PCR is routinely performed with standard Polymerases, yielding a final PCR product with few errors. Although Polymerases vary in fidelity, the inherent Mutation rate of PCR is insufficient to generate the high frequency of mutations necessary to generate a mutant library. The addition of nucleotide analogues to the PCR reaction reduces Polymerase fidelity increased substitution frequency in the PCR products.
PickMutant™ Random Mutagenesis (by Nucleotide Analog dPTP) Kit is based on the incorporation of Mutagenic dPTP, into an amplified DNA fragment by PCR. The Mutagenic dPTPs are eliminated by a second PCR step in the presence of the four natural dNTPs only, resulting in a rate of Mutagenesis of up to 20% (Zaccolo et. Al).
dPTP is an excellent substrate for Taq Polymerase (Km =22 mM versus Km = 9.5 mM for TTP); it is incorporated in place of TTP and, with a ≈fourfold lower efficiency, in place of dCTP. dPTP produces four transitions (A→G, T→C, G→A and C→T). Two transitions (A→G and T→C) occur at higher frequency than the other two (G→A and C→T) (Zaccolo et. Al).
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