Sara Henriksson

Twelve-gel slide format optimised for comet assay and fluorescent in situ hybridisation

The comet assay is widely used to measure DNA damage and repair in basic research, genotoxicity testing and human biomonitoring. The conventional format has 1 or 2 gels on a microscope slide, 1 sample per slide. To increase throughput, we have designed and tested a system with 12 smaller gels on one slide, allowing incubation of individual gels with different reagents or enzymes. Thus several times more samples can be analysed with one electrophoresis run, and fewer cells and smaller volumes of test solutions are required. Applications of the modified method include treatment with genotoxic agents at different concentrations; simultaneous analysis of different lesions using a range of enzymes; analysis of cell extracts for DNA repair activity; and fluorescent in situ hybridisation (FISH) to comet DNA with specific labelled probes.

Development of an in situ assay for simultaneous detection of the genomic and replicative form of PCV2 using padlock probes and rolling circle amplification

BackgroundIn this study we utilized padlock probes and rolling circle amplification as a mean to detect and study the replication of porcine circovirus type 2 (PCV2) in cultured cells and in infected tissue. Porcine circovirus type 2 is a single-stranded circular DNA virus associated with several severe diseases, porcine circovirus diseases (PCVD) in pigs, such as postweaning multisystemic wasting syndrome. The exact reason and mechanisms behind the trigger of PCV2 replication that is associated with these diseases is not well-known. The virus replicates with rolling circle replication and thus also exists as a double-stranded replicative form.ResultsBy applying padlock probes and rolling circle amplification we could not only visualise the viral genome but also discriminate between the genomic and the replicative strand in situ. The genomic strand existed in higher numbers than the replicative strand. The virus accumulated in certain nuclei but also spread into the cytoplasm of cells in the surrounding tissue. In cultured cells the average number of signals increased with time after infection.ConclusionsWe have developed a method for detection of both strands of PCV2 in situ that can be useful for studies of replication and in situ detection of PCV2 as well as of DNA viruses in general.

In situ rolling circle amplification detection of Crimean Congo hemorrhagic fever virus (CCHFV) complementary and viral RNA

Crimean Congo hemorrhagic fever virus (CCHFV) is a human pathogen that causes a severe disease with high fatality rate for which there is currently no specific treatment. Knowledge regarding its replication cycle is also highly limited. In this study we developed an in situ technique for studying the different stages during the replication of CCHFV. By integrating reverse transcription, padlock probes, and rolling circle amplification, we were able to detect and differentiate between viral RNA (vRNA) and complementary RNA (cRNA) molecules, and to detect viral protein within the same cell. These data demonstrate that CCHFV nucleocapsid protein (NP) is detectable already at 6 hours post infection in vRNA- and cRNA-positive cells. Confocal microscopy showed that cRNA is enriched and co-localized to a large extent with NP in the perinuclear area, while vRNA has a more random distribution in the cytoplasm with only some co-localize with NP. However, vRNA and cRNA did not appear to co-localize directly.

Study of gene-specific DNA repair in the comet assay with padlock probes and rolling circle amplification

We used padlock probes to study the rate of gene specific repair of three genes, OGG1 (8-oxoguanine-DNA glycosylase-1), XPD (xeroderma pigmentosum group D), and HPRT (hypoxanthine-guanine phosphoribosyltransferase) in human lymphocytes, in relation to the repair rate of Alu repeats and total genomic DNA. Padlock probes offer highly specific detection of short target sequences by combining detection by ligation and signal amplification. In this approach only genes in sequences containing strand breaks, which become single-stranded in the tail, are available for hybridisation. Thus the total number of signals from the padlock probes per comet gives a direct measure of the amount of damage (strand-breaks) present and allows the repair process to be monitored. This method could provide insights on the organisation of genomic DNA in the comet tail. Alu repeat containing DNA was repaired rapidly in comparison with total genomic DNA, and the studied genes were generally repaired more rapidly than the Alu repeats.

Finding cells, finding molecules, finding patterns

Many modern molecular labelling techniques result in bright point signals. Signals from molecules that are detected directly inside a cell can be captured by fluorescence microscopy. Signals repres ...

Padlock Probes and Rolling Circle Amplification for Detection of Repeats and Single-Copy Genes in the Single-Cell Comet Assay

Padlock probes and rolling circle amplification are techniques which can be used for detection of DNA sequences in situ with high specificity and high signal to noise. The single-cell gel electrophoresis assay is used to measure DNA damage and repair in cells. Here, we describe how padlock probes and rolling circle amplification can be used to detect DNA sequences within comet preparations.

Application of Padlock and Selector Probes in Molecular Medicine

Abstract Padlock and selector probes are versatile tools that have been used and developed as great alternatives to molecular methods for the detection of nucleic acid sequences in the context of medicine. Their application has been proven to be useful in different areas from targeted sequencing, genotyping, and molecular diagnostics to the development of novel biosensors and techniques. This chapter describes the molecular functioning of these probes, in combination with DNA ligase-assisted specific circularization and isothermal rolling circle amplification, their properties, and their main applications in the field of medicine to date. It shows the potential of these approaches for developing assays that can be integrated into assays or instruments that will enable and improve access to information valuable for molecular medicine research and for improving medical diagnostics.

Chapter 10 – Application of Padlock and Selector Probes in Molecular Medicine

Padlock and selector probes are versatile tools that have been used and developed as great alternatives to molecular methods for the detection of nucleic acid sequences in the context of medicine. Their application has been proven to be useful in different areas from targeted sequencing, genotyping, and molecular diagnostics to the development of novel biosensors and techniques. This chapter describes the molecular functioning of these probes, in combination with DNA ligase-assisted specific circularization and isothermal rolling circle amplification, their properties, and their main applications in the field of medicine to date. It shows the potential of these approaches for developing assays that can be integrated into assays or instruments that will enable and improve access to information valuable for molecular medicine research and for improving medical diagnostics.