降落式PCR和SSCP检测淋巴细胞白血病单克隆T细胞受体γ基因重排

南方医科大学学报 ›› 2004, Vol. 24 ›› Issue (02): 188-191.

降落式PCR和SSCP检测淋巴细胞白血病单克隆T细胞受体γ基因重排

韩西群¹, 齐宗利¹, 贺莉¹, 陆地², 赵彤¹

1. 第一军医大学病理学教研室, 广东, 广州, 510515;
2. 第一军医大学细胞生物学教研室, 广东, 广州, 510515

出版日期:2004-02-20 发布日期:2004-02-20
基金资助:
收稿日期:2003-10-11。
基金项目:广东省科技计划项目(B30101)
作者简介:韩西群(1971-),男,第一军医大学在读博士研究生,医师,电话:020-61363263,E-mail:hanxiqun@fimmu.com

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

HAN Xi-qun¹, QI Zong-li¹, HE Li¹, LU Di², ZHAO Tong¹

1. 第一军医大学病理学教研室, 广东, 广州, 510515;
2. 第一军医大学细胞生物学教研室, 广东, 广州, 510515

Online:2004-02-20 Published:2004-02-20

摘要/Abstract

摘要： 目的以降落式PCR和单链构型多态性(SSCP)方法检测淋巴细胞白血病单克隆T细胞受体(TCR)γ基因重排,探讨其在淋巴细胞白血病诊断中的价值。方法盐析法提取淋巴细胞白血病患者外周血白细胞DNA,TCR-γ基因重排通用引物和降落式PCR扩增基因重排。分别采用琼脂糖凝胶电泳、SSCP和DNA直接测序方法检测PCR扩增产物。阳性细胞系DNA模板与反应增生性淋巴组织DNA按不同比例混合后扩增,检测降落式PCR的灵敏度。结果琼脂糖电泳中,18例T淋巴细胞白血病中有15例、4例B淋巴细胞白血病中有2例为阳性,阳性标本经SSCP进一步分析,呈不连续条带。DNA直接测序证实扩增产物为TCR-γ基因重排。阳性对照模板占1%以上时可得到阳性扩增结果。结论 TCR-γ基因重排通用引物结合降落式PCR可有效扩增T淋巴细胞白血病基因重排,可用于T淋巴细胞白血病的辅助诊断。

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.

中图分类号:

韩西群¹, 齐宗利¹, 贺莉¹, 陆地², 赵彤¹. 降落式PCR和SSCP检测淋巴细胞白血病单克隆T细胞受体γ基因重排[J]. 南方医科大学学报, 2004, 24(02): 188-191.

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.

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