Practice question

During two-step overlap extension mutagenesis, initial PCR reactions Q1 with Q3 and Q2 with Q4 generate overlapping fragments each carrying introduced mutation via internal primers. Denaturation and annealing of these fragments allow overlapping mutated region to hybridize, extending to form full-length template. Final amplification with only flanking primers Q1 and Q4 yields 1 kb product containing desired mutations at both targeted sites, while wild-type parental template remains largely undigested or removed by DpnI treatment. Shorter products like 700 bp or 0.5 kb would indicate incomplete fusion or nonspecific amplification, not successful mutagenesis.