Isolation and Characterization of Plant Genomic DNA Sequences via (Inverse) PCR Amplification

Abstract

Today the isolation and characterization of a gene of interest from any organism has become a standard procedure. One of the milestones that have facilitated this procedure in particular, has been the development of in vitro techniques for amplification of specific RNA or DNA sequences. By far the most important amplification technique, the polymerase chain reaction (PCR), was proposed more than 20 years ago (1), but only reached its practical form in 1985 (2). The basic principle is that following heat denaturing of the DNA sample two oligodeoxynucleotide primers are allowed to anneal to their target DNA sequences located at the opposite DNA strand. In the presence of deoxynucleotide triphosphates, DNA polymerase synthesizes new strands from the 3′ hydroxyl ends of the primers, doubling the number of copies of the target DNA segment. Repeated cycles of denaturation, primer annealing and extension eventually result m accumulation of the DNA segment defined by the primers. In 1985 Saiki et al. (2) used the Klenow fragment of Escherichiu coli DNA polymerase I, which because of its heat-sensitivity had to be added to the reaction mixture after each denaturation step. The discovery and application of the heat stable DNA polymerase from the thermophilic bacterium Thermus aquaticus for PCR and the automation of the procedure (3) laid the foundation for the development of numerous and diverse PCR methods (4).