SINGLE NUCLEOTIDE DETECTION METHOD

摘要

SINGLE NUCLEOTIDE DETECTION METHOD A method of sequencing a nucleic acid such as DNA or RNA is provided. It is characterised by the steps of (1) generating a stream of single nucleoside triphosphates by progressive pyrophosphorolysis of the nucleic acid; (2) producing at least one substantially double-stranded oligonucleotide used probe by reacting in the presence of a polymerase and a ligase at least one of the single nucleoside triphosphates with a corresponding probe system comprising (a) a first single-stranded oligonucleotide labelled with characteristic detectable elements in an undetectable state and (b) second and third single-stranded oligonucleotides capable of hybridising to complementary regions on the first oligonucleotide; (3) digesting the used probe with an enzyme having double-stranded exonucleolytic activity to yield the detectable elements in a detectable state and a single-stranded fourth oligonucleotide which is at least in part the sequence complement of the first oligonucleotide; (4) reacting the fourth oligonucleotide with another first oligonucleotide to produce a substantially double-stranded oligonucleotide product corresponding to the used probe; (5) repeating steps (3) and (4) in a cycle and (6) detecting the characteristic detectable elements released in each iteration of step (3). Suitably the detectable elements are fluorophores. The method of the present invention generates a stronger fluorescence signal from a single nucleoside triphosphate than has been described previously. Suitable probe systems are also disclosed.