stickyend

Sticky end, or cohesive end, refers to a short single-stranded DNA overhang created when double-stranded DNA is cut by certain restriction endonucleases. These overhangs, typically 4 to 6 nucleotides long, are complementary to matching overhangs on other DNA fragments cut with the same enzyme or a compatible enzyme. The base-pairing between overhangs allows fragments to align with high specificity, facilitating subsequent ligation by DNA ligase to produce a continuous molecule.

Formation and use in cloning: Sticky ends arise from staggered restriction enzyme cuts, producing either 5' or 3' overhangs. After annealing between complementary ends, the fragments are joined by ligase, which seals the remaining phosphodiester backbone. In genetic engineering, scientists exploit sticky ends for directional cloning by using two different restriction enzymes so that the insert can only ligate in one orientation. To reduce unwanted vector self-ligation, researchers often dephosphorylate the vector ends or use non-compatible ends.

Limitations and context: Not all restriction enzymes generate sticky ends; blunt-ended fragments require ligation without base-pairing and are generally less efficient. Compatibility of overhangs is crucial, and mismatches or repetitive sequences can hinder correct ligation. Modern DNA assembly methods sometimes extend the concept of sticky ends, using defined overhangs (as in type IIS enzyme strategies) or overlapping sequences to guide precise assembly beyond traditional cloning.