Nucleic acid amplification

Patent metadata

Field	Value
Publication number	US-10131939-B2
Application number	US-201514850608-A
Country	US
Kind code	B2
Filing date	Sep 10, 2015
Priority date	Mar 15, 2013
Publication date	Nov 20, 2018
Grant date	Nov 20, 2018

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 as nucleic acid polymerases and primers.

First claim

We claim:

1. A method of generating a concatemer comprising at least two copies of a double stranded nucleic acid template, the method comprising:
incubating in a reaction mixture at least a polymerase, a first template molecule, and a second template molecule, wherein:
the first template molecule comprises a first nucleic acid strand and a second nucleic acid strand, wherein:
the first nucleic acid strand of the first template molecule comprises a nucleotide sequence having the general structure in the 5′ to 3′ direction of: H′-S-Y 1 -H′, wherein: H′ represents the nucleotide sequence of a first homology sequence, S represents the nucleotide sequence of a first strand of the double stranded nucleic acid template, and Y 1 represents any number and sequence of nucleotides; and
the second nucleic acid strand of the first template molecule comprises a nucleotide sequence having the general structure in the 5′ to 3′ direction of: H-Y 1 ′-S′-H, wherein: H represents the nucleotide sequence of a second homology sequence, wherein the first homology sequence and second homology sequence are complementary to each other, Y 1 ′ represents a nucleotide sequence which is complementary to the nucleotide sequence of Y 1 , and S′ represents the nucleotide sequence of a second strand of the double stranded nucleic acid template, wherein the first strand and second strand of the double stranded nucleic acid template are complementary to each other; and

the second template molecule comprises a first nucleic acid strand and a second nucleic acid strand, wherein:
the first nucleic acid strand of the second template molecule comprises a nucleotide sequence having the general structure in the 5′ to 3′ direction of: H′-S-Y 2 -H′, wherein: H′ represents the nucleotide sequence of the first homology sequence, S represents the nucleotide sequence of the first strand of the double stranded nucleic acid template, and Y 2 represents any number and sequence of nucleotides; and
the second nucleic acid strand of the first template molecule comprises a nucleotide sequence having the general structure in the 5′ to 3′ direction of: H-Y 2 ′-S′-H, wherein: H represents the nucleotide sequence of the second homology sequence, Y 2 ′ represents a nucleotide sequence which is complementary to the nucleotide sequence of Y 2 , and S′ represents the nucleotide sequence of the second strand of the double stranded nucleic acid template; and

upon incubation of the first template molecule with the second template molecule in the reaction mixture, at least one concatemer comprising at least two copies of the double stranded nucleic acid template is formed, wherein the concatemer comprises a first concatemer strand and a second 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: H′-S-Y 2 -H′-S-Y 1 -H′, wherein each of H′, Y 1 , S, and Y 2 represent nucleotide sequences as described above; and wherein the second concatemer strand comprises a 5′ end and a 3′ end, and comprises a sequence having the general structure in the 5′ to 3′ direction of: H-Y 1 ′-S′-H-Y 2 ′-S′-H, wherein each of H′, Y 1 , S, and Y 2 represent nucleotide sequences as described above.

2. The method of claim 1 , wherein at least one of Y 1 and Y 2 represents 0 nucleotides.

3. The method of claim 2 , wherein both of Y 1 and Y 2 represent 0 nucleotides.

4. The method of claim 1 , wherein Y 1 contains a sequence having the general structure in the 5′ to 3′ direction of [(H′-S) N1 ] wherein H′ represents the nucleotide sequence of a first homology sequence, S represents the nucleotide sequence of a first strand of the double stranded nucleic acid template, and N1 is any integer between 0 and 2000.

5. The method of claim 1 , wherein Y 1 contains a sequence having the general structure in the 5′ to 3′ direction of [(H′-S) N1 ] wherein H′ represents the nucleotide sequence of a first homology sequence, S represents the nucleotide sequence of a first strand of the double stranded nucleic acid template, and N1 is any integer between 0 and 2000, and wherein Y 2 contains a sequence having the general structure in the 5′ to 3′ direction of [(H′-S) N2 ] wherein H′ represents the nucleotide sequence of a first homology sequence, S represents the nucleotide sequence of a first strand of the double stranded nucleic acid template, and N2 is any integer between 0 and 2000.

6. The method of claim 5 , wherein N1 and N2 are different integers.

7. The method of claim 6 , wherein N1 is 0, and N2 is an integer between 1 and 2000.

8. The method of claim 1 , wherein the first template molecule and second template molecule are both double-stranded DNA molecules.

9. The method of claim 1 , wherein the first homology sequence contains between 4 and 25 nucleotides.

10. A method for generating a concatemer comprising two or more copies of a polynucleotide template or an analogous sequence thereof, the method comprising,
(A) treating a primary nucleic acid comprising the polynucleotide template with a first copy of a first primer and a polymerase under conditions such that an extension product of the first copy of the first primer is synthesized which is annealed to the polynucleotide template,
wherein
the first primer comprises a 5′ terminal nucleotide, a 3′ terminal nucleotide, and two regions:
(i) a tail region comprising
(a) the 5′ terminal nucleotide of the primer
(b) an innermost nucleotide, wherein the innermost nucleotide is downstream from the 5′ terminal nucleotide
(c) a middle section between the 5′ terminal nucleotide and the innermost nucleotide, comprising one or more nucleotides, and

(ii) a template-binding region comprising
(a) the 3′ terminal nucleotide of the primer
(b) an innermost nucleotide, wherein the innermost nucleotide is upstream from the 3′ terminal nucleotide
(c) a middle section between the 3′ terminal nucleotide and the innermost nucleotide, comprising one or more nucleotides, and the template-binding region of the first copy of the first primer anneals to the polynucleotide template,

(B) treating the extension product of the first copy of the first primer of step (A) with a second primer and a polymerase under conditions such that an extension product of the second primer is synthesized which is annealed to the extension product of the first copy of the first primer of step (A),
wherein
the second primer comprises a 5′ terminal nucleotide, a 3′ terminal nucleotide, and two regions:
(i) a tail region comprising
(a) the 5′ terminal nucleotide of the primer
(b) an innermost nucleotide, wherein the innermost nucleotide is downstream from the 5′ terminal nucleotide
(c) a middle section between the 5′ terminal nucleotide and the innermost nucleotide, comprising one or more nucleotides

(ii) a template-binding region comprising
(a) the 3′ terminal nucleotide of the primer
(b) an innermost nucleotide, wherein the innermost nucleotide is upstream from the 3′ terminal nucleotide
(c) a middle section between the 3′ terminal nucleotide and the innermost nucleotide, comprising one or more nucleotides,

the tail region of the second primer contains a nucleotide sequence which is complementary to the nucleotide sequence of the tail region of the first primer,
the template-binding region of the second primer anneals to the extension product of the first copy of the first primer of step (A), and
the extension product of the second primer contains a 5′ terminal nucleotide, a 3′ terminal nucleotide, and a 3′ terminal region comprising the 3′ terminal nucleotide, wherein the 3′ terminal region contains the same nucleotide sequence as the nuc

Assignees

• Theranos Ip Co Llc

Inventors

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

Classifications

• 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

• C07H1/00

  Processes for the preparation of sugar derivatives · CPC title

• C12Q2531/119

  Strand displacement amplification [SDA] · CPC title