SINGLE NUCLEOTIDE DETECTION METHOD

摘要

The present invention provides a microfluidic nucleic acid sequencing device characterised by comprising:
€¢ a first zone for containing the nucleic acid to be sequenced and an enzyme for pyrophosphorolysing the nucleic acid progressively into its constituent nucleotide triphosphates;
€¢ first and second microfluidic pathways for respectively introducing a first aqueous stream into the first chamber and removing a second ordered aqueous stream of the nucleotide triphosphates;
€¢ a second zone connected to said second microfluidic pathway comprising a droplet head for creating microdroplets of the second aqueous stream in a water-immiscible carrier solvent, at least some of which contain a single nucleotide triphosphate;
€¢ third and fourth microfluidic pathways for respectively introducing the carrier solvent into the second chamber and removing a first stream of the microdroplets in the carrier solvent therefrom;
€¢ at least one contact means to contact the single nucleotide triphosphate in each microdroplet in the first stream with one or more of a pyrophosphatase, a ligase, a polymerase, a fluorophore-labelled capture system selective for at least one of the nucleotide triphosphate types characteristic of the nucleic acid and an exonuclease or polymerase having exonuclease activity and
€¢ a fluorescence-detection system attached to said fourth microfluidic pathway comprising a light source adapted to impinge on each droplet in the first stream of microdroplets in turn and a detector to detect fluorescence emitted therefrom. The present invention also provides the use of the microfluidic sequencing device to sequence DNA or RNA.