Nucleic acid amplification
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.
1. A method for generating a concatemer comprising two or more copies of a double-stranded nucleic acid template, the method comprising,
(A) annealing a primary nucleic acid comprising the double-stranded nucleic acid template to a first primer comprising a 5′ terminal nucleotide, a 3′ terminal nucleotide, and two regions, wherein the first primer comprises:
(a) a tail region comprising
(1) the 5′ terminal nucleotide of the primer
(2) an innermost nucleotide, wherein the innermost nucleotide is downstream from the 5′ terminal nucleotide
(3) a middle section between the 5′ terminal nucleotide and the innermost nucleotide, comprising one or more nucleotides, and
(b) a template-binding region comprising
(1) the 3′ terminal nucleotide of the primer
(2) an innermost nucleotide, wherein the innermost nucleotide is upstream from the 3′ terminal nucleotide
(3) a middle section between the 3′ terminal nucleotide and the innermost nucleotide, comprising one or more nucleotides,
wherein the template-binding region is complementary to a first strand of the nucleic acid template,
(B) synthesizing a first extension product from the first primer in the presence of an isolated nucleic acid polymerase;
(C) annealing the first extension product to the a second primer comprising a 5′ terminal nucleotide, a 3′ terminal nucleotide, and two regions, wherein the second primer comprises:
(a) a tail region comprising
(1) the 5′ terminal nucleotide of the primer
(2) an innermost nucleotide, wherein the innermost nucleotide is downstream from the 5′ terminal nucleotide
(3) a middle section between the 5′ terminal nucleotide and the innermost nucleotide, comprising one or more nucleotides, and
(b) a template-binding region comprising
(1) the 3′ terminal nucleotide of the primer
(2) an innermost nucleotide, wherein the innermost nucleotide is upstream from the 3′ terminal nucleotide
(3) a middle section between the 3′ terminal nucleotide and the innermost nucleotide, comprising one or more nucleotides, and
wherein the template-binding region is complementary to a second strand of the nucleic acid template, and
wherein 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, if the sequences are aligned such that the 5′ terminal nucleotide of the second primer is aligned with the innermost nucleotide of the tail region of the first primer and the 5′ terminal nucleotide of the first primer is aligned with the innermost nucleotide of the tail region of the second primer,
(D) synthesizing a second extension product from the second primer in the presence of the isolated nucleic acid polymerase; and
repeating steps (A)-(D) to generate multiple copies of the double-stranded nucleic acid template.
2. The method of claim 1 , wherein all steps of the method are performed at a temperature of no greater than 70° C.
3. The method of claim 1 , wherein all steps of the method are performed at a temperature of no greater than 60° C.
4. The method of claim 1 , wherein all steps of the method are performed at a temperature of no greater than 50° C.
5. The method of claim 1 , wherein all steps of the method are performed at a temperature of no greater than 40° C.
6. The method of claim 1 , wherein two or more steps of the method are performed simultaneously.
7. The method of claim 1 , wherein the nucleic acid template is amplified at least 10-fold within 60 minutes of initiation of the method.
8. The method of claim 1 , wherein the isolated nucleic acid polymerase is a DNA polymerase.
9. The method of claim 1 , wherein the isolated nucleic acid polymerase is a reverse transcriptase.
10. The method of claim 1 , wherein the vessel or kit comprises both a DNA polymerase and a reverse transcriptase.
11. The method of claim 1 , wherein the isolated nucleic acid polymerase has strand displacement activity.
12. The method of claim 1 , further comprising treating one or more of the reaction components with a nucleic acid dye.
13. The method of claim 1 , wherein the nucleic acid template is a DNA molecule.
14. The method of claim 12 , wherein the nucleic acid template is an RNA molecule.
15. The method of claim 14 , wherein the tail region of the first and second primer comprises at least 6 nucleotides.
16. The method of claim 1 , wherein the tail region of the first and second primer comprises at least 10 nucleotides.
17. The method of claim 16 , wherein the tail region of the first and second primer comprises no more than 30 nucleotides.
18. The method of claim 17 , wherein the template binding region of the first and second primer comprises at least 6 nucleotides.
