Rapid screening of T-cell receptor (TCR) variable gene usage by multiplex PCR: Application for assessment of clonal composition
Rapid screening of T-cell receptor (TCR) variable gene usage by multiplex PCR: Application for assessment of clonal composition
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DOI：
10.1034/j.1399-0039.1999.530202.x
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发表时间：
1999-02-01
期刊：
影响因子：
--
通讯作者：
Hansen, JA
中科院分区：
文献类型：
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作者：
Akatsuka, Y;Martin, EG;Hansen, JA
胸腺内分化过程中各种T细胞受体（TCR）基因家族的选择和复杂的重排为成熟T细胞表达抗原特异性提供了基础。TCR β可变（TCRBV）转录物可以通过RT PCR鉴定，但是需要多个反应来检测TCRBV亚家族的所有基因。我们在这里描述了一种多重PCR方法，该方法扩增46个功能基因，比较5个反应中的23个TCRBV家族，其中每个反应包含4个IV 7特异性引物以及单个荧光标记的TCR β恒定区引物。23个TCRBV家族中的每一个家族内的8至10种不同亚型可以通过分析CDR 3长度来鉴定。从琼脂糖凝胶分离的多重PCR产物可以进行直接测序以确认和必要的确定性克隆分型。本文所示的数据表明，多重PCR技术可用于筛选TCRBV的使用，并且可以容易地适用于分析T细胞群体中的克隆组成。
The selection of various T-cell receptor (TCR) gene families and complex rearrangements during intra-thymic differentiation provide the basis for the expression of antigen specificity by mature T cells. TCR beta variable (TCRBV) transcripts can be identified by RT PCR, but multiple reactions are required to detect ail genes of the TCRBV subfamilies. We describe here a multiplex PCR method that amplifies 46 functional genes comparing 23 TCRBV families in 5 reactions where each reaction contains 4 iv 7 specific primers together with a single fluorescence-tagged TCR beta constant region primer. Between 8 and 10 distinct subtypes within each of the 23 TCRBV families can be identified by analysis of the CDR3 length. Multiplex PCR products isolated from agarose gels can be subjected to direct sequencing fur confirmation and definitive clonotyping ii necessary. The data illustrated here show that the multiplex PCR technique is useful for screening TCRBV usage and can be easily adapted for analysis of clonal composition in T-cell populations.