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International audience; The REvolutionary Approaches and Devices for Nucleic Acid analysis (READNA) project received 12 million s funding under the European Union Framework Programme 7 from 1st June 2008 to 30th November 2012. The 19 project partners from both academia and industry from in total 7 countries had a project budget of 16 Ms with which they have discovered, created and developed a huge body of insights into nucleic acid analysis. Results have been presented widely in publications and in innumerous public presentations. Results have been moved to spin-offs such as the Olink enrichment kits (now sold by Agilent as Haloplex) and are findingtheir way to the market, such as the Oxford Nanopore MinIon sequencer that was first released to early-access user sites in 2014.

Active microbes likely have larger impact on gut health status compared to inactive or dormant microbes. We investigate the composition of active and total mucosal microbiota of treatment-naïve ulcerative colitis (UC) patients to determine the microbial picture at the start-up phase of disease, using both a 16S rRNA transcript and gene amplicon sequencing. DNA and RNA were isolated from the same mucosal colonic biopsies. Our aim was to identify active microbial members of the microbiota in early stages of disease and reveal which members are present, but do not act as major players. We demonstrated differences in active and total microbiota of UC patients when comparing inflamed to non-inflamed tissue. Several taxa, among them the Proteobacteria phyla and families therein, revealed lower transcriptional activity despite a high presence. The Bifidobacteriaceae family of the Actinobacteria phylum showed lower abundance in the active microbiota, although no difference in presence was detected. The most abundant microbiota members of the inflamed tissue in UC patients were not the most active. Knowledge of active members of microbiota in UC patients could enhance our understanding of disease etiology. The active microbial community composition did not deviate from the total when comparing UC patients to non-IBD controls.

Epigenetic alterations may provide important insights into gene-environment interaction in inflammatory bowel disease (IBD). Here we observe epigenome-wide DNA methylation differences in 240 newly-diagnosed IBD cases and 190 controls. These include 439 differentially methylated positions (DMPs) and 5 differentially methylated regions (DMRs), which we study in detail using whole genome bisulphite sequencing. We replicate the top DMP (RPS6KA2) and DMRs (VMP1, ITGB2 and TXK) in an independent cohort. Using paired genetic and epigenetic data, we delineate methylation quantitative trait loci; VMP1/microRNA-21 methylation associates with two polymorphisms in linkage disequilibrium with a known IBD susceptibility variant. Separated cell data shows that IBD-associated hypermethylation within the TXK promoter region negatively correlates with gene expression in whole-blood and CD8+ T cells, but not other cell types. Thus, site-specific DNA methylation changes in IBD relate to underlying genotype and associate with cell-specific alteration in gene expression., Epigenetic perturbations may be an important factor in diseases where both genes and environment play a role. Here, Ventham and colleagues show that DNA methylation changes in inflammatory bowel disease are related to the underlying genotype, and are associated with cell-specific changes to gene expression.

Improved methods are needed for in situ characterization of post-translational modifications in cell lines and tissues. For example, it is desirable to monitor the phosphorylation status of individual receptor tyrosine kinases in samples from human tumors treated with inhibitors to evaluate therapeutic responses. Unfortunately the leading methods for observing the dynamics of tissue post-translational modifications in situ, immunohistochemistry and immunofluorescence, exhibit limited sensitivity and selectivity. Proximity ligation assay is a novel method that offers improved selectivity through the requirement of dual recognition and increased sensitivity by including DNA amplification as a component of detection of the target molecule. Here we therefore established a generalized in situ proximity ligation assay to investigate phosphorylation of platelet-derived growth factor receptor beta (PDGFRbeta) in cells stimulated with platelet-derived growth factor BB. Antibodies specific for immunoglobulins from different species, modified by attachment of DNA strands, were used as secondary proximity probes together with a pair of primary antibodies from the corresponding species. Dual recognition of receptors and phosphorylated sites by the primary antibodies in combination with the secondary proximity probes was used to generate circular DNA strands; this was followed by signal amplification by replicating the DNA circles via rolling circle amplification. We detected tyrosine phosphorylated PDGFRbeta in human embryonic kidney cells stably overexpressing human influenza hemagglutinin-tagged human PDGFRbeta in porcine aortic endothelial cells transfected with the beta-receptor, but not in cells transfected with the alpha-receptor, and also in immortalized human foreskin fibroblasts, BJ hTert, endogenously expressing the PDGFRbeta. We furthermore visualized tyrosine phosphorylated PDGFRbeta in tissue sections from fresh frozen human scar tissue undergoing wound healing. The method should be of great value to study signal transduction, screen for effects of pharmacological agents, and enhance the diagnostic potential in histopathology.

