Touch-down PCR and single-strand conformation polymorphism for detecting clonal T cell receptor γ gene rearrangement in lymphoid leukemia

Abstract

Abstract: Objective To explore the value of detecting clonal T cell receptor γ (TCR-γ) gene rearrangement with touch-down PCR and single-strand conformational polymorphism analysis (SSCP) in the diagnosis of lymphoid leukemia. Methods The DNA of peripheral blood leucocytes from lymphoid leukemia patients were extracted for amplification of the TCR-γ gene rearrangement with the consensus primers and touch-down PCR. The PCR products were analyzed by agarose gel electrophoresis, direct DNA sequencing and SSCP analysis. The positive control cell line DNA was mixed in different proportions with the DNA extracted from reactive lymphoid tissue to test the sensitivity of the touch-down PCR. Results Fifteen of 18 T lymphoid leukemia and 2 of the 4 B lymphoid leukemia patients were identified to be positive by agarose electrophoresis. The positive PCR products were further analyzed by SSCP analysis, which showed discrete bands. Direct DNA sequencing confirmed the clones to be TCR-γ gene rearrangement, and the sensitivity of touch-down PCR was 1%. Conclusion Consensus primers for studying TCR-γ gene rearrangement in combination with touch-down PCR can effectively amplify the clonal TCR-γ gene rearrangement in T lymphoid leukemia.

CLC Number:

HAN Xi-qun¹, QI Zong-li¹, HE Li¹, LU Di², ZHAO Tong¹. Touch-down PCR and single-strand conformation polymorphism for detecting clonal T cell receptor γ gene rearrangement in lymphoid leukemia[J]. Journal of Southern Medical University, 2004, 24(02): 188-191.

References

[1] Macintyre EA, Delabesse E. Molecular approaches to the diagnosis and evaluation of lymphoid malignancies[J]. Semin Hematol, 1999,36(4): 373-89.
[2] Gebhard S, Benhattar J, Bricod C, et al. Polymerase chain reaction in the diagnosis of T-cell lymphoma in paraffin-embedded bone marrow biopsies: a comparative study[J]. Histopathology, 2001, 38(1): 37-44.
[3] Kneba M, Bolz I, Linke B, et al. Characterization of clone-specific rearrangement T-cell receptor gamma-chain genes in lymphomas and leukemias by the polymerase chain reaction and DNA sequencing[J]. Blood, 1994, 84(2): 574-81.
[4] Strominger JL. Developmental biology of T cell receptors[J].Science, 1989, 26, 244(4907): 943-50.
[5] Zhu D, Kadin ME, Samoszuk M. Detection of clonal T-cell receptorgamma gene rearrangement by PCR/temporal temperature gradient gel electrophoresis[J]. Am J Clin Pathol, 2001, 116(4): 527-34.
[6] Fodinger M, Buchmayer H, Schwarzinger I, et al. Multiplex PCR for rapid detection ofT-cell receptor-gamma chain gene rearrangements in patients with lymphoproliferative diseases[J]. Br J Haematol,1996, 94(1): 136-9.
[7] Larsen HH, Masur H, Kovacs JA, et al. Development and evaluation of a quantitative, touch-down, real-time PCR assay for diagnosing Pneumocystis carinii pneumonia[J]. J Clin Microbiol, 2002, 40(2):490-4.
[8] Wack A, Montagna D, Dellabona P, et al. An improved PCR-heteroduplex method permits high-sensitivity detection of clonal expansions in complex T cell populations[J]. J Immunol Methods,1996, 196(2): 181-92.
[9] Langerak AW, Szczepanski T, van der Burg M, et al. Heteroduplex PCR analysis ofrearrangod T cell receptor genes for clonality assessment in suspect T cell proliferations[J]. Leukemia, 1997, 11 (12): 2192-9.
[10] Crisi GM, Emanuel JR, Johnson C, et al. Semireannealing, singlestranded conformational polymorphism: a novel and effective tool for the diagnosis of T-cell locality[J]. Diagn Mol Pathol, 2002, 11(2): 67-74.
[11] Trainor KJ, Brisco MJ, Wan JH, et al. Gene rearrangement in B-and T-lymphoproliferative disease detected by the polymerase chain reaction[J]. Blood, 1991, 78(1): 192-6.

