Introduction to the Workshop
URLs Provided by Attendees

Abstracts

Mapping

Informatics

Sequencing

Instrumentation

Ethical, Legal, and Social Issues

Infrastructure

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.

Toward an Automated System for High-Throughput DNA Sequencing: 1. Primer Walking from a Hexamer Library

Jan Kieleczawa, Shiping Zhang, John J. Dunn and F. William Studier
Biology Department, Brookhaven National Laboratory, Upton, New York 11973

Strings of three adjacent hexamers can prime DNA sequencing reactions specifically and efficiently when the template DNA is saturated with a single-stranded DNA-binding protein (SSB) [1]. The SSB prevents individual hexamers from priming at isolated complementary sites, but base stacking between hexamers bound at adjacent complementary sites drives formation of a priming complex. A library of all 4096 hexamers provides ready access to primers, and priming is effective using Sequenase and E. coli SSB at 0 °C with templates at least as large as 40 kbp.

Priming by strings of three hexamers has been tested at more than 2000 sites in M13 single-stranded DNAs of 6.4 kb or 7.3 kb, and at more than 1000 sites in T7 DNA, a double stranded DNA of 40 kbp, in radioactively labeled sequencing reactions. More than half of the 4096 possible hexamers have been sampled, and it appears that almost all hexamers will be able to participate effectively in these priming reactions, although hexamers containing only A and T seem to be less effective at an outside position than internally in a priming string. Priming is prevented within template hairpin structures that are too stable to be disrupted by SSB, but such structures are expected to be recognizable and infrequent. Outside of such hairpins, about three-fourths of the hexamer strings tested on M13 DNA and more than half of those tested on T7 DNA primed easily readable sequence. The ability to select hexamer strings that prime effectively should improve as the factors that affect priming are defined and controlled.

Instant access to primers at negligible cost opens the prospect of being able to sequence by primer walking on multiple templates as fast as sequencing reactions can be assembled. Taking full advantage of such capacity requires a matching capacity to read the sequence from the products of the sequencing reactions. The four-color fluorescent terminators supplied by ABI allow rapid, automated readout of the sequence, and we find that priming by strings of three hexamers can be as effective as priming by long primers in generating sequence information with these terminators. This poster will present results of fluorescent sequencing with an ABI Sequencer, where reactions were primed with strings of three hexamers at many different sites in M13 templates. Progress toward developing a multiple capillary electrophoresis system using a replaceable matrix and full spectral detection, and in developing vectors for supplying 40-kbp templates in quantities suitable for primer walking, will be presented in accompanying posters. The ultimate goal is to develop a fully automated system for high-throughput DNA sequencing that uses primers supplied from a hexamer library for primer walking on multiple templates in parallel.

[1] Kieleczawa, J., Dunn, J. J., and Studier, F. W. (1992) DNA sequencing by primer walking with strings of contiguous hexamers. Science, 258, 1787-1791.