Cellular processes can only be understood as the dynamic interplay of molecules. There is a need for techniques to monitor interactions of endogenous proteins directly in individual cells and tissues to reveal the cellular and molecular architecture and its responses to perturbations. Here we report our adaptation of the recently developed proximity ligation method to examine the subcellular localization of protein-protein interactions at single-molecule resolution. Proximity probes-oligonucleotides attached to antibodies against the two target proteins-guided the formation of circular DNA strands when bound in close proximity. The DNA circles in turn served as templates for localized rolling-circle amplification (RCA), allowing individual interacting pairs of protein molecules to be visualized and counted in human cell lines and clinical specimens. We used this method to show specific regulation of protein-protein interactions between endogenous Myc and Max oncogenic transcription factors in response to interferon-gamma (IFN-gamma) signaling and low-molecular-weight inhibitors.

During the past two years, significant breakthroughs have been achieved in genetic analyses through the application of technologies based on analytical DNA-circularization reactions. Padlock probes and molecular inversion probes have enabled parallel, high-throughput single nucleotide polymorphism (SNP) genotyping at increased scales, whereas, at the other end of the analysis spectrum, DNA molecules in individual cells have been genotyped, in situ, using padlock probes and rolling-circle amplification (RCA). This review describes the recent developments in the technologies that use specific DNA circularization, coupled to DNA amplification through PCR or rolling-circle amplification, and addresses the great potential of these tools.

Procedures and reagents are needed to specifically detect all the macromolecules that are being identified in the course of genome projects. We discuss how this challenge may be met using a set of ligation-based reagents termed padlock probes and proximity ligation probes. These probes include elements with affinity for specific nucleic acid and protein molecules, respectively, along with unique identifier DNA sequence elements that encode the identity of the recognized target molecules. The information content of DNA strands that form in the detection reactions are recorded after amplification, allowing the recognized target molecules to be identified. The procedures permit highly specific solution-phase or localized analyses of large sets of target molecules as required in future molecular analyses.

We present a tightly controlled process for strand-specific amplification of circularized DNA molecules. Tandem repeated complements of DNA circles are generated by rolling-circle replication, and converted to monomer circles of opposite polarity to that of the starting material. These circles are then subjected to one more round of rolling-circle replication and circularization, and the process can be further repeated. The method can be directed to produce single-stranded circular or linear monomers, or linear concatemers of the desired polarity. The reaction is not product inhibited, and can yield ≈100-fold higher concentrations of monomer products than PCR. Each generation of the amplification process proceeds in a linear fashion, ensuring precise quantification. The procedure is suitable for parallel amplification of large numbers of DNA circles, because the few cycles and the robust reaction mechanism preserves the proportion of amplified molecules. We demonstrate the utility of the method for multiplexed genotyping of polymorphic loci and for quantitative DNA analysis.

Highly specific high-throughput assays will be required to take full advantage of the accumulating information about the macromolecular composition of cells and tissues, in order to characterize biological systems in health and disease. We discuss the general problem of detection specificity and present the approach our group has taken, involving the reformatting of analogue biological information to digital reporter segments of genetic information via a series of DNA ligation assays. The assays enable extensive, coordinated analyses of the numbers and locations of genes, transcripts and protein.

