Direct sequencing of bacterial artificial chromosomes (BACs) and prokaryotic genomes by biotin-capture PCR

Holmberg, Anders (author)

Alexandersson, G (author)

(creator_code:org_t)

1998
1998
English.
In: Journal of Biotechnology. - 0168-1656 .- 1873-4863. ; 60:1-2, s. 119-129

Related links:: https://urn.kb.se/re...; show more...; https://doi.org/10.1...; show less...

Abstract Subject headings

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.

By the author/editor: Sterky, Fredrik; Holmberg, Anders; Alexandersson, G; Lundeberg, Joaki ...; Uhlen, Mathias

About the subject

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