Nucleic acid amplification

Patent metadata

Field	Value
Publication number	US-11649487-B2
Application number	US-202016986557-A
Country	US
Kind code	B2
Filing date	Aug 6, 2020
Priority date	Mar 15, 2013
Publication date	May 16, 2023
Grant date	May 16, 2023

Abstract

Methods and compositions for the amplification of nucleic acids and generation of concatemers are disclosed. Amplification methods provided herein may be performed under isothermal conditions. Methods and compositions may include reagents such nucleic acid polymerases and primers.

First claim

We claim:

1. A method of copying a polynucleotide template, the method comprising:
(A) annealing a nucleic acid comprising the polynucleotide template to a first primer, wherein: the polynucleotide template comprises in the 5′ to 3′ direction a second nucleotide sequence and a first nucleotide sequence; and the first primer comprises in the 5′ to 3′ direction a first nucleotide sequence and a second nucleotide sequence, wherein the second nucleotide sequence of the first primer comprises a nucleotide sequence which is complementary to the first nucleotide sequence-of the polynucleotide template;
(B) synthesizing a first extension product from the first primer in the presence of a polymerase;
(C) annealing a second primer to the first extension product, wherein the second primer comprises in the 5′ to 3′ direction a first nucleotide sequence and a second nucleotide sequence, wherein the second nucleotide sequence of the second primer comprises a nucleotide sequence which is complementary to a partner nucleotide sequence, wherein the partner nucleotide sequence is a nucleotide sequence complementary to the second nucleotide sequence of the polynucleotide template, wherein the first nucleotide sequence of the first and second primers are complementary to each other;
(D) synthesizing a second extension product from the second primer in the presence of the polymerase;
(E) annealing the first primer to the 3′ end of the second extension product and synthesizing a third expression product from the first primer in the presence of the polymerase;
(F) generating a double-stranded nucleic acid comprising the third extension product and the second extension product;
(G) repeating at least steps (E)-(F) one or more additional times to generate at least a first copy and a second copy of the double-stranded nucleic acid comprising the third extension product and the second extension product;
(H) annealing the 3′ terminal region of the third extension product from the first copy of the double-stranded nucleic acid to the 3′ terminal region of the second extension product from the second copy of the double-stranded nucleic acid to produce a cross-over structure;
(I) synthesizing a fourth extension product from the third extension product of step (H) and a fifth extension product from the second extension product of step (H) in the presence of the polymerase; and
(J) producing a concatemer comprising at least two copies of the polynucleotide template, wherein the concatemer comprises the fourth extension product and the fifth extension product, and wherein the concatemer comprises a first concatemer strand,
wherein the first concatemer strand comprises a 5′ end and a 3′ end, and comprises a nucleotide sequence having the general structure in the 5′ to 3′ direction of: C′-T-C′-T-C′, wherein: C′ represents the nucleotide sequence of the first region of the second primer, and T represents the nucleotide sequence of the polynucleotide template or an analogous sequence thereof;
and wherein steps (A)-(J) are performed in a reaction mixture at a substantially isothermal temperature and wherein the reaction mixture does not comprise a recombinase enzyme;
wherein the polynucleotide template is double stranded.

2. The method of claim 1 , wherein the reaction mixture further comprises a DNA polymerase having strand-displacement activity.

3. The method of claim 2 , wherein the reaction mixture further comprises a reverse transcriptase.

4. The method of claim 1 , wherein the polynucleotide template is an RNA molecule.

5. The method of claim 1 , wherein the polynucleotide template comprises one strand of double-stranded nucleic acid template.

6. The method of claim 1 , wherein the first region of the first primer contains between 4 and 25 nucleotides.

7. The method of claim 1 , wherein the first region of the first primer and the first region of the second primer contain the same number of nucleotides.

8. The method of claim 1 , wherein the polynucleotide template contains between 10 and 1000 nucleotides.

9. The method of claim 1 , wherein the reaction mixture further comprises a nucleic acid dye.

10. The method of claim 1 , wherein the number of copies of the polynucleotide template in the reaction mixture is increased at least 10-fold within 60 minutes of initiation of the method.

11. A method of copying a polynucleotide template, the method comprising:
(A) annealing a nucleic acid comprising the polynucleotide template to a first primer, wherein: the polynucleotide template comprises in the 5′ to 3′ direction a second nucleotide sequence and a first nucleotide sequence; and the first primer comprises in the 5′ to 3′ direction a first nucleotide sequence and a second nucleotide sequence, wherein the second nucleotide sequence of the first primer comprises a nucleotide sequence which is complementary to the first nucleotide sequence-of the polynucleotide template;
(B) synthesizing a first extension product from the first primer in the presence of a polymerase;
(C) annealing a second primer to the first extension product, wherein the second primer comprises in the 5′ to 3′ direction a first nucleotide sequence and a second nucleotide sequence, wherein the second nucleotide sequence of the second primer comprises a nucleotide sequence which is complementary to a partner nucleotide sequence, wherein the partner nucleotide sequence is a nucleotide sequence complementary to the second nucleotide sequence of the polynucleotide template, wherein the first nucleotide sequence of the first and second primers are complementary to each other;
(D) synthesizing a second extension product from the second primer in the presence of the polymerase;
(E) annealing the first primer to the 3′ end of the second extension product and synthesizing a third expression product from the first primer in the presence of the polymerase;
(F) generating a double-stranded nucleic acid comprising the third extension product and the second extension product;
(G) repeating at least steps (E)-(F) one or more additional times to generate at least a first copy and a second copy of the double-stranded nucleic acid comprising the third extension product and the second extension product;
(H) annealing the 3′ terminal region of the third extension product from the first copy of the double-stranded nucleic acid to the 3′ terminal region of the second extension product from the second copy of the double-stranded nucleic acid to produce a cross-over structure;
(I) synthesizing a fourth extension product from the third extension product of step (H) and a fifth extension product from the second extension product of step (H) in the presence of the polymerase; and
(J) producing a concatemer comprising at least two copies of the polynucleotide template, wherein the concatemer comprises the fourth extension product and the fifth extension product, and wherein the concatemer comprises a first concatemer strand,
wherein the first concatemer strand comprises a 5′ end and a 3′ end, and comprises a nucleotide sequence having the general structure in the 5′ to 3′ direction of: C′-T-C′-T-C′, wherein: C′ represents the nucleotide sequence of the first region of the second primer, and T represents the nucleotide sequence of a DNA sequence which is analogous to the RNA sequence of the polynucleotide template;
and wherein steps (A)-(J) are performed in a reaction mixture at a substantially isothermal temperature and wherein the reaction mixture does not comprise a recombinase enzyme;
wherein the polynucleotide template is double stranded.

12. A method of copying a polynucleotide template, the method comprising:
(A) annealing a nucleic acid comprisin

Assignees

• Labrador Diagnostics Llc

Inventors

• Belhocine Kamila
• Lee Josephine
• Patel Pranav
• Richardson Aaron
• Tabakman Scott

Classifications

• C07H1/00

  Processes for the preparation of sugar derivatives · CPC title

• C12Q1/6844 Primary

  Nucleic acid amplification reactions · CPC title

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title

• C12P19/34 Primary

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

• C12Q2525/151

  repeat or repeated sequences, e.g. VNTR, microsatellite, concatemer · CPC title