Enzyme system for improving the detection limit in pyrosequencing

Highly sensitive real-time pyrosequencing seems promising for constructing an inexpensive and small DNA sequencer with a low running cost. A DNA sample of a picomole level is usually used in the conventional pyrosequencing based on a luciferase assay coupled with an APS-ATP surfurylase reaction for producing ATP from pyrophosphate (PPi). Although the luminescence intensity could be increased by increasing the amount of luciferase, it was impossible to reduce the target DNA amount because of a large background luminescence due to the luciferase-APS reaction. In this report, a novel approach using a new conversion reaction of PPi to ATP is proposed. This method has a very low background and can produce high signals in the presence of a large amount of luciferase; thus, the sample amount required for sequencing is significantly reduced. The ATP production from PPi is catalyzed with pyruvate orthophosphate dikinase ( PPDK) using AMP and phosphoenolpyruvate as the substrates, which are inactive for the luciferase-catalyzed reaction. All of the components in the AMP-PPDK-based pyrosequencing system are suitable for highly sensitive DNA sequencing in one tube. Real-time DNA sequencing with a readable length up to 70 bases was successfully demonstrated by using this system. By increasing the amount of luciferase, as low as 2.5 fmol of DNA templates was accurately sequenced by the proposed method with a novel simple and inexpensive DNA sequencer having a photodiode array as a sensor instead of a PMT or CCD camera. A sample amount as low as 2 orders of magnitude smaller than that used in the conventional pyrosequencer can be used.