Coamplification of hprt cDNA and γ T-cell receptor sequences from 6-thioguanine resitant human T-lymphocytes

Abstract

The nature of mutation at the HPRT locus in human T-lymphocytes in vivo is currently a subject of considerable interest. Determination of clonality in individual mutant T-lymphocytes is essential for the proper interpretation. This requires the molecular analysis of their respective T-cell receptors (TCR). We have developed a polymerase chain reaction (PCR)-based method for coamplification of hprt cDNA and the rearranged gamma T-cell receptor genes from crude cell lysates of individual 6-thioguanine resistant human T-lymphocytes. Following reverse transcription to produce hprt cDNA, the crude cell lysate is treated with proteinase K and subjected to a primary PCR with two sets of amplification primers, one specific for the hprt cDNA and the other for the rearranged gamma TCR gene. A secondary round of PCR, employing appropriate sets of nested amplification primers, are then used to produce sufficient quantities of DNA for both the sequencing and restriction fragment length analysis, of the hprt cDNA and gamma TCR gene respectively.