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Development of the Non‐Palindromic Adaptor Polymerase Chain Reaction (NPA‐PCR) for the Amplification of Alpha‐and Beta‐Chain T‐Cell Receptor cDNAs
Author(s) -
CHEN P.F.,
PLATSOUCAS C. D.
Publication year - 1992
Publication title -
scandinavian journal of immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.934
H-Index - 88
eISSN - 1365-3083
pISSN - 0300-9475
DOI - 10.1111/j.1365-3083.1992.tb03253.x
Subject(s) - microbiology and biotechnology , ecori , biology , primer (cosmetics) , complementary dna , primer extension , plasmid , dna , chemistry , genetics , gene , rna , organic chemistry
We have developed a highly efficient new method for the amplification of α‐ and β‐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 mono‐nuclear leucocytes, using either an oligo (dT) 15 ‐ Not 1 or a Cα‐ Not 1 or a Cβ‐ Not 1 primer and RNase H‐negative reverse transcriptase. The double‐stranded cDNA was ligated with the non‐palindromic adaptors EcoRI‐Xmn I [d(ATTCGAACCCCTTCG)] and Xmn I G strand [d(pCGAAGGGGTTCG)] (phosphorylated), which resulted in the addition of the EcoR I‐ Xmn I site in both 5′ and 3′ ends. These two non‐palindromic adaptors, EcoR I‐ Xmn I and Xmn I G strand, are complementary to each other and both are required for ligation. The EcoRI‐Xmn I adaptor was removed from the 3′ end by treatment with Not I restriction nuclease, whereas it was retained al the 5′end. The non‐palindromic adaptor EcoR I‐ Xmn I was used as the 5′ amplitication primer. Cα or Cβ constant region primers were used as 3′ amplification primers. The amplified cDNAs were cloned and the plasmids were used to transform DH5α competent cells. Over 1000 white colonies per 0.1–0.25 μg of total RNA or per 10,000 to 50,000 human peripheral blood mononuclear cells were obtained after amplification of either the α‐ or the β‐chain TCR cDNAs. Between 40 and 62% of the colonies (range from live donors) were positive after screening with either a Cα or a Cβ probe, located 5′ to the Cα and Cβ amplification primers. A total of 50 amplified α‐ or β‐chain cDNA positive clones from two normal donors were randomly chosen and sequenced, and the sequences obtained were typical of αβ TCR. Two new Jα gene segments were identified. Approximately 30% of the α‐chain positive clones have 5′ untranslated region, and most of the remaining α‐ or β‐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 Vα and Vβ families, a large number of different 5′ end primers are required for amplification of αβ TCR cDNAs by conventional PCR.