Suicide polymerase endonuclease restriction, a novel technique for enhancing PCR amplification of minor DNA templates.
PCR-based molecular analyses can be hindered by the presence of unwanted or dominant DNA templates that reduce or eliminate detection of alternate templates. We describe here a reaction in which such templates can be exclusively digested by endonuclease restriction, leaving all other DNAs unmodified. After such a modification, the digested template is no longer available for PCR amplification, while nontarget DNAs remain intact and can be amplified. We demonstrate the application of this method and use denaturing gradient gel electrophoresis to ascertain the removal of target DNA templates and the subsequent enhanced amplification of nondigested DNAs. Specifically, plastid 16S rRNA genes were exclusively digested from environmental DNA extracted from plant roots. In addition, pure culture and environmental DNA extracts were spiked with various amounts of genomic DNA extracted from Streptomyces spp., and selective restriction of the Streptomyces 16S rRNA genes via the suicide polymerase endonuclease restriction PCR method was employed to remove the amended DNA.
Green
Stefan J
SJ
Institute of Soil, Water, and Environmental Sciences, Agriculture Research Organization, The Volcani Center, P.O. Box 6, Bet-Dagan 50-250, Israel.
Minz
Dror
D
D010367:Arachis / Q000235:genetics; D018553:Cucumis sativus / Q000235:genetics; D004247:DNA / Q000378:metabolism*; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000235:genetics; D004586:Electrophoresis / Q000379:methods; D005821:Genetic Techniques; D018087:Plastids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D013302:Streptomyces / Q000235:genetics; D019914:Taq Polymerase / Q000378:metabolism*; D013698:Templates, Genetic
D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D012336:RNA, Ribosomal, 16S; D004247:DNA; D019914:Taq Polymerase; D004262:DNA Restriction Enzymes