Detection of clonal T cell populations by high resolution PCR using fluorescently labelled nucleotides; evaluation using conventional LIS-SSCP

Abstract

AIMS: To detect clonal T cell populations by high resolution polymerase chain reaction (PCR) using fluorescently labelled nucleotides and analysis on an ABI 377 DNA sequencer, and to evaluate this method using low ionic strength single strand conformation polymorphism (LIS-SSCP) analysis.

METHODS: DNA samples from 11 patients diagnosed with a T cell disease and 15 with no known T cell disorder were amplified using four multiplex T cell receptor gamma (TCR gamma) PCR reactions containing fluorescently labelled nucleotides. PCR products were analysed using both LIS-SSCP electrophoresis and an ABI 377 DNA sequencer using Genescan software. A Jurkat T cell leukaemia cell line was used to determine the sensitivity of the two methods.

RESULTS: Clonal TCR gamma populations were detected in all 11 samples from patients with a T cell disease and no clonal populations were detected in samples from patients without a T cell disorder, using both LIS-SSCP and DNA sequencer analysis. Although the sensitivity of the two methods was comparable, the data generated by the sequencer were easier to interpret than the LIS-SSCP gels, and allowed accurate size determination of every product, which was not possible using LIS-SSCP.

CONCLUSIONS: The use of fluorescent labelled nucleotides provides a more flexible and economical alternative to end labelled fluorescent primers for the detection of clonal TCR gamma gene rearrangements. This method allows clonal populations to be sized accurately and reproducibly, permitting the detection of identical clonal populations in different samples, and providing a method of monitoring disease progression and response to treatment.