Padlock probes are molecular tools that combine highly specific target sequence recognition with the potential for multiplexed analysis of large sets of target DNA or RNA sequences. In this brief review, we exemplify the ability of these probes to distinguish single-nucleotide target sequence variants. We further discuss means to detect the location of target sequences in situ, and to amplify reacted padlock probes via rolling-circle replication, as well as to sort reaction products on tag-arrays. We argue that the probes have the potential to render high-throughput genetic analyses precise and affordable.

Background A pelvic examination is the most common procedure in gynecological practice. A majority of women have negative experiences of such examinations. The aim of the present study was to explore attitudes to and experiences of pelvic examinations, as well as possible background factors to such attitudes and experiences. Methods A postal inquiry was sent to 788 randomly selected Swedish women, of fertile age. Sixty-seven per cent answered the questionnaire, which had 56 items and covered, inter alia, attitudes to and experiences of pelvic examinations, as well as possible background factors. Results The women had positive, uniform attitudes to pelvic examinations in general, but negative experiences of the specific parts of the procedure. Women's attitudes to and experiences of pelvic examinations correlated. The experience of the first pelvic examination was more negative than the experience of the last. A negative experience in general and the experience of pain during the first pelvic examination correlated. The first pelvic examination emerged as a statistically powerful background factor for subsequent attitudes to pelvic examinations. Conclusions Swedish women have positive attitudes to pelvic examination in spite of negative previous experiences. A powerful background factor for subsequent attitudes to pelvic examination was the experience of the first one. A woman's first pelvic examination should therefore be used as an opportunity to condition positive emotions and behaviors to the examination situation, as a basis for future positive experiences.

The heterogeneous nature of cancer results in highly variable therapeutic responses even among patients with identical stages and grades of a malignancy. The move towards personalised medicine in cancer therapy has therefore been motivated by a need to customise therapy according to molecular features of individual tumours. Companion diagnostics serves to support early drug development, it can provide surrogate markers in clinical trials, and also guide selection of individual therapies and monitoring of responses in routine clinical care. The era of companion diagnostics can be said to have begun with the introduction of the HercepTest - a first-of-a-kind diagnostic tool developed by DakoCytomation in 1998 to select patients for therapy with the anticancer drug Herceptin (trastuzumab). Herceptin and the paired test proved that companion diagnostics can help guide patient-tailored therapies. We will discuss herein technologies to analyse companion diagnostics markers at the level of DNA, RNA or protein, focusing on a series of methods developed in our laboratory that can facilitate drug development and help stratify patients for therapy.

Proximity Extension Assay technology identifies novel serum biomarkers for predicting Inflammatory Bowel Disease : IBD Character Consortium

Western blotting is a powerful and widely used method, but limitations in detection sensitivity and specificity, and dependence upon high quality antibodies to detect targeted proteins, are hurdles to overcome. The in situ proximity ligation assay, based on dual antibody recognition and powerful localized signal amplification, offers increased detection sensitivity and specificity, along with an ability to identify complex targets such as phosphorylated or interacting proteins. Here we have applied the in situ proximity ligation assay mechanism in Western blotting. This combination allowed the use of isothermal rolling circle amplification of DNA molecules formed in target-specific ligation reaction, for 16-fold or greater increase in detection sensitivity. The increased specificity because of dual antibody recognition ensured highly selective assays, detecting the specific band when combinations of two cross-reactive antitubulin antibodies were used (i.e. both producing distinct nonspecific bands in traditional Western blotting). We also demonstrated detection of phosphorylated platelet-derived growth factor receptor β by proximity ligation with one antibody directed against the receptor and another directed against the phosphorylated tyrosine residue. This avoided the need for stripping and re-probing the membrane or aligning two separate traditional blots. We demonstrate that the high-performance in situ proximity ligation-based Western blotting described herein is compatible with detection via enhanced chemiluminescence and fluorescence detection systems, and can thus be readily employed in any laboratory.

