In situ genotyping individual DNA molecules by target-primed rolling-circle amplification of padlock probes

Larsson, Chatarina; Koch, Jørn; Nygren, Anders O.H.; Janssen, George M.C.; Raap, Anton K.; Landegren, Ulf; Nilsson, Mats

doi:10.1038/nmeth723

Cited by 280 publications

(275 citation statements)

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“…There are many different methods for in situ detection of sub-cellular structures, e.g., immunofluorescence staining, in situ Proxim-ity Ligation Assay (in situ PLA) [1], FISH (fluorescent in situ hybridization) and padlock-probing [2]. Although the procedures for these methods differ, the images produced share many similarities.…”

Section: Introductionmentioning

confidence: 99%

BlobFinder, a tool for fluorescence microscopy image cytometry

Allalou

Wählby

2009

Computer Methods and Programs in Biomedicine

119

106

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Pages 8 c o m p u t e r m e t h o d s a n d p r o g r a m s i n b i o m e d i c i n e x x x ( 2Images can be acquired at high rates with modern fluorescence microscopy hardware, giving rise to a demand for high-speed analysis of image data. Digital image cytometry, i.e., automated measurements and extraction of quantitative data from images of cells, provides valuable information for many types of biomedical analysis. There exists a number of different image analysis software packages that can be programmed to perform a wide array of useful measurements. However, the multi-application capability often compromises the simplicity of the tool. Also, the gain in speed of analysis is often compromised by time spent learning complicated software. We provide a free software called BlobFