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

Academic Article

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 IgG1/κ, IgG2b/κ and IgM/κ isotypes. © 1989.
  • Published In

  • Gene  Journal
  • Digital Object Identifier (doi)

    Pubmed Id

  • 20876659
  • Author List

  • LeBœuf RD; Galin FS; Hollinger SK; Peiper SC; Blalock JE
  • Start Page

  • 371
  • End Page

  • 377
  • Volume

  • 82
  • Issue

  • 2