Assessment of T-cell receptor beta-chain diversity by heteroduplex analysis.
The aim of this work was to search for a simple and alternative approach to the currently used methodologies for the analysis of T-cell receptor repertoire diversity. To this end we studied whether the heteroduplex analysis could be adapted to study the clonality of the T-cell receptor beta chain (TCRBV). We therefore analyzed, by sequencing, the molecular characteristics of the V-D-J junctions of numerous TCRBV chains from a variety of patients and from normal individuals, and compared the results with those obtained with the heteroduplex analysis. The latter procedure involves the amplification of the target TCRBV chains and the denaturation and renaturation of the amplified product to permit the random association of the distinct DNA strands encoding the different junctional regions. Whereas amplified material from polyclonal lymphoid cells migrates on a polyacrylamide gel as a "smear" of bands composed of different-sized polyclonal PCR fragments, the mismatched chains derived from oligoclonal populations migrate as discrete "heteroduplexes" and can be separated from the matched "homoduplex" obtained from homogeneous clonal cells. Our results provide evidence demonstrating that heteroduplex analysis can successfully be applied to the analysis of T-cell clonality in a variety of samples and can be complementary or substitute for the standard approach of TCR cloning and multiple sequencing of junctional regions. Thus, the procedure should facilitate the implementation of the analysis of TCR in diagnostic routine and should find applications in numerous physiologic and pathologic conditions.
Sottini
A
A
Institute of Chemistry, Medical School, Spedali Civili, Brescia, Italy.
Quiròs-Roldan
E
E
Albertini
A
A
Primi
D
D
Imberti
L
L
D000595:Amino Acid Sequence; D001483:Base Sequence / Q000235:genetics*; D002999:Clone Cells; D003001:Cloning, Molecular; D006801:Humans; D008969:Molecular Sequence Data; D009692:Nucleic Acid Heteroduplexes / Q000032:analysis*; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic / Q000235:genetics*; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000032:analysis* / Q000235:genetics*; D016176:T-Lymphocyte Subsets / Q000276:immunology
D009692:Nucleic Acid Heteroduplexes; D016693:Receptors, Antigen, T-Cell, alpha-beta