Stochastic events in the amplification of HTLV-I integration sites by linker-mediated PCR

Abstract

Human T-cell leukaemia virus type I (HTLV-I) proviral integration sites from an asymptomatic carrier and from the MT4 cell line were analysed by linker-mediated PCR (LMPCR) and inverse PCR (IPCR). LMPCR was more sensitive, allowing detection of a greater number of integrated proviruses. Reconstruction experiments using a cloned integrated HTLV-1 provirus indicated that > 100 copies were necessary to be detected frequently by LMPCR. To circumvent this problem, the LMPCR analysis was performed approximately 20 times per sample. Thus, for the MT4 cell line, the seven major integration sites were accompanied by approximately 20 clones of lesser frequency. For an asymptomatic HTLV-I carrier, nine integration sites were identified in a single amplfication, while a further 9 followed from 14 additional reactions. These findings show that there is a stochastic element to sampling HTLV-I integration sites by LMPCR, which tends to underestimate the actual number of HTLV-I bearing clones. Accordingly, those detected in at least two reactions represent the most abundant clones.