Method enabling pyrosequencing on double-stranded DNA.

T Nordström; K Nourizad; M Ronaghi; P Nyrén

doi:10.1006/abio.2000.4603

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PubMed · 10873272

Method enabling pyrosequencing on double-stranded DNA.

T Nordström·K Nourizad·M Ronaghi·P Nyrén

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Abstract

Pyrosequencing is a new nonelectrophoretic, single-tube DNA sequencing method that takes advantage of co-operativity between four enzymes to monitor DNA synthesis (M. Ronaghi, M. Uhlén, and P. Nyrén, Science 281, 363-365). Pyrosequencing has so far only been performed on single-stranded DNA. In this paper different enzymatic strategies for template preparation enabling pyrosequencing on double-stranded DNA were studied. High quality data were obtained with several different enzyme combinations: (i) shrimp alkaline phosphatase and exonuclease I, (ii) calf intestine alkaline phosphatase and exonuclease I, (iii) apyrase and inorganic pyrophosphatase together with exonuclease I, and (iv) apyrase and ATP sulfurylase together with exonuclease I. In many cases, when the polymerase chain reaction was efficient exonuclease I could be omitted. In certain cases, additives such as dimethyl sulfoxide, single-stranded DNA-binding protein, and Klenow DNA polymerase improved the sequence quality. Apyrase was the fastest and most efficient of the three different nucleotide degrading enzymes tested. The data quality obtained on double-stranded DNA was comparable with that on single-stranded DNA. Pyrosequencing data for more than 30 bases could be generated on both long and short templates, as well as on templates with high GC content.

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Alkaline Phosphatase Apyrase Base Sequence DNA DNA, Single-Stranded Exodeoxyribonucleases Inorganic Pyrophosphatase Luciferases Polymerase Chain Reaction Pyrophosphatases Sequence Analysis, DNA Sulfate Adenylyltransferase Templates, Genetic Comparative Study Journal Article Research Support, Non-U.S. Gov't

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T Nordström, K Nourizad, M Ronaghi, P Nyrén. 2000-07-01. Method enabling pyrosequencing on double-stranded DNA.. https://doi.org/10.1006/abio.2000.4603

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