Clonal amplification of nucleic acid on solid surface with template walking

Patent metadata

Field	Value
Publication number	US-2015275284-A1
Application number	US-201514692706-A
Country	US
Kind code	A1
Filing date	Apr 21, 2015
Priority date	Dec 17, 2010
Publication date	Oct 1, 2015
Grant date	—

Abstract

Novel methods of generating a localized population of immobilized clonal amplicons on a support are provided.

First claim

What is claimed:

1 . A method for amplifying nucleic acids, comprising:
a) providing a plurality of forward primers immobilized on a support, wherein the plurality of forward primers includes a first forward primer and a second forward primer, wherein the plurality of forward primers have substantially identical sequences; b) providing a nucleic acid reverse strand having a forward primer-binding sequence that can hybridize to any one of the plurality of forward primers, and the nucleic acid reverse strand having a sequence that is complementary to a first reverse primer; c) hybridizing the first forward primer to the forward primer-binding sequence on the nucleic acid reverse strand; d) generating an extended first forward strand that is a full-length complement of the nucleic acid reverse strand and is hybridize thereto, by primer extension of the first forward primer using the reverse strand as a template; and e) hybridizing the second forward primer to the forward primer-binding sequence on the nucleic acid reverse strand, wherein a portion of the nucleic acid reverse strand is also hybridized to the extended first forward strand.

2 . The method of claim 1 , wherein the support is immobilized only with primers having sequences that are substantially identical to the plurality of forward primers.

3 . The method of claim 1 , wherein step (e) comprises: partially denaturing the forward primer-binding sequence on the nucleic acid reverse strand from the extended first forward strand and re-hybridizing the forward primer-binding sequence to the second forward primer.

4 . The method of claim 1 , wherein at least 60% of the bases of the plurality of forward primers are adenine, thymine or uracil, or are complementary to adenine, thymine or uracil.

5 . The method of claim 1 , further comprising: generating an extended second forward strand that is full-length complement of the reverse strand and is hybridized thereto, by primer extension of the second forward primer using the reverse strand as a template.

6 . The method of claim 5 , wherein primer extension of the second forward primer using the reverse strand as a template results in displacing the extended first forward strand that is hybridized to the nucleic acid reverse strand.

7 . The method of claim 5 , further comprising: hybridizing the forward primer-binding sequence on the nucleic acid reverse strand to a third forward primer immobilized to the support, wherein a portion of the reverse strand is also hybridized to the extended second forward strand, and wherein the third primer includes a sequence that is substantially identical to the plurality of forward primers sequences.

8 . The method of claim 7 , wherein the hybridizing the forward primer-binding sequence on the nucleic acid reverse strand to a third forward primer comprises: partially denaturing the forward primer-binding sequence on the nucleic acid reverse strand from the extended second forward strand and re-hybridizing the forward primer-binding sequence to the third forward primer.

9 . The method of claim 8 , further comprising: generating an extended third forward strand that is a full-length complement of the reverse strand and is hybridize thereto, by primer extension of the third forward primer using the reverse strand as a template.

10 . The method of claim 1 , further comprising:
a) hybridizing a first reverse primer to the reverse primer-binding sequence on the extended first forward strand, by partially denaturing the reverse primer-binding sequence on the extended first forward strand from the nucleic acid reverse strand, wherein the first reverse primer is a soluble primer; and b) extending the first reverse primer using the extended first forward strand as a template.

11 . The method of claim 1 , wherein the amplifying is performed under an isothermal condition.

12 . The method of claim 11 , wherein the isothermal condition includes a temperature that is higher than the Tm of all forward primers, and lower than the Tm of the nucleic acid reverse strand.

13 . The method of claim 1 , wherein the plurality of forward primers are immobilized adjacent to each other on the support.

14 . The method of claim 1 , wherein the plurality of forward primers are immobilized adjacent to each other at an immobilization site on the support.

15 . The method of claim 14 , wherein the support includes a plurality of spatially-separate immobilization sites.

16 . The method of claim 1 , wherein at least 10 6 forward primers are immobilized to the support.

17 . The method of claim 15 , wherein the amplifying generates a clonal population of nucleic acid strands immobilized on at least one immobilization site of the plurality of separate immobilization sites.

18 . The method of claim 1 , wherein the 5′ ends of the plurality of the forward primers are attached to the support.

19 . The method of claim 1 , further comprising contacting the first forward primer of step (c) with a recombinase or helicase.

20 . The method of claim 1 , further comprising contacting the second forward primer of step (e) with a recombinase or helicase.

Assignees

• Life Technologies Corp

Inventors

• Li Bin
• Lao Kai Qin
• O'Neil Jennifer
• Kunkel Jennifer
• Haley Kellie
• Kasinskas Rachel
• Ma Zhaochun
• Brzoska Pius

Classifications

• C12Q2531/119

  Strand displacement amplification [SDA] · CPC title

• C12P19/34

  Polynucleotides, e.g. nucleic acids, oligoribonucleotides · CPC title

• C12Q1/46

  involving cholinesterase · CPC title

• C12Q1/6834

  Enzymatic or biochemical coupling of nucleic acids to a solid phase · CPC title

• C12Q1/6846 Primary

  Common amplification features · CPC title