Megaprimer Mutagenesis Using Very Long Primers
نویسندگان
چکیده
منابع مشابه
Megaprimer mutagenesis using very long primers.
It is well known that some genetic mutations have a critical impact on protein structure and function. To study the effects of these mutations, site-directed mutagenesis allows the introduction of a defined substitution of specific residues. For the past few years, numerous PCR-based methods have been employed for achieving site-directed mutagenesis. Among these methods, megaprimer PCR has been...
متن کاملCreating random mutagenesis libraries using megaprimer PCR of whole plasmid.
The conventional method for cloning a DNA fragment is to insert it into a vector and ligate it. Although this method is commonly used, it is labor intensive because the ratio and concentrations of the DNA insert and the vector need optimizing. Even then, the resultant library is often plagued with unwanted plasmids that have no inserts or multiple inserts. These species have to be eradicated to...
متن کامل"Megaprimer" method of PCR-based mutagenesis: the concentration of megaprimer is a critical factor.
The technique of site-directed mutagenesis (SDM) is widely used in the field of molecular biology to introduce mutations into DNA (5). The development of polymerase chain reaction (PCR)-based SDM has eliminated the need to clone DNA, therefore simplifying the technique and enabling the desired results to be achieved more quickly (3). The “megaprimer” method of PCR-based SDM incorporates three p...
متن کاملSimplified one-tube "megaprimer" polymerase chain reaction mutagenesis.
Site-directed mutagenesis has become an essential tool in the investigation of gene structure and function as well as the products of gene expression, namely RNA and protein. Selected positions in a DNA sequence are changed, and the eVects of the mutations are studied in vitro and/or in vivo. Although many diVerent mutagenic strategies are available [1], the simplicity, cost eVectiveness, and a...
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...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
ژورنال
عنوان ژورنال: BioTechniques
سال: 2003
ISSN: 0736-6205,1940-9818
DOI: 10.2144/03341bm07