Repeated GenomiPhi, phi29 DNA polymerase-based rolling circle amplification, is useful for generation of large amounts of plasmid DNA.
The GenomiPhi DNA Amplification Kit (Amersham Biosciences Co., Piscataway, NJ, USA) uses a novel, isothermal DNA amplification technique for production of microgram quantities of DNA from picograms of starting material. In this study, we tested whether repeated reactions using the kit would enhance high-fidelity amplification of a small amount (10 ng) of plasmid DNA containing enhanced green fluorescent protein (EGFP) cDNA. Approximately 4.7 x 10(9)-fold amplification of plasmid DNA was achieved after 3 repeated reactions, and the RCA products were sufficient for EGFP expression in cells. Based on the preliminary results, repeated reactions using the kit appears feasible for amplification of the entire genome of organisms or cells (such a sperms and oocytes) carrying very small amounts of DNA.
Sato
Masahiro
M
The Institute of Medical Sciences, Tokai University, Bohseidai, Isehara, Kanagawa 259-1193, Japan.
Ohtsuka
Masato
M
Ohmi
Yuhsuke
Y
D000818:Animals; D017103:Bacillus Phages / Q000201:enzymology*; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D021141:Nucleic Acid Amplification Techniques; D010957:Plasmids / Q000096:biosynthesis*; D013552:Swine; D014162:Transfection
D004247:DNA; D004259:DNA-Directed DNA Polymerase