Universal cloning and direct sequencing of rearranged antibody V genes using C region primers, biotin-captured cDNA and one-side PCR.
Polymerase chain reaction (PCR) cloning has greatly facilitated the cloning of heavy and light chain genes from B cells and hybridomas and has been critical for the generation of natural antibody gene libraries for expression in bacteria and on filamentous phages. There remain difficulties, however, in cloning VH and VL genes from a number of mouse and rat hybridoma lines and from B cells from several other species due to insufficient sequence information. Here we describe a rapid and 'universal' strategy for cloning rearranged antibody genes from any species for which the sequence of the C segment(s) are known. First strand synthesis is primed with a biotinylated C region primer and full length cDNA is captured on streptavidin-coated magnetic beads for tailing with dGTP and terminal deoxynucleotidyl transferase. After tailing, the cDNA is captured again, amplified using polyC primers and used for direct sequencing or cloning. The use of C region primers and cDNA capture ensures that this one-side PCR procedure is efficient and rapid as well as being entirely independent of the sequence of the V segment. We demonstrate its application to the direct sequencing or cloning of the H and L chain genes from six mouse and rat hybridomas and propose that the method described will find applications in three areas: (i) cloning rearranged antibody genes in all cases in which cloning with V-J primers is not possible; (ii) repertoire studies in which an unbiased cloning procedure is required for accurate estimate of gene usage; and (iii) generation of VH and VL gene libraries from immunised animals.
CRF Cell Interactions Laboratory, Cambridge University Medical School, UK.
A
A
Heinrichs
C
C
Milstein
E
E
Gherardi
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000138:chemical synthesis / Q000302:isolation & purification; D015322:Gene Rearrangement, B-Lymphocyte / Q000235:genetics*; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*
D017931:DNA Primers; D018076:DNA, Complementary; D007135:Immunoglobulin Variable Region