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Escherichia coli single-stranded DNA-binding a molecular tool for improved sequence protein quality in pyrosequencing
KTH, Superseded Departments, Biotechnology.
KTH, Superseded Departments, Biotechnology.ORCID iD: 0000-0002-4657-8532
KTH, Superseded Departments, Biotechnology.ORCID iD: 0000-0003-4313-1601
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2002 (English)In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 23, no 19, 3289-3299 p.Article in journal (Refereed) Published
Abstract [en]

Pyrosequencing is a four-enzyme bioluminometric DNA sequencing technique based on a DNA sequencing by synthesis principle. Currently, the technique is limited to analysis of short DNA sequences exemplified by single-nucleotide polymorphism analysis. In order to expand the field for pyrosequencing, the read length needs to be improved and efforts have been made to purify reaction components as well as add single-stranded DNA-binding protein (SSB) to the pyrosequencing reaction. In this study, we have performed a systematic effort to analyze the effects of SSB by comparing the pyrosequencing result of 103 independent complementary DNA (cDNA) clones. More detailed information about the cause of low quality sequences on templates with different characteristics was achieved by thorough analysis of the pyrograms. Also, real-time biosensor analysis was performed on individual cDNA clones for investigation of primer annealing and SSB binding on these templates. Results from these studies indicate that templates with high performance in pyrosequencing without SSB possess efficient primer annealing and low SSB affinity. Alternative strategies to improve the performance in pyrosequencing by increasing the primer-annealing efficiency have also been evaluated.

Place, publisher, year, edition, pages
2002. Vol. 23, no 19, 3289-3299 p.
Keyword [en]
biacore, DNA template length, read length, single-stranded DNA-binding protein, pyrosequencing, real-time pyrophosphate, tumor-suppressor gene, nucleotide polymorphisms, skeletal-muscle, mutation, overexpression, identification, amplification, purification, inhibitors
Identifiers
URN: urn:nbn:se:kth:diva-21986ISI: 000178746700007OAI: oai:DiVA.org:kth-21986DiVA: diva2:340684
Note
QC 20100525Available from: 2010-08-10 Created: 2010-08-10 Last updated: 2017-12-12Bibliographically approved

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Hober, Sophia

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