PCR Amplification of Specific Sequences from a cDNA Library

Abstract

Until recently, the most commonly used procedures in gene isolation required the establishment of a cDNA library from tissue or cellular RNAs. The gene of interest was then identified by screening the library with antibody¹ or DNA probes.² This approach has been very successful in cloning a great number of genes. However, construction and screening of a cDNA library is quite time-consuming, labor intensive and requires considerable protein sequence information for screening with oligonucleotide probes. The polymerase chain reaction (PCR) procedure^3,4 has enabled the specific amplification of a DNA species many million fold and has become an invaluable tool in molecular cloning and diagnosis. Most recently, the availability of Taq DNA polymerase has greatly simplified the PCR procedure. • This enzyme has a broad temperature optimum centered around 75°C, and can survive repeated incubations at <95°C. More importantly, this enzyme is very processive and lacks 3’ exonuclease activity.⁵ It is, therefore, theoretically possible to clone out any gene from total RNA or DNA without going through all the steps of library construction and screening. To test this hypothesis, we selected a gene from which only limited protein sequence information was available as a model to develop a quick and simple procedure for gene isolation. We now describe the approach and methods used in the identification of an exocrine protein from the salivary glands of a South American bat.