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Direct sequencing o...
Direct sequencing of bacterial artificial chromosomes (BACs) and prokaryotic genomes by biotin-capture PCR
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- Sterky, Fredrik (author)
- KTH,Bioteknologi
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Holmberg, Anders (author)
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Alexandersson, G (author)
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- Lundeberg, Joakim (author)
- KTH,Bioteknologi
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- Uhlen, Mathias (author)
- KTH,Bioteknologi
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(creator_code:org_t)
- 1998
- 1998
- English.
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In: Journal of Biotechnology. - 0168-1656 .- 1873-4863. ; 60:1-2, s. 119-129
- Related links:
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https://urn.kb.se/re...
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https://doi.org/10.1...
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Abstract
Subject headings
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- 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.
Subject headings
- TEKNIK OCH TEKNOLOGIER -- Industriell bioteknik (hsv//swe)
- ENGINEERING AND TECHNOLOGY -- Industrial Biotechnology (hsv//eng)
Keyword
- genome sequencing
- biotin capture
- solid-phase technology
- PCF
- Bioengineering
- Bioteknik
Publication and Content Type
- ref (subject category)
- art (subject category)
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