Contiguous strings of strongly binding short oligonucleotides as a useful tool for completing sequencing experiments.

Abstract

Large-scale DNA sequencing is currently based mostly on the shotgun approach. Although widely used, it is not free of shortcomings: the total length of randomly sequenced subclones appear to be five to eight times more than the total DNA length; such a high statistical redundancy of random sequences cannot guarantee the absence of unsequenced gaps. We calculated that these problems could be alleviated if the average length of the subcloned random fragments were increased. In this paper we present the primer walking approach based on the use of contiguous modified hexamer strings strongly binding to complementary templates as segmented primers. The approach exhibits the following characteristic features: 1) Single-stranded templates appear to have advantage over double-stranded ones. 2) With single-stranded templates, an overall sequencing success rate is about 80%. 3) A relatively small library of 1400 hexanucleotides selected according to the most frequent occurrence in the human genome is sufficient for sequencing all clones. The approach allows one to reduce the number of templates necessary for the shotgun strategy and also to decrease the number of gaps between sequenced contigs. This should be kept in mind when considering the ways for enhancement of the present strategy of large-scale sequencing.