Koch Lab:Protocols/Fork unzipping constructs

Principle
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DNA tethers suitable for short unzipping can easily be formed by hybridizing two DNA oligonucleotides with partial complementarity, and partial non-complementarity -- forming a fork construct. The non-complementary parts are best formed from poly(dT) stretches. The initial length of the tether will be the length of the two poly(dT) stretches, and due to limitations of synthesis length, will be shorter than in the anchoring segment method. Among many challenges, one you need to look out for is ionic repulsion between microspheres and surface, which can inhibit tether formation.

Unzipping forks can incorporate protein binding sites directly, or a cohesive end for ligation of long DNA unzipping fragments.