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Sökning: onr:"swepub:oai:DiVA.org:uu-183141" > Detection and Seque...

Detection and Sequencing of Amplified Single Molecules

Ke, Rongqin (författare)
Uppsala universitet,Molekylära verktyg,Mats Nilsson
Nilsson, Mats, Professor (preses)
Uppsala universitet,Institutionen för immunologi, genetik och patologi
Landegren, Ulf (preses)
Uppsala universitet,Institutionen för immunologi, genetik och patologi
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Drmanac, Radoje (opponent)
Complete Genomics
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 (creator_code:org_t)
ISBN 9789155485085
Uppsala : Acta Universitatis Upsaliensis, 2012
Engelska 48s.
Serie: Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, 1651-6206 ; 829
  • Doktorsavhandling (övrigt vetenskapligt)
Abstract Ämnesord
Stäng  
  • Improved analytical methods provide new opportunities for both biological research and medical applications. This thesis describes several novel molecular techniques for nucleic acid and protein analysis based on detection or sequencing of amplified single molecules (ASMs). ASMs were generated from padlock probe assay and proximity ligation assay (PLA) through a series of molecular processes.In Paper I, a simple colorimetric readout strategy for detection of ASMs generated from padlock probe assay was used for highly sensitive detection of RNA virus, showing the potential of using padlock probes in the point-of-care diagnostics. In Paper II, digital quantification of ASMs, which were generated from padlock probe assay and PLA through circle-to-circle amplification (C2CA), was used for rapid and sensitive detection of nucleic acids and proteins, aiming for applications in biodefense. In Paper III, digital quantification of ASMs that were generated from PLA without C2CA was shown to be able to improve the precision and sensitivity of PLA when compared to the conventional real-time PCR readout. In Paper IV, a non-optical approach for detection of ASMs generated from PLA was used for sensitive detection of bacterial spores. ASMs were detected through sensing oligonucleotide-functionalized magnetic nanobeads that were trapped within them.Finally, based on in situ sequencing of ASMs generated via padlock probe assay, a novel method that enabled sequencing of individual mRNA molecules in their natural context was established and presented in Paper V. Highly multiplex detection of mRNA molecules was also achieved based on in situ sequencing. In situ sequencing allows studies of mRNA molecules from different aspects that cannot be accessed by current in situ hybridization techniques, providing possibilities for discovery of new information from the complexity of transcriptome. Therefore, it has a great potential to become a useful tool for gene expression research and disease diagnostics.

Ämnesord

MEDICAL AND HEALTH SCIENCES  -- Medical Biotechnology -- Biomedical Laboratory Science/Technology (hsv//eng)
MEDICIN OCH HÄLSOVETENSKAP  -- Medicinsk bioteknologi -- Biomedicinsk laboratorievetenskap/teknologi (hsv//swe)
NATURAL SCIENCES  -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)
NATURVETENSKAP  -- Biologiska vetenskaper -- Biokemi och molekylärbiologi (hsv//swe)
NATURVETENSKAP  -- Biologi (hsv//swe)
MEDICIN OCH HÄLSOVETENSKAP  -- Medicinsk bioteknologi (hsv//swe)
NATURAL SCIENCES  -- Biological Sciences (hsv//eng)
MEDICAL AND HEALTH SCIENCES  -- Medical Biotechnology (hsv//eng)

Nyckelord

padlock probes
proximity ligation assay
rolling circle amplification
circle-to-circle amplification
single molecule detection
amplified single molecules
sequencing
in situ
single cell
Molecular Biotechnology
Molekylär bioteknik

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