APPLICATIONS OF THE LONG AND ACCURATE POLYMERASE CHAIN-REACTION METHOD IN YEAST MOLECULAR-BIOLOGY - DIRECT SEQUENCING OF THE AMPLIFIED DNA AND ITS INTRODUCTION INTO YEAST

A DNA fragment longer than 10 kb can be amplified by the long and accu rate polymerase chain reaction (LA-PCR) method. We demonstrate here ap plications of this technique in molecular biological studies of Saccha romyces cerevisiae. We have shown that DNA fragments amplified by LA-P CR can be directly used as a template in the chain-termination sequenc ing protocol, making it possible to quickly identify the DNA insert of yeast genomic library clones, We have also shown that the amplified y east DNA can easily be introduced into yeast by co-transformation with linearized vector DNA. Overlapping DNA between the amplified yeast fr agment and the vector must be more than 20 bp long in order to obtain 90% or more correct recombinant plasmids. These results suggest that s imple amplification of yeast clones by LA-PCR can replace the previous procedures of yeast clone recovery, consisting of transformation of E scherichia coli, propagation of plasmids in E. coli and preparation of plasmid DNA. (C) 1997 by John Wiley & Sons, Ltd.