Cloning of size-selected human immunoglobulin heavy-chain rearrangements from third complementarity-determining region fingerprint profiles.
Methods have been developed to rapidly visualize the size distribution of third complementarity-determining regions (CDR3) in immunoglobulin (Ig) and T-cell receptor (TCR) molecules. DNA fragments spanning the Ig or TCR CDR3 are generated by PCR using primers at fixed positions in the variable and constant segments. These fragments differ in length due to size variation of the CDR3s. Visualization of the amplification products in polyacrylamide gels as a "CDR3 fingerprint profile" is a rough measure for the complexity of the Ig and TCR antigen-binding specificities. We report an adaptation of this method for the analysis of human Ig heavy-chain genes that incorporates silver staining, which allows for the fine analysis of specific regions of the profiles. This is especially useful for the study of low-abundant transcripts.
Raaphorst
F M
FM
University of Texas Health Science Center at San Antonio 78284-7874, USA. raaphorst@uthscsa.edu
Tami
J
J
Sanz
I E
IE
D000328:Adult; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D016172:DNA Fingerprinting / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D005333:Fetus / Q000276:immunology; D015326:Gene Rearrangement, B-Lymphocyte, Heavy Chain; D005803:Genes, Immunoglobulin; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012834:Silver; D013194:Staining and Labeling
D017931:DNA Primers; D018076:DNA, Complementary; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D012834:Silver