An automated method for the analysis of T-cell receptor repertoires. Rapid RT-PCR fragment length analysis of the T-cell receptor beta chain complementarity-determining region 3.
The examination of T-cell receptor (TCR) repertoires has an important role in the study of lymphoproliferative disorders and autoimmune diseases. Analysis of the complementarity-determining region 3 (CDR3) of the TCR beta chain is used to assess the clonality of T-cell populations. We developed a rapid fluorescence-based method for CDR3 length analysis of expressed TCR gene families. TCR beta chain complementary DNA is amplified by a nested polymerase chain reaction with V beta family-specific oligonucleotide primers and a fluorochrome-labeled C beta primer. The polymerase chain reaction products were analyzed on a compact automated DNA sequencing system (OpenGene system, Visible Genetics, Toronto, Ontario). To demonstrate the usefulness of our technique, we examined the CDR3 length distribution of peripheral blood T cells from a healthy subject, intestinal T cells from a patient with ulcerative colitis, and the T-cell leukemia cell line Jurkat. The analysis revealed polyclonal, oligoclonal, and monoclonal CDR3 distributions, respectively, for the 3 T-cell populations. Our new method shows virtually identical CDR3 length patterns compared with the traditional radioisotope-based method. The new technique offers the convenience of rapid throughput, nonradioactive labeling, and quality data analysis.
Lue
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Department of Medicine, University of Arkansas for Medical Sciences, Little Rock 72205, USA.
Mitani
Y
Y
Crew
M D
MD
George
J F
JF
Fink
L M
LM
Schichman
S A
SA
D001331:Automation; D001773:Blood Cells / Q000502:physiology; D003093:Colitis, Ulcerative / Q000235:genetics / Q000473:pathology; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D007422:Intestines / Q000473:pathology / Q000503:physiopathology; D019169:Jurkat Cells / Q000502:physiology; D015458:Leukemia, T-Cell / Q000235:genetics; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics* / Q000276:immunology; D012016:Reference Values; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013601:T-Lymphocytes / Q000502:physiology; D014158:Transcription, Genetic / Q000502:physiology
D007135:Immunoglobulin Variable Region; D016693:Receptors, Antigen, T-Cell, alpha-beta