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Randomize Gene Sequences with New PCR Mutagenesis Kit

a superior alternative to Taq-based random mutagenesis methods. The benefits of this kit include the unique characteristics of the error-prone Mutazyme DNA polymerase, synthesis of high product yields over a broad range of amplicon sizes (0.1-10 kb), efficient mutagenesis rates of one to seven bases per kb per PCR, incorporation of all mutation types with minimal bias, and a simplified protocol with one set of robust reaction conditions. The GeneMorph kit is a finely tuned system for achieving desired mutation frequencies and, producing a unique spectrum of mutations.

Methods

Error-prone PCR conditions: DNA sequences were amplified as described in the manual for the GeneMorph PCR mutagenesis kit. Briefly, an amount of plasmid DNA corresponding to 1 pg to 100 ng of amplicon was amplified in PCR reactions (50 ml) containing 1X Mutazyme reaction buffer, 200 mM each dNTP, 125 ng of each primer, and 2.5 U Mutazyme DNA polymerase. PCR reactions were performed with Taq DNA polymerase using identical conditions, except that 1X Taq reaction buffer (1.5 mM MgCl2) was employed. Error-prone PCR reactions contained 5 U of Taq DNA polymerase, 1X Taq reaction buffer (supplemented with 0.5 mM MnCl2), and a nucleotide mixture of 200 mM dGTP, 200 mM dATP, 1 mM dCTP, and 1 mM TTP.2

Sequential PCR amplifications: To obtain high mutation levels (>0.7%), PCR products were reamplified in a second PCR reaction. A small portion of a PCR reaction (e.g., amplicon synthesized from 0.1 ng-1 ng of DNA target) was first diluted 1:1000 in TE buffer. One ml of diluted amplicon was then reamplified as described above.


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