Cloning and sequencing of imniunoglobulin variable-region genes using degenerate oligodeoxy-ribonucleotides and polymerase chain reaction

Abstract

A procedure is described for using the polymerase chain reaction (PCR) to amplify and clone the cDNA from mouse immunoglobulin (Ig) variable (V) regions. This method uses a set of universal 5′-oligodeoxy-ribonucleotide primers that are degenerate and allow for the amplification of Ig V-region sequences from γ and μ heavy chains and from κ light chains. Selective first-strand cDNA synthesis is performed using Ig constant region primers and then a PCR is achieved by using the appropriate universal 5′-primer. The universal Ig heavy-chain primer was used to amplify the V-region cDNA from γ and μ isotypes and the universal light-chain primer was used to amplify three separate κ light V-region sequences. This procedure was used to obtain Ig V-region gene sequences from hybridomas secreting IgG 1/κ, IgG 2b/κ and IgM/κ isotypes.