PCR-Based Full-Length cDNA Cloning Utilizing the Universal-Adaptor/Specific DOS Primer-Pair Strategy

Abstract

Biological systems are often influenced by molecules that are neither present in vast quantities or easily purified to homogeneity from other cellular constituents. The development of simple, efficient molecular cloning systems coupled with the relative ease of DNA sequence determination has made nucleic acid sequence determination the choice methodology when sequence determination of the complete biological molecule is indicated. However, one bottleneck that impedes direct access to sequence determination is the necessity to screen recombinant libraries containing a large number of clones for the low-abundance member. Advances in protein microsequencing techniques combined with application of the polymerase chain reaction (PCR) help simplify this process (1-3). Utilizing only the N-terminal protein sequence information, we have developed a protocol for the selective amplification and subsequent cloning of specific full-length cDNAs, which in some instances may circumvent the time-consuming, tedious process of library screening. This approach, as illustrated in Fig. 1, employs the following steps: