M13 Cloning of Mung Bean Nuclease Digested PCR Fragments as a Means of Gap Closure Within A/T-rich, Genome Sequencing Projects

Obtaining the complete DNA sequence of a genome is often not straightforward. After standard shotgun sequencing strategies have been employed there are often gaps remaining and these can be the most intractable regions, frequently containing repeat sequences, “uncloneable” sequences and/or regions of potential secondary structure or differential base composition. In genomes with a high A/T content, such as Plasmodium falciparum and Dictyostelium discoideum, solving these gaps is a particularly difficult problem as the sequences concerned are “fragile” and easily denatured, commonly uncloneable and have a paucity of good oligonucleotide priming sites. Reported here is a simple, yet reliable method for determining the sequence of A/T-rich gap-spanning PCR products. This method relies on the slippage of the specificity of mung bean nuclease so that it digests A/T-rich double-stranded DNA into a set of deletion fragments that can then be cloned into M13, sequenced and the original sequence assembled therefrom.