In a site-directed mutagenesis, Q2 and Q3 primers contain:
In overlap extension site-directed mutagenesis, primers Q2 and Q3 are internal mutagenic primers designed to be complementary to each other and harbor desired nucleotide substitution, insertion, or deletion at central position. They anneal to opposite strands of template and introduce specific mutation during first-round PCR amplifications generating two half fragments. These primers do not encode exon deletions, epigenetic modifications, or ligand moieties. Their overlapping mutated region allows subsequent fusion PCR with flanking primers Q1 and Q4 to produce full-length product carrying targeted change, enabling precise protein engineering studies.
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.