Inverse PCR

Abstract

Since the first report on cDNA cloning in 1972 (1), this technology has developed into a powerful and universal tool in isolation, characterization, and analysis of both eukaryotic and prokaryotic genes. However, the conventional methods of cDNA cloning require much effort to generate a library that is packaged in phage or plasmid, and then surveyed in a large number of recombinant phages or plasmids. There are three major limitations in these methods. First, a substantial amount (at least 1 mg) of purified mRNA is needed as starting material to generate libraries of sufficient diversity (2). Second, the intrinsic difficulty of multiple sequential enzymatic reactions required for cDNA cloning often leads to low yields and truncated clones (3). Finally, screening of a library with hybridization technique is time-consuming. PCR technology can simplify and improve cDNA cloning. Using PCR with two gene-specific primers, a piece of known sequence cDNA can be specifically and efficiently amplified and isolated from very small numbers (<10⁴) of cells (4). However, it is often difficult to isolate full-length cDNA copies of mRNA on the basis of very limited sequence information. The unknown sequence flanking a small stretch of the known sequence of DNA cannot be amplified by the conventional PCR. Recently, anchored PCR (5–7) and inverse PCR (8–10) have been developed to resolve this problem. Anchored PCR techniques have the common point that DNA cloning goes from a small stretch of known DNA sequence to the flanking unknown sequence region with the aid of a gene-specific primer at one end and a universal primer at other end. Because of only one gene-specific primer in the anchored PCR it is easier to get a high level of nonspecific amplification by PCR than with two gene-specific primers (10,11). The major advantage of inverse PCR (IPCR) is to amplify the flanking unknown sequence by using two gene-specific primers.