Emulsion PCR using nonmagnetic DNA capture beads
Highly parallel sample preparation methods are need for massively parallel DNA sequencing and detection. Emulsion PCR-based clone is one of the most promising techniques which can generate millions different amplicons each from a single template in a tube. In this paper, we used carboxylic acid beads as DNA capture beads carrying out emulsion PCR. Beads covalently coated with carboxylic acid group are bound to 5-amino-modified primer. An aqueous mix containing all the necessary components for PCR plus primer-bound beads and template DNA are stirred together with an oil mix to create microemulsions. The microemulsions are temperature-cycled as in a conventional PCR. To enrich the amplified beads, biotinylated enrichment primer and streptavidin-coat beads were used to capture the amplified beads. The results of amplification of emulsion and enrichment of beads were detected by single Cy5-labeled nucleotide extension on beads arrays immobilized into acrylamide gel. The results show that the optimal amount of template for emulsion PCR is at 1-5pM and the nonmagnetic beads is facilely enriched by a magnetic capture beads.
emulsion PCR beads array
Chao Tang Xiaolong Shi Zuhong Lu
State Key Laboratory of Bioelectronics,The School of Basic Medical Sciences,Southeast University, Na State Key Laboratory of Bioelectronics,Southeast University, Nanjing 210096, Jiangsu，China
国际会议
The 5th International Forum on Post-genome Technologies(5IFPT)(第五届国际后基因组生命科学技术学术论坛)
苏州
英文
2007-09-10（万方平台首次上网日期，不代表论文的发表时间）