Curry J, Skandalis A, Holcroft J, de Boer J, Glickman B
University of Victoria, Center for Environmental Health, Sidney, BC, Canada.
Mutat Res. 1993 Aug;288(2):269-75. doi: 10.1016/0027-5107(93)90094-v.
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.
体内人类T淋巴细胞中次黄嘌呤磷酸核糖转移酶（HPRT）基因座的突变性质目前是一个备受关注的课题。确定单个突变T淋巴细胞的克隆性对于正确解读至关重要。这需要对它们各自的T细胞受体（TCR）进行分子分析。我们开发了一种基于聚合酶链反应（PCR）的方法，用于从单个对6-硫鸟嘌呤耐药的人类T淋巴细胞的粗细胞裂解物中共扩增hprt cDNA和重排的γ T细胞受体基因。在逆转录产生hprt cDNA后，将粗细胞裂解物用蛋白酶K处理，并使用两组扩增引物进行一次PCR，一组对hprt cDNA特异，另一组对重排的γ TCR基因特异。然后进行第二轮PCR，使用适当的嵌套扩增引物组，分别产生足够量的DNA用于hprt cDNA和γ TCR基因的测序和限制性片段长度分析。
