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Science 11 December 1992:
Vol. 258. no. 5089, pp. 1787 - 1791
DOI: 10.1126/science.1465615
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Articles

Science, Vol 258, Issue 5089, 1787-1791
Copyright © 1992 by American Association for the Advancement of Science

articles

DNA sequencing by primer walking with strings of contiguous hexamers

J Kieleczawa, JJ Dunn, and FW Studier

Biology Department, Brookhaven National Laboratory, Upton, NY 11973.

When template DNA is saturated with a single-stranded DNA binding protein (SSB), strings of three or four contiguous hexanucleotides (hexamers) can cooperate through base-stacking interactions to prime DNA synthesis specifically from the 3' end of the string. Under the same conditions, priming by individual hexamers is suppressed. Strings of three of four hexamers representing more than 200 of the 4096 possible hexamers primed easily readable sequence ladders at more than 75 different sites in single-stranded or denatured double-stranded templates 6.4 kilobases to 40 kilobase pairs long, with a success rate of 60 to 90 percent. A synthesis of 1 micromole of hexamer supplies enough material for thousands of primings, so multiple libraries of all 4096 hexamers could be distributed at a reasonable cost. Such libraries would allow rapid and economical sequencing. Automating this strategy could increase the speed and efficiency of large-scale DNA sequencing by at least an order of magnitude.


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