the same pool for each pool. It can be seen that the probability of forming primer dimers (e.g., primer dimers resulting from complementarity between different primers) increases with increasing number of primers.

Table 1: the relationship between primer number and dimer length.

For the data in table 1, 100% indicates that each primer in the pool of primers forms a dimer with at least one other primer in the same pool of primers, and that the dimer is not shorter in length than the number shown; 20% means that 20% of the primers in the pool form dimers in the pool, and the dimers are not shorter in length than the indicated number.

Table 2: relationship between primer number and dimer length in primer 3' end

For the data in table 2, 100% indicates that each primer in the pool of primers forms a dimer with at least one other primer in the same pool of primers starting from its 3' end, and the dimer is not shorter in length than the indicated number; 10% means that 10% of the primers in the pool of primers form dimers in the pool of primers shown, and the dimers are not shorter in length than the indicated number.

In some embodiments, there are no more than 20 complementary nucleotide pairs between any two primers. In some embodiments, there are no more than 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7,6, or 5 complementary nucleotide pairings between any two primers. In some embodiments, there are no more than 20 consecutive complementary nucleotide pairs between any two primers. In some embodiments, there are no more than 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7,6, or 5 consecutive complementary nucleotide pairs between any two primers. In some embodiments, the complementary nucleotide pair is located in the region from the 1 st nucleotide at the 3 'end of the primer to the 20 th, 19 th, 18 th, 17 th, 16 th, 15 th, 14 th, 13 th, 12 th, 11 th, 10 th, 9 th or 8 th nucleotide from the 3' end of the primer. In some embodiments, there are no more than 7,6, or 5 consecutive complementary nucleotide pairings within the region from the 1 st nucleotide at the 3 'end of the primer to the 20 th, 19 th, 18 th, 17 th, 16 th, 15 th, 14 th, 13 th, 12 th, 11 th, 10 th, 9 th, or 8 th nucleotide from the 3' end of the primer. In some embodiments, when counting the number of pairings between two primers, common tail sequences are not counted.

In some embodiments, there are no more than 20 complementary nucleotide pairings and no more than 50% sequence complementarity between any two primers. In some embodiments, there are no more than 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7,6, or 5 complementary nucleotide pairs and no more than 45%, 40%, 35%, 30%, 25%, or 20% sequence complementarity between any two primers. In some embodiments, when calculating the percent complementarity between the two primers, the common tail sequence is not included.

The term "nucleotide complementarity" or "complementarity" as used in this disclosure when referring to nucleotides refers to a nucleotide on a nucleic acid strand that is capable of base pairing with another nucleotide on another nucleic acid strand. For example, in DNA, adenine (A) is complementary to thymine (T) and guanine (G) is complementary to cytosine (C). For another example, in RNA, adenine (A) is complementary to uracil (U), and guanine (G) is complementary to cytosine (C).

The term "percent complementarity" as used in this disclosure refers to the percentage of nucleotide residues in a nucleic acid molecule that are complementary to nucleotide residues of another nucleic acid molecule when the nucleic acid molecule is annealed to the other nucleic acid molecule. The percent complementarity is calculated by dividing the number of nucleotides in the first nucleic acid that are complementary to the nucleotides at the corresponding position in the second nucleic acid by the total length of the first nucleic acid.

The percent complementarity of a nucleic acid or the number of nucleotides in a nucleic acid that are complementary to another nucleic acid can also be determined routinely using the BLAST program (basic local alignment search tool) and the PowerBLAST program known in the art (Altschul et al, J.Mol.biol.,215, 403-.

For example, primer 1 will have 90% sequence complementarity in the following cases: 18 of the 20 nucleotides of the primer 1 have complementarity with 18 nucleotides of the primer 2. In this example, the complementary nucleotides can be adjacent to each other or interspersed with non-complementary nucleotides (intercrossed).