The existence of G protein-coupled receptor (GPCR) dimers and/or oligomers has been demonstrated in heterologous systems using a variety of biochemical and biophysical assays. While these interactions are the subject of intense research because of their potential role in modulating signaling and altering pharmacology, evidence for the existence of receptor interactions in vivo is still elusive because of a lack of appropriate methods to detect them. Here, we adapted and optimized a proximity ligation assay (PLA) for the detection in brain slices of molecular proximity of two antigens located on either the same or two different GPCRs. Using this approach, we were able to confirm the existence of dopamine D2 and adenosine A2A receptor complexes in the striatum of mice ex vivo.

The activity of proteins is typically regulated by secondary modifications and by interactions with other partners, resulting in the formation of protein complexes whose functions depend on the participating proteins. Accordingly, it is of central importance to monitor the presence of interaction complexes as well as their localization, thus providing information about the types of cells where the proteins are located and in what sub-cellular compartment these interactions occur. Several methods for visualizing protein interactions in situ have been developed during the last decade. These methods in most cases involve genetic constructs, and they have been successfully used in assays of living cell maintained in tissue culture, but they cannot easily be implemented in studies of clinical specimens. For such samples, affinity reagents like antibodies can be used to target the interacting proteins. In this review we will describe the in situ proximity ligation assays (in situ PLA), a method that is suitable for visualizing protein interactions in both tissue sections and in vitro cell lines, and we discuss research tasks when this or other method may be selected.

Knowledge about the total human genome sequence now provides opportunities to study its myriad gene products. However, the presence of alternative splicing, post-translational modifications, and innumerable protein-protein interactions among proteins occurring at widely different concentrations, all combine to place extreme demands on the specificity and sensitivity of assays. The choice of method also depends on matters such as whether proteins will be analyzed in body fluids and lysates, or localized inside single cells. In this review we discuss commonly used detection methods and compare these to the recently-developed proximity ligation technique.

Introduction Advances in protein profiling using proximity extension assays (PEA) have transformed our ability to compare concentrations of multiple proteins across biological samples. PEA utilises the specificity of antibody proximity and the sensitivity of polymerase chain reaction (qPCR) to detect proteins of interest. As part of the EC funded IBD Character initiative to discover biomarkers for clinical use using multiomic technologies, we performed high-throughput prospective case-control serum profiling to identify protein biomarkers that can predict Inflammatory Bowel Disease (IBD). Method Utilising Proseek Multiplex panels (Olink Bioscience, Sweden), serum profiling was performed in patients with a new diagnosis of IBD. Our control group consisted of symptomatic individuals. Phenotypic data was captured including age, sex, diagnosis and IBD medications. Statistical analysis was performed using R. Data were normalised and then batch corrected using ComBAT. Linear models were created for each protein including age and sex as covariates. After quality control, data from 186 proteins was available for analysis. Results A total of 245 patient serum samples (n = 153) newly diagnosed IBD, n = 92 symptomatic controls) from 4 IBD centres from Norway, United Kingdom and Sweden were included in the study from December 2012 to June 2014. 70 had Crohn’s disease (CD), 70 ulcerative colitis (UC) and 13 Inflammatory Bowel Disease Unclassified (IBDU). The mean age of the entire cohort was 33 years (range 0–79 years) and 54% were female. Multivariable analysis identified a set of 48 protein markers that were significantly associated with IBD. The 5 most significant protein markers were MMP-12 (Holm-adjusted p = 3.3 × 10 –13 ), OSM (p = 2.4 × 10 –12 ), CXCL9 (p = 1.7 × 10 –9 ), MMP10 (p = 1.7 × 10 –9 ) and EGFR (p = 1.8 × 10 –9 ). Of these five, all except EGFR were upregulated in IBD. The top 2 markers, MMP-12 and OSM were able to discriminate IBD from controls with an area under the receiver operator characteristics curve of 0.81 and 0.75 respectively. Using linear discriminant analysis, a combined biomarker consisting of MMP-12 and OSM was able to discriminate IBD from controls with a sensitivity and specificity of 80% and 72% respectively. Conclusion We have identified serum biomarkers that can predict IBD. These data demonstrate a potential for a PEA multiplex technology in IBD diagnostics and its ability to identify novel proteins that may be relevant in disease pathogenesis. Disclosure of interest R. Kalla Grant/Research Support from: IBD Character, N. Kennedy Grant/Research Support from: Wellcome Trust, Conflict with: Abbvie, MSD, Warner Chilcott, Ferring speaker fees, F. Hjelm: None Declared, E. Modig: None Declared, M. Sundell: None Declared, J. Soderholm: None Declared, B. Andreassen: None Declared, D. Bergemalm: None Declared, N. Ventham Grant/Research Support from: IBD BIOM, H. Hjortswang: None Declared, R. Petr: None Declared, M. Vatn: None Declared, J. Halfvarson: None Declared, M. Gullberg: None Declared, J. Satsangi Grant/Research Support from: EC Grants, Wellcome, CSO, MRC, Consultant for: Takeda, Conflict with: MSD speaker fees.

