AMPLIFICATION AND SEQUENCE-ANALYSIS OF DNA FLANKING INTEGRATED PROVIRUSES BY A SIMPLE 2-STEP POLYMERASE CHAIN-REACTION METHOD

We describe a two-step polymerase chain reaction method that can be us ed for the amplification of cellular DNA sequences adjacent to an inte grated retroviral provirus. The technique involves a partly degenerate , arbitrary primer that will hybridize in the provirus-flanking cellul ar DNA. By using this primer in combination with a biotinylated provir us-specific primer, a provirus-cellular DNA junction fragment can be i solated from the nonspecific amplification products by using streptavi din-coated magnetic beads. A second amplification employing a nested p rovirus-specific primer and a biotinylated nondegenerate primer derive d from the partly degenerate primer followed by purification with stre ptavidin-coated beads enhances the specificity and the efficiency of r ecovery 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 ups tream or downstream from a known sequence.