[Constuction of an improved system for the determination of fidelity of polymerase in PCR].
A system used for the determination of fidelity of DNA polymerase in PCR was developed in E.coli and was used to determine the fidelity of FD DNA polymerase in PCR amplication. Frame shift and base substitution mutations were created in vitro in the lacZ gene in pUC118 and pUC119. As a result, a set of six derived plasmids namely pFDFM118 and pFDFM119 (-1 frame shift), pFDFP118 and pFDFP119 (+1 frame shift), pFDFU118 and pFDFU119 (base substitution) were obtained. All of them failed to carry out lacZ alpha-complementation in E.coli MV1184 and the colonies appeared white on medium with X-Gal and IPTG consequently. PCR reaction was carried out using these derived plasmids as templates and the PCR products were ligated to specially constructed cloning vectors pFDFL118 or pFDFL119, and the ligated products were used to transform MV1184. If any back mutation happens to occur during PCR, the transformants would appear blue on medium with X-Gal and IPTG. By scoring the number of blue and white colonies, the fidelity of DNA polymerase can be calculated. With this system the error of replication of the FD DNA polymerase was found to be 10(-5)-10(-6).
Du
H
H
Institute of Genetics, Fudan University Shanghai.
Xu
M
M
Ji
C
C
Mao
Y
Y
D001483:Base Sequence; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*
D004259:DNA-Directed DNA Polymerase