Amplification and sequence analysis of DNA flanking integrated proviruses by a simple two-step polymerase chain reaction method.
We describe a two-step polymerase chain reaction method that can be used for the amplification of cellular DNA sequences adjacent to an integrated retroviral provirus. The technique involves a partly degenerate, arbitrary primer that will hybridize in the provirus-flanking cellular DNA. By using this primer in combination with a biotinylated provirus-specific primer, a provirus-cellular DNA junction fragment can be isolated from the nonspecific amplification products by using streptavidin-coated magnetic beads. A second amplification employing a nested provirus-specific primer and a biotinylated nondegenerate primer derived from the partly degenerate primer followed by purification with streptavidin-coated beads enhances the specificity and the efficiency of recovery of a fragment(s) containing the unknown flanking sequences. In addition to being relevant in studies of viral integration sites, the method should be generally useful to analyze DNA sequences either upstream or downstream from a known sequence.
Sørensen
A B
AB
Department of Molecular Biology, University of Aarhus, Denmark.
Duch
M
M
Jørgensen
P
P
Pedersen
F S
FS
D000818:Animals; D001426:Bacterial Proteins; D001483:Base Sequence; D001710:Biotin; D002846:Chromatography, Affinity; D017931:DNA Primers; D004279:DNA, Viral / Q000302:isolation & purification; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D011533:Proviruses / Q000235:genetics*; D012190:Retroviridae / Q000235:genetics*; D017422:Sequence Analysis, DNA; D019809:Streptavidin; D016662:Virus Integration / Q000235:genetics*
D001426:Bacterial Proteins; D017931:DNA Primers; D004279:DNA, Viral; D001710:Biotin; D019809:Streptavidin