ANALYSIS OF REARRANGED T-CELL RECEPTOR BETA-CHAIN GENES BY POLYMERASE CHAIN-REACTION (PCR) DNA-SEQUENCING AND AUTOMATED HIGH-RESOLUTION PCR FRAGMENT ANALYSIS
ANALYSIS OF REARRANGED T-CELL RECEPTOR BETA-CHAIN GENES BY POLYMERASE CHAIN-REACTION (PCR) DNA-SEQUENCING AND AUTOMATED HIGH-RESOLUTION PCR FRAGMENT ANALYSIS
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DOI：
10.1182/blood.v86.10.3930.bloodjournal86103930
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发表时间：
1995-11-15
期刊：
影响因子：
20.3
通讯作者：
HIDDEMANN, W
中科院分区：
文献类型：
--
作者：
KNEBA, M;BOLZ, I;HIDDEMANN, W
聚合酶链式反应 (PCR) 引导的重排免疫基因扩增和测序，用于鉴定克隆特异性标记物，越来越多地用于急性淋巴细胞白血病 (ALL) 和非霍奇金淋巴瘤 (NHL) 患者，而不是耗时且费力的 Southern 分析。 在之前的报告中，尚未鉴定出单一共同的 V β 和 J β 序列，可以可靠地扩增大多数重排 T 细胞抗原受体 (TCR)-β V-D-J 连接。在 DNA 水平上，由于重排过程涉及相对大量的可能的 TCR-β 可变 (V beta) 和连接 (J beta) 基因片段，在本研究中，我们设计了针对 J beta 基因保守序列的高度简并 PCR 引物，与之前发表的共有 V beta 引物相结合，这些 J beta 引物特异性扩增来自基因组 DNA 的 TCR-β V-N(D)N-J 连接。使用这种方法，我们研究了从 9 名 T 细胞淋巴增殖性疾病患者、1 名 c-ALL 患者和 5 名非恶性疾病患者的活检材料中提取的 DNA，T 细胞系 Molt 3、Jurkat 和 HM 2 作为单克隆对照，克隆后对各个 PCR 产物进行测序，确定了 96 个随机选择的重组载体的核苷酸序列，在多克隆对照中，所有分析的克隆的 TCR-β 均不同V-N(D)N-J 连接，在 T 细胞系、所有 T 细胞恶性肿瘤和 c-ALL 中，可以通过克隆限制性 V-N(D)N-J 连接的演示来鉴定单克隆 PCR 产物，在高分辨率聚丙烯酰胺凝胶中分离后，通过自动荧光定量和 PCR 产物大小测定来确认 PCR 结果，该程序允许快速、具体地表征来自基因组 DNA 的克隆 TCR-P 重排和将显着简化当前在 T 谱系 NHL 和 ALL 患者的初始分期和随访期间识别和定量恶性 T 细胞的实验方法。 (C) 1995 年，美国血液学会。
Polymerase chain reaction (PCR)-directed amplification and sequencing of rearranged immune genes for identification of clone-specific markers are increasingly being used in acute lymphoblastic leukemia (ALL) and non-Hodgkin's lymphoma (NHL) patients instead of the time consuming and labor intensive Southern analysis, In previous reports, no single common V beta and J beta sequence had been identified that allowed reliable amplification of the majority of rearranged T-cell antigen receptor (TCR)-beta V-D-J junctions at the DNA level because of the relatively large number of possible TCR-beta variable (V beta) and joining (J beta) gene segments involved in the rearrangement processes, In the present study we designed highly degenerate PCR primers directed against conserved sequences of the J beta genes, In combination with a previously published consensus V beta primer, these J beta primers specifically amplify TCR-beta V-N(D)N-J junctions from genomic DNA. Using this approach we studied DNA extracted from biopsy material of nine patients with T-cell lymphoproliferative disorders, one c-ALL patient, and five patients with nonmalignant diseases, T-cell lines Molt 3, Jurkat, and HM 2 served as monoclonal controls, Individual PCR products were sequenced after cloning, The nucleotide sequences of 96 randomly chosen recombinant vectors were determined, In the polyclonal controls all analyzed clones differed in their TCR-beta V-N(D)N-J junctions, In the T-cell lines, in all of the T-cell malignancies, and in the c-ALL, monoclonal PCR products could be identified by demonstration of clonally restricted V-N(D)N-J junctions, The PCR results were confirmed by automated fluorescence quantification and size determination of PCR products after separation in a high-resolution polyacrylamide gel, The procedure allows rapid and specific characterization of clonal TCR-P rearrangements from genomic DNA and will significantly simplify current experimental approaches to identify and to quantitate malignant T cells during initial staging and follow-up of T-lineage NHL and ALL patients. (C) 1995 by The American Society of Hematology.