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Nucleic Acids Research, 1988, Vol. 16, No. 21 10301-10321
© 1988

MOLECULAR BIOLOGY

Kinetic analysis for optimization of DNA ligation reactions

Dennis Revie+, Douglas W. Smith* and Thomas W. Yee§

Department of Biology B-022, University of California at San Diego La Jolla, CA 92093, USA

* To whom correspondence should be addressed

Received October 16, 1987. Accepted October 6, 1988.

Kinetic equations describing ligation of DNA to circular recombinant forms were developed and solved for tour types of reactions: (a) a homogeneous population of singly restricted DNA fragments, (b) Insertion of singly restricted Insert into vector, (C) forced directional Insertion of doubly restricted insert into vector, and (d) Insertion of singly restricted insert Into pliosphatased vector. The effects of varying vector and Insert sizes, starting concentrations, and phosphatase treatment on the yield of cIrcular 1:1 recombinants were analyzed. Selected theoretical predictions were experimentally tested and verified. Our suggestions on optimizing ligation reactions in several cases are at variance with common practice. For example, optimum conditions in case (b) and (d) ligatlons are best specified as Individual insert and vector concentrations rather than as insert/vector molar ratios, except in case (d) ligations Involving very small Insert size. In case (c) ligatlons, highest efficiencies are obtained when both vector and Insert are at relatively iow concentration.

Present addresses: +Chemistry Department, University of San Diego, San Diego, CA 92110

Present addresses: §Biology Department, FO-31, University of Texas at Dallas, Richardson, TX 75083, USA


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