Gene Cloning and Expression Profiling by Rapid Amplification of Gene Inserts with Universal Vector Primers.

Isolation of a full-length gene and analysis of expression profiling are fundamental and challenging in the current molecular biology. A great deal of effort is needed to detect unknown gene sequences by screening cDNA or genomic libraries by nucleic acid or protein probes. As the complete genome sequences of many organisms have been reported, this has raised the most challenging issue that how global gene expression patterns can be detected. Recently, various PCR methods have been developed for cloning of unknown genes and analysis of expression profiling (1-10). Several PCR-based strategies such as iAFLP (introduced amplified fragment length polymorphism) and TOGA (total gene-expression analysis) are currently available for global gene-expression analysis (8-10). An outline of gene cloning and expression profiling by anchored-PCR with vector primers is illustrated in Fig. 1B. In this chapter, we focus on how to use a gene library and anchored-PCR for cloning unknown gene sequences (see fig. 1 A). Friedmann et al. (1) 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 can not be amplified by this method. [ABSTRACT FROM AUTHOR]