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 for amplifying a double stranded nucleic acid template, comprising,
(A) preparing a reaction mixture comprising:
(i) a double-stranded nucleic acid comprising at least one copy of the double-stranded nucleic acid template, wherein the double-stranded nucleic acid comprises a first strand and a second strand,
(ii) an isolated nucleic acid polymerase,
(iii) an isolated nucleic acid ligase,
(iv) a first primer, wherein the first primer is complementary to the first strand of the double-stranded nucleic acid template,
(v) a second primer, wherein the second primer is complementary to the second strand of the double-stranded nucleic acid template, and
(B) incubating the reaction mixture at a temperature of no greater than 70 C for at least 5 minutes,
wherein
a plurality of concatemers comprising at least two copies of the double-stranded nucleic acid template are generated, and
the double-stranded nucleic acid template is amplified at least 100-fold within 60 minutes of initiation of the method.
2 . The method of claim 1 , wherein the reaction mixture is incubated at a temperature of no greater than 65 C.
3 . The method of claim 2 , wherein the reaction mixture is incubated at a temperature of no greater than 60 C.
4 . A vessel, comprising in fluid communication therein:
(A) an isolated nucleic acid polymerase,
(B) an isolated nucleic acid ligase,
(C) a double-stranded nucleic acid template comprising a first strand and a second strand,
(D) a first primer, wherein the first primer is complementary to the first strand of the double-stranded nucleic acid template,
(E) a second primer, wherein the second primer is complementary to the second strand of the double-stranded nucleic acid template,
5 . A kit for detecting a target nucleic acid of interest comprising at least a first strand, the kit comprising two or more fluidically isolated containers, the containers collectively comprising:
(A) an isolated nucleic acid polymerase,
(B) an isolated nucleic acid ligase,
(C) a first primer, wherein the first primer is complementary to the first strand of the target nucleic acid of interest,
(D) a second primer, wherein the second primer is complementary to a sequence complementary the first strand of the target nucleic acid of interest.
6 . The kit of claim 5 , further comprising the target nucleic acid of interest.
7 . The vessel of claim 4 , further comprising a nucleic acid dye.
8 - 9 . (canceled)
10 . The vessel of claim 4 , wherein the vessel comprises both a DNA polymerase and a reverse transcriptase.
11 . (canceled)
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 first primer and second primer each comprise at least 6 nucleotides.
14 . The method of claim 13 , wherein the first primer and second primer each comprise at least 8 nucleotides.
15 . The method of claim 1 , wherein the first primer and second primer each comprise no more than 30 nucleotides.
16 . The kit of claim 5 , further comprising a nucleic acid dye.
17 . The kit of claim 5 , wherein the kit comprises both a DNA polymerase and a reverse transcriptase.
18 . The vessel of claim 4 , wherein the first primer and second primer each comprise at least 6 nucleotides.
19 . The vessel of claim 18 , wherein the first primer and second primer each comprise at least 8 nucleotides.
20 . The kit of claim 5 , wherein the first primer and second primer each comprise at least 6 nucleotides.
21 . The kit of claim 20 , wherein the first primer and second primer each comprise at least 8 nucleotides.
22 . The vessel of claim 4 , wherein the first primer and second primer each comprise no more than 30 nucleotides.
23 . The kit of claim 5 , wherein the first primer and second primer each comprise no more than 30 nucleotides.