An improved PCR-heteroduplex method permits high-sensitivity detection of clonal expansions in complex T cell populations.
We have modified the PCR-heteroduplex technique to render it more suitable for the study of the clonal make up of complex T cell populations. This technique is based on separate PCR amplifications of all the TCR V beta genes expressed by a polyclonal T cell sample, followed by a heteroduplex reaction of the PCR products and a gel separation. Our modification involves performing each heteroduplex reaction in the presence of excess carrier DNA, which is the PCR product of a cloned TCR V beta cDNA having the same variable and constant region of the amplified V beta family, but a different N region. In this way, every clonotypic V beta chain that is amplified in the polyclonal mixture forms a unique and reproducible pair of heteroduplex bands with the carrier DNA. This molecular footprint permits the identification of a given T cell clone over time, or in different anatomical sites. The specificity and sensitivity of the detection of T cell clones can be further increased by hybridising the blotted heteroduplex gel with oligonucleotides specific for either a TCR V beta N region or the carrier DNA. In conclusion, we have developed a simple and reproducible technique that permits the simultaneous detection of the expanded T cell clones present in heterogeneous T cell populations in a very specific and sensitive manner.
Wack
A
A
Unità di Immunochimica, DIBIT, Istituto Scientifico H.S. Raffaele, Milan, Italy.
Montagna
D
D
Dellabona
P
P
Casorati
G
G
D000368:Aged; D001483:Base Sequence; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D002352:Carrier Proteins / Q000235:genetics; D002999:Clone Cells; D004247:DNA / Q000737:chemistry; D006801:Humans; D007252:Influenza Vaccines / Q000276:immunology; D017493:Leukocyte Common Antigens / Q000032:analysis / Q000235:genetics; D008213:Lymphocyte Activation; D008969:Molecular Sequence Data; D009692:Nucleic Acid Heteroduplexes / Q000032:analysis*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011948:Receptors, Antigen, T-Cell / Q000235:genetics; D012680:Sensitivity and Specificity; D016176:T-Lymphocyte Subsets / Q000166:cytology / Q000276:immunology*
D002352:Carrier Proteins; D007252:Influenza Vaccines; D009692:Nucleic Acid Heteroduplexes; D011948:Receptors, Antigen, T-Cell; D004247:DNA; D017493:Leukocyte Common Antigens