A novel and rapid cloning method for the T-cell receptor variable region sequences

Abstract

Conventional polymerase chain reactions (PCR) require sequence information on both ends of the DNA to be amplified. The novel technique described here allows the amplification of cDNA fragments with sequence information from one end only. Blunt-ended double-strand cDNA is prepared, circularized with T4 DNA ligase and used as a PCR template. The two PCR primers are designed to hybridize to the known region in an outward orientation allowing the amplification of the unknown sequence. The method was established using the alpha-chain of T-cell antigen receptors (Tcr) as an example. The cDNA synthesized from 1 microgram of total RNA from human peripheral lymphocytes was amplified and cloned resulting in a library of 1-2 x 10(6) Tcr-specific clones. The method should also be useful for cloning full-length cDNA or for the identification of new members of a gene family that share a conserved domain.