The term "x nucleotide pairings" as used in the present disclosure refers to the number of nucleotide residues in a nucleic acid molecule that are complementary to corresponding nucleotides in another nucleic acid molecule when the nucleic acid molecule is annealed to the other nucleic acid molecule. For example, "18 nucleotide pairings" refers to 18 nucleotide residues of a first nucleic acid molecule being complementary to 18 nucleotide residues of a second nucleic acid molecule. In this example, the complementary nucleotides can be adjacent to each other or interspersed with non-complementary nucleotides.

Nucleic acid polymerases

In some embodiments, the nucleic acid polymerase can be selected from a family of DNA polymerases such as e.coli DNA polymerase I (e.g., e.coli DNA polymerase I, Taq DNA polymerase, Tth DNA polymerase, TfI DNA polymerase, and the like). The polymerase may contain naturally occurring wild-type sequences or modified variants and fragments thereof.

In some embodiments, the nucleic acid polymerase can be selected from modified DNA polymerases of the DNA polymerase family (e.g., e.coli DNA polymerase I), such as N-terminally deleted DNA polymerases (e.g., Klenow fragment of e.coli DNA polymerase I), N-terminally deleted Taq polymerases (including Stoffel fragment, Klentaq-235, and Klentaq-278 of Taq DNA polymerase), and the like.

In some embodiments, the nucleic acid polymerase includes, but is not limited to, a thermostable DNA polymerase. Examples of thermostable DNA polymerases include, but are not limited to: tth DNA polymerase, TfI DNA polymerase, Taq DNA polymerase, N-terminal deleted Taq polymerase (e.g., Stoffel fragment, Klentaq-235, and Klentaq-278 of DNA polymerase). Other DNA polymerases include Klenaqi, Taquenase^TM(Amersham)、Ad-vanTaq^TM(Clontech), GoTaq Flexi (Promega) and KlenaTaq-S DNA polymerase.

In some embodiments, the nucleic acid polymerase can be a commercially available DNA polymerase mix, including, but not limited to, TaqLA, TthLA, or Expand High Fidelity typing Enzyme Blend (Roche); TthXL Klen TaqLA (Perkin-Elmer);(Takara Shuzo)；(Life Technologies)；Advantage^TMKlenTaq，Advantage^TMtth and Advantage2^TM(Clontech)；TaqExtender^TM(Stratagene)；Expand^TMLong Template and Expand^TMHigh Fidelity (Boehringer Mannheim); and TripleMaster^TM Enzyme Mix(Eppendorf)。

To further reduce amplification of unwanted nucleic acids, one or more additional polymerases can be added to the reaction mixture. In some embodiments, the reaction mixture comprises a high fidelity polymerase. In some embodiments, the high fidelity polymerase is PFU DNA polymerase, Klentaq-1, Vent, or Deep Vent.

Deblocking agent

The deblocking agent may be selected based on the blocking group contained in the blocking primer. The deblocking agent can be any such agent: when the nucleotide in the blocking primer having the blocking group is complementary to the corresponding nucleotide in the target nucleic acid, it can result in deblocking of the blocking group in the blocking primer under conditions in which the target nucleic acid is amplified. In some embodiments, the deblocking agent is pyrophosphate, CS5 DNA polymerase, the CS5 DNA polymerase having a mutation selected from G46E, L329A, Q601R, D640G, I669F, S671F, E678G, or a combination thereof. In some embodiments, the deblocking agent is ampliTaq or KlenTaq polymerase with the F667Y mutation, or RNase H2.

In some embodiments, when the blocking group is a 2',3' -dideoxynucleotide, the deblocking agent is pyrophosphate. In some embodiments, when the blocking group is 2' -O-PO₃Nucleotide, the deblocking agent is CS5 DNA polymerase, the CS5 DNA polymerase has a mutation selected from G46E, L329A, Q601R, D640G, I669F, S671F, E678G, or a combination thereof (e.g., the DNA polymerase shown in U.S. patent No. 20070154914). In some embodiments, when the blocking group is 2' -O-PO₃When the nucleotide, the deblocking agent is ampliTaq or Klena having the F667Y mutationq polymerase. In some embodiments, when the blocking group is a ribonucleotide residue, the deblocking agent is RNaseH 2.

