Nucleic acid amplification
Methods and compositions for the amplification of nucleic acids are disclosed. Amplification methods provided herein may be performed under isothermal conditions. Methods and compositions may include reagents such as nucleic acid polymerases, ligases, and primers.
1. A method of replicating a double stranded nucleic acid template, the method comprising,
incubating in a reaction mixture a first primer, a DNA polymerase, a nucleic acid ligase, a first copy of a single template linear double-stranded nucleic acid, a second copy of the single template linear double-stranded nucleic acid, a third copy of the single template linear double-stranded nucleic acid, a first copy of a multiple template linear double-stranded nucleic acid, and a second copy of the multiple template linear double-stranded nucleic acid for at least 5 minutes at a temperature of no greater than 80° C., wherein:
the DNA polymerase has strand-displacement activity,
the nucleic acid ligase has activity on blunt-ends of double-stranded nucleic acids,
each copy of the single template linear double-stranded nucleic acids contains a single copy of the double stranded nucleic acid template,
each copy of the single template linear double-stranded nucleic acid and multiple template linear double-stranded nucleic acid comprises: i) a first strand and a second strand, each strand comprising a 5′ end and a 3′ end, and ii) a first end and a second end, the first end comprising the 5′ end of the first strand and the 3′ end of the second strand, and the second end comprising the 5′ end of the second strand and the 3′ end of the first strand, and
during the incubation of the reaction mixture,
i) the first primer anneals to the first strand of the first copy of the single template linear double-stranded nucleic acid and serves as a primer for the generation of an extension product of the first primer, wherein the extension product of the first primer is complementary to the first strand of the single template linear double stranded nucleic acid;
ii) the second end of the second copy of the single template linear double-stranded nucleic acid is ligated to the first end of the third copy of the single template linear double-stranded nucleic acid; and
iii) a cross-over structure comprising the second strand of the first copy of the multiple template linear double-stranded nucleic acid annealed to the first strand of the second copy of the multiple template linear double-stranded nucleic acid is formed, wherein the 3′ end of the first strand of the second copy multiple template linear double stranded nucleic acid is annealed to the second strand of the first copy multiple template linear double-stranded nucleic acid past the 5′ end of the second strand of the first copy multiple template linear double-stranded nucleic acid;
wherein the multiple template linear double-stranded nucleic acid comprises a repeating unit of nucleic acid regions, wherein each of said nucleic acid regions comprises nucleic acid sequences, each of which constitutes a single template.
2. The method of claim 1 , wherein the reaction mixture is incubated at a first temperature and a second temperature, wherein during incubation at the first temperature, the first copy of a single template linear double-stranded nucleic acid undergoes a ligation reaction by the nucleic acid ligase to produce a second double-stranded nucleic acid, and during incubation at the second temperature, the first copy of a single template linear double-stranded nucleic acid and/or the second double-stranded nucleic acid undergoes a polymerase reaction by the isolated nucleic acid polymerase, and wherein the first temperature and the second temperature are no greater than about 70° C.
3. The method of claim 2 , wherein the ligation reaction is an end-to-end ligation reaction.
4. The method of claim 2 , wherein the polymerase reaction is a primer-based replication reaction and/or a cross-over strand replication reaction.
5. The method of claim 2 , wherein the second double-stranded nucleic acid comprises at least two copies of the first copy of a single template linear double-stranded nucleic acid.
