Amplification of Gene Ends from Gene Libraries by Polymerase Chain Reaction with Single-Sided Specificity

Abstract

Isolation of a full-length gene on the basis of a limited sequence information is often troublesome and challenging. Tremendous effort is needed to isolate a specific gene by screening cDNA or genomic libraries by oligonucleotide or nucleic acid probes. In those methods, basically nucleic acid probes are used in a screening process to check whether or not a plaque or a colony contains the sequence of interest. There have been attempts to isolate specific DNA fragments using immobilized DNA, in which particular DNA fragments were enriched by hybrid selection and then the concentrated library was screened by a specific DNA probe (1,2). Recently, polymerase chain reaction (PCR) has been applied to the cloning of genes. Friedmann et al. (3) first used PCR to screen λgt11 library with two gene-specific primers. This protocol can be effectively used to isolate a particular DNA fragment between two specific primers or to generate nucleic acid probe from cDNA libraries. The unknown sequences flanking the fragment between the two specific primers cannot be amplified by this method.