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.
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 may 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.
Ozawa
Tatsuhiko
T
University of Toyama, Toyama, Toyama, Japan.
Kishi
Hiroyuki
H
Muraguchi
Atsushi
A
D001402:B-Lymphocytes / Q000378:metabolism*; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000032:analysis* / Q000235:genetics*; D006801:Humans; D007147:Immunoglobulin Light Chains / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*
D018076:DNA, Complementary; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region