Double-step and inverse polymerase chain reaction for sensitive detection and cloning of T cell receptor variable region sequences

Abstract

We have developed a sensitive and rapid method for detection and cloning of cDNA amplified by double-step and inverse polymerase chain reaction (PCR) techniques. The blunt-ended double-strand cDNA libraries were circularized with T4 ligase and subsequently amplified by double-step PCR with two sets of primers of outward orientation (at amounts of 1-10 pmol in the first step and 100 pmol in the second step) which hybridize with the known region of the target DNA. This method is useful for analysis of the repertoires of TCR or immunoglobulins, in particular TCR alpha-chains, which are encoded by a single known constant region gene and greater than 100 unknown variable and joining region gene segments. By using this method, we detected at least 10(4) copies of TCR alpha-chain transcripts in the original samples which are equivalent to 10(3) T cells. The use of 1-10 ng of cytoplasmic RNA allowed us to make approximately 10(3) independent TCR alpha-chain libraries and to determine the sequence of unknown TCR alpha-chain cDNA by this method. We also show the frequency of a TCR alpha-chain usage in naive spleen and tumor-infiltrating lymphocytes.