AN EXPERIMENTALLY VALIDATED PANEL OF SUBFAMILY-SPECIFIC OLIGONUCLEOTIDE PRIMERS (V-ALPHA-1-W29/V-BETA-1-W24) FOR THE STUDY OF HUMAN T-CELL RECEPTOR VARIABLE V-GENE SEGMENT USAGE BY POLYMERASE CHAIN-REACTION
AN EXPERIMENTALLY VALIDATED PANEL OF SUBFAMILY-SPECIFIC OLIGONUCLEOTIDE PRIMERS (V-ALPHA-1-W29/V-BETA-1-W24) FOR THE STUDY OF HUMAN T-CELL RECEPTOR VARIABLE V-GENE SEGMENT USAGE BY POLYMERASE CHAIN-REACTION
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DOI：
10.1002/eji.1830220522
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发表时间：
1992-05-01
影响因子：
5.4
通讯作者：
HERCEND, T
中科院分区：
文献类型：
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作者：
GENEVEE, C;DIU, A;HERCEND, T
我们在这里报告了一系列T细胞受体(TCR)V(α)或V(β)亚家族特异性寡核苷酸引物的特征。指导设计每个寡核苷酸的标准包括适当的热力学参数以及与相关和不相关的靶序列的差异碱基配对分数。用聚合酶链式反应(PCR)对一系列TCR编码的质粒DNA和克隆性T细胞群体进行检测，以检测V(α)或V(β)亚家族寡核苷酸的特异性。与不相关的亚家族基因片段相对应的质粒序列发生了意外的交叉反应。这导致合成了额外的一系列寡核苷酸，以获得相关的小组。最终选择了一系列V(α)1-W29/V(β)1-W24 TCR亚家族特异性寡核苷酸，其产生的交叉反应很少，如果有的话。使用C(α)或C(β)引物扩增内部阳性对照模板(即用于V(α)系列的C(β)和用于V(β)系列的C(α))已经用来自外周血淋巴细胞的cDNA进行了PCR检测；这被证明不会改变V亚家族特异性DNA片段的扩增。这组寡核苷酸将有助于研究TCRV基因片段的使用，从而可能导致更好地表征T细胞反应，从而可能导致更好地表征T细胞在生理和病理情况下的反应。
We report here the characterization of a series of T cell receptor (TcR) V(alpha) or V(beta) subfamily-specific oligonucleotide primers. Criteria that have guided the design of each oligonucleotide include appropriate thermodynamic parameters as well as differential base-pairing scores with related and unrelated target sequences. The specificity of the oligonucleotides for each V(alpha) or V(beta) subfamily was tested by polymerase chain reaction (PCR) on both a series of TcR encoding plasmid DNA and clonal T cell populations. Unexpected cross-reactivities were observed with plasmid cDNA sequences corresponding to unrelated subfamily gene segments. This led to the synthesis of additional series of oligonucleotides to obtain a relevant panel. A series of V(alpha)1-w29/V(beta)1-w24 TcR subfamily-specific oligonucleotides was eventually selected which generates little, if any, cross-reactivity. The use of C(alpha) or C(beta) primers for the amplification of internal positive control templates (i.e. C(beta) for the V(alpha) series and C(alpha) for the V(beta) series) has been tested in PCR performed with cDNA derived from peripheral blood lymphocytes; it was shown not to alter the amplification of the V subfamily-specific DNA fragments. This panel of oligonucleotides will be helpful in the study of TcR V gene segment usage and, thus, may lead to a better characterization of T cell response and, thus, may lead to a better characterization of T cell responses in physiological and pathological situations.