Mutagenic PCR.

PCR Methods and ApplicationsMost practitioners of PCR prefer to carry out DNA amplification in an accurate manner, introducing as few base substitutions as possible.This is especially critical when one is studying clonal isolates and must distinguish natural variation from artifactual variation that is introduced by polymerase error.Fortunately, thermostable DNA polymerases are available that operate with high fidelity because of an intrinsic 3'--~ 5' exonuclease activity (for review, see ref. 1).Manipulation of PCR conditions can lead to further improvement of copying accuracy.Here, we consider the other side of the fidelity issue--those instances where promiscuity is a virtue.Oftentimes, in probing the structure or function of a protein or nucleic acid, one wishes to generate a library of mutants and apply a screening method to isolate individuals that exhibit a particular property.For mutations over a short stretch of nucleotides within a cloned gene, it is appropriate to replace a portion of the gene with a synthetic DNA fragment that contains random or partially randomized nucleotides.(2-s) For mutations over a longer segment, up to the size of an entire gene, it may be preferable to scatter random mutations over the entire sequence, typically at a frequency of one or a few mutations per molecule.In such cases, it is most convenient to introduce random mutations through inaccurate copying by a DNA polymerase, especially if the polymerase is a thermostable enzyme that can operate in the context of the PCR.Each pass of the polymerase during the PCR allows for the possibility of mutation, so that the cumulative error rate can become substantial.