A general solution for opening double-stranded DNA for isothermal amplification.
Nucleic acid amplification is the core technology of molecular biology and genetic engineering. Various isothermal amplification techniques have been developed as alternatives to polymerase chain reaction (PCR). However, most of these methods can only detect single stranded nucleic acid. Herein, we put forward a simple solution for opening double-stranded DNA for isothermal detection methods. The strategy employs recombination protein from E. coli (RecA) to form nucleoprotein complex with single-stranded DNA, which could scan double-stranded template for homologous sites. Then, the nucleoprotein can invade the double-stranded template to form heteroduplex in the presence of ATP, resulting in the strand exchange. The ATP regeneration system could be eliminated by using high concentration of ATP, and the 3'-OH terminal of the invasion strand can be recognized by other DNA modifying enzymes such as DNA polymerase or DNA ligase. Moreover, dATP was found to be a better cofactor for RecA, which make the system more compatible to DNA polymerase. The method described here is a general solution to open dsDNA, serving as a platform to develop more isothermal nucleic acids detection methods for real DNA samples based on it.
Natural Products Research Center, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, P. R. China.
Gangyi
G
Chen
Natural Products Research Center, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, P. R. China.
Juan
J
Dong
Natural Products Research Center, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, P. R. China.
Yi
Y
Yuan
Natural Products Research Center, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, P. R. China.
Na
N
Li
Natural Products Research Center, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, P. R. China.
Xin
X
Huang
Natural Products Research Center, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, P. R. China.
Xin
X
Cui
Natural Products Research Center, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, P. R. China.
Zhuo
Z
Tang
Nat Genet. 1998 Jul;19(3):225-32
9662393
Biochemistry. 2001 Feb 13;40(6):1779-86
11327840
EMBO Rep. 2004 Aug;5(8):795-800
15247927
DNA Repair (Amst). 2010 Dec 10;9(12):1264-72
20971042
Nat Protoc. 2008;3(5):877-82
18451795
J Biol Chem. 1988 Aug 15;263(23):11029-32
3042763
Genome Res. 2004 Dec;14(12):2478-85
15574826
J Biomed Nanotechnol. 2013 Feb;9(2):267-73
23627053
J Mol Biol. 1993 Apr 5;230(3):1015-24
8478916
J Forensic Sci. 2006 Jul;51(4):865-73
16882231
Chem Rev. 2015 Nov 25;115(22):12491-545
26551336
Mol Biochem Parasitol. 1990 Apr;40(1):129-36
2348829
Biochemistry. 1986 Apr 8;25(7):1482-94
2939874
Nucleic Acids Res. 1992 Apr 11;20(7):1691-6
1579461
Nucleic Acids Res. 1997 Jun 1;25(11):2106-13
9153309
J Biochem. 1982 May;91(5):1767-75
7047518
Nature. 1991 Mar 7;350(6313):91-2
1706072
J Biol Chem. 1987 Jan 25;262(3):1337-43
3543003
Proc Natl Acad Sci U S A. 1981 Jun;78(6):3433-7
7022448
Proc Natl Acad Sci U S A. 1992 Jan 1;89(1):392-6
1309614
Transfusion. 2004 Jan;44(1):97-103
14692974
J Mol Biol. 1992 Sep 5;227(1):334-46
1522597
J Biol Chem. 1993 Oct 25;268(30):22525-30
8226762
PLoS Biol. 2006 Jul;4(7):e204
16756388
J Biol Chem. 1990 Oct 5;265(28):17050-4
2211610
Clin Chem Lab Med. 2016 Sep 1;54(9):1427-33
26845722
J Virol Methods. 2014 Jun;202:101-5
24642236
EMBO J. 1993 Jan;12(1):9-16
8428597
CMAJ. 2000 Aug 8;163(3):301-9
10951731
Clin Chem. 2000 Dec;46(12):1987-9
11106331
Science. 2015 Aug 28;349(6251):977-81
26315438
Genes Dev. 1990 Nov;4(11):1951-63
2276627
Nature. 2012 Nov 8;491(7423):274-8
23103864
J Biol Chem. 1982 Dec 10;257(23):13981-6
6754721
J Biol Chem. 1983 May 25;258(10):6073-7
6222041
Methods Mol Biol. 1994;30:263-79
8004201
Lancet Infect Dis. 2004 Jun;4(6):337-48
15172342
J Clin Microbiol. 2000 Dec;38(12):4499-502
11101586
J Biol Chem. 1992 May 5;267(13):9315-20
1533628
Annu Rev Microbiol. 1996;50:349-73
8905084
PLoS One. 2014 Feb 13;9(2):e88845
24551178
Cell. 1980 May;20(1):223-35
7388943
J Biol Chem. 1985 Jan 25;260(2):1196-202
3838171
Proc Natl Acad Sci U S A. 1980 May;77(5):2606-10
6994103
Trends Mol Med. 2002 Jun;8(6):257-60
12067606