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Direct sequencing of bacterial artificial chromosomes (BACs) and prokaryotic genomes by biotin-capture PCR
KTH, Superseded Departments, Biotechnology.ORCID iD: 0000-0003-3281-8088
KTH, Superseded Departments, Biotechnology.ORCID iD: 0000-0003-4313-1601
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1998 (English)In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 60, no 1-2, 119-129 p.Article in journal (Refereed) Published
Abstract [en]

Determination of unknown DNA sequences adjacent to known segments is an important task in genome-related research. We have applied the methodology of biotin-capture PCR for direct sequencing of bacterial artificial chromosomes (BACs) and bacterial genomes. The strategy involves extension of a biotinylated primer from a known locus into unknown regions of the template to yield single-stranded DNA, which is immobilised onto paramagnetic beads. An arbitrary primer initiates extension from the unknown region and back towards the known locus. The arbitrary primer contains a universal primer 'handle', which is utilised for subsequent amplification. The PCR products are then directly sequenced by solid-phase or cycle sequencing. The fact that BACs or bacterial chromosomes can be sequenced without prior purification or subcloning might be useful in numerous applications, such as gap-filling, sequencing of regulatory regions upstream known genes and determination of intron/exon-boundaries.

Place, publisher, year, edition, pages
1998. Vol. 60, no 1-2, 119-129 p.
Keyword [en]
genome sequencing, biotin capture, solid-phase technology, PCF
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-24088ISI: 000073047100012OAI: oai:DiVA.org:kth-24088DiVA: diva2:343309
Note
QC 20100813Available from: 2010-08-13 Created: 2010-08-13 Last updated: 2010-08-13Bibliographically approved
In thesis
1. Sequencing of genes and genomes
Open this publication in new window or tab >>Sequencing of genes and genomes
1998 (English)Doctoral thesis, comprehensive summary (Other scientific)
Place, publisher, year, edition, pages
Stockholm: KTH, 1998. 63 p.
Keyword
DNA sequencing, genome analysis, expressed sequence tag, expression analysis, complementary DNA, biotin-capture, solid-phase technology, automation, poplar
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-2714 (URN)91-7170-315-2 (ISBN)
Public defence
1998-11-01, 00:00
Note
QC 20100813Available from: 2000-01-01 Created: 2000-01-01 Last updated: 2010-08-13Bibliographically approved

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Sterky, FredrikHolmberg, AndersUhlen, Mathias

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