Failure to Cut Near DNA Termini (Restriction Enzyme Digest)

Question

Accepted Answer

Restriction enzyme fails to cleave recognition sites located within 6 bp of linear DNA ends, such as PCR product termini. Full-length uncut product observed. Common causes: 1) Recognition site positioned <6 nucleotides from DNA end lacks required flanking sequence 2) PCR primer design did not include sufficient buffer nucleotides beyond restriction site 3) Enzyme requires 6–12 bp flanking sequence for efficient binding and cleavage near termini Solutions: 1) Add ≥6 nucleotides to PCR primer 5' ends beyond the restriction recognition site 2) Consult NEB enzyme-specific data for exact flanking requirements (typically 6–12 bp) 3) Redesign primers to position restriction site further from DNA termini 4) Consider using Type IIS enzymes that cleave outside recognition site if end-cutting required