An automated method for the analysis of T-cell receptor repertoires: Rapid RT-PCR fragment length analysis of the T-cell receptor chain complementarity-determining region 3

Cummins Lue, Yuichi Mitani, Mark D. Crew, James F. George, Louis M. Fink, Steven A. Schichman

Research output: Contribution to journalArticlepeer-review

11 Scopus citations

Abstract

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 β 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 β chain complementary DNA is amplified by a nested polymerase chain reaction with Vβ family-specific oligonucleotide primers and a fluorochrome-labeled Cβ 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.

Original languageEnglish
Pages (from-to)683-690
Number of pages8
JournalAmerican Journal of Clinical Pathology
Volume111
Issue number5
DOIs
StatePublished - 1999
Externally publishedYes

Keywords

  • Complementarity-determining region 3
  • DNA fragment analysis
  • Jurkat cell line
  • Oligoclonality
  • Polyacrylamide gel electrophoresis
  • Polymerase chain reaction
  • T-cell receptor
  • Ulcerative colitis

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