Mutagenesis and PCR

Synthetic oligonucleotides are very important tools in the study and manipulation of DNA, including such techniques as site-directed mutagenesis and DNA amplification by the polymerase chain reaction (PCR). The techniques for chemical synthesis of oligonucleotides have been highly developed. Very efficient and automated methodologies based on synthesis on a solid support are used widely in fields that depend on the availability of defined DNA sequences.152... 

Illustration of a general method of mutagenesis using PCR. Primers are represented as short lines with arrowheads pointing in the 3 direction. The bump in primers 2 and 3 and their products represent a mismatched base, a deliberate alteration in base sequence from that present in the starting DNA. Of the four major products resulting from step 3 only D is extendable by DNA polymerase. 

Site-directed mutagenesis is one of the most powerful tools available to the biochemist. What are some of the applications of this technique How can the PCR method be used to do site-directed mutagenesis, and what is the advantage of this method ... 

G. Cortopassi, and D. J. Gales, A simple method for site-directed mutagenesis using PCR, Nucleic Acid Res. 1989, 17, 6545-6551. 

In addition to saturation mutagenesis and related protocols, shuffling-based methods have also benefited from easy and reliable synthesis of DNA. The original gene-shuffling method that hinges upon random fragmentation followed by PCR... 

Normal sequential error-prone PCR mutagenesis and DNA shuffling can not efficiently recombine or dissect two or more mutations if they are very close to each other [18]. In contrast, the Walk-Through approach allows recombination to occur at every position of templates, and therefore provides the possibihty of recombining or dissecting two or more mutations, although they may be very close to each other. 

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