Failure to Cut Near DNA Termini

Symptom

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.

1. 1 Recognition site positioned <6 nucleotides from DNA end lacks required flanking sequence
2. 2 PCR primer design did not include sufficient buffer nucleotides beyond restriction site
3. 3 Enzyme requires 6–12 bp flanking sequence for efficient binding and cleavage near termini

1. 1 Add ≥6 nucleotides to PCR primer 5' ends beyond the restriction recognition site
2. 2 Consult NEB enzyme-specific data for exact flanking requirements (typically 6–12 bp)
3. 3 Redesign primers to position restriction site further from DNA termini
4. 4 Consider using Type IIS enzymes that cleave outside recognition site if end-cutting required

Related Video (3)

New England Biolabs ★ 82

Episode #56: Molecular Cloning Series: Restriction Enzymes 101

"Explains restriction enzyme fundamentals, directly relevant to understanding why enzymes cut (or fail to cut) at recognition sites."

New England Biolabs ★ 78

Cloning With Restriction Enzymes

"Covers practical guidelines for choosing and using restriction enzymes in cloning, the exact technique context of this failure."

Addgene ★ 70

Restriction Digest Analysis

"Demonstrates a restriction digest and gel analysis, showing how uncut vs cut products are evaluated — the symptom described here."