Restriction-PCR fingerprinting of the immunoglobulin VH repertoire: direct detection of an immune response and global analysis of B cell clonality.
Here we describe a method for fingerprinting the mouse immunoglobulin heavy chain variable region (VH) repertoire. Using a novel combination of existing techniques, large numbers of expressed VH genes can be simultaneously displayed as a fingerprint of VH gene fragments on a sequencing gel. This is achieved using isotype-specific reverse transcription-PCR amplification, restriction digestion and end-labeled primer run-offs. This technique (Res-PCR) allows analysis of the immune response, in this case to phenyloxazolone, in several different tissues and enables molecular cloning and sequencing of the VH genes involved in vivo. In addition, with Res-PCR, a "global" picture of expressed immunoglobulin genes is represented. This elucidates B cell clonality in different tissues and isotypes and detects a preference for shorter complementarity-determining region 3 in IgM-expressing cells. Res-PCR is a rapid and revealing method which will allow analysis of the complexity and sequence composition of the B cell immunoglobulin repertoire and so perhaps better define the B cell memory compartment and immune responses in vivo.
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University College London, Department of Oncology and Imperial Cancer Research Fund, Tumour Immunology Unit, GB. harry@the-angel.demon.co.uk
D000595:Amino Acid Sequence; D000818:Animals; D001402:B-Lymphocytes / Q000276:immunology*; D001483:Base Sequence; D016172:DNA Fingerprinting / Q000379:methods*; D018076:DNA, Complementary; D005260:Female; D005803:Genes, Immunoglobulin; D006241:Haptens / Q000276:immunology / Q000494:pharmacology; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007132:Immunoglobulin Isotypes / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010081:Oxazolone / Q000031:analogs & derivatives / Q000276:immunology / Q000494:pharmacology; D016133:Polymerase Chain Reaction / Q000379:methods*; D015183:Restriction Mapping / Q000379:methods*
D018076:DNA, Complementary; D006241:Haptens; D007143:Immunoglobulin Heavy Chains; D007132:Immunoglobulin Isotypes; D007135:Immunoglobulin Variable Region; C018361:2-phenyloxazolone; D010081:Oxazolone