DEVELOPMENT OF THE NON-PALINDROMIC ADAPTER POLYMERASE CHAIN-REACTION (NPA-PCR) FOR THE AMPLIFICATION OF ALPHA-CHAIN AND BETA-CHAIN T-CELL RECEPTOR CDNAS

We have developed a highly efficient new method for the amplification of alpha and beta-chain human T-cell receptor (TCR) cDNAs. This method is designated non-palindromic adaptor polymerase chain reaction (NPA-PCR). cDNA was synthesized from total RNA isolated from mononuclear leucocytes. using either an oligo (dT)15-NotI or a C-alpha-NotI or a C-beta-NotI primer and RNase H-negative reverse transcriptase. The double-stranded cDNA was ligated with the nonpalindromic adaptors EcoRI-XmnI [d(ATTCGAACCCCTTCG)] and XmnI G strand [d(pCGAAGGGGTTCG)] (phosphorylated), which resulted in the addition of the EcoRI-XmnI site in both 5' and 3' ends. These two non-palindromic adaptors, EcoRI-XmnI and XmnI G strand, are complementary to each other and both are required for ligation. The EcoRI-XmnI adaptor was removed from the 3' end by treatment with NotI restriction nuclease, whereas it was retained at the 5' end. The non-palindromic adaptor EcoRI-XmnI was used as the 5' amplification primer. C-alpha or C-beta-constant region primers were used as 3' amplification primers. The amplified cDNAs were cloned and the plasmids were used to transform DH5-alpha competent cells. Over 1000 white colonies per 0.1 0.25-mu-g of total RNA or per 10,000 to 50,000 human peripheral blood mononuclear cells were obtained after amplification of either the alpha- or the beta-chain TCR cDNAs. Between 40 and 62% of the colonies (range from five donors) were positive after screening with either a C-alpha or a C-beta probe, located 5' to the C-alpha and C-beta amplification primers. A total of 50 amplified alpha- or beta-chain cDNA positive clones from two normal donors were randomly chosen and sequenced, and the sequences obtained were typical of alpha-beta-TCR. Two new J-alpha gene segments were identified. Approximately 30%, of the alpha-chain positive clones have 5' untranslated region, and most of the remaining alpha- or beta-chain TCR clones started from the initiation codon or near the 5' end. NPA-PCR has several advantages over existing PCR methods for the amplification of cDNAs with unknown or variable 5' end, such as the T-cell antigen receptors and the immunoglobulins. Among these advantages is that only one 5' end extension primer is required. Because of the large number of TCR V-alpha and V-beta families, a large number of different 5' end primers are required for amplification of alpha-beta TCR cDNAs by conventional PCR.