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Method enabling pyrosequencing on double-stranded DNA
KTH, Superseded Departments, Biochemistry and Biotechnology.
2000 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 282, no 2, 186-193 p.Article in journal (Refereed) Published
Abstract [en]

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. Uhlen, and P. Nyren, 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.

Place, publisher, year, edition, pages
2000. Vol. 282, no 2, 186-193 p.
Keyword [en]
pyrosequencing, DNA sequencing, template preparation, luciferase, apyrase, ATP sulfury-lase, DNA polymerase, single-stranded DNA-binding protein, alkaline phosphatase, exonuclease I, double-stranded DNA, inorganic pyrophosphatase, nucleotides, luciferin, polymerase chain-reaction, hybridization, pyrophosphate, primer
URN: urn:nbn:se:kth:diva-19882ISI: 000088047200003OAI: diva2:338574
QC 20100525Available from: 2010-08-10 Created: 2010-08-10Bibliographically approved

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