-
[show abstract]
[hide abstract]
ABSTRACT: Padlock rolling circle amplification (RCA) is a powerful analytical method for ultrasensitive DNA detection. While there are some advantages to bead-based RCA, a detailed study of the relationship between the bead material and the efficiency of bead-based RCA has not been reported. Here, we compared the reaction efficiencies of bead-based RCA performed on 2 types of bead materials, agarose and polystyrene. Agarose was a more suitable material for on-bead RCA. The calibration curve showed linearity between 0.05 and 1 nM, and the limit of detection was 9 pM (9 amol) for Salmonella DNA determination.
Analytical Biochemistry 03/2013; · 3.00 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Analysis at the single-cell level is essential for the understanding of cellular responses in heterogeneous cell populations, but it has been difficult to perform because of the strict requirements put on detection methods with regard to selectivity and sensitivity (i.e., owing to the cross-reactivity of probes and limited signal amplification). Here we describe a 1.5-d protocol for enumerating and genotyping mRNA molecules in situ while simultaneously obtaining information on protein interactions or post-translational modifications; this is achieved by combining padlock probes with in situ proximity ligation assays (in situ PLA). In addition, we provide an example of how to design padlock probes and how to optimize staining conditions for fixed cells and tissue sections. Both padlock probes and in situ PLA provide the ability to directly visualize single molecules by standard microscopy in fixed cells or tissue sections, and these methods may thus be valuable for both research and diagnostic purposes.
Nature Protocol 01/2013; 8(2):355-72. · 8.36 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Control of the global epidemic tuberculosis is severely hampered by the emergence of drug-resistant Mycobacterium tuberculosis strains. Molecular methods offer a more rapid means of characterizing resistant strains than phenotypic drug susceptibility testing. We have developed a molecular method for detection of rifampicin-resistant M. tuberculosis based on padlock probes and magnetic nanobeads. Padlock probes were designed to target the most common mutations associated with rifampicin resistance in M. tuberculosis, i.e. at codons 516, 526 and 531 in the gene rpoB. For detection of the wild type sequence at all three codons simultaneously, a padlock probe and two gap-fill oligonucleotides were used in a novel assay configuration, requiring three ligation events for circularization. The assay also includes a probe for identification of the M. tuberculosis complex. Circularized probes were amplified by rolling circle amplification. Amplification products were coupled to oligonucleotide-conjugated magnetic nanobeads and detected by measuring the frequency-dependent magnetic response of the beads using a portable AC susceptometer.
PLoS ONE 01/2013; 8(4):e62015. · 4.09 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
New Biotechnology 05/2012; 29(6):634-40. · 2.76 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A magnetic-nanobead-based, substrate-free method for the sensitive detection of spores in an immunoassay format is presented. The method is shown to detect Bacillus globigii spores, the non-pathogenic simulant of Bacillus anthracis, with a limit-of-detection of 50 spores with a reaction time of 135 min. The study shows the versatility of magnetic nanobeads for detection of biological molecules other than DNA.
Small 04/2012; 8(14):2174-7. · 8.35 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A tumor does not consist of a homogenous population of cancer cells. Therefore, to understand cancer, the tumor microenvironment and the interplay between the different cell types present in the tumor has to be taken into account, and how this regulates the growth and survival of the cancer cells. To achieve a full picture of this complex interplay, analysis of tumor tissue should ideally be performed with cellular resolution, providing activity status of individual cells in this heterogeneous population of different cell-types. In addition, in situ analysis provides information on the architecture of the tissue wherein the cancer cells thrive, providing information of the identity of neighboring cells that can be used to understand cell-cell communication. Herein we describe how padlock probes and in situ PLA can be used for visualization of nucleic acids and protein activity, respectively, directly in tissue sections, and their potential future role in personalized medicine.
EPMA Journal, The 01/2012; 3(1):7.
