Yurovsky V V, Schulze D H, White B
Department of Medicine, University of Maryland School of Medicine, Baltimore 21201.
J Immunol Methods. 1994 Oct 14;175(2):227-36. doi: 10.1016/0022-1759(94)90365-4.
A sensitive, highly resolvable, and quantitative method was designed to analyse the diversity of polymerase chain reaction (PCR)-amplified transcripts which possess length polymorphism. A reverse transcriptase-PCR technique was used to amplify rearranged T cell antigen receptor (TCR) transcripts isolated from human blood. Oligonucleotide primers specific for conserved TCR V and C region sequences were used in PCR, with one of the primers end-labeled with 32P. Amplified cDNA products were analysed by polyacrylamide sequencing gel electrophoresis with an M13mp18 sequencing ladder as a size marker. 32P-labeled products were detected by either autoradiography or PhosphorImager. The method allowed determination of the sizes of PCR products with the precision of one nucleotide. The resolution using this technique was much higher than by electrophoresis in agarose gel with ethidium bromide staining. The sizes of PCR products determined by sequencing gel electrophoresis were consistent with the lengths of nucleotide sequences obtained after subcloning PCR products in competent bacterial cells. Analysis of PCR products by sequencing gel electrophoresis was more rapid and as accurate as nucleotide sequence analysis in determining the relative ratios of TCR mRNA in mixtures of T cell clones. The method is applicable for analysis of both rearranged TCR and immunoglobulin genes.
设计了一种灵敏、高分辨率且定量的方法，用于分析具有长度多态性的聚合酶链反应（PCR）扩增转录本的多样性。采用逆转录 - PCR技术扩增从人血中分离的重排T细胞抗原受体（TCR）转录本。PCR中使用了针对保守TCR V区和C区序列的寡核苷酸引物，其中一个引物用32P进行末端标记。扩增的cDNA产物通过聚丙烯酰胺测序凝胶电泳进行分析，以M13mp18测序梯作为大小标记。通过放射自显影或磷屏成像检测32P标记的产物。该方法能够以一个核苷酸的精度确定PCR产物的大小。使用该技术的分辨率远高于用溴化乙锭染色的琼脂糖凝胶电泳。通过测序凝胶电泳确定的PCR产物大小与在感受态细菌细胞中对PCR产物进行亚克隆后获得的核苷酸序列长度一致。在确定T细胞克隆混合物中TCR mRNA的相对比例时，通过测序凝胶电泳分析PCR产物比核苷酸序列分析更快速且同样准确。该方法适用于分析重排的TCR和免疫球蛋白基因。
