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) treating a primary double-stranded nucleic acid comprising the double-stranded nucleic acid 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 a first strand of the double-stranded nucleic acid 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 first strand of the double-stranded nucleic acid 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, 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,
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 of the first primer and second primer 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,
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 nucleotide sequence of the tail region of the second primer read in the 5′ to 3′ direction,
(C) treating the extension product of the second primer of step (B) with a second copy of the first primer and a polymerase under conditions such that an extension product of the second copy of the first primer is synthesized which is annealed to the extension product of the second primer of step (B), to produce a first copy of a secondary nucleic acid comprising the extension product of the second primer of step (B) and the extension product of the second copy of the first primer,
wherein
the extension product of the second copy of the first 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 nucleotide sequence of the tail region of the first primer read in the 5′ to 3′ direction,
(D) repeating steps (A)-(C) one or more addition times to generate at least a second copy of the secondary nucleic acid of step (C),
(E) treating the first copy of the secondary nucleic acid of step (C) and the second copy of the secondary nucleic acid of step (D) under conditions such that the 3′ terminal region of the extension product of the second copy of the first primer of the first copy of the secondary nucleic acid anneals to the 3′ terminal region of the extension product of the second primer of the second copy of the secondary nucleic acid, to produce a cross-over structure comprising the extension product of the second copy of the first primer of the first copy of the secondary nucleic acid and the extension product of the second primer of the second copy of the secondary nucleic acid,
(F) treating the cross-over structure of step (E) with a polymerase under conditions such that an extension product of the extension product of the second copy of the first primer of the first copy of the secondary nucleic acid is synthesized and an extension product of the extension product of the second primer of the second copy of the secondary nucleic acid is synthesized, to produce a concatemer comprising two copies of the double-stranded nucleic acid template of step (A), wherein the concatemer comprises the extension product of the extension product of the second copy of the first primer of the first copy of the secondary nucleic acid and the extension product of the extension product of the second primer of the second copy of the secondary nucleic acid.
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 two or more steps of the method are performed simultaneously.
4. The method of claim 1 , wherein the nucleic acid template is amplified at least 10-fold within 60 minutes of initiation of the method.
5. The method of claim 1 , further comprising treating one or more of the reaction components with a nucleic acid dye.
6. The method of claim 1 , wherein the tail region of each of the first primer and second primer comprises at least 6 nucleotides.
7. The method of claim 1 , wherein the tail region of each of the first primer and second primer comprises no more than 30 nucleotides.
8. The method of claim 1 , wherein the polymerase is a DNA polymerase.
9. The method of claim 1 , wherein all steps of the method are performed under isothermal conditions.
10. The method of claim 9 , wherein all steps of the method are performed at a temperature of no greater than 60° C.
11. The method of claim 9 , wherein all steps of the method are performed at a temperature of no greater than 50° C.
12. The method of claim 9 , wherein all steps of the method are performed at a temperature of no greater than 40° C.
13. The method of claim 1 , wherein the tail region of each of the first and second primer comprises at least 8 nucleotides.
14. The method of claim 1 , wherein the tail region of each of the first and second primer comprises at least 9 nucleotides.
15. The method of claim 1 , wherein the tail region of each of the first and second primer comprises at least 10 nucleotides.
16. The method of claim 1 , wherein the template binding region of each of the first and second primer comprises at least 6 nucleotides.
17. The method of claim 1 , wherein the template binding region of each of the first and second primer comprises at least 8 nucleotides.
18. The method of claim 1 , wherein the template binding region of each of the first and second primer comprises at least 9 nucleotides.
19. The method of claim 1 , wherein the template binding region of each of the first and second primer comprises at least 10 nucleotides.
20. The method of claim 1 , wherein the template binding region of each of the first and second primer comprises no more than 30 nucleotides.