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

J Biotechnol. 1998 Feb 5;60(1-2):119-29. doi: 10.1016/s0168-1656(97)00196-x.

Abstract

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.

MeSH terms

  • Base Sequence
  • Biotin*
  • Chromosomes, Bacterial / chemistry*
  • DNA, Bacterial / chemistry*
  • Electrophoresis, Agar Gel
  • Escherichia coli / genetics
  • Molecular Sequence Data
  • Polymerase Chain Reaction / methods*
  • Sequence Analysis, DNA / methods*
  • beta-Galactosidase / genetics

Substances

  • DNA, Bacterial
  • Biotin
  • beta-Galactosidase