Misligation

Misligation is the unintended joining of DNA fragments during enzymatic ligation, producing constructs that differ from the planned design. In cloning workflows, misligation most often involves either ligating an insert into a vector in an incorrect fashion or allowing the vector to re-ligate without an insert.

Common forms of misligation include: vector re-ligation, where the vector closes on itself without incorporating the desired insert; incorrect orientation or multiple inserts, resulting in frameshifts, fusion junctions, or unorientated inserts; and ligation of fragments with compatible ends that produce unexpected junctions, such as blunt-end ligations or repetitive sequences joining in undesired ways. Misligation can create nonfunctional plasmids, truncated genes, or misleading screening results.

Several factors increase the risk of misligation. Incomplete or partial digestion can leave ends that are capable of rejoining inappropriately. Insufficient dephosphorylation of the vector can promote self-ligation. High insert-to-vector ratios or poorly controlled ligation conditions can favor multiple inserts or incorrect junctions. The use of identical or compatible cohesive ends without directional strategies also raises misligation chances.

Detection and verification typically involve screening methods such as colony PCR, restriction digest analysis, and sequencing to confirm the presence, orientation, and integrity of the insert. Prevention relies on strategies such as dephosphorylating the vector, using directional cloning with two different enzymes, ensuring complete digestion and clean fragment preparation, purifying products, and implementing thorough screening of clones.