Multiplex polymerase chain reaction-based analysis of T-cell receptor γ gene rearrangements for the determination of T-lymphocyte clonality
✍ Scribed by Hongbao Ma; Darlene H. Smith; Abraham W. Hsie; Jonathan B. Ward Jr.
- Publisher
- John Wiley and Sons
- Year
- 2000
- Tongue
- English
- Weight
- 117 KB
- Volume
- 35
- Category
- Article
- ISSN
- 0893-6692
No coin nor oath required. For personal study only.
✦ Synopsis
Determination of the frequency of mutations at hprt or other loci in human lymphocytes provides a useful biomarker for human exposure to mutagens. One problem, however, is distinguishing between unique mutants and sibling mutants arising as progeny of an earlier mutant cell. We have developed a multiplex polymerase chain reaction (PCR)based method to analyze T-cell receptor (TCR) ␥ gene rearrangements for determination of T-cell clonality in mutational spectrum analysis. PCR primers for different subgroups of the V gene segment of the TCR ␥ gene were selected at different sites in the TCR ␥ gene so that the size of PCR products could define which V subgroup was involved in rearranged TCR ␥ genes; ␥ genes involving different V and J subgroups could be deter-mined directly by PCR. Mutant T-lymphocytes with rearranged TCR ␥ genes containing the same V and J subgroups were analyzed using PCR-based denaturing polyacrylamide gel electrophoresis. All of the 161 hprt mutant clones analyzed contained rearranged TCR ␥ genes. Rearrangements among all subgroups of the V and J gene segments of the TCR ␥ gene could be detected. V␥I and J␥1/2 subgroups were involved in 69 and 71% of rearranged TCR ␥ genes, respectively. This PCR-based analysis of TCR ␥ gene rearrangements provides a simple and comprehensive method for identifying the clonality of mutant T-lymphocytes in human hprt mutant lymphocyte assay and mutational spectrum analysis.