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Genotyping and Mutation Detection In Situ: Development and application of single-molecule techniques
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. (Molecular Diagnostics)
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The human body is composed of trillions of cells closely working together to maintain a functional organism. Every cell is unique in molecular composition and can acquire genetic variations that might cause it to turn pathological. It is essential to develop improved tools to better understand the development of normal and disease tissue, ideally enabling single-cell expression studies in preserved context of complex tissue with single-nucleotide resolution. This thesis presents the development and application of a new in situ method for localized detection and genotyping of individual transcripts directly in cells and tissues. The described technique utilizes padlock probes and target-primed rolling circle amplification and is highly suitable for sensitive in situ analysis.

First, a new strategy for directed cleavage of single stranded DNA was investigated, e.g. nucleic acid targets with extended 3´ ends, for successful initiation of rolling circle amplification. The presented cleavage strategy is simple and applicable for subsequent enzymatic reactions, e.g. ligation and polymerization. Specific cleavage of long target overhangs was demonstrated in synthetic oligonucleotides and in genomic DNA and the detection efficiency was substantially increased.

For multiplex detection and genotyping of individual transcripts in single cells, a new in situ method was developed. The technique showed a satisfactorily detection efficiency and was later applied as a general mutation analysis tool for detection of KRAS point mutations in complex tumor tissue sections, e.g. formalin-fixed, paraffin-embedded tumor tissues and cytologic tumor imprints. Mutation status was assessed in patient samples by in situ padlock probe detection and results were confirmed by DNA-sequencing.  Finally, the method was adapted for simultaneous detection of individual mRNA molecules and endogenous protein modifications in single cells using padlock probes and in situ PLA. This assay will be useful for gene expression analysis and exploration of new drugs with vague effector sites.

To our knowledge, no other technique exists today that offers in situ transcript detection with single-nucleotide resolution in heterogeneous tissues. The method will especially be suitable for discrimination of highly similar transcripts, e.g. splice variants, SNPs and point mutations, within gene expression studies and for cancer diagnostics.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2011. , 59 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 656
Keyword [en]
padlock probes, in situ, rolling circle amplification, mRNA, genotyping, mutation detection, cancer, tissue sections, diagnostics, single-molecule, single-cell, microscopy
National Category
Medical Genetics
Research subject
Molecular Medicine
Identifiers
URN: urn:nbn:se:uu:diva-149776ISBN: 978-91-554-8034-9OAI: oai:DiVA.org:uu-149776DiVA: diva2:405873
Public defence
2011-05-06, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2011-04-14 Created: 2011-03-23 Last updated: 2011-05-05Bibliographically approved
List of papers
1. Glycosylases and AP-cleaving enzymes as a general tool for probe-directed cleavage of ssDNA targets
Open this publication in new window or tab >>Glycosylases and AP-cleaving enzymes as a general tool for probe-directed cleavage of ssDNA targets
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2010 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 38, no 7, e99- p.Article in journal (Refereed) Published
Abstract [en]

The current arsenal of molecular tools for site-directed cleavage of single-stranded DNA (ssDNA) is limited. Here, we describe a method for targeted DNA cleavage that requires only the presence of an A nucleotide at the target position. The procedure involves hybridization of a complementary oligonucleotide probe to the target sequence. The probe is designed to create a deliberate G:A mismatch at the desired position of cleavage. The DNA repair enzyme MutY glycosylase recognizes the mismatch structure and selectively removes the mispaired A from the duplex to create an abasic site in the target strand. Addition of an AP-endonuclease, such as Endonuclease IV, subsequently cleaves the backbone dividing the DNA strand into two fragments. With an appropriate choice of an AP-cleaving enzyme, the 3'- and 5'-ends of the cleaved DNA are suitable to take part in subsequent enzymatic reactions such as priming for polymerization or joining by DNA ligation. We define suitable standard reaction conditions for glycosylase/AP-cleaving enzyme (G/AP) cleavage, and demonstrate the use of the method in an improved scheme for in situ detection using target-primed rolling-circle amplification of padlock probes.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-136603 (URN)10.1093/nar/gkp1238 (DOI)000276744600007 ()
Available from: 2010-12-13 Created: 2010-12-13 Last updated: 2011-05-05Bibliographically approved
2. In situ detection and genotyping of individual mRNA molecules
Open this publication in new window or tab >>In situ detection and genotyping of individual mRNA molecules
2010 (English)In: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 7, no 5, 395-397 p.Article in journal (Refereed) Published
Abstract [en]

Increasing knowledge about the heterogeneity of mRNA expression within cell populations highlights the need to study transcripts at the level of single cells. We present a method for detection and genotyping of individual transcripts based on padlock probes and in situ target-primed rolling-circle amplification. We detect a somatic point mutation, differentiate between members of a gene family and perform multiplex detection of transcripts in human and mouse cells and tissue.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-137786 (URN)10.1038/NMETH.1448 (DOI)000277175100023 ()20383134 (PubMedID)
Note

De 2 första författarna delar förstaförfattarskapet.

