Genomic DNA sequencing by SPEL-6 primer walking using hexamer ligation

DNA sequencing by SPEL-6 (Sequential Primer Elongation by Ligation of 6-mer s) primer walking is based on the rapid assembly of true primers by ligatio n of several (three to 10) contiguous hexamers complementary to a DNA templ ate saturated with Escherichia coli single-stranded DNA-binding protein. To prove the usefulness and to check the reliability of this method, a 3-kb D NA fragment carrying the genes encoding the EcoVIII restriction-modificatio n (RM) system was sequenced with low redundancy (2.8). The use of both sing le-stranded (ss) and double-stranded (ds) DNA templates was compared. For t his project, 27 primers were assembled by hexamer ligation to form 18-30-nt strings of three to five hexamers. Each primer was designed based on nucle otide sequence determined in a previous run, and was produced in a matter o f minutes. The overall length of the easily readable sequencing ladders was about 300-450 nt. We found that strong secondary structures in the ss DNA tend to interfere with its template function for the primer assembly by hex amer ligation, especially when they overlap the 3'-end of such a primer. Th is was easily overcome either by avoiding such hairpin regions or by using longer strings of hexamers, since we show that their ligation is highly coo perative, and ligation efficiency increases with the length of the string ( Kaczorowski and Szybalski, 1996a). Some general rules for successful primer assembly and prospects for using the SPEL-6 method for large-scale, fully automated fluorescent sequencing of large genomes are discussed. (C) 1998 P ublished by Elsevier Science B.V. All rights reserved.