What is the purpose of mixing PCR products from Q1-Q3 and Q2-Q4 before second round?
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
Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.
