( oy mutation frequencies of one to four ...)Randomize Gene Sequences with New PCR Mutagenesis Kit

oy mutation frequencies of one to four amino acid changes (2-7 nucleotide changes) per gene.^3-6 Proteins with improved activities have also been isolated from highly mutagenized libraries exhibiting 20 point mutations per gene.⁷ Error-prone PCR methods commonly employ Taq DNA polymerase, as it lacks proofreading activity. To achieve useful mutation frequencies, the error rate of Taq DNA polymerase is further increased by employing PCR reaction buffers containing Mn²⁺ and unbalanced dNTP concentrations.^1,2, These changes, however, can lead to lower PCR product yields⁴ and a strong bias in the types of mutations produced (mutational spectra). ²

The GeneMorph PCR mutagenesis kit overcomes the limitations of Taq-based random mutagenesis methods by using an inherently error-prone PCR enzyme and varying DNA template concentration to achieve the desired mutation frequency.

Mutazyme DNA Polymerase

Fig.1

A key component of the GeneMorph kit is Mutazyme DNA polymerase, a novel DNA polymerase mutant developed at Stratagene. As shown in Figure 1, Mutazyme DNA polymerase introduces mutations much more frequently than Taq DNA polymerase. Moreover, high mutation frequencies are achieved under optimal PCR reaction conditions for the enzyme (MgCl₂, balanced nucleotides). Despite its inherent high error rate, Mutazyme DNA polymerase produces high PCR product yields over a broad range of DNA template concentrations.

PCR Mutation Frequency

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