Amplification of T-cell receptor alpha- and beta-chain transcripts from mouse spleen lymphocytes by the nonpalindromic adaptor-polymerase chain reaction.

Abstract

We employed the nonpalindromic adaptor-PCR (NPA-PCR) method to amplify T-cell receptor (TCR) alpha- and beta-chain transcripts from the spleen of normal SJL mice. The NPA-PCR method has been specifically designed for the amplification of transcripts with variable or unknown 5' ends, such as TCRs and immunoglobulins (Ig). This method has certain distinct advantages over existing two-sided PCR methods for the amplification of TCR transcripts. Two NPA-PCR amplifications are sufficient to amplify all the TCR transcripts (one for the alpha-chain and another one for the beta-chain). Amplification of TCR transcripts by classical two-sided PCR requires a minimum of 45 amplification reactions for the murine TCR (20 for the V alpha families and 25 for the V beta families), using 45 different V-family-specific amplification primers. cDNA was synthesized from spleen RNA, using oligonucleotides complementary to sequences of either the murine TCR C alpha or C beta regions. The NotI restriction site was conjugated to these primers and therefore, a NotI restriction site was incorporated at the 3' end of the cDNA. A double-stranded nonpalindromic adaptor (EcoRI-XmnI strand and XmnI G strand, which are complementary to each other) was ligated onto both ends of the double-stranded cDNA. The adaptor was removed from the 3' end by NotI nuclease digestion whereas the adaptor was retained at the 5' end. Two rounds of PCR amplification were carried out. In the first, the EcoRI-XmnI adaptor was used as 5' end amplification primer; an antisense C region primer, designated mC alpha 2 or mC beta 2 (for the alpha- and beta-chain, respectively), was used as 3' amplification primer. In the second round of PCR amplification the same 5' end primer and a 3' end antisense primer, designated mC alpha 1 or mC beta 1, were used. These mC alpha 1 and mC beta 1 primers are located 5' to the mC alpha 2/mC beta 2 primers that were used for the first amplification. The amplified transcripts were cloned. Colonies were screened using a 32P-labeled probe, either C alpha or C beta, located 5' to those used for the last amplification and many positive clones were isolated and sequenced. All clones were unique when compared to each other, as anticipated for polyclonal T-cell populations. Comparison of the sequences obtained to those in the GENBANK/EMBL database revealed that they were typical of mouse alpha- or beta-chain TCR. With the exception of two beta-chain TCR transcripts, all the sequences shown here (36 alpha-chain and 20 beta-beta chain) have not been previously reported to the GENBANK/EMBL database.