Effectiveness of capillary electrophoresis using fluorescent-labeled primers in detecting T-cell receptor gamma gene rearrangements.
We describe the use of fluorescent-labeled primers to analyze T-cell receptor gamma gene rearrangements (TCR gamma GR) 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 TCR gamma GR 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 TCR gamma GR (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 TCR gamma GR (38 of 41) identified by DGGE. The reproducible sensitivity for detecting TCR gamma GR 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 TCR gamma GR. The effectiveness of capillary electrophoresis is similar to DGGE.
Greiner
Timothy C
TC
Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska 68198-3135, USA. tgreiner@unmc.edu
Rubocki
Ronald J
RJ
D001483:Base Sequence; D017931:DNA Primers; D019075:Electrophoresis, Capillary / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D015334:Gene Rearrangement, gamma-Chain T-Cell Antigen Receptor; D006801:Humans; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D017931:DNA Primers; D005456:Fluorescent Dyes