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Oligonucleotide gap-fill ligation for mutation detection and sequencing in situ
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Show others and affiliations
(English)Manuscript (preprint) (Other academic)
National Category
Biomedical Laboratory Science/Technology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-109696OAI: oai:DiVA.org:su-109696DiVA: diva2:766478
Available from: 2014-11-27 Created: 2014-11-27 Last updated: 2016-01-29Bibliographically approved
In thesis
1. In situ Sequencing: Methods for spatially-resolved transcriptome analysis
Open this publication in new window or tab >>In situ Sequencing: Methods for spatially-resolved transcriptome analysis
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

It is well known that cells in tissues display a large heterogeneity in gene expression due to differences in cell lineage origin and variation in the local environment at different sites in the tissue, a heterogeneity that is difficult to study by analyzing bulk RNA extracts from tissue. Recently, genome-wide transcriptome analysis technologies have enabled the analysis of this variation with single-cell resolution. In order to link the heterogeneity observed at molecular level with the morphological context of tissues, new methods are needed which achieve an additional level of information, such as spatial resolution.

In this thesis I describe the development and application of padlock probes and rolling circle amplification (RCA) as molecular tools for spatially-resolved transcriptome analysis. Padlock probes allow in situ detection of individual mRNA molecules with single nucleotide resolution, visualizing the molecular information directly in the cell and tissue context. Detection of clinically relevant point mutations in tumor samples is achieved by using padlock probes in situ, allowing visualization of intra-tumor heterogeneity. To resolve more complex gene expression patterns, we developed in situ sequencing of RCA products combining padlock probes and next-generation sequencing methods. We demonstrated the use of this new method by, for the first time, sequencing short stretches of transcript molecules directly in cells and tissue. By using in situ sequencing as read-out for multiplexed padlock probe assays, we measured the expression of tens of genes in hundreds of thousands of cells, including point mutations, fusions transcripts and gene expression level.

These molecular tools can complement genome-wide transcriptome analyses adding spatial resolution to the molecular information. This level of resolution is important for the understanding of many biological processes and potentially relevant for the clinical management of cancer patients.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2014. 49 p.
Keyword
Padlock probes, rolling circle amplification, in situ sequencing, spatially-resolved transcriptomics, molecular diagnostics
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-110057 (URN)978-91-7649-066-2 (ISBN)
Public defence
2015-01-23, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.

Available from: 2014-12-29 Created: 2014-12-05 Last updated: 2014-12-15Bibliographically approved
2. Padlock Probe-Based Assays for Molecular Diagnostics
Open this publication in new window or tab >>Padlock Probe-Based Assays for Molecular Diagnostics
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Treatment success often depends on the availability of accurate and reliable diagnostic assays to guide clinical practitioners in their treatment choices. An optimal test must excel in specificity and sensitivity, and depending on the application area time, low-cost and simplicity are equally important. For instance, time is essential in infectious diagnostics but this is less important in non-invasive prenatal testing (NIPT). In NIPT, specificity and sensitivity are the most important parameters.

In this thesis I describe the development of four different methods, all based on padlock probes and rolling circle amplification, intended for molecular diagnostics. Application areas range from infectious disease diagnostics to NIPT and oncology. The methods described have in common that they overcome certain limitations of currently available assays. This thesis includes two new assays targeting infectious agents: one assay specifically detecting a highly variable double stranded RNA virus and the second assay demonstrating a new format of antibiotic susceptibility testing, which is rapid and generally applicable to different pathogens. Furthermore, I describe the development of a method that uses methylation markers to enrich fetal DNA, accurately quantify chromosome ratios and thus, detecting trisomy 21 and 18. The fourth method described in this thesis uses gap-fill ligation of padlock probes to detect diagnostic relevant point mutations with high specificity in situ.

The assays presented have the potential, after automation and successful validation and verification studies, to be implemented into clinical practice. Furthermore, these assays demonstrate the wide applicability of padlock probes which, due to their properties in regard to specificity and multiplexity, are useful tools for nucleic acid detection in vitro as well as in situ.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2015. 53 p.
Keyword
Padlock probes; rolling circle amplification; molecular diagnostics; in situ mutation detection
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-116214 (URN)978-91-7649-155-3 (ISBN)
Public defence
2015-06-12, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.

Available from: 2015-05-21 Created: 2015-04-14 Last updated: 2015-07-08Bibliographically approved

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