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 accurate polymerase chain reaction (LA-PCR) method. We demonstrate here applications of this technique in molecular biological studies of Saccharomyces cerevisiae. We have shown that DNA fragments amplified by LA-PCR can be directly used as a template in the chain-termination sequencing protocol, making it possible to quickly identify the DNA insert of yeast genomic library clones. We have also shown that the amplified yeast DNA can easily be introduced into yeast by co-transformation with linearized vector DNA. Overlapping DNA between the amplified yeast fragment and the vector must be more than 20 bp long in order to obtain 90% or more correct recombinant plasmids. These results suggest that simple amplification of yeast clones by LA-PCR can replace the previous procedures of yeast clone recovery, consisting of transformation of Escherichia coli, propagation of plasmids in E. coli and preparation of plasmid DNA.
Takita
Y
Y
Department of Biological Sciences, Graduate School of Science, University of Tokyo, Japan.
Takahara
M
M
Nogami
S
S
Anraku
Y
Y
Ohya
Y
Y
D001483:Base Sequence; D004271:DNA, Fungal / Q000737:chemistry*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D017422:Sequence Analysis, DNA
D004271:DNA, Fungal