19. A method for generating a concatemer comprising two or more copies of a double-stranded nucleic acid template, the method comprising,
(A) annealing a primary nucleic acid comprising the double-stranded nucleic acid template to a first primer comprising a 5′ terminal nucleotide, a 3′ terminal nucleotide, and two regions, wherein the first primer comprises:
(a) a tail region comprising
(1) the 5′ terminal nucleotide of the primer
(2) an innermost nucleotide, wherein the innermost nucleotide is downstream from the 5′ terminal nucleotide
(3) a middle section between the 5′ terminal nucleotide and the innermost nucleotide, comprising one or more nucleotides, and
(b) a template-binding region comprising
(1) the 3′ terminal nucleotide of the primer
(2) an innermost nucleotide, wherein the innermost nucleotide is upstream from the 3′ terminal nucleotide
(3) a middle section between the 3′ terminal nucleotide and the innermost nucleotide, comprising one or more nucleotides,
wherein the template-binding region is complementary to a first strand of the nucleic acid template,
(B) synthesizing a first extension product from the first primer in the presence of an isolated nucleic acid polymerase;
(C) annealing the first extension product to the a second primer comprising a 5′ terminal nucleotide, a 3′ terminal nucleotide, and two regions, wherein the second primer comprises:
(a) a tail region comprising
(1) the 5′ terminal nucleotide of the primer
(2) an innermost nucleotide, wherein the innermost nucleotide is downstream from the 5′ terminal nucleotide
(3) a middle section between the 5′ terminal nucleotide and the innermost nucleotide, comprising one or more nucleotides, and
(b) a template-binding region comprising
(1) the 3′ terminal nucleotide of the primer
(2) an innermost nucleotide, wherein the innermost nucleotide is upstream from the 3′ terminal nucleotide
(3) a middle section between the 3′ terminal nucleotide and the innermost nucleotide, comprising one or more nucleotides, and
wherein the template-binding region is complementary to a second strand of the nucleic acid template, and
wherein 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, if the sequences are aligned such that the 5′ terminal nucleotide of the second primer is aligned with the innermost nucleotide of the tail region of the first primer and the 5′ terminal nucleotide of the first primer is aligned with the innermost nucleotide of the tail region of the second primer,
(D) synthesizing a second extension product from the second primer in the presence of the isolated nucleic acid polymerase; and
repeating steps (A)-(D) to generate multiple copies of the double-stranded nucleic acid template; wherein the template binding region of the first and second primer comprises at least 10 nucleotides.
20. A method for generating a concatemer comprising two or more copies of a double-stranded nucleic acid template, the method comprising,
(A) annealing a primary nucleic acid comprising the double-stranded nucleic acid template to a first primer comprising a 5′ terminal nucleotide, a 3′ terminal nucleotide, and two regions, wherein the first primer comprises:
(a) a tail region comprising
(1) the 5′ terminal nucleotide of the primer
(2) an innermost nucleotide, wherein the innermost nucleotide is downstream from the 5′ terminal nucleotide
(3) a middle section between the 5′ terminal nucleotide and the innermost nucleotide, comprising one or more nucleotides, and
(b) a template-binding region comprising
(1) the 3′ terminal nucleotide of the primer
(2) an innermost nucleotide, wherein the innermost nucleotide is upstream from the 3′ terminal nucleotide
(3) a middle section between the 3′ terminal nucleotide and the innermost nucleotide, comprising one or more nucleotides,
wherein the template-binding region is complementary to a first strand of the nucleic acid template,
(B) synthesizing a first extension product from the first primer in the presence of an isolated nucleic acid polymerase;
(C) annealing the first extension product to the a second primer comprising a 5′ terminal nucleotide, a 3′ terminal nucleotide, and two regions, wherein the second primer comprises:
(a) a tail region comprising
(1) the 5′ terminal nucleotide of the primer
(2) an innermost nucleotide, wherein the innermost nucleotide is downstream from the 5′ terminal nucleotide
(3) a middle section between the 5′ terminal nucleotide and the innermost nucleotide, comprising one or more nucleotides, and
(b) a template-binding region comprising
(1) the 3′ terminal nucleotide of the primer
(2) an innermost nucleotide, wherein the innermost nucleotide is upstream from the 3′ terminal nucleotide
(3) a middle section between the 3′ terminal nucleotide and the innermost nucleotide, comprising one or more nucleotides, and
wherein the template-binding region is complementary to a second strand of the nucleic acid template, and
wherein 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, if the sequences are aligned such that the 5′ terminal nucleotide of the second primer is aligned with the innermost nucleotide of the tail region of the first primer and the 5′ terminal nucleotide of the first primer is aligned with the innermost nucleotide of the tail region of the second primer,
(D) synthesizing a second extension product from the second primer in the presence of the isolated nucleic acid polymerase; and
repeating steps (A)-(D) to generate multiple copies of the double-stranded nucleic acid template; wherein the template binding region of the first and second primer comprises no more than 30 nucleotides.