Practice question

Site-directed mutagenesis via overlap extension employs internal primers carrying desired substitutions. First round PCR amplifies upstream fragment using primers Q1 and mutated Q3, and downstream fragment using mutated Q2 and Q4, each intermediate bearing single mutation in overlapping region. Mixing these products allows complementary mutated ends to anneal and extend as primers for each other during second round amplification. This deliberate assembly forces incorporation of point mutations into full-length construct. Purpose is to introduce specific nucleotide changes into gene, not merely to increase specificity or prevent primer dimers.