6. The method of claim 2 , wherein the first temperature and second temperature are different.
7. The method of claim 2 , wherein the first temperature is about 42° C.
8. The method of claim 2 , wherein the second temperature is about 56° C.
9. The method of claim 2 , wherein the second temperature is about 65° C.
10. The method of claim 2 , wherein the first temperature and second temperature are the same.
11. A method of replicating a double stranded nucleic acid template, the method comprising,
incubating in a reaction mixture a first primer, a DNA polymerase, a nucleic acid ligase, a first copy of a single template linear double-stranded nucleic acid, a second copy of the single template linear double-stranded nucleic acid, a third copy of the single template linear double-stranded nucleic acid, a first copy of a multiple template linear double-stranded nucleic acid, and a second copy of the multiple template linear double-stranded nucleic acid for at least 5 minutes at a temperature of no greater than 80° C., wherein:
the DNA polymerase has strand-displacement activity,
the nucleic acid ligase has activity on blunt-ends of double-stranded nucleic acids,
each copy of the single template linear double-stranded nucleic acids contains a single copy of the double stranded nucleic acid template,
each copy of the single template linear double-stranded nucleic acid and multiple template linear double-stranded nucleic acid comprises: i) a first strand and a second strand, each strand comprising a 5′ end and a 3′ end, and ii) a first end and a second end, the first end comprising the 5′ end of the first strand and the 3′ end of the second strand, and the second end comprising the 5′ end of the second strand and the 3′ end of the first strand, and
during the incubation of the reaction mixture,
i) the first primer anneals to the first strand of the first copy of the single template linear double-stranded nucleic acid and serves as a primer for the generation of an extension product of the first primer, wherein the extension product of the first primer is complementary to the first strand of the single template linear double stranded nucleic acid;
ii) the second end of the second copy of the single template linear double-stranded nucleic acid is ligated to the first end of the third copy of the single template linear double-stranded nucleic acid; and
iii) a cross-over structure comprising the second strand of the first copy of the multiple template linear double-stranded nucleic acid annealed to the first strand of the second copy of the multiple template linear double-stranded nucleic acid is formed, wherein the 3′ end of the first strand of the second copy multiple template linear double stranded nucleic acid is annealed to the second strand of the first copy multiple template linear double-stranded nucleic acid past the 5′ end of the second strand of the first copy multiple template linear double-stranded nucleic acid;
wherein the multiple template linear double-stranded nucleic acid comprises a repeating unit of nucleic acid regions, wherein each of said nucleic acid regions comprises nucleic acid sequences, each of which constitutes a single template;
wherein the reaction mixture comprises a second primer, wherein the second primer is complementary to the second strand of the first copy of the single template linear double-stranded nucleic acid.
12. A method of replicating a double stranded nucleic acid template, the method comprising,
incubating in a reaction mixture a first primer, a DNA polymerase, a nucleic acid ligase, a first copy of a single template linear double-stranded nucleic acid, a second copy of the single template linear double-stranded nucleic acid, a third copy of the single template linear double-stranded nucleic acid, a first copy of a multiple template linear double-stranded nucleic acid, and a second copy of the multiple template linear double-stranded nucleic acid for at least 5 minutes at a temperature of no greater than 80° C., wherein:
the DNA polymerase has strand-displacement activity,
the nucleic acid ligase has activity on blunt-ends of double-stranded nucleic acids,
each copy of the single template linear double-stranded nucleic acids contains a single copy of the double stranded nucleic acid template,
each copy of the single template linear double-stranded nucleic acid and multiple template linear double-stranded nucleic acid comprises: i) a first strand and a second strand, each strand comprising a 5′ end and a 3′ end, and ii) a first end and a second end, the first end comprising the 5′ end of the first strand and the 3′ end of the second strand, and the second end comprising the 5′ end of the second strand and the 3′ end of the first strand, and
during the incubation of the reaction mixture,
i) the first primer anneals to the first strand of the first copy of the single template linear double-stranded nucleic acid and serves as a primer for the generation of an extension product of the first primer, wherein the extension product of the first primer is complementary to the first strand of the single template linear double stranded nucleic acid;
ii) the second end of the second copy of the single template linear double-stranded nucleic acid is ligated to the first end of the third copy of the single template linear double-stranded nucleic acid; and
iii) a cross-over structure comprising the second strand of the first copy of the multiple template linear double-stranded nucleic acid annealed to the first strand of the second copy of the multiple template linear double-stranded nucleic acid is formed, wherein the 3′ end of the first strand of the second copy multiple template linear double stranded nucleic acid is annealed to the second strand of the first copy multiple template linear double-stranded nucleic acid past the 5′ end of the second strand of the first copy multiple template linear double-stranded nucleic acid;
wherein the multiple template linear double-stranded nucleic acid comprises a repeating unit of nucleic acid regions, wherein each of said nucleic acid regions comprises nucleic acid sequences, each of which constitutes a single template;
wherein the reaction mixture further comprises a reverse transcriptase.
13. The method of claim 12 , further comprising providing a single-stranded RNA and reverse transcribing said single-stranded RNA and producing the first single template linear double-stranded nucleic acid.