Direct sequencing of bacterial artificial chromosomes (BACs) and prokaryotic genomes by biotin-capture PCR
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
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.
genome sequencing, biotin capture, solid-phase technology, PCF
IdentifiersURN: urn:nbn:se:kth:diva-24088ISI: 000073047100012OAI: oai:DiVA.org:kth-24088DiVA: diva2:343309
QC 201008132010-08-132010-08-132010-08-13Bibliographically approved