APPLICATION OF AN IMPROVED HIGH-SENSITIVITY PYROSEQUENCING SYSTEM FOR A PORTABLE BIOLUMINESCENCE ANALYZER
Pyrosequencing technique is so ideal to analyze short DNA sequences that it has been widely applied in SNP analysis, microbial typing, CpG methylation analysis, and so on. The conventional pyrosequencing system requires a DNA sample at a picomole level, and the sensitivity cannot be improved by increasing the amount of luciferase due to a high background generated by the APS-luciferase reactioa In this report, an improved high-sensitivity pyrosequencing system with large amount of ATP sulfurylase and luciferase is proposed. Because ATP sulfurylase can competitively inhibit the combination of APS and luciferase, the luminescence intensity can be increased by using high concentration of luciferase with a very low background. It becomes possible to reduce the DNA sample to several tens of femtomoles even by a portable bioluminescence analyzer equipped with inexpensive photodiode detector. An avian influenza A virus sample was sequenced to determine its subtype by using the improved system, and 1μL LATE-PCR products were directly detected by pyrosequencing for SNP analysis.
Pyrosequencing high-sensitivity portable bioluminescence analyzer
Wu Haiping Chen Zhiyao Wu Wenjuan Zhou Gitorfua
China Pharmaceutical University, Nanjing 210009, China Huadong Research Institute for Medicine and B Huadong Research Institute for Medicine and Biotechnics, Nanjing, 210002, China
国际会议
The 6th International Forum on Post-genome Technologies(6IFPT)(第六届国际后基因组生命科学技术学术论坛)
北京
英文
188-192
2009-09-17（万方平台首次上网日期，不代表论文的发表时间）