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Table 4 Comparison of the single-primer and double-primer PCR reactions.

From: Simple and efficient site-directed mutagenesis using two single-primer reactions in parallel to generate mutants for protein structure-function studies

  Single-primer PCR Double-primer PCR
Number of reactions 2 (1 for each primer) 1
Plasmid template ~500 ng 5–50 ng
Primers Forward OR reverse
(each in a separate tube)
Forward AND reverse together
Primer-primer annealing during PCR - +
Number of cycles 30 12–18
Fold amplification of DNA 30 4,096–262,144
Probability of mutation during PCR Low* High*
DNA polymerase Pwo PfuTurbo
  1. * A function of the fold amplification of DNA.