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Methodological improvements of pyrosequencing technology
Stanford Univ, Stanford Genome Technol Ctr.
KTH, School of Biotechnology (BIO), Biochemistry.
KTH, School of Biotechnology (BIO), Biochemistry.
Univ Örebro, Dept Caring Sci, Div Biomed.
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2006 (English)In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 124, no 3, 504-511 p.Article in journal (Refereed) Published
Abstract [en]

Pyrosequencing technology is a rather novel DNA sequencing method based on the sequencing-by-synthesis principle. This bioluminometric, real-time DNA sequencing technique employs a cascade of four enzymatic reactions producing sequence peak signals. The method has been proven highly suitable for single nucleotide polymorphism analysis and sequencing of short stretches of DNA. Although the pyrosequencing procedure is relatively straightforward, users may face challenges due to varying parameters in PCR and sequencing primer design, sample preparation and nucleotide dispensation; such challenges are labor and cost intensive. In this study, these issues have been addressed to increase signal quality and assure sequence accuracy.

Place, publisher, year, edition, pages
2006. Vol. 124, no 3, 504-511 p.
Keyword [en]
DNA sequencing; primer-dimers; pyrosequencing technology; sample preparation; single-strand binding protein; SINGLE-NUCLEOTIDE POLYMORPHISM; TUMOR-SUPPRESSOR GENE; STRANDED DNA-BINDING; SEQUENCING PRIMERS; MUTATION; PROTEIN
National Category
Biochemistry and Molecular Biology
URN: urn:nbn:se:kth:diva-8158DOI: 10.1016/j.jbiotec.2006.01.025ISI: 000239156700004ScopusID: 2-s2.0-33745504382OAI: diva2:13407

QC 20100624

Available from: 2008-04-03 Created: 2008-04-03 Last updated: 2013-02-26Bibliographically approved
In thesis
1. Nucleic Acid Based Pathogen Diagnostics
Open this publication in new window or tab >>Nucleic Acid Based Pathogen Diagnostics
2008 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

Pathogenic organisms are transmitted to the host organism through all possible connected pathways, and cause a myriad of diseases states. Commonly occurring curable infectious diseases still impose the greatest health impacts on a worldwide perspective. The Bill & Melinda Gates Foundation partnered with RAND Corporation to form the Global Health Diagnostics Forum, with the goal of establishing and interpreting mathematical models for what effects a newly introduced point-of-care pathogen diagnostic would have in developing countries. The results were astonishing, with potentially millions of lives to be saved on an annual basis.

Golden standard for diagnostics of pathogenic bacteria has long been cultureable medias. Environmental biologists have estimated that less than 1% of all bacteria are cultureable. Genomic-based approaches offer the potential to identify all microbes from all the biological kingdoms. Nucleic acid based pathogen diagnostics has evolved significantly over the past decades. Novel technologies offer increased potential in sensitivity, specificity, decreased costs and parallel sample management. However, most methods are confined to core laboratory facilities. To construct an ultimate nucleic acid based diagnostic for use in areas of need, potential frontline techniques need to be identified and combined.

The research focus of this doctoral thesis work has been to develop and apply nucleic acid based methods for pathogen diagnostics. Methods and assays were applied to the two distinct systems i) screening for antibiotic resistance mutations in the bacterial pathogen Neisseria gonorrhoeae, and ii) genotype determination of the cancer causative Human Papillomavirus (HPV). The first part of the study included development of rapid, direct and multiplex Pyrosequencing nucleic acid screenings. With improved methodology in the sample preparation process, we could detect an existence of multiple co-infecting HPV genotypes at greater sensitivities than previously described, when using the same type of methodology. The second part of the study focused on multiplex nucleic acid amplification strategies using Molecular Inversion Probes with end-step Pyrosequencing screening. The PathogenMip assay presents a complete detection schematic for virtually any known pathogenic organism. We also introduce the novel Connector Inversion Probe, a padlock probe capable of complete gap-fill reactions for multiplex nucleic acid amplifications.

Abstract [sv]

Patogena organismer smittas till värd organismen genom alla möjliga kontaktnätverk och skapar en mångfald olika sjukdomstillstånd. Dock är det fortfarande vanligt förekommande behandlingsbara infektiösa sjukdomar som orsakar den största hälsoförlusten, sett från ett globalt perspektiv. Bill och Melinda Gates Stiftelsen samarbetade med RAND kooperation för att forma “The Global Health Diagnostics Forum”. Deras mål var att etablera och analysera matematiska modeller för vilka effekter en ny diagnostisk metod utrustat för fältarbete skulle ha i utvecklingsländer. Resultaten var häpnadsveckande, med potentiellt miljoner av liv som skulle kunna räddas på en årlig basis.

Den etablerade standarden för diagnostik av patogena bakterier har länge varit kultiveringsmedia baserad. Miljö specialiserade biologer har estimerat att mindre än 1 % av alla bakterie arter går att kultivera. Dock erbjuder genetiska analyser potentialen att kunna identifiera alla mikrober från alla de biologiska rikena. Nukleinsyrebaserade diagnostiska metoder har märkbart förbättrats över de senaste årtionden. Nya tekniker erbjuder utökad sensitivitet, selektivitet, sänkta kostnader och parallella analyser av patient prover. Dock är de flesta metoderna begränsade till standardiserade laboratoriemiljöer. För att konstruera en väl fungerande diagnostisk fältutrustning för användning i problem områden, behöver världsledande tekniker identifieras och kombineras.

Fokuseringsområdet för denna doktorsavhandling har varit att utveckla och utföra nukleinsyrebaserade metoder för patogen diagnostik. Metoder och experimentella utförande applicerades på två distinkta system i) sökning av antibiotika resistens relaterade mutationer i den patogena bakterien Neisseria gonorrhoeae och ii) genotypning av det cancer orsakande Humana Papillomaviruset (HPV). Den första delen av studien inriktade sig mot utveckling av snabba, direkta och multiplexa Pyrosekvenserings baserade nukleinsyreanalyser. Med förbättrad provprepareringsmetodologi kunde vi detektera multipla HPV infektioner med högre sensitivitet än vad tidigare beskrivits med liknande metodologi. Den andra delen av studien fokuserades på multiplexa nukleinsyre amplifikationer med “Molecular Inversion Probe” tekniken med sista steg Pyrosekvenserings analys. “PathogenMip assay” erbjuder ett komplett detektionsprotokoll för alla kända patogena organismer. Vi introducerar även den nya “Connector Inversion Probe”, en “Padlock Probe” kapabel att genomföra kompletta gap fyllningar för multiplex nukleinsyre amplifiering.

Place, publisher, year, edition, pages
Stockholm: KTH, 2008. [8], 51 p.
Trita-BIO-Report, ISSN 1654-2312 ; 2008:4
pathogen diagnostics, antibiotic resistance, connector inversion probes (CIPer), human papillomavirus (HPV), genotyping, global health diagnostic forum, molecular inversion probe (MIP), neisseria gonorrhoeae, padlock probes, pyrosequencing
National Category
Biochemistry and Molecular Biology
urn:nbn:se:kth:diva-4684 (URN)978-91-7178-899-3 (ISBN)
Public defence
2008-04-18, FR4, AlbaNova University Center, Roslagstullsbacken 21, Stockholm, 13:00
QC 20100624Available from: 2008-04-03 Created: 2008-04-03 Last updated: 2010-06-30Bibliographically approved

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