[Applications of PCR in molecular immunology]

Abstract

Since a wide panel of monoclonal antibodies specific for antigen receptor variable (V) regions is unavailable, PCR has emerged as the primary analytical method for determining the expressed repertoire of T- and B-lymphocyte populations. Furthermore, PCR amplification of immunoglobulin V-region genes is exploited for the production of monoclonal antibodies by repertoire cloning as a replacement of the B-cell-hybridoma technique and for the humanization of murine monoclonal antibodies. The heavy and light chains of immunoglobulins as well as the transmembrane-protein chains which build the heterodimeric T-cell receptors consist of a constant and of an antigen-binding variable segment. Conventional PCR makes use of two specific oligonucleotide primers that flank a sequence of interest to prime replication of the bracketed DNA. In the case of antigen receptor encoding DNA one flanking sequence of the variable region is unknown or heterogeneous. Therefore, modifications of the conventional PCR had to be developed for the cloning and analysis of antigen-receptor DNA: (1) the usage of known V-gene segments by a lymphocyte population can be analyzed by quantitative PCR with panels of V-specific primers; (2) since PCR primers do not have to be completely complementary to the target DNA, it is possible to amplify a variety of different V regions with degenerate primers for relatively conserved sequences of the V genes; (3) enzymatic addition of a homopolymeric dG tail to the variable end of the antigen receptor DNA makes the amplification completely independent of the structure of the variable region because the dG-tailed DNA can be amplified with a nonspecific poly-dC oligonucleotide combined with a specific primer for the constant gene segment of interest.