Method of amplifying a target nucleic acid
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) multiple primer pairs, wherein at least one primer of each type of primer pairs 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) nucleic acid polymerase, and (iv) de-blocking agent capable of enabling polymerization of the target nucleic acid by said nucleic acid polymerase using the blocking primers; and (b) incubating the reaction mixture under a condition for amplification of the target nucleic acid and a kit used for the method. The present disclosure further provides a method of sequencing a target nucleic acid and a kit used for the method.
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 pairs is complementary to a portion of the target nucleic acid, and each primer pair has two blocking primers comprising a blocking group capable of blocking polymerase extension, wherein the blocking group is 2′, 3′-dideoxynucleotide and the blocking group is at 3′ terminal of each blocking primer, (iii) nucleic acid polymerase, and (iv) de-blocking agent capable of enabling polymerization of the target nucleic acid by said nucleic acid polymerase using the blocking primers; and
(b) incubating the reaction mixture under a condition for amplification of the target nucleic acid.
2. The method of claim 1 , wherein the blocking primer is further modified to decrease the amplification of undesired nucleic acid.
3. The method of claim 2 , wherein the modification is introduction of at least one mismatched nucleotide in the primer.
4. The method of claim 3 , wherein the mismatched nucleotide is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 bp away from the nucleotide with the blocking group.
5. The method of claim 3 , wherein the mismatched nucleotide base is located on the 5′ side of the nucleotide with the blocking group.
6. The method of claim 2 , wherein the modification is a modification to form an extra bridge connecting the 2′ oxygen and 4′ carbon of at least one nucleotide of the blocking primer.
7. 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.
8. The method of claim 1 , wherein the different types of primers pairs can complementarily bind to different target nucleic acids or different sequences in the same target nucleic acid.
9. The method of claim 1 , wherein the target nucleic acid is double strand DNA ligated with single or double adaptor tags or single stranded DNA ligated with single adaptor tag.
10. The method of claim 1 , wherein the target nucleic acid is double stranded DNA comprising single or double molecular index tag or single stranded DNA comprising single molecular index tag.
11. The method of claim 10 , wherein the molecular index tag comprises unique identifier nucleic acid sequence and an adaptor tag.
12. The method of claim 1 , wherein the primers have common tailing sequence at or near 5′ terminal of the primers.
13. The method of claim 12 , wherein the common tailing sequence can be used as molecular index tag, sample index tag or adaptor tag or combinations of three tags.
14. The method of claim 1 , wherein the nucleic acid other than the target nucleic acid is not amplified in step (b) substantially.
15. The method of claim 1 , wherein the method is used for selective enrichment of mutant nucleic acid in a sample comprising wildtype nucleic acid.
16. The method of claim 15 , wherein at least one blocking primer is complementary to the mutant nucleic acid at the mutant residues and the nucleotide of the blocking primer corresponding to a mutant residue has the blocking group.
17. 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 pairs is complementary to a portion of the target nucleic acid, and each primer pair has two blocking primers comprising a blocking group capable of blocking polymerase extension, wherein the blocking group is 2′, 3′-dideoxynucleotide and the blocking group is at 3′ terminal of each blocking primer, (iii) nucleic acid polymerase, and (iv) de-blocking agent capable of enabling polymerization of the target nucleic acid by said nucleic acid polymerase using the blocking primers;
(b) incubating the reaction mixture under a condition for amplification of the target nucleic acid;
(c) adding adaptor tag, molecular index tag and/or sample index tag to the reaction products obtained from step (b); and
(d) determining the sequence of the reaction products obtained from step (c).