AMPLIFICATION OF T-CELL RECEPTOR ALPHA-CHAIN AND BETA-CHAIN TRANSCRIPTS FROM MOUSE SPLEEN LYMPHOCYTES BY THE NONPALINDROMIC ADAPTER-POLYMERASE CHAIN-REACTION

We employed the nonpalindromic adaptor-PCR (NPA-PCR) method to amplify T-cell receptor (TCR) alpha- and beta-chain transcripts from the sple en of normal SJL mice. The NPA-PCR method has been specifically design ed for the amplification of transcripts with variable or unknown 5' en ds, such as TCRs and immunoglobulins (Ig). This method has certain dis tinct advantages over existing two-sided PCR methods for the amplifica tion of TCR transcripts. Two NPA-PCR amplifications are sufficient to amplify all the TCR transcripts (one for the alpha-chain and another o ne 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 familie s), using 45 different V-family-specific amplification primers, cDNA w as synthesized from spleen RNA, using oligonucleotides complementary t o sequences of either the murine TCR C alpha or C beta regions. The No tI restriction site was conjugated to these primers and therefore, a N oti restriction site was incorporated at the 3' end of the cDNA. A dou ble-stranded nonpalindromic adaptor (EcoRI-XmnI strand and XmnI G stra nd, which are complementary to each other) was ligated onto both ends of the double-stranded cDNA. The adaptor was removed from the 3' end b y NotI nuclease digestion whereas the adaptor was retained at the 5' e nd. Two rounds of PCR amplification were carried out. In the first, th e EcoRI-XmnI adaptor was used as 5' end amplification primer; art anti sense C region primer designated mC alpha 2 or mC beta 2 (for the alph a- 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 alph a 2/mC beta 2 primers that were used for the first amplification. The amplified transcripts were cloned. Colonies were screened using a P-32 -labeled probe, either C alpha or C beta, located 5' to those used for the last amplification and many positive clones were isolated and seq uenced. All clones were unique when compared to each other as anticipa ted for polyclonal T-cell populations. Comparison of the sequences obt ained to those in the GENBANK/EMBL database revealed that they were ty pical of mouse alpha- or beta-chain TCR. With the exception of two bet a-chain TCR transcripts, all the sequences shown here (36 alpha-chain and 20 beta-beta chain) have not been previously reported to the GENBA NK/EMBL database.