Direct sequencing of bacterial artificial chromosomes (BACs) and prokaryotic genomes by biotin-capture PCR.
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.
Sterky
F
F
Department of Biochemistry and Biotechnology, KTH, Royal Institute of Technology, Stockholm, Sweden.
Holmberg
A
A
Alexandersson
G
G
Lundeberg
J
J
Uhlén
M
M
D001483:Base Sequence; D001710:Biotin; D002876:Chromosomes, Bacterial / Q000737:chemistry*; D004269:DNA, Bacterial / Q000737:chemistry*; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*; D001616:beta-Galactosidase / Q000235:genetics
D004269:DNA, Bacterial; D001710:Biotin; D001616:beta-Galactosidase