Levy Ulanovsky, Lev Kotler, Mugasimangalam Raja, Maya Shmulevitz and Alexander Beskin
Dept. of Structural Biology, Weizmann Institute of Science, Rehovot, 76100 Israel. Phone: +972-8-343547; FAX: +972-8-344105, E-mail: BPLEVY@WEIZMANN.WEIZMANN.AC.IL
We are developing DNA sequencing technology using modular primers, which eliminate the primer synthesis, the main bottleneck in primer walking. Modular primers are assembled from three 5-mer, 6-mer or 7-mer modules selected from a presynthesized library of as few as 1000 oligonucleotides . The three modules anneal contiguously at the selected template site and prime there uniquely, even though each is not unique for the most part when used alone. This technique is expected to speed up primer walking 20 to 50 fold, and reduce the sequencing cost by a factor of 5 to 15. Time and expense will be saved not only on primer synthesis itself but, more important, because the instant availability of the primers enables closed-loop automation of the complete cycle of walking sequencing. Apart from saving time and cost, the closed-loop automation would largely eliminate the need for human intervention between the walks, as a source of errors and complications.
We have found that modular primers can be used with dyeterminators of the ABI 373A automated sequencer. Also, reactions with the modular primers and dye terminators were run successfully on replaceable matrix capillaries of Barry Karger (Northeastern University) in 60 min. and read beyond 500 bases with as few as 2 base-calling errors. Because no protein needs to be removed, no precipitation or phenol extraction (obstacle to closed-end automation) is required. The success rate and quality of automated sequencing with modular primers are similar to those with conventional primers 17-20 base long. For the most part few, if any, base-calling errors are found within the first 400 bases of the sequence run. We currently prefer pentamer-based primers of the 5+7+7 structure with Pu-Pu base stacking between the 5-mer (to be extended) and adjacent 7-mer. Both heptamers are 3'-end modified to prevent their extension by the polymerase; each has two degenerate positions and thus the same size library as the pentamers (512 sequences).
*Supported by DOE grant De-FG02-94ER61831.