-
Anton K Raap,
Roshan S Jahangir Tafrechi,
Frans M van de Rijke,
Angela Pyle,
Carolina Wählby,
Karoly Szuhai,
Raimond B G Ravelli,
René F M de Coo,
Harsha K Rajasimha, Mats Nilsson,
Patrick F Chinnery,
David C Samuels,
George M C Janssen
[show abstract]
[hide abstract]
ABSTRACT: Many pathogenic mitochondrial DNA mutations are heteroplasmic, with a mixture of mutated and wild-type mtDNA present within individual cells. The severity and extent of the clinical phenotype is largely due to the distribution of mutated molecules between cells in different tissues, but mechanisms underpinning segregation are not fully understood. To facilitate mtDNA segregation studies we developed assays that measure m.3243A>G point mutation loads directly in hundreds of individual cells to determine the mechanisms of segregation over time. In the first study of this size, we observed a number of discrete shifts in cellular heteroplasmy between periods of stable heteroplasmy. The observed patterns could not be parsimoniously explained by random mitotic drift of individual mtDNAs. Instead, a genetically metastable, heteroplasmic mtDNA segregation unit provides the likely explanation, where stable heteroplasmy is maintained through the faithful replication of segregating units with a fixed wild-type/m.3243A>G mutant ratio, and shifts occur through the temporary disruption and re-organization of the segregation units. While the nature of the physical equivalent of the segregation unit remains uncertain, the factors regulating its organization are of major importance for the pathogenesis of mtDNA diseases.
PLoS ONE 01/2012; 7(12):e52080. · 4.09 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We developed a molecular diagnostic method for detection of RNA virus based on padlock probes and colorimetric readout. The feasibility of our approach was demonstrated by using detection of Crimean-Congo hemorrhagic fever (CCHF) virus as a model. Compared with conventional PCR-based methods, our approach does not require advanced equipment, involves easier assay design, and has a sensitivity of 10(3) viral copies/ml. By using a cocktail of padlock probes, synthetic templates representing different viral strain variants could be detected. We analyzed 34 CCHF patient samples, and all patients were correctly diagnosed when the results were compared to those of the current real-time PCR method. This is the first time that highly specific padlock probes have been applied to detection of a highly variable target sequence typical of RNA viruses.
Journal of clinical microbiology 09/2011; 49(12):4279-85. · 4.16 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Here, the volume-amplified magnetic nanobead detection assay (VAM-NDA) is for the first time applied for detection of rolling circle amplified (RCA) DNA molecules in a portable, commercial AC susceptometer that operates at ambient temperatures and with an analysis time of about 20 min. The performance of the assay is investigated using three different magnetic nanobead sizes: 50, 130 and 250nm. The performance of the assay using the AC susceptometer is compared to the performance achieved using a superconducting quantum interference device (SQUID). It is found that the performance of the assay is comparable in the two setups with a quantitative detection limit of ∼4pM for all bead sizes under study. The findings show that the VAM-NDA holds promise for future wide-spread implementation in commercial AC susceptometer setups thus opening up for the possibility to perform magnetic bead-based DNA detection in point-of-care and outpatient settings.
Biosensors & bioelectronics 08/2011; 29(1):195-9. · 5.43 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The rolling circle amplification (RCA) is a versatile DNA amplification method in which a DNA molecule is amplified using a single DNA primer, allowing the product to be counted as a single dot. Circular templates for RCA can arise from padlock probes in highly specific DNA target-mediated ligation reactions. However, improvement of detection efficiency represents an important challenge. In homogeneous assays, the detection efficiency is generally only under 0.1%, mainly because the sample volume is too large compared with the detection volume. Here, we used microchannel surfaces in a glass microchip for DNA detection in small volume samples. First, DNA patterning on glass surfaces in microchannels was demonstrated using chemical surface patterning by UV light. By using a photochemical reaction, we realized DNA patterning in a closed space. Second, RCA was demonstrated using dilutions of target molecules, and a calibration curve was obtained. The highest detection efficiency was 22.5% by virtue of the reduced sample volumes from several hundred microliters to 5.0 nL. Accordingly, a countable number of DNA molecules was successfully detected. This method is suitable for analysis of very small volume samples such as single cells, especially by using extended-nanochannels with dimensions of 10-1000 nm.
Analytical Chemistry 04/2011; 83(9):3352-7. · 5.86 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Toxicology Letters 02/2011; 202(2):142-7. · 3.23 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
PLoS ONE 01/2011; 6(5):e20148. · 4.09 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: In 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.
By 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.
We 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.
