REAL-TIME DNA-SEQUENCING USING DETECTION OF PYROPHOSPHATE RELEASE

An approach for real-time DNA sequencing without the need for electrop horesis has been developed. The approach relies on the detection of DN A polymerase activity by an enzymatic luminometric inorganic pyrophosp hate (PPi) detection assay (ELIDA) (Nyren, P. (1987) Anal. Biochem. 16 7, 235-238), The PPI formed in the DNA polymerase reaction is converte d to ATP by ATP sulfurylase and the ATP production is continuously mon itored by the firefly luciferase. In the sequencing procedure, immobil ized single-stranded template was used in a repeated cycle of deoxynuc leotide extension. Real-time signals in the ELIDA, proportional to the amount of incorporated nucleotide, were observed when complementary b ases were incorporated. An increased signal-to-noise ratio was obtaine d by substitution of deoxyadenosine alpha-thiotriphosphate (dATP alpha S) for the natural deoxyadenosine triphosphate, dATP alpha S is effic iently used by the DNA polymerase, but is not recognized by the lucife rase. As a model, 15 bases of a single-stranded PCR product were seque nced. The possibility for parallel processing of many samples in an au tomated manner is discussed. (C) 1996 Academic Press, Inc.