Inverse PCR. Genomic DNA cloning.

Abstract

AbstractInverse PCR (IPCR) was designed for amplifying anonymous flanking genomic DNA regions (1 2). The technique involves the digestion of source DNA, circulation of restriction fragments, and amplification using oligonucleotides that prime the DNA synthesis directed away from the core region of a known sequence, i.e., opposite of the direction of primers used in normal or standard PCR Fig. 1). Prior to the invention of the polymerase chain reaction (PCR), the acquisition of a specific DNA fragment usually entailed the construction and screening of DNA libraries, and the traditional “walking” into flanking DNA fragments involved the successive probing of libraries with clones obtained in the prior screening. These time-consuming procedures could be replaced by IPCR. Because IPCR can be used to efficiently and rapidly amplify regions of unknown sequence flanking any identified segment of cDNA or genomic DNA, researchers do not need to construct and screen DNA libraries to obtain additional unidentified DNA sequence information using this technique. Some recombinant phage or plasmids may be unstable in bacteria and amplified libraries tend to lose them. IPCR eliminates this problem. Diagram of IPCR for genomic DNA cloning. The procedure consists of four steps: genomic DNA isolation, circularization of double-stranded DNA, reopening of the circular DNA, and amplification of reverse DNA fragment. The black and open bars represent the known and unknown sequence regions of double-stranded cDNA, respectively. RE: restriction enzymesite; 65p: gene-specific primer. KeywordsStandard Polymerase Chain ReactionRecombinant PhageInverse Polymerase Chain ReactionPolymerase Chain Reaction MutagenesisIntramolecular LigationThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.