Co-operativity of hexamer ligation

Gene
T Kaczorowski, W Szybalski

Abstract

The SPEL-6 (sequential rimer elongation by ligation of 6-mers) procedure is based on the assembly of DNA primers by ligation of three or more hexamers taken from a library of 4096 hexamers. In this way, the synthesized primers enable DNA sequencing by primer walking. Ligation by both T4 DNA ligase and Rhodothermus marinus thermophilic DNA ligase is highly cooperative. Sequencing ladders obtained with 18-60-nucleotide (nt) primers (produced by ligation of three to ten hexamers using T4 DNA ligase) were all of high quality, with no spurious bands. R. marinus DNA ligase requires at least seven hexamers for successful primer synthesis. Long primers (up to 60 nt), which are easy to obtain, especially by automated ligation, offer a definite advantage in DNA priming in regions with pronounced secondary structure. Moreover, the SPEL-6 procedure for DNA sequencing reduces the sequencing effort manifold. An additional application of hexamer ligation is the detection of point mutants, as described here.

References

Dec 1, 1977·Proceedings of the National Academy of Sciences of the United States of America·F SangerA R Coulson
Nov 1, 1990·Proceedings of the National Academy of Sciences of the United States of America·D A NickersonU Landegren
Apr 27, 1989·Nature·W R Brown
Apr 1, 1986·Analytical Biochemistry·E C StraussL E Hood
Aug 26, 1988·Science·U LandegrenL Hood
Apr 1, 1996·Molecular Biotechnology·T Kaczorowski, M Sektas

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Citations

Jun 15, 1999·Molecular Biotechnology·R Maldonado-RodriguezK L Beattie

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