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 as nucleic acid polymerases and primers.
1. A method for copying a polynucleotide template, the method comprising:
(A) annealing a nucleic acid comprising the polynucleotide template to a first primer, wherein the first primer comprises a first region and a second region, wherein the first region comprises a 5′ end of the primer, the second region comprises a 3′ end of the primer, and the second region is complementary to a first portion of the polynucleotide template, wherein the polynucleotide template further comprises a third portion, wherein the third portion is situated in the polynucleotide template between the first portion and the second portion of the polynucleotide template, and wherein the first region of the first primer is complementary to the third portion of the polynucleotide template;
(B) synthesizing a first extension product from the first primer;
(C) annealing a second primer to the first extension product, wherein the second primer comprises a first region and a second region, wherein the first region comprises a 5′ end of the primer, the second region comprises a 3′ end of the primer, and the second region is complementary to a sequence which is complementary to a second portion of the polynucleotide template;
the first region of the first primer is complementary to the first region of the second primer;
(D) synthesizing a second extension product from the second primer; and
repeating steps (A)-(D) to generate multiple copies of the polynucleotide template.
2. The method of claim 1 , wherein the polynucleotide template further comprises a third portion, wherein the third portion is situated in the polynucleotide template between the first portion and the second portion, and wherein the first region of the second primer is complementary to the third portion of the polynucleotide template.
3. A method for amplifying a polynucleotide template, the method comprising
(A) annealing a nucleic acid comprising the polynucleotide template to a first primer, wherein the polynucleotide template comprises a first portion, a second portion and a third portion, wherein the third portion is situated in the polynucleotide template between the first portion and the second portion, wherein the first primer comprises a first region and a second region, wherein the second region of the first primer is complementary to the first portion of the polynucleotide template, wherein the polynucleotide template further comprises a third portion, wherein the third portion is situated in the polynucleotide template between the first portion and the second portion, and wherein the first region of the first primer is complementary to the third portion of the polynucleotide template;
(B) synthesizing a first extension product from the first primer;
(C) annealing a second primer to the first extension product, wherein the second primer comprises a first region and a second region, wherein the second region of the second primer is complementary to a sequence which is complementary to the second portion of the polynucleotide template, the first region of the second primer is complementary to the first region of the first primer, and the first region of the second primer is complementary to the third portion of the polynucleotide template.
4. The method of claim 2 , further comprising:
A) providing copies of the polynucleotide template in each of at least a first reaction mixture and a second reaction mixture, wherein:
the first reaction mixture comprises copies of the polynucleotide template, the first primer, and the second primer,
the second reaction mixture comprises copies of the polynucleotide template, a third primer, and a fourth primer, wherein:
the third primer comprises a first region and a second region, wherein the first region comprises a 5′ end of the primer, the second region comprises a 3′ end of the primer, and the second region is complementary to the first portion of the polynucleotide template;
the fourth primer comprises a first region and a second region, wherein the first region comprises a 5′ end of the primer, the second region comprises a 3′ end of the primer, and the second region is complementary to a sequence which is complementary to the second portion of the polynucleotide template;
the first region of the third primer is complementary to the first region of the fourth primer; and
the first region of the fourth primer is complementary to the third portion of the polynucleotide template; and
the nucleotide sequence of the first region of the second primer differs from the nucleotide sequence of first region of the fourth primer by a single nucleotide, wherein the position of the different nucleotide in the second and fourth primers corresponds to a position of a nucleotide of interest in the third portion of the polynucleotide template if the nucleotide sequence of the first region of the second primer or fourth primer is oriented with the nucleotide sequence of the third portion of the polynucleotide template for maximum complementation of the sequences;
B) incubating the first reaction mixture and second reaction mixture under conditions without thermocycling; and
C) comparing the rate or amount of amplification of the polynucleotide template in the first reaction mixture to the rate or amount of amplification of the polynucleotide template in the second reaction mixture, wherein the rate or amount of amplification of the polynucleotide template is indicative of the degree of complementation between first region of the second or fourth primer and the nucleotide sequence of the third portion of the polynucleotide template.
5. The method of claim 1 , wherein the polynucleotide template is a DNA strand.
6. The method of claim 1 , wherein the polynucleotide template is an RNA strand.
7. The method of claim 1 , wherein the first portion and second portion of the polynucleotide template are each between 6 and 30 nucleotides in length.
8. The method of claim 2 , wherein the third portion of the polynucleotide template is between 4 and 14 nucleotides in length.
9. 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.
10. A method for amplifying a double stranded nucleic acid molecule, the method comprising
A) annealing a nucleic acid comprising the double stranded nucleic acid molecule to a first primer, wherein the double stranded nucleic acid molecule comprises a first strand and a second strand, wherein the first strand comprises a first portion and a third portion and the second strand comprises a second portion, wherein the first primer comprises a first region and a second region, wherein the second region of the first primer is complementary to the first portion of the first strand, wherein the polynucleotide template further comprises a third portion, wherein the third portion is situated in the polynucleotide template between the first portion and the second portion, and wherein the first region of the first primer is complementary to the third portion of the polynucleotide template;
(B) synthesizing a first extension product from the first primer;
(C) annealing a second primer to the first extension product, wherein the second primer comprises a first region and a second region, wherein the second region of the second primer is complementary to the second portion of the second strand, the first region of the second primer is complementary to the third portion of the first strand, and the first region of the second primer is complementary to the first region of the first primer.
11. The method of claim 3 , wherein the reaction mixture is incubated without thermocycling.
12. The method of claim 3 , wherein the polynucleotide template is a DNA strand.
13. The method of claim 3 , wherein the polynucleotide template is an RNA strand.
14. The method of claim 3 , wherein the first portion and second portion of the polynucleotide template are each between 6 and 30 nucleotides in length.
15. The method of claim 3 , wherein the third portion of the polynucleotide template is between 4 and 14 nucleotides in length.
16. The method of claim 3 , 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.
17. The method of claim 10 , wherein the reaction mixture is incubated without thermocycling.