1Department of Dermatology, Wayne State University, Detroit, Michigan 48201; 2Department of Dermatology, Niigata University, Niigata, Japan As the application of PCR has expanded, more amplified products have been used for cloning and mutagenesis. Although Taq polymerase (from eubacterium Thermus aquaticus) is the most convenient and intensively studied enzyme to date, one of its drawbacks is a...

The use of a "direct PCR" DNA polymerase enables PCR amplification without any prior DNA purification from blood samples due to the enzyme's resistance to inhibitors present in blood components. Such DNA polymerases are now commercially available. We compared the PCR performance of six direct PCR-type DNA polymerases (KOD FX, Mighty Amp, Hemo KlenTaq, Phusion Blood II, KAPA Blood, and BIOTAQ) i...

We describe a convenient PCR-based protocol for in vitro recombination of homologous genes, thereby minimizing the rate of associated point mutations. High-fidelity recombination conditions were obtained using Vent DNA polymerase, which, in contrast to Taq DNA polymerase, shows significant proofreading activity and ranges among the slowest thermostable DNA polymerases, allowing tight control of...

One of the key points in the genome project is finding ways to reduce the running cost in DNA sequencing. One way is to use a highly-sensitive fluorescent DNA sequencer, where only trace amounts of template DNA and reagents are needed. An experimental protocol optimized for the trace amounts of DNA analysis was established by using the hybridization reaction rate coefficient of primers on templ...

DNA polymerases are desired that incorporate modified nucleotides into DNA with diminished pausing, premature termination and infidelity. Reported here is a simple in vitro assay to screen for DNA polymerases that accept modified nucleotides based on a set of primer extension reactions. In combination with the scintillation proximity assay (SPA[trade]), this allows rapid and simple screening of...

lysates of the recombinant P. pastoris strains KM71 (pPIC9-MYO)-MutS and GS115 (pPIC9-MYO)-Mut+, which contained the integration of the myostatin gene to chromosomal DNA, was completed. When Taq DNA polymerase was used for amplification, we observed either no or nonspecific amplification products. The positive control sample yielded the 831-bp specific fragment. When the HOTStar Taq DNA polymer...

Large-scale DNA sequencing projects can be limited by various technical bottlenecks. As more DNA templates are generated and successfully processed, the mechanisms and technologies continue to be improved and optimized. Fluorescence-based automated sequencing (1) has replaced radioisotope-based manual DNA sequencing (2) as the primary approach in large-scale genome projects. For this approach, ...