An improved PCR-heteroduplex method permits high-sensitivity detection of clonal expansions in complex T cell...

We have modified the PCR-heteroduplex technique to render it more suitable for the study of the clonal make up of complex T cell populations. This technique is based on separate PCR amplifications of all the TCR V beta genes expressed by a polyclonal T cell sample, followed by a heteroduplex reaction of the PCR products and a gel separation. Our modification involves performing each heteroduplex reaction in the presence of excess carrier DNA, which is the PCR product of a cloned TCR V beta cDNA having the same variable and constant region of the amplified V beta family, but a different N region. In this way, every clonotypic V beta chain that is amplified in the polyclonal mixture forms a unique and reproducible pair of heteroduplex bands with the carrier DNA. This molecular footprint permits the identification of a given T cell clone over time, or in different anatomical sites. The specificity and sensitivity of the detection of T cell clones can be further increased by hybridising the blotted heteroduplex gel with oligonucleotides specific for either a TCR V beta N region or the carrier DNA. In conclusion, we have developed a simple and reproducible technique that permits the simultaneous detection of the expanded T cell clones present in heterogeneous T cell populations in a very specific and sensitive manner.