Sequencing method

Abstract

Disclosed is a method for determining the sequence of nucleotide bases in a polynucleotide analyte characterised by the steps of: a. generating a stream of droplets at least some of which contain a single nucleotide and wherein the order of single nucleotides in the droplet stream corresponds to the sequence of nucleotides in the analyte; b. introducing into each droplet a plurality of biological probe types each type (i) comprising a different detectable element in an undetectable state and (ii) being adapted to capture a different complimentary single nucleotide from which the analyte is constituted; c. causing the single nucleotide contained in the droplet to bind to its complimentary probe to create a used probe; and d. causing the detectable element to be released from the used probe in a detectable state. Typically the biological probe employed comprises a single-stranded nucleotide region the ends of which are attached to two different oligonucleotide regions wherein at least one of the oligonucleotide regions comprises detectable elements having a characteristic detection property and wherein the detectable elements are so arranged on the oligonucleotide region that the detectable property is essentially undetectable in the probe's unused state. In a most preferred embodiment the probe is labelled with multiple fluorophores and further comprises a restriction enzyme recognition site generated by the binding of the target single nucleotide to the single-stranded nucleotide region. Suitably, step c is carried out in the presence of a polymerase and ligase and step d in the presence of a restriction enzyme and an exonuclease. Typically the flow rate of the droplets is 100 to 2000 droplets per second.