Chen P F, Platsoucas C D
Department of Immunology, University of Texas, M.D. Anderson Cancer Center, Houston 77030.
Scand J Immunol. 1992 May;35(5):539-49. doi: 10.1111/j.1365-3083.1992.tb03253.x.
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 non-palindromic 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 micrograms 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.
我们开发了一种高效的新方法，用于扩增人α和β链T细胞受体（TCR）的cDNA。该方法被命名为非回文衔接子聚合酶链反应（NPA-PCR）。使用寡聚（dT）15-NotI、Cα-NotI或Cβ-NotI引物以及RNase H阴性逆转录酶，从单核白细胞中分离的总RNA合成cDNA。双链cDNA与非回文衔接子EcoRI-XmnI [d(ATTCGAACCCCTTCG)]和XmnI G链[d(pCGAAGGGGTTCG)]（磷酸化）连接，这导致在5'和3'末端都添加了EcoRI-XmnI位点。这两个非回文衔接子EcoRI-XmnI和XmnI G链相互互补，连接时两者都需要。通过用NotI限制性核酸酶处理，从3'末端去除EcoRI-XmnI衔接子，而它保留在5'末端。非回文衔接子EcoRI-XmnI用作5'扩增引物。Cα或Cβ恒定区引物用作3'扩增引物。扩增的cDNA被克隆，质粒用于转化DH5α感受态细胞。在扩增α链或β链TCR cDNA后，每0.1 - 0.25微克总RNA或每10,000至50,000个人外周血单核细胞可获得超过1000个白色菌落。在用位于Cα和Cβ扩增引物5'端的Cα或Cβ探针筛选后，40%至62%的菌落（来自五个供体的范围）呈阳性。从两个正常供体中随机选择总共50个扩增的α链或β链cDNA阳性克隆并进行测序，获得的序列是αβTCR的典型序列。鉴定出两个新的Jα基因片段。大约30%的α链阳性克隆具有5'非翻译区，其余大多数α链或β链TCR克隆从起始密码子或靠近5'端开始。与现有的用于扩增5'端未知或可变的cDNA（如T细胞抗原受体和免疫球蛋白）的PCR方法相比，NPA-PCR有几个优点。其中一个优点是只需要一个5'端延伸引物。由于大量的TCR Vα和Vβ家族，通过常规PCR扩增αβTCR cDNA需要大量不同的5'端引物。