Step of incubating the reaction mixture under conditions for amplification of the target nucleic acid

The incubation of the reaction mixture of the present invention can be performed in a multi-cycle process that employs several alternating heating and cooling steps to amplify the DNA (see U.S. Pat. No. 4,683,202 and U.S. Pat. No. 4,683,195). In some embodiments, the incubating comprises the steps of: denaturing the target nucleic acid; annealing the primer to the target nucleic acid to allow formation of a target nucleic acid-primer hybrid; and incubating the target nucleic acid-primer hybrid to allow a nucleic acid polymerase to amplify the target nucleic acid.

An example of an amplification process is briefly described below. First, the reaction mixture is heated to a temperature sufficient to denature the double-stranded target DNA into its two single strands. The temperature of the reaction mixture is then reduced to allow the specific single stranded primers to anneal to their respective complementary single stranded target DNAs. After the annealing step, the temperature is maintained or adjusted to the optimal temperature of the DNA polymerase used, which allows incorporation of complementary nucleotides at the 3' end of the annealed oligonucleotide primer, thereby reconstituting the double-stranded target DNA. By using a thermostable DNA polymerase, the cycle consisting of denaturation, annealing and extension can be repeated as many times as necessary to produce the desired product, and no polymerase is added after each thermal denaturation (see "Current Protocols in molecular biology", f.m. ausubel et al, John Wiley and Sons, inc., 1998).

In some embodiments, the target nucleic acid denaturation is performed at about 90 ℃ -100 ℃ for about 10 seconds to 10 minutes (preferably about 1 to 8 minutes for the first cycle). In some embodiments, annealing of the primer to the target nucleic acid is performed at about 5 ℃ -60 ℃ for about 3 seconds to 10 minutes. In some embodiments, the nucleic acid-primer hybrid is incubated such that amplification of the target nucleic acid by the nucleic acid polymerase is performed at about 60 ℃ -90 ℃ for about 1 minute to 15 minutes.

In some embodiments, the incubating step is repeated at least 1, 5, 10, 15, 20, 25, 30, 35, or 40 times. In some embodiments, the incubating step is repeated from about 20 times to about 50 times.

In some embodiments, nucleic acids other than the target nucleic acid are not substantially amplified in step (b). In some embodiments, the molar percentage of undesired nucleic acids in the product obtained after the incubating step is less than 20%, 15%, 10%, 5%, 3%, 2%, or 1%.

The amplification methods described in the present disclosure can be used to construct DNA sequencing libraries. In some embodiments, the product obtained from the incubation step can be used directly as a DNA sequencing library without the need for enzymatic digestion to reduce unwanted amplification products. In some embodiments, the product obtained from the incubation step can be used as a DNA sequencing library after ligation with an adaptor tag without the need for enzymatic digestion to reduce unwanted amplification products.

The "DNA sequencing library" described in the present disclosure refers to a collection of DNA fragments having a quantity that can be sequenced, wherein one or both ends of each fragment in the collection of DNA fragments comprises a specific sequence that is partially or completely complementary to a primer used in sequencing, so that it can be used directly for subsequent DNA sequencing.

Some examples of the construction of DNA sequencing libraries are shown in FIGS. 1-4 and 6-7.

In some embodiments, the method is for selectively enriching for mutant nucleic acids in a sample comprising wild-type and mutant nucleic acids.

Some examples of selective enrichment of mutant nucleic acids are shown in FIGS. 5-7.

Another aspect of the disclosure provides a method of sequencing a target nucleic acid, wherein the method comprises: (a) providing a reaction mixture comprising: (i) a nucleic acid sample, (ii) at least 20 different primer pairs, wherein at least one primer of each primer pair is complementary to a portion of the target nucleic acid and comprises a blocking group capable of blocking polymerase extension ("blocking primer"), (iii) a nucleic acid polymerase, and (iv) a deblocking agent capable of polymerizing the target nucleic acid by the nucleic acid polymerase; (b) incubating the reaction mixture under conditions for amplification of the target nucleic acid; (c) determining the sequence of the product obtained from step (b).

