Fig. 4

HIV integration site loop amplification (ISLA) assay workflow. HIV DNA copy numbers are quantified from extracted nucleic acid and diluted to an endpoint prior to linear extension using primers in HIV env and HIV nef, then random decamers (blue) tailed with an HIV LTR U5-specific sequence (red) are annealed to the linear template and extended, the single stranded DNA downstream of the random decamer primer is removed and the U5-specific region anneals to its complementary sequence in the HIV LTR forming a loop which is then amplified, the resulting loop contains U5 sequence that is flanked by the host genome, using primers complementary to U5 the integration site can be mapped. Integration sites identified more than once indicate clonal expansion