Endre søk
RefereraExporteraLink to record
Permanent link

Direct link
Referera
Referensformat
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Annet format
Fler format
Språk
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Annet språk
Fler språk
Utmatningsformat
  • html
  • text
  • asciidoc
  • rtf
Viral and microbial genotyping by a combination of multiplex competitive hybridization and specific extension followed by hybridization to generic tag arrays
KTH, Tidigare Institutioner                               , Bioteknologi.
KTH, Tidigare Institutioner                               , Bioteknologi.
KTH, Tidigare Institutioner                               , Bioteknologi.
KTH, Tidigare Institutioner                               , Bioteknologi.ORCID-id: 0000-0002-3627-6899
Vise andre og tillknytning
2003 (engelsk)Inngår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 31, nr 22, s. e146-Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

 Detection and identification of microbial pathogens are important for disease diagnosis, treatment and prophylaxis measurements. By introducing an innovative technique, we show a robust, reliable and accurate microarray-based method for identification of microbial pathogens. The technique utilizes a unique combination of multiplex competitive hybridization, which enhances hybridization accuracy of oligonucleotides to the specific target, and apyrase-mediated allele-specific extension, which improves specific extension. As a model system, different clinically relevant human papillomaviruses were selected for this study. The method generated accurate results and proves to be promising for specific and correct microbial and viral typing.

sted, utgiver, år, opplag, sider
2003. Vol. 31, nr 22, s. e146-
Emneord [en]
article, DNA microarray, female, genetics, genotype, human, methodology, nucleic acid hybridization, Papilloma virus, polymerase chain reaction, reproducibility, sensitivity and specificity, virology, virus infection
HSV kategori
Identifikatorer
URN: urn:nbn:se:kth:diva-8906DOI: 10.1093/nar/gng147ISI: 000186590600045OAI: oai:DiVA.org:kth-8906DiVA, id: diva2:14389
Merknad
QC 20101005. Uppdaterad från In press till Published (20101005).Tilgjengelig fra: 2005-12-09 Laget: 2005-12-09 Sist oppdatert: 2017-12-14bibliografisk kontrollert
Inngår i avhandling
1. Arrayed identification of DNA signatures
Åpne denne publikasjonen i ny fane eller vindu >>Arrayed identification of DNA signatures
2005 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

In this thesis techniques are presented that aim to determine individual DNA signatures by controlled synthesis of nucleic acid multimers. Allele-specific extension reactions with an improved specificity were applied for several genomic purposes. Since DNA polymerases extend some mismatched 3’-end primers, an improved specificity is a concern. This has been possible by exploiting the faster extension of matched primers and applying the enzymes apyrase or Proteinase K. The findings were applied to methods for resequencing and viral and single nucleotide polymorphism (SNP) genotyping.

P53 mutation is the most frequent event in human cancers. Here, a model system for resequencing of 15 bps in p53 based on apyrase-mediated allele-specific extension (AMASE) is described, investigated and evaluated (Paper I). A microarray format with fluorescence detection was used. On each array, four oligonucleotides were printed for each base to resequence. Target PCR products were hybridized and an AMASE-reaction performed in situ to distinguish which of the printed oligonucleotides matched the target. The results showed that without the inclusion of apyrase, the resulting sequence was unreadable. The results open the possibilities for developing large-scale resequencing tools.

The presence of certain types of human papillomaviruses (HPV) transforms normal cells into cervical cancer cells. Thus, HPV type determination is clinically important. Also, multiple HPV infections are common but difficult to distinguish. Therefore, a genotyping platform based on competitive hybridization and AMASE is described, used on clinical sample material and evaluated by comparison to Sanger DNA sequencing (Papers II and III). A flexible tag-microarray was used for detection and the two levels of discrimination gave a high level of specificity. Easy identification of multiple infections was possible which provides new opportunities to investigate the importance of multiply infected samples.

To achieve highly multiplexed allele-specific extension reactions, large numbers of primers will be employed and lead to spurious hybridizations. Papers IV to VI focus on an alternative approach to control oligomerization by using protease mediated allele-specific extension (PrASE). In order to maintain stringency at higher temperatures, Proteinase K, was used instead of apyrase, leading to DNA polymerase degradation and preventing unspecific extensions. An automated assay with tag-array detection for SNP genotyping was established. First PrASE was introduced and characterized (Paper IV), then used for genotyping of 10 SNPs in 442 samples (Paper V). A 99.8 % concordance to pyrosequencing was found. PrASE is a flexible tool for association studies and the results indicate an improved assay conversion rate as compared to plain allele-specific extension.

The highly polymorphic melanocortin-1 receptor gene (MC1R) is involved in melanogenesis. Twenty-one MC1R variants were genotyped with PrASE since variants in the gene have been associated to an increased risk of developing melanoma. A pilot study was performed to establish the assay (Paper VI) and subsequently a larger study was executed to investigate allele frequencies in the Swedish population (Paper VII). The case and control groups consisted of 1001 and 721 samples respectively. A two to sevenfold increased risk of developing melanoma was observed for carriers of variants.

sted, utgiver, år, opplag, sider
Stockholm: KTH, 2005. s. 67
Emneord
apyrase, allele-specific extension, competitive hybridization, DNA sequencing, genotyping, human papillomavirus (HPV), MC1R, microarray, mutation, p53, protease
HSV kategori
Identifikatorer
urn:nbn:se:kth:diva-549 (URN)91-7178-219-2 (ISBN)
Disputas
2005-12-16, Sal D2, Lindstedtsvägen 5, Stockholm, 10:00
Opponent
Veileder
Merknad
QC 20101028Tilgjengelig fra: 2005-12-09 Laget: 2005-12-09 Sist oppdatert: 2011-09-06bibliografisk kontrollert

Open Access i DiVA

Fulltekst mangler i DiVA

Andre lenker

Forlagets fulltekst

Personposter BETA

Uhlén, Mathias

Søk i DiVA

Av forfatter/redaktør
Gharizadeh, BabackKäller, MaxNyrén, PålAndersson,, Anders F.Uhlén, MathiasLundeberg, JoakimAhmadian, Afshin
Av organisasjonen
I samme tidsskrift
Nucleic Acids Research

Søk utenfor DiVA

GoogleGoogle Scholar

doi
urn-nbn

Altmetric

doi
urn-nbn
Totalt: 121 treff
RefereraExporteraLink to record
Permanent link

Direct link
Referera
Referensformat
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Annet format
Fler format
Språk
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Annet språk
Fler språk
Utmatningsformat
  • html
  • text
  • asciidoc
  • rtf