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

100%

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.

Genome sequencing and progress in biotechnology

75%

Przybecki Z., Pawelkowicz M. E., Woycicki R.

Biotechnologia

2010

issue 4

9-23

As a result of the Human Genome Project and announcement of the project: Archon X Prize for Genomics 'genome for 1000$', the sequencing technology made huge progress. The purpose was to develop radically new technology that would dramatically reduce the time and cost of sequencing and resequencing eukaryotic genomes (or large region), and transcriptomes. This progress has influenced many areas also widely understood biotechnology. Owing to comparative genomic and sequenced genomes, new opportunities have occurred such as acceleration of genomic polymorphism identification, genes and genome structure prediction. It has also stimulated the development of assignation of functional effect of SNP, CNP or INDEL polymorphism and enabled functional and structural annotation of genome, establishment of a net of genome interaction (netoms) and netom's controlling. Genetic and physical mapping of genome elements methods undergo considerable alterations, thus they become critical in association methodology of genome and their function. The analysis of metabolic network and its elements is basic in planning activities in new biotechnology. Here we present the achievements in cucumber which were possible due to sequencing the genome of this species by our team.

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2 Biotechnologia

1 2010

1 2000

1 Kaczorowski T.

1 Pawelkowicz M. E.

1 Przybecki Z.

1 Szybalski W.

1 Woycicki R.

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

Genome sequencing and progress in biotechnology