SplinkBES: a splinkerette-based method for generating long end sequences from large insert DNA libraries.
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 (BESs): ungapped and gapped. The average sequence length for ungapped and gapped BESs was 698 and 1055 nucleotides, respectively, with an overall average length of 838 nucleotides. This 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 BESs are more informative, since they had more matches to the GenBank database and contained more simple sequence repeats (SSRs). A pilot high-throughput procedure is proposed for splinkerette-based end sequencing (SplinkBES). This method may contribute to generating more robust BES analysis and provide a richer source of BES-derived markers for genomics, mapping, and breeding.
Cavagnaro
Pablo F
PF
Department of Horticulture, University of Wisconsin, Madison, WI 53706, USA.
Senalik
Douglas
D
Simon
Philipp W
PW
D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics*; D004251:DNA Transposable Elements / Q000235:genetics*; D018744:DNA, Plant / Q000235:genetics*; D018552:Daucus carota / Q000235:genetics*; D015723:Gene Library; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA / Q000379:methods*
D017931:DNA Primers; D004251:DNA Transposable Elements; D018744:DNA, Plant