Fusion PCR, a one-step variant of the "megaprimer" method of mutagenesis.
نویسنده
چکیده
منابع مشابه
A new approach to 'megaprimer' polymerase chain reaction mutagenesis without an intermediate gel purification step
BACKGROUND Site-directed mutagenesis is an efficient method to alter the structure and function of genes. Here we report a rapid and efficient megaprimer-based polymerase chain reaction (PCR) mutagenesis strategy that by-passes any intermediate purification of DNA between two rounds of PCR. RESULTS The strategy relies on the use of a limiting concentration of one of the flanking primers (reve...
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1.Barik, S. and M.S. Galinski. 1991. “Megaprimer” method of PCR: increased template concentration improves yield. BioTechniques 10:489-490. 2.Colosimo, A., Z. Xu, G. Novelli, B. Dallapiccola, and D.C. Gruenert. 1999. Simple version of “megaprimer” PCR for site-directed mutagenesis. BioTechniques 26:870-873. 3.Datta, A.K. 1995. Efficient amplification using “megaprimer” by asymmetric polymerase ...
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3.Higuchi, R., B. Krummel and R.K. Saiki. 1988. A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res. 16:7351-7367. 4.Sarkar, G. and S.S. Sommer. 1990. The “megaprimer” method of site-directed mutagenesis. BioTechniques 8:404-407. 5.Smith, M. 1985. In vitro mutagenesis. Annu. Rev. Genet. 19:423-462. 6.Upende...
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We have developed a novel three-primer, one-step PCR-based method for site-directed mutagenesis. This method takes advantage of the fact that template plasmid DNA cannot be efficiently denatured at its reannealing temperature (T(ra)), which is otherwise a troublesome problem in regular PCR. Two flanking primers and one mutagenic primer with different melting temperatures (T(m)) are used togethe...
متن کاملSite-directed mutagenesis by double polymerase chain reaction : megaprimer method.
The "megaprimer" method (1) based on polymerase chain reaction (PCR) is one of the simplest and most versatile procedures of site-specific in vitro mutagenesis available to date. The method utilizes three oligonucleotide primers and two rounds of PCR performed on a DNA template containing the cloned gene that is to be mutated. The rationale of the method is shown schematically in Fig. 1 where A...
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ورودعنوان ژورنال:
- BioTechniques
دوره 24 5 شماره
صفحات -
تاریخ انتشار 1998