Available from: 2010-12-15 Created: 2010-12-15 Last updated: 2013-03-14Bibliographically approved
3. Diagnostic mutation testing in situ in routine FFPE tissue sections for treatment prediction in clinical oncology
Open this publication in new window or tab >>Diagnostic mutation testing in situ in routine FFPE tissue sections for treatment prediction in clinical oncology
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Activating mutations in the KRAS gene are present in different cancer types and are strongly associated with resistance to epidermal growth factor receptor (EGFR) inhibitor therapy. Hence there is a requirement for sensitive KRAS mutation analysis to determine the most suitable treatment for the patients. Also, little is known about the impact of tumor heterogeneity with regard to KRAS mutation status in different sub-clones during tumorigenesis, and if this is important for treatment response and prognosis. To improve the diagnostic accuracy, we developed an RNA-based genotyping assay that targets KRAS-mutations in codon 12 and 13 in situ on tissue samples by the use of multiple mutation specific padlock probes and rolling-circle amplification. Thus, the distribution of wild-type (green rolling-circle products) and mutated (red rolling-circle products) KRAS alleles can be determined for single cancer cells in different parts of a heterogeneous tumor without the use of microdissection. We demonstrate reliable detection of KRAS point mutations on cytologic tumor imprints as well as on fresh frozen and formalin-fixed paraffin-embedded tissue sections from colorectal and lung cancer. This in situ method offers single cell mutation detection for diagnostics and holds great promise as a tool to investigate the role of oncogenic mutations in complex tumor tissues.

Keyword
mutation, KRAS, padlock probes, rolling circle amplification, in situ, FFPE, touch imprints, cancer, diagnostics
National Category
Medical Genetics Medical Genetics Cancer and Oncology
Research subject
Molecular Medicine; Oncology
Identifiers
urn:nbn:se:uu:diva-149822 (URN)
Available from: 2011-03-24 Created: 2011-03-24 Last updated: 2011-05-05
4. Simultaneous visualization of both signaling cascade activity and end-point gene expression in single cells
Open this publication in new window or tab >>Simultaneous visualization of both signaling cascade activity and end-point gene expression in single cells
2011 (English)In: PLoS ONE, ISSN 1932-6203, Vol. 6, no 5, e20148- p.Article in journal (Refereed) Published
Abstract [en]

We have developed an approach for simultaneous detection of individual endogenous protein modifications and mRNA molecules in single cells in situ. For this purpose we combined two methods previously developed in our lab: in situ proximity ligation assay for the detection of individual protein interactions and -modifications and in situ detection of single mRNA molecules using padlock probes. As proof-of-principle, we demonstrated the utility of the method for simultaneous detection of phosphorylated PDGFRβ and DUSP6/MKP-3 mRNA molecules in individual human fibroblasts upon PDGF-BB stimulation. Further we applied drugs disrupting the PDGFRβ signaling pathway at various sites to show that this combined method can concurrently monitor the molecular effect of the drugs, i.e. inhibition of downstream signaling from the targeted node in the signaling pathway. Due to its ability to detect different types of molecules in single cells in situ the method presented here can contribute to a deeper understanding of cell-to-cell variations and can be applied to e.g. pinpoint effector sites of drugs in a signaling pathway.

Keyword
in situ PLA, padlock probe, rolling circle amplification, single molecule, single cell
National Category
Medical Genetics
Research subject
Molecular Medicine
Identifiers
urn:nbn:se:uu:diva-149877 (URN)10.1371/journal.pone.0020148 (DOI)000291006500029 ()21647446 (PubMedID)
Note

De 2 första författarna delar förstaförfattarskapet.

Available from: 2011-03-24 Created: 2011-03-24 Last updated: 2013-03-08Bibliographically approved

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