Mutagenic PCR

Mutagenic PGR. More recendy, methods have been developed to use the PCR reaction to randomly mutagenize a defined sequence (25). The Taq polymerase used in PCR misincorporates nucleotides in a random fashion if manganese dichloride [7773-01-5], MnC, is included in the reaction buffer during PCR. The library of mutagenized PCR products can be screened for the desired phenotype. 

In principle, the product of a mutagenic PCR may be used for successive rounds of random mutagenization. Therefore, a small aliquot of the first reaction may be employed to seed the next one. We found that it is necessary to gel-purify the product of the first PCR before proceeding with another amplification cycle. Otherwise, the PCR yielded exclusively nonspecific amplification products. 

Routinely run a mutagenic PCR in parallel with the respective standard PCR and analyze a portion of these by agarose gel electrophoresis If no PCR product is observed with the mutagenic PCR, try varying (mostly, increasing) the Mg2+ concentration in steps of 2 pL (= 2 mM). 

With genes consisting of more than 1000 bp, it might be difficult to obtain amplification products after mutagenic PCR. If so, the template DNA may be divided into two or more fragments that are amplified separately. 

Random pools can also be generated by using various forms of mutagenic PCR (Section 8.3.1.4). The pools derived from these methods do not have the same... 

Cadwell, R.C., Joyce, G.F. Mutagenic PCR. PCR Methods Appl. 3, S136-S140 (1994). 

Kore et have used mutagenic PCR with the triphosphates dPTP (155) and 8-oxo-dGTP to isolate purine-specific hammerhead ribozymes. After five rounds of selection, new ribozymes were isolated with up to 90 times higher in trans cleavage than the starting ribozyme. 

It is possible to expand the number of RNA molecules in an ongoing selection process by altering the conditions during the PCR step, causing an increase in the mutation rate.42 Mutagenic PCR is used to introduce mutations into already selected sequences and is therefore a powerful technique for sampling the local sequence space. 

Cadwell RC, Joyce GF (1995) Mutagenic PCR. In Dieffenbach CW, Dveksler GS (eds) PCR Primer a laboratory manual. CSHL Press, Cold Spring Harbor, p 583... 

The amplified mutagenic DNA can be gel purified to remove the original template from mutagenized PCR product. Practical experience has shown contamination ofWT scFv pYDl plasmid has not been problematic due to minimal amount of intact plasmid and relatively low transformation frequency of yeast. However, gel purification should be used if PCR does not generate a discrete product band of appropriate size or one wants to eliminate all WT plasmid. 

A single selection round of a PAR mutant library generated by mutagenic PCR... 

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