Open AccessCloning of Size-Selected Human Immunoglobulin Heavy-Chain Rearrangements from Third Complementarity-Determining Region Fingerprint Profiles
Author(s) -
Frank M. Raaphorst,
Joseph A. Tami,
Igñacio Sanz
Publication year - 1996
Publication title -
biotechniques/biotechniques
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.617
H-Index - 131
eISSN - 1940-9818
pISSN - 0736-6205
DOI - 10.2144/96201st02
Subject(s) - complementarity determining region , t cell receptor , biology , microbiology and biotechnology , cloning (programming) , immunoglobulin light chain , antibody , complementarity (molecular biology) , gene , immunoglobulin heavy chain , genetics , computational biology , t cell , immune system , computer science , programming language
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.