Representation of DNA sequences in recombinant DNA libraries prepared by restriction enzyme partial digestion

Abstract

We present a theoretical study of the fraction of sequences incorporated in a recombinant DNA partial digest library as a function of the size of the library. The fraction incorporated depends on the degree of restriction enzyme partial digestion. If all restriction sites in the target DNA can be cleaved with the same rate, optimum incorporation of sequences is observed when the number average length of the digested DNA equals the desired average length of the cloned insert. Overdigestion severely reduces the fraction of sequences present in a sample of clones. Heterogeneity in restriction enzyme cleavage rates also reduces the fraction incorporated, and underdigestion improves sequence representation in the face of cleavage rate heterogeneity. Practical methods for determining the number average length of partially digested DNAs are also presented.