Submitted to: Biotechniques
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 30, 2009
Publication Date: August 1, 2009
Citation: Simon, P.W., Cavagnaro, P.F., Senalik, D.A. 2009. SplinkBES - A Splinkerette-Based Method for Generating Long End Sequences From Large Insert DNA Libraries. Biotechniques. 47:681-690. Interpretive Summary: Large pieces of DNA from an organism have served a pivotal role for a number of genetic and genomic purposes, including sequencing of entire genomes, and also a source of molecular markers useful for creating genetic maps and crop breeding. Here we report on the development of a novel method, suitable for high throughput application, for generating longer sequences from large pieces of DNA, using carrot as a model, and testing this method on other well-characterized genomes. This method gives significantly longer sequences than other methods, which are more informative in characterizing all the DNA of an organism. This research is of interest to plant researchers, biotechnology and seed companies, and genomics scientists.
Technical Abstract: We report on the development of a novel splinkerette-based method for generating long end-sequences from large insert library clones, using a carrot (Daucus carota L.) BAC library as a model. The procedure involves digestion of the BAC DNA with a 6-bp restriction enzyme, followed by ligation of splinkerette adaptors that serve as primer-annealing-sites for PCR amplification of the BAC-ends. The resulting amplicons are sequenced from both directions, and when overlapping, the pairs of sequences are assembled, originating two types of BAC-end sequences (BES), ungapped and gapped. The average sequence length for ungapped and gapped BES was 698 and 1,055 nt, respectively, with an overall average length of 838 nt, which is considerably higher than the average length typically obtained by direct end-sequencing. Through the analysis of actual and in silico-generated BES of different lengths, from carrot and five model organisms, we demonstrated that longer BES are more informative, as they had more matches to the GenBank database and contained more SSRs. A pilot high throughput procedure is proposed for splinkerette-based end sequencing (SplinkBES). We hope that this method will contribute in generating more robust BES analysis and provide a richer source of BES-derived markers for genomics, mapping and breeding.