Are some ligations more difficult than others?

Ligation of blunt ends and single-base overhangs require optimized reaction conditions.


Greg Lohman:

Blunt ends and single base overhangs are common difficult to ligate substrates. In these cases the ends don't appreciably anneal on their own, so the ligase has to bind two separate fragments in order to catalyze the reaction. So having to bind two fragments means that you need higher concentrations of the ligase, lower reaction volumes, or specialized buffers in order to get these reactions to go efficiently.

So by contrast, cohesive or sticky ends, or sealing a nick in a double-stranded DNA duplex, are much easier as the ligase only has to bind one substrate. In these cases, regular concentration ligase is usually sufficient.

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