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A new approach for DNA sequencing based on assembly of primers by hexamer ligation

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Kaczorowski T., Szybalski W.

Biotechnologia

2000

issue 3

151-163

Present-day sequencing of large genomes (usually 3-3000 Mb) requires two cloning steps: (i) cloning of large (30-100 kb) DNA fragments in the appropriate cosmid, P1, BAC or YAC vectors; and (ii) subcloning of various segments of such large clones in M13-like vectors next to the site recognized by the universal sequencing primer. These steps must be followed by the actual sequencing of 300- to 600-nt DNA segments and their assembly into contigs. An alternative approach is based on primer walking strategy. The method presented in this article is a variant of the latter approach. In principle, the method is based on the assembly of any primer by ligation on a DNA template of three or more complementary, contigous hexamers taken from a library of 4096 hexamers or 1024 singly degenerate hexamers. The presence of Escherichia coli single-stranded DNA binding protein (SSB) is essential for effective hexamer ligation. e Due to its speed and suitability, this method may dramatically change the face of contemporary DNA sequencing by speeding up efforts focused on deciphering of particular genomes. The presented method is fully compatible with automated DNA sequencing applying ABI sequencers and fluorescent label.

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1 Biotechnologia

1 2000

1 Kaczorowski T.

1 Szybalski W.

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A new approach for DNA sequencing based on assembly of primers by hexamer ligation