Gene cloning and polymerase chain reaction with proliferating cell nuclear antigen from Thermococcus kodakaraensis KOD1.
The gene encoding the proliferating cell nuclear antigen (PCNA), a sliding clamp of DNA polymerases, was cloned from an euryarchaeote, Thermococcus kodakaraensis KOD1. The PCNA homologue, designated Tk-PCNA, contained 249 amino acid residues with a calculated molecular mass of 28,200 Da and was 84.3% identical to that from Pyrococcus furiosus. Tk-PCNA was overexpressed in Escherichia coli and purified. This protein stimulated the primer extension abilities of the DNA polymerase from T. kodakaraensis KOD1 'KOD DNA polymerase'. The stimulatory effect of Tk-PCNA was observed when a circular DNA template was used and was equally effective on both circular and linear DNA. The Tk-PCNA improved the sensitivity of PCR without adverse effects on fidelity with the KOD DNA polymerase. This is the first report in which a replication-related factor worked on PCR.
Kitabayashi
Masao
M
Tsuruga Institute of Biotechnology, Toyobo Co., Ltd., 10-24 Toyo-cho, Tsuruga, Fukui 914-0047, Japan. masao_kitabayashi@bio.toyobo.co.jp
Nishiya
Yoshiaki
Y
Esaka
Muneharu
M
Itakura
Mitsuo
M
Imanaka
Tadayuki
T
D003001:Cloning, Molecular; D017931:DNA Primers; D004269:DNA, Bacterial / Q000096:biosynthesis / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial / Q000235:genetics*; D008970:Molecular Weight; D010957:Plasmids / Q000235:genetics; D018809:Proliferating Cell Nuclear Antigen / Q000096:biosynthesis / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019712:Thermococcus / Q000235:genetics* / Q000378:metabolism
D017931:DNA Primers; D004269:DNA, Bacterial; D018809:Proliferating Cell Nuclear Antigen; D011994:Recombinant Proteins; D004259:DNA-Directed DNA Polymerase