A final product from Q1-Q4 PCR with Q2 and Q3 mutated will have:
Overlap extension mutagenesis with mutagenic primers Q2 and Q3 produces two first-round fragments each carrying complementary mutation in overlapping region. Upon mixing, overlap anneals and serves as priming site for extension, followed by amplification with outer flanking primers Q1 and Q4 to generate full-length one kilobase product. Resulting double-stranded molecule retains original size but incorporates two designed point mutations at targeted codons, converting wild-type sequence to mutant duplex. It cannot be one kilobase wild-type DNA nor truncated 0.7 or 0.5 kilobase species because
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.