[1]	ZHANG San-quan¹, PENG Hong², JIANG Hui-yong¹, HU Hai¹, ZHANG Jin-hua¹, YAO Kai-tai¹, ZHAO Tong¹. Cloning of human KIAA1173 gene and biological characterization of transfected 6-10B cells [J]. Journal of Southern Medical University, 2005, 25(10): 1216-1220.
[2]	LI Rong, ZHENG Hang, LUO Rong-cheng. Cloning and expression of the HER2 gene in MCF-7 cells [J]. Journal of Southern Medical University, 2005, 25(10): 1264-1267.
[3]	WANG Chun-xia, HOU Lian-bing, LIU Shi-ting. Effect of Changtong oral liquid on serum cytokine concentrations in rats with postoperative intestinal adhesion [J]. Journal of Southern Medical University, 2005, 25(06): 601-604.
[4]	JIANG Ai-da¹, YU Lin-zhong¹, GONG Xiao-wei², DENG Peng², MA Xiao-dong³. Effect of Liangge San on lipopolysaccharide-induced nuclear factor kappa B activation in cultured mouse macrophages [J]. Journal of Southern Medical University, 2005, 25(06): 619-622.
[5]	HONG Quan, WU Di, CHEN Xiang-mei, HOU Kai. Cloning and sequence analysis of human uric acid transporter gene [J]. Journal of Southern Medical University, 2005, 25(06): 623-625,629.
[6]	WANG Li-jun, MA Hong, CAI Yi-ming, HE Jian-gui, LIAO Xin-xue, ZENG Wu-tao, WANG Li-chun, LIU Jun. Angiotensin-(1-7) modulates fibrinolytic imbalance induced by oxidized low-density lipoprotein in cultured human umbilical vein endothelial cells [J]. Journal of Southern Medical University, 2005, 25(06): 633-637.
[7]	WANG Shi-jie, GUO Xiong, ZUO Hong, ZHANG Yin-gang, XU Peng, PING Zhi-guang, ZHANG Zeng-tie, GENG Dong. Chondrocyte apoptosis and the expression of Bcl-2, Bax, Fas and iNos in articular cartilage in Kashin-Beck disease [J]. Journal of Southern Medical University, 2005, 25(06): 643-646.
[8]	YUE Feng¹, MA Wen-li¹, SONG Yan-bin¹, SHI Rong¹, ZHANG Bao¹, ZHENG Wen-ling². c-myc gene silencing in K562 cells with RNA interference [J]. Journal of Southern Medical University, 2005, 25(06): 647-650.
[9]	LI Xiao-hua, LI Xiang-ping, LI Gang, LIU Xiong. Adeno-associated virus-mediated expression of human endostatin and its biological activity in vitro [J]. Journal of Southern Medical University, 2005, 25(06): 651-654.
[10]	LIU Ling, GUO Zhi-gang, WANG Qi-guang, LIU Sheng-lin, LAI Wen-yan, TU Yan. Significance of -191G/C single nucleotide polymorphisms in the promoter region of ATP-binding cassette transporter gene in coronary artery disease [J]. Journal of Southern Medical University, 2005, 25(06): 660-662,666.
[11]	ZHOU Hai-kuan¹, ZHAO Yong-bin², ZHENG Shao-bin¹, CHEN Tong¹, MAO Xiang-ming¹, QI Huan¹, WU Rong-juan¹. Association of S100A4 mRNA expression with differentiation and metastasis of renal cell carcinoma [J]. Journal of Southern Medical University, 2005, 25(06): 712-714,717.
[12]	LI Zhi-Feng, NIE Jun, DAI Ying-Chun, LI Jian-dong, CHEN Qing, YU Shou-yi. Bioinformatic analysis of Vibrio parahaemolyticus thermolabile hemolysin gene [J]. Journal of Southern Medical University, 2004, 24(10): 1153-1155.
[13]	LI Zhi-feng, NIE Jun, DAI Ying-chun, CHEN Qing, YU Shou-yi. Renaturation of thermolabile hemolysin inclusion bodies expressed by Vibrio parahaemolyticus [J]. Journal of Southern Medical University, 2004, 24(09): 995-997.
[14]	CHEN Jian-fa¹, HUNAG Zong-hai¹, YU Jin-long¹, LI Zhou¹, CHE Xiao-yan². Construction of recombinant adenoviruses containing cytosine deaminase gene driven by the vascular endothelial growth factor promoter using an AdEasier-1 system [J]. Journal of Southern Medical University, 2004, 24(06): 623-627.
[15]	MIAO Pei-hong¹, LIU Bei-yi², ZHENG Shan-gen², PANG Jian-xin¹, XU Jiang-ping¹. Screening and identification of human telomerase reverse transcriptase epitopes from random phage display peptide library [J]. Journal of Southern Medical University, 2004, 24(03): 252-255.