Effectiveness of capillary electrophoresis using fluorescent-labeled primers in detecting T-cell receptor γ gene rearrangements

Timothy Charles Greiner, Ronald J. Rubocki

Research output: Contribution to journalArticle

46 Citations (Scopus)

Abstract

We describe the use of fluorescent-labeled primers to analyze T-cell receptor γ gene rearrangements (TCRγGR) using capillary electrophoresis in the ABI Prism 310 Genetic Analyzer. We also compare the performance with denaturing gradient gel electrophoresis (DGGE). In a single multiplex polymerase chain reaction (PCR) we amplified TCRγGR with primers for all known groups of variable region genes, and joining region genes described in lymphoid neoplasms. Ten reactive samples, followed by five cell lines and 25 tumor samples with 41 individual TCRγGR (due to many biallelic rearrangements) previously identified by DGGE, were analyzed to validate the technique. The capillary electrophoresis protocol has 92% concordance for both TCR clonal status (23 of 25) and 95% concordance in the number of individual TCRγGR (38 of 41) identified by DGGE. The reproducible sensitivity for detecting TCRγGR diluted in reactive lymphoid DNA is 2% in clinical applications. Discrimination of predominant rearrangements requires a minimum ratio of two times the height of the normal distribution of polyclonal peaks. Capillary electrophoresis can provide results within 60 minutes for each specimen after PCR is complete. Capillary electrophoresis provides a faster result than sequence-based separation methods and gives an archival electronic record. Fluorescent labeling allows the identification of both the variable and joining gene segments used in a TCRγGR. The effectiveness of capillary electrophoresis is similar to DGGE.

Original languageEnglish (US)
Pages (from-to)137-143
Number of pages7
JournalJournal of Molecular Diagnostics
Volume4
Issue number3
DOIs
StatePublished - Jan 1 2002

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T-Lymphocyte Gene Rearrangement
T-Cell Receptor Genes
Capillary Electrophoresis
Denaturing Gradient Gel Electrophoresis
Genes
Multiplex Polymerase Chain Reaction
Normal Distribution
Tumor Cell Line
Polymerase Chain Reaction
DNA

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Molecular Medicine

Cite this

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abstract = "We describe the use of fluorescent-labeled primers to analyze T-cell receptor γ gene rearrangements (TCRγGR) using capillary electrophoresis in the ABI Prism 310 Genetic Analyzer. We also compare the performance with denaturing gradient gel electrophoresis (DGGE). In a single multiplex polymerase chain reaction (PCR) we amplified TCRγGR with primers for all known groups of variable region genes, and joining region genes described in lymphoid neoplasms. Ten reactive samples, followed by five cell lines and 25 tumor samples with 41 individual TCRγGR (due to many biallelic rearrangements) previously identified by DGGE, were analyzed to validate the technique. The capillary electrophoresis protocol has 92{\%} concordance for both TCR clonal status (23 of 25) and 95{\%} concordance in the number of individual TCRγGR (38 of 41) identified by DGGE. The reproducible sensitivity for detecting TCRγGR diluted in reactive lymphoid DNA is 2{\%} in clinical applications. Discrimination of predominant rearrangements requires a minimum ratio of two times the height of the normal distribution of polyclonal peaks. Capillary electrophoresis can provide results within 60 minutes for each specimen after PCR is complete. Capillary electrophoresis provides a faster result than sequence-based separation methods and gives an archival electronic record. Fluorescent labeling allows the identification of both the variable and joining gene segments used in a TCRγGR. The effectiveness of capillary electrophoresis is similar to DGGE.",
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