The term "sequencing" as used in this disclosure refers to any and all biochemical methods that can be used to determine the identity and order of nucleotide bases (including but not limited to adenine, guanine, cytosine, and thymine) in one or more DNA molecules. In some embodiments, the methods are used for sequencing by capillary electrophoresis, PCR, or high-throughput sequencing (e.g., Next Generation Sequencing (NGS)).

Yet another aspect of the disclosure provides a method of sequencing a target nucleic acid, wherein the method comprises: (a) providing a reaction mixture comprising: (i) a nucleic acid sample, (ii) at least 20 different primer pairs, wherein at least one primer of each primer pair is complementary to a portion of the target nucleic acid and comprises a blocking group capable of blocking polymerase extension ("blocking primer"), (iii) a nucleic acid polymerase, and (iv) a deblocking agent capable of polymerizing the target nucleic acid by the nucleic acid polymerase; (b) incubating the reaction mixture under conditions for amplification of the target nucleic acid; (c) adding an adapter tag, a molecular index tag and/or a sample index tag to the reaction product obtained from step (b); (d) determining the sequence of the product obtained from step (c).

In some embodiments, in step (c), an adapter tag, a molecular index tag, and/or a sample index tag is attached to the target nucleic acid obtained from step (b). The linker tag, molecular index tag and/or sample index tag may be attached according to the methods described above.

Yet another aspect of the present disclosure provides a kit for amplifying a target nucleic acid, wherein the kit comprises: (i) at least 20 different primer pairs, wherein at least one primer of each primer pair is complementary to a portion of the target nucleic acid and comprises a blocking group capable of blocking polymerase extension, (ii) a nucleic acid polymerase, and (iii) a deblocking agent capable of polymerizing the target nucleic acid by the nucleic acid polymerase.

In some embodimentsThe kit further comprises one or more reagents selected from the group consisting of: dNTPs, Mg²⁺(e.g. MgCl)₂) Bovine serum albumin, pH buffer (e.g., Tris HCL), glycerol, DNase inhibitor, RNase, SO42^-、Cl^-、K⁺、Ca²⁺、Na⁺And (NH)₄)⁺。

In some embodiments, the kit further comprises instructions showing how to perform amplification of the target nucleic acid (e.g., showing the methods of the present disclosure).

Yet another aspect of the present disclosure provides a method of amplifying a target nucleic acid, wherein the method comprises: (a) providing a reaction mixture comprising: (i) a nucleic acid sample, (ii) at least one primer complementary to a portion of the target nucleic acid and comprising a blocking group capable of blocking polymerase extension ("blocking primer"), wherein the blocking primer is modified to reduce amplification of unwanted nucleic acids, (iii) a nucleic acid polymerase, and (iv) a deblocking agent capable of polymerizing the target nucleic acid by the nucleic acid polymerase; (b) incubating the reaction mixture under conditions for amplifying the target nucleic acid.

Yet another aspect of the present disclosure provides a kit for amplifying a target nucleic acid, wherein the kit comprises: (i) at least one primer complementary to a portion of the target nucleic acid and comprising a blocking group capable of blocking polymerase extension ("blocking primer"), wherein the blocking primer is modified to reduce amplification of unwanted nucleic acids, (ii) a nucleic acid polymerase, and (iii) a deblocking agent capable of polymerizing the target nucleic acid by the nucleic acid polymerase.

Any embodiment following any aspect of the present disclosure may be applied to other aspects of the present disclosure as long as the resulting embodiments are possible or reasonable to those skilled in the art.

It should be understood that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "bridge probe" means one or more bridge probes and includes equivalents thereof known to those skilled in the art, and so forth.

All publications and patents cited in this specification are herein incorporated by reference in their entirety.

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