Isothermal in vitro amplification of DNA by a restriction enzyme/DNA polymerase system - PMC Skip to main content Official websites use .gov A .gov website belongs to an official government organization in the United States. Secure .gov websites use HTTPS A lock ( Lock Locked padlock icon ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites. Search PMC Full-Text Archive Search in PMC Journal List User Guide PERMALINK Copy As a library, NLM provides access to scientific literature. Inclusion in an NLM database does not imply endorsement of, or agreement with, the contents by NLM or the National Institutes of Health. Learn more: PMC Disclaimer | PMC Copyright Notice Proc Natl Acad Sci U S A . 1992 Jan 1;89(1):392–396. doi: 10.1073/pnas.89.1.392 Isothermal in vitro amplification of DNA by a restriction enzyme/DNA polymerase system. G T Walker G T Walker 1 Department of Molecular Biology, Becton Dickinson Research Center, Research Triangle Park, NC 27709. Find articles by G T Walker 1 , M C Little M C Little 1 Department of Molecular Biology, Becton Dickinson Research Center, Research Triangle Park, NC 27709. Find articles by M C Little 1 , J G Nadeau J G Nadeau 1 Department of Molecular Biology, Becton Dickinson Research Center, Research Triangle Park, NC 27709. Find articles by J G Nadeau 1 , D D Shank D D Shank 1 Department of Molecular Biology, Becton Dickinson Research Center, Research Triangle Park, NC 27709. Find articles by D D Shank 1 Author information Copyright and License information 1 Department of Molecular Biology, Becton Dickinson Research Center, Research Triangle Park, NC 27709. PMC Copyright notice PMCID: PMC48243 PMID: 1309614 Abstract An isothermal in vitro DNA amplification method was developed based upon the following sequence of reaction events. Restriction enzyme cleavage and subsequent heat denaturation of a DNA sample generates two single-stranded target DNA fragments (T1 and T2). Present in excess are two DNA amplification primers (P1 and P2). The 3' end of P1 binds to the 3' end of T1, forming a duplex with 5' overhangs. Likewise, P2 binds to T2. The 5' overhangs of P1 and P2 contain a recognition sequence (5'-GTTGAC-3') for the restriction enzyme HincII. An exonuclease-deficient form of the large fragment of Escherichia coli DNA polymerase I (exo- Klenow polymerase) [Derbyshire, V., Freemont, P. S., Sanderson, M. R., Beese, L., Friedman, J. M., Joyce, C. M. & Steitz, T. A. (1988) Science 240, 199-201] extends the 3' ends of the duplexes using dGTP, dCTP, TTP, and deoxyadenosine 5'-[alpha-thio]triphosphate, which produces hemiphosphorothioate recognition sites on P1.T1 and P2.T2. HincII nicks the unprotected primer strands of the hemiphosphorothioate recognition sites, leaving intact the modified complementary strands. The exo- Klenow polymerase extends the 3' end at the nick on P1.T1 and displaces the downstream strand that is functionally equivalent to T2. Likewise, extension at the nick on P2.T2 results in displacement of a downstream strand functionally equivalent to T1. Nicking and polymerization/displacement steps cycle continuously on P1.T1 and P2.T2 because extension at a nick regenerates a nickable HincII recognition site. Target amplification is exponential because strands displaced from P1.T1 serve as targets for P2 and strands displaced from P2.T2 serve as targets for P1. A 10(6)-fold amplification of a genomic sequence from Mycobacterium tuberculosis or Mycobacterium bovis was achieved in 4 h at 37 degrees C. Full text PDF Images in this article Image on p.395 Selected References These references are in PubMed. This may not be the complete list of references from this article. Barany F. Genetic disease detection and DNA amplification using cloned thermostable ligase. 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