Amplification and analysis of cDNA generated from a single cell by 5′-RACE:: application to isolation of antibody heavy and light chain variable gene sequences from single B cells
Amplification and analysis of cDNA generated from a single cell by 5′-RACE:: application to isolation of antibody heavy and light chain variable gene sequences from single B cells
复制标题
DOI：
10.2144/000112123
复制
发表时间：
2006-04-01
期刊：
影响因子：
2.7
通讯作者：
Muraguchi, A
中科院分区：
文献类型：
--
作者：
Ozawa, T;Kishi, H;Muraguchi, A
cDNA末端的5 ′-快速扩增（5 ′-RACE）技术广泛用于扩增信使RNA（mRNA）5 ′末端的未知序列。然而，由于一个细胞中存在少量mRNA，常规的5 '-RACE不适合于从单个细胞产生cDNA。在这项研究中，我们报告了一种改进的5 '-RACE方法，该方法适合从单细胞中生成cDNA。在该方法中，从单个细胞直接合成第一链cDNA，并且加尾反应和第二链cDNA合成在同一管中进行，而不纯化cDNA样品。使用这种方法，我们能够从超过50%的单个B细胞中扩增免疫球蛋白（IG）可变区基因的cDNA。扩增的cDNA片段含有全长IG可变区，包括5“-非翻译区、前导序列和起始密码子。因此，该方法可应用于人和动物中淋巴细胞库的IG可变基因的综合分析，从而有助于开发用于感染性疾病的基于抗体的治疗剂。
The technique of 5'-rapid amplification of cDNA ends (5'-RACE) is widely used to amplify unknown sequences at the 5' end of a messenger RNA (mRNA). However, conventional 5'-RACE is inappropriate for producing cDNAs from a single cell due to the small quantity of mRNA present in one cell. In this study, we report an improved 5'-RACE method that is suitable for generating cDNA from a single cell. In this method, the first-strand cDNA was directly synthesized from a single cell, and both the tailing reaction and second-strand cDNA synthesis were performed in the same tube without purifying the cDNA sample. Using this method, we were able to amplify the cDNA of the immunoglobulin (Ig) variable region gene from more than 50% of single B cells. The amplified cDNA fragment contained a full-length Ig variable region including a 5''-untranslated region, a leader sequence, and an initiation codon. This method mail thus be applicable for a comprehensive analysis of the Ig variable genes of the lymphocyte repertoire in humans and animals, thereby contributing to the development of antibody-based therapeutics for infectious diseases.