PCR mutagenesis

The technique described earher can be modified for many different purposes. One way in which we regularly use a slight modification of the technique is to clean up dirty DNA. Often, when cDNAs are cloned from a hbrary, additional extraneous DNA is associated with the cDNA of interest. After subcloning into a vector, this extra DNA can interfere with the expression of the clone and the extra size that it adds to the insert can make the DNA difficult to manipulate. Additionally, the cDNA may be in a vector that is not suitable for expression and may not have the correct restriction endonucleases cut sites to allow it to be subcloned into the vector of choice. PCR mutagenesis can be useful to remove the extraneous DNA and to insert restriction sites at convenient positions. 

K. Aalbers, and C. T. Lutz, Unwanted mutations in PCR mutagenesis avoiding the predictable, PCR Methods Appl. 1993, 2, 53-57. 

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. 

Figure 3.18 PCR mutagenesis. This is the simple way to engineer the primary structure of a protein of interest. Where desired mutations are near the 5 -terminus or the 3 -terminus or the sense strand, then the mismatched primer technique is used. In the illustrated case, the desired mutation is near the 3 -terminus so a normal sense strand primer is combined with a mismatched complementary strand primer containing a mutation (blue). When the PCR reaction is allowed to proceed with the template DNA, then the mismatch in the complementary strand primer forces a mismatch to appear in both sense and complementary strands of the final PCR amplification product resulting in a PCR mutant gene. Restriction cutting and ligation of the mutant PCR product into a cloning or expression vector generates a mutant recombinant pDNA construct ready for transformation and selection, then DNA purification and sequencing of correct mutant recombinant DNA.

A number of methods developed for PCR mutagenesis (substitution, insertion, and deletion) are based on overlap-extension strategies (28—31). The prototype consists of the following steps (Fig. A.5) First, two sets of DNA fragments are amplified separately using two (inner and outer) pairs of oligonucleotide primers ... 

FIGURE A.5 A general scheme for standard PCR mutagenesis which employs two mutagenic primers (forward and reverse, m( and m respectively) and two universal (or amplification) primers (Uf and u,). The two PCR products are joined by annealing at the overlap sequences and are further extended and PCR amplified. The overlap-extension strategy produces a homoduplex mutant cassette which is then inserted into a suitable expression vector. REl and RE2 indicate two available (natural or engineered) restriction sites. 

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