APPLICATION OF AN IMPROVED HIGH-SENSITIVITY PYROSEQUENCING SYSTEM FOR A PORTABLE BIOLUMINESCENCE ANALYZER

摘要

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.