Analysis of diversity of T cell antigen receptor genes using polymerase chain reaction and sequencing gel electrophoresis.

ABSTRACT

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.