Knowledge about the total human genome sequence now provides opportunities to study its myriad gene products. However, the presence of alternative splicing, post-translational modifications, and innumerable protein-protein interactions among proteins occurring at widely different concentrations, all combine to place extreme demands on the specificity and sensitivity of assays. The choice of method also depends on matters such as whether proteins will be analyzed in body fluids and lysates, or localized inside single cells. In this review we discuss commonly used detection methods and compare these to the recently-developed proximity ligation technique.

We present a method to specifically select large sets of DNA sequences for parallel amplification by PCR using target-specific oligonucleotide constructs, so-called selectors. The selectors are oligonucleotide duplexes with single-stranded target-complementary end-sequences that are linked by a general sequence motif. In the selection process, a pool of selectors is combined with denatured restriction digested DNA. Each selector hybridizes to its respective target, forming individual circular complexes that are covalently closed by enzymatic ligation. Non-circularized fragments are removed by exonucleolysis, enriching for the selected fragments. The general sequence that is introduced into the circularized fragments allows them to be amplified in parallel using a universal primer pair. The procedure avoids amplification artifacts associated with conventional multiplex PCR where two primers are used for each target, thereby reducing the number of amplification reactions needed for investigating large sets of DNA sequences. We demonstrate the specificity, reproducibility and flexibility of this process by performing a 96-plex amplification of an arbitrary set of specific DNA sequences, followed by hybridization to a cDNA microarray. Eighty-nine percent of the selectors generated PCR products that hybridized to the expected positions on the array, while little or no amplification artifacts were observed.

Efficient and precise detection techniques, along with extensive repertoires of specific binding reagents, will be needed to meet the challenges of proteome analyses. The recently established proximity ligation mechanism enables sensitive high-capacity protein measurements by converting the detection of specific proteins to the analysis of DNA sequences. Proximity probes containing oligonucleotide extensions are designed to bind pairwise to target proteins and to form amplifiable tag sequences by ligation when brought in proximity. In our previous report, both the ligatable arms and the protein binders were DNA molecules. We now generalize the method by providing simple and convenient protocols to convert any polyclonal antibodies or matched pair of monoclonal antibodies to proximity probe sets through the attachment of oligonucleotide sequences. Sufficient reagent for >100,000 proximity ligation assays can be prepared from 1 μg of antibody. The technique is applied to measure cytokines in a homogenous test format with femtomolar detection sensitivities in 1-μl samples, and we exemplify its utility in situations when only minute sample amounts are available.

This communication describes the development of a controlled microwave methodology for rapid milliliter-scale PCR.

Publisher Summary This chapter discusses that with the completion of the Human Genome Project, all macromolecules encoded in human genomes are accessible for analysis and novel molecular procedures and sets of specific reagents are needed. A fundamental challenge of in situ analyses concerns the dual requirement for extremely high specificity and sensitivity to detect most biomolecules. Some molecular procedures can bring the required dual-recognition to localized detection of either target nucleic acid or protein molecules. These strategies are padlock probes as dual-recognition in situ probes; proximity probes for detection of proteins; and signal amplification through rolling-circle replication. The combination of highly specific DNA-, RNA-, or protein-dependent probe ligation by padlock and proximity probes with amplification of ligation products by rolling-circle replication (RCR) constitutes an integral series of reaction steps that preserve specificity and discourage the appearance of nonspecific signals. New in situ analysis methods will offer radical means of investigating the many ways by which the genome orchestrates cellular functions. A new generation of dual-specificity in situ probing mechanisms, resulting in the formation of amplifiable reaction products, promises to permit highly specific detection of even single target DNA, RNA, or protein molecules in situ, in individual samples, or in arrays of samples. For diagnostic purposes, the ability to monitor all macromolecules and their interactions will expand the scope from mere measurement of markers of disease to analyses of their functions and their correlation to disease.

Highly specific and sensitive procedures will be required to evaluate proteomes. Proximity ligation is a recently introduced mechanism for protein analysis. In this technique, the convergence of sets of protein-binding reagents on individual target molecules juxtaposes attached nucleic acid sequences. Through a ligation reaction a DNA reporter sequence is created, which can be amplified. The procedure thus encodes detected proteins as specific nucleic acid sequences in what may be viewed as a reverse translation reaction.

The advent of in vitro DNA amplification has enabled rapid acquisition of genomic information. We present here an analogous technique for protein detection, in which the coordinated and proximal binding of a target protein by two DNA aptamers promotes ligation of oligonucleotides linked to each aptamer affinity probe. The ligation of two such proximity probes gives rise to an amplifiable DNA sequence that reflects the identity and amount of the target protein. This proximity ligation assay detects zeptomole (40 x 10(-21) mol) amounts of the cytokine platelet-derived growth factor (PDGF) without washes or separations, and the mechanism can be generalized to other forms of protein analysis.

Duolink® offers a user-friendly solution for studying protein-protein interactions. Duolink utilizes a pair of antibodies that are capable of quantitatively reporting even weak and transient protein-protein interactions in natively expressing cells either as countable, bright fluorescent spots for a standard fluorescence microscope or via chromogenic detection for brightfield microscopes. It offers all the benefits of traditional coimmunoprecipitation (Co-IP) and western blotting but with better quantitative precision and more information about cell-to-cell variability and target localization. It is also amenable to high throughput in 96 or 384 wells.

Olink Bioscience has developed the Duolink® assay, which allows precise detection and quantification of proteins, protein interactions and modifications in fixed cells and tissue samples in their correct cellular context at physiologically relevant expression levels. This tool may ultimately be used in conjunction with new-generation anti-cancer therapies for inhibiting specific receptor dimerizations.

Olink Bioscience has developed a protein detection assay that allows precise detection and quantification of proteins, protein interactions and modifications in fixed cells and tissue samples. Duolink® immunoassay reagents are based on in situ proximity ligation assays (PLA™), and the assay reports proteins or protein complexes as countable distinct fluorescent or chromogenic spots visualized by standard microscopy. This allows for robust validation of protein-interaction data to show an interaction in the correct cellular context at physiologically relevant expression levels.

Olink Bioscience has developed a new protein detection assay that allows precise detection and quantification of proteins, protein interactions and protein modifications in fixed cells and tissue samples. The Duolink® immunoassay reagents are based on the in situ proximity ligation assay, capable of reporting proteins as countable distinct fluorescent or chromogenic spots visualized by standard microscopy. The assay exhibits a very high specificity for detection of phosphorylated proteins owing to a dual recognition format.

We describe a method to monitor rolling-circle replication of circular oligonucleotides in dual-color and in real-time using molecular beacons. The method can be used to study the kinetics of the polymerization reaction and to amplify and quantify circularized oligonucleotide probes in a rolling-circle amplification (RCA) reaction. Modified molecular beacons were made of 2′-O-Me-RNA to prevent 3′ exonucleolytic degradation by the polymerase used. Moreover, the complement of one of the stem sequences of the molecular beacon was included in the RCA products to avoid fluorescence quenching due to inter-molecular hybridization of neighboring molecular beacons hybridizing to the concatemeric polymerization product. The method allows highly accurate quantification of circularized DNA over a broad concentration range by relating the signal from the test DNA circle to an internal reference DNA circle reporting in a distinct fluorescence color.