COAMPLIFICATION OF HPRT CDNA AND GAMMA T-CELL RECEPTOR SEQUENCES FROM6-THIOGUANINE RESISTANT HUMAN T-LYMPHOCYTES

The nature of mutation at the HPRT locus in human T-lymphocytes in viv o is currently a subject of considerable interest. Determination of cl onality in individual mutant T-lymphocytes is essential for the proper interpretation. This requires the molecular analysis of their respect ive T-cell receptors (TCR). We have developed a polymerase chain react ion (PCR)-based method for coamplification of hprt cDNA and the rearra nged gamma T-cell receptor genes from crude cell lysates of individual 6-thioguanine resistant human T-lymphocytes. Following reverse transc ription to produce hprt cDNA, the crude cell lysate is treated with pr oteinase K and subjected to a primary PCR with two sets of amplificati on primers, one specific for the hprt cDNA and the other for the rearr anged gamma TCR gene. A secondary round of PCR, employing appropriate sets of nested amplification primers, are then used to produce suffici ent quantities of DNA for both the sequencing and restriction fragment length analysis, of the hprt cDNA and gamma TCR gene respectively.