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Real-Time DNA Sequencing Using Detection of Pyrophosphate Release

Ronaghi, Mostafa ; Karamohamed, Samer ; Pettersson, Bertil ; Uhlén, Mathias ; Nyrén, Pål

Analytical biochemistry, 1996-11, Vol.242 (1), p.84-89 [Periódico revisado por pares]

United States: Elsevier Inc

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Título:
Real-Time DNA Sequencing Using Detection of Pyrophosphate Release
Autor: Ronaghi, Mostafa ; Karamohamed, Samer ; Pettersson, Bertil ; Uhlén, Mathias ; Nyrén, Pål
Assuntos: Adenosine Triphosphate - analysis ; Base Sequence ; Deoxyadenine Nucleotides ; Diphosphates - analysis ; DNA - analysis ; DNA - biosynthesis ; DNA, Single-Stranded - analysis ; DNA-Directed DNA Polymerase - analysis ; Luciferases ; Luminescent Measurements ; Molecular Sequence Data ; Sequence Analysis, DNA - methods ; Thionucleotides
É parte de: Analytical biochemistry, 1996-11, Vol.242 (1), p.84-89
Notas: ObjectType-Article-1
SourceType-Scholarly Journals-1
ObjectType-Feature-2
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Descrição: An approach for real-time DNA sequencing without the need for electrophoresis has been developed. The approach relies on the detection of DNA polymerase activity by an enzymatic luminometric inorganic pyrophosphate (PPi) detection assay (ELIDA) (Nyrén, P. (1987)Anal. Biochem.167, 235–238). The PPiformed in the DNA polymerase reaction is converted to ATP by ATP sulfurylase and the ATP production is continuously monitored by the firefly luciferase. In the sequencing procedure, immobilized single-stranded template was used in a repeated cycle of deoxynucleotide extension. Real-time signals in the ELIDA, proportional to the amount of incorporated nucleotide, were observed when complementary bases were incorporated. An increased signal-to-noise ratio was obtained by substitution of deoxyadenosine α-thiotriphosphate (dATPαS) for the natural deoxyadenosine triphosphate, dATPαS is efficiently used by the DNA polymerase, but is not recognized by the luciferase. As a model, 15 bases of a single-stranded PCR product were sequenced. The possibility for parallel processing of many samples in an automated manner is discussed.
Editor: United States: Elsevier Inc
Idioma: Inglês

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Real-Time DNA Sequencing Using Detection of Pyrophosphate Release

Ronaghi, Mostafa ; Karamohamed, Samer ; Pettersson, Bertil ; Uhlén, Mathias ; Nyrén, Pål

United States: Elsevier Inc

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