STOCHASTIC EVENTS IN THE AMPLIFICATION OF HTLV-I INTEGRATION SITES BYLINKER-MEDIATED PCR

Human T-cell leukaemia virus type I (HTLV-I) proviral integration site s from an asymptomatic carrier and from the MT4 cell line were analyse d by linker-mediated PCR (LMPCR) and inverse PCR (LPCR). LMPCR was mor e sensitive, allowing detection of a greater number of integrated prov iruses. Reconstruction experiments using a cloned integrated HTLV-I pr ovirus indicated that > 100 copies were necessary to be detected frequ ently by LMPCR. To circumvent this problem, the LMPCR analysis was per formed similar to 20 times per sample. Thus, for the MT4 cell line, th e seven major integration sites were accompanied by similar to 20 clon es of lesser frequency. For an asymptomatic HTLV-I carrier, nine integ ration sites were identified in a single amplification, while a furthe r 9 followed from 14 additional reactions. These findings show that th ere is a stochastic element to sampling HTLV-I integration sites by LM PCR, which tends to underestimate the actual number of HTLV-I bearing clones. Accordingly, those detected in at least two reactions represen t the most abundant clones.