Virology Journal 01/2011; 8:37. · 2.34 Impact Factor
-
H Johansson,
M Isaksson,
E Falk Sörqvist,
F Roos,
J Stenberg,
T Sjöblom,
J Botling,
P Micke,
K Edlund,
S Fredriksson,
H Göransson Kultima,
Olle Ericsson, Mats Nilsson
[show abstract]
[hide abstract]
ABSTRACT: Targeted genome enrichment is a powerful tool for making use of the massive throughput of novel DNA-sequencing instruments. We herein present a simple and scalable protocol for multiplex amplification of target regions based on the Selector technique. The updated version exhibits improved coverage and compatibility with next-generation-sequencing (NGS) library-construction procedures for shotgun sequencing with NGS platforms. To demonstrate the performance of the technique, all 501 exons from 28 genes frequently involved in cancer were enriched for and sequenced in specimens derived from cell lines and tumor biopsies. DNA from both fresh frozen and formalin-fixed paraffin-embedded biopsies were analyzed and 94% specificity and 98% coverage of the targeted region was achieved. Reproducibility between replicates was high (R(2) = 0, 98) and readily enabled detection of copy-number variations. The procedure can be carried out in <24 h and does not require any dedicated instrumentation.
Nucleic Acids Research 11/2010; 39(2):e8. · 8.03 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The present work provides the first real-space analysis of nanobead-DNA coil interactions. Immobilization of oligonucleotide-functionalized magnetic nanobeads in rolling circle amplified DNA-coils was studied by complex magnetization measurements and transmission electron microscopy (TEM), and a statistical analysis of the number of beads hybridized to the DNA-coils was performed. The average number of beads per DNA-coil using the results from both methods was found to be around 6 and slightly above 2 for samples with 40 and 130 nm beads, respectively. The TEM analysis supported an earlier hypothesis that 40 nm beads are preferably immobilized in the interior of DNA-coils whereas 130 nm beads, to a larger extent, are immobilized closer to the exterior of the coils. The methodology demonstrated in the present work should open up new possibilities for characterization of interactions of a large variety of functionalized nanoparticles with macromolecules, useful for gaining more fundamental understanding of such interactions as well as for optimizing a number of biosensor applications.
The Journal of Physical Chemistry B 10/2010; 114(41):13255-62. · 3.70 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: This work presents sensitive detection of bacterial genomic DNA using a magnetic nanoparticle-based substrate-free method. For the first time, such a method is employed for detection of a clinically relevant analyte by implementing a solid-phase-based molecular probing and amplification protocol that can be executed in 80 min. The molecular detection and amplification protocol is presented and verified on samples containing purified genomic DNA from Escherichia coli cells, showing that as few as 50 bacteria can be detected. This study moves the use of volume-amplified magnetic nanoparticles one step further toward rapid, sensitive, and selective infectious diagnostics.
Analytical Chemistry 10/2010; 82(22):9138-40. · 5.86 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Alternative splicing creates diverse mRNA isoforms from single genes and thereby enhances complexity of transcript structure and of gene function. We describe a method called spliceotyping, which translates combinatorial mRNA splicing patterns along transcripts into a library of binary strings of nucleic acid tags that encode the exon composition of individual mRNA molecules. The exon inclusion pattern of each analyzed transcript is thus represented as binary data, and the abundance of different splice variants is registered by counts of individual molecules. The technique is illustrated in a model experiment by analyzing the splicing patterns of the adenovirus early 1A gene and the beta actin reference transcript. The method permits many genes to be analyzed in parallel and it will be valuable for elucidating the complex effects of combinatorial splicing.
Nucleic Acids Research 09/2010; 38(16):e163. · 8.03 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The sensitive detection and quantification of DNA targets in the food industry and in environmental and clinical settings are issues of utmost importance in ensuring contamination-free food, monitoring the environment, and battling disease. Selective probes coupled with powerful amplification techniques are therefore of major interest. In this study, we set out to create an integrated microchemical chip that benefits from microfluidic chip technology in terms of sensitivity and a strong detection methodology provided jointly by padlock probes and rolling circle amplification (RCA). Here, we have integrated padlock probes and RCA into a microchip. The chip uses solid phase capture in a microchannel to enable washing cycles and decrease analytical area, and employs on-bead RCA for single-molecule amplification and detection. We investigated the effects of reagent concentration and amount of padlock probes, and demonstrated the feasibility of detecting Salmonella.
Lab on a Chip 05/2010; 10(10):1262-6. · 5.67 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Nature Methods 04/2010; 7(5):395-7. · 19.28 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Nucleic Acids Research 04/2010; 38(7):e99. · 8.03 Impact Factor
