Introduction to the Workshop
URLs Provided by Attendees

Abstracts

Mapping

Informatics

Sequencing

Instrumentation

Ethical, Legal, and Social Issues

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The electronic form of this document may be cited in the following style:
Human Genome Program, U.S. Department of Energy, DOE Human Genome Program Contractor-Grantee Workshop IV, 1994.

Abstracts scanned from text submitted for November 1994 DOE Human Genome Program Contractor-Grantee Workshop. Inaccuracies have not been corrected.

Modular Primers in Automated DNA Sequencing

Levy Ulanovsky, Irina Sobolev & Lev Kotler
Dept. of Structural Biology, Weizmann Institute of Science, Rehovot, 76100 Israel.

Recently, in two independent findings, hexamers and even pentamers were shown to assemble into long unique primers upon their contiguous annealing to the template with no ligation required [1, 2]. Studier and colleagues used SSB to suppress alternative priming (l), while we found that adjacently annealed pentamers, hexamers and heptamers could uniquely prime sequencing reactions without the difficult-to-remove SSB [2].

Here we describe our progress in the development of the technology of DNA sequencing by modular primer walking, eliminating the primer synthesis bottleneck. 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 site in the template, and, in a striking way, prime there uniquely, while each of them is not unique for the most part, when used separately. This technology is expected to reduce the time per walk by a factor of 20 to 50, and the cost of DNA sequencing 5 to 15 fold. Both time and expense will be saved not only on the primer synthesis per se but, more importantly, as a result of the automation of the complete cycle of walking sequencing, made possible by the instant availability of the primers.

In our most recent advance we show that modular primers can now be used with dye terminators on the ABI 373A automated sequencer [3]. The single most important requirement is that of the three modules, the two upstream ones should have their 3'-ends modified to prevent their extension by the polymerase. The success rate and the quality of the automated sequencing with modular primers are similar to those with the conventional 17-20 base long primers, and for the most part few if any base-calling errors are found within the first 400 bases of the sequence run, with no stretch-liner, even though little optimization has yet been done for the reaction conditions. The protocol is only improved in that no precipitation or phenol extraction (obstacle to closed-end automation) is required. We currently use the pentamer-based primers of the 5+7+7 structure with Pu-Pu base-stacking between the 5-mer (to be extended) and the adjacent 7-mer. Both 3'-blocked heptamers have two degenerate positions each, and thus the same size library as the pentamer (512 sequences).

[1.] Kieleczawa, J., Dunn, J. & Studier, F. (1992), Science 258: 1789-1791.
[2.] Kotler, L., Zevin-Sonkin, D., Sobolev, I., Beskin, A. & Ulanovsky, L. (1993), Proc. Natl. Acad. Sci. USA 90: 4241-4245.
[3.] Kotler, L., Sobolev, I. & Ulanovsky, L. (1994), BioTechniques (in press).