Abstract
Southern blot analysis and the polymerase chain reaction (PCR) are powerful tools for detecting clonal antigen receptor gene rearrangements. However, a number of limitations restrict the predictive value of the results obtained by these techniques as they are commonly used. We describe a new method, automated high-resolution PCR fragment analysis, that can partially overcome many of the limitations of analyzing the T-cell receptor (TCR) gamma-chain gene. Analysis of TCR-gamma is performed using PCR with four sets of primers, previously described by others, specific for all variable (V) and joining (J) regions of the TCR gamma-chain gene. In addition, the four V region primers are 5' end-labeled with a fluorescent compound, 5-carboxyfluorescein. After amplification, the labeled PCR products are separated with an automated sequencing system, ABI 373 (Applied Biosystems, Weiterstadt, Germany). With the help of the Gene-Scan software ABI 672 (Applied Biosystems) and fluorescent-labeled DNA length markers, the exact size of each peak can be displayed and analyzed. The resolution of this method allows separation of PCR products differing in length by as little as 1 bp. Semiquantitative estimation of specific clones also can be performed. Infiltrate-specific gene rearrangement patterns can be identified and recognized in different tissue specimens at the time of diagnosis or in subsequent biopsy specimens. We conclude that automated high-resolution PCR fragment analysis allows more accurate and convenient analysis of the TCR gamma-chain gene.
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- Bottaro M., Berti E., Biondi A., Migone N., Crosti L. Heteroduplex analysis of T-cell receptor gamma gene rearrangements for diagnosis and monitoring of cutaneous T-cell lymphomas. Blood. 1994 Jun 1;83(11):3271–3278. [PubMed] [Google Scholar]
- Graf A., Kaudewitz P., Simon M., Kind P., Sander C. A. Nachweis von Klonalität in kutanen T-Zellymphomen mittels der Polymerasekettenreaktion. Pathologe. 1996 Nov;17(6):446–450. doi: 10.1007/s002920050184. [DOI] [PubMed] [Google Scholar]
- Greiner T. C., Raffeld M., Lutz C., Dick F., Jaffe E. S. Analysis of T cell receptor-gamma gene rearrangements by denaturing gradient gel electrophoresis of GC-clamped polymerase chain reaction products. Correlation with tumor-specific sequences. Am J Pathol. 1995 Jan;146(1):46–55. [PMC free article] [PubMed] [Google Scholar]
- Kneba M., Bolz I., Linke B., Hiddemann W. Analysis of rearranged T-cell receptor beta-chain genes by polymerase chain reaction (PCR) DNA sequencing and automated high resolution PCR fragment analysis. Blood. 1995 Nov 15;86(10):3930–3937. [PubMed] [Google Scholar]
- Pannetier C., Even J., Kourilsky P. T-cell repertoire diversity and clonal expansions in normal and clinical samples. Immunol Today. 1995 Apr;16(4):176–181. doi: 10.1016/0167-5699(95)80117-0. [DOI] [PubMed] [Google Scholar]
- Shibata D. K., Arnheim N., Martin W. J. Detection of human papilloma virus in paraffin-embedded tissue using the polymerase chain reaction. J Exp Med. 1988 Jan 1;167(1):225–230. doi: 10.1084/jem.167.1.225. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Volkenandt M., Wienecke R., Koch O. M., Buer J., Probst M., Atzpodien J., Horikoshi T., Danenberg K., Danenberg P., Bertino J. R. Conformational polymorphisms of cRNA of T-cell-receptor genes as a clone-specific molecular marker for cutaneous lymphoma. J Invest Dermatol. 1993 Oct;101(4):514–516. doi: 10.1111/1523-1747.ep12365889. [DOI] [PubMed] [Google Scholar]
- Wood G. S., Tung R. M., Haeffner A. C., Crooks C. F., Liao S., Orozco R., Veelken H., Kadin M. E., Koh H., Heald P. Detection of clonal T-cell receptor gamma gene rearrangements in early mycosis fungoides/Sezary syndrome by polymerase chain reaction and denaturing gradient gel electrophoresis (PCR/DGGE). J Invest Dermatol. 1994 Jul;103(1):34–41. doi: 10.1111/1523-1747.ep12389114. [DOI] [PubMed] [Google Scholar]