Biomedical subjects
Publications and source records attributed to Yuhsuke Ohmi.
The GenomiPhi DNA Amplification Kit employs rolling circle amplification (RCA) using phi29 polymerase, dNTPs, and random hexamers. We demonstrated that repeated RCA (at least three times) is useful for high-fidelity amplification of large amounts of plasmid DNA.
Type IIS or interrupted palindrome restriction endonuclease digestion produces unique cohesive ends. Utilization of this feature of BglI, SfiI and BstXI has led to direct subcloning of 28.5-kb and 22.9-kb regions, which contains candidate genes for medaka Double anal fin (Da) mutant, from a 229-kb BAC insert. This method does not contain gel-fractionation of the fragments and time-consuming screening process using hybridization. Non-recombinant backgrounds were distinguished from recombinant clones by introducing GFPuv gene into the subcloning vector. This strategy was successfully applicable even when the target region was cut into three fragments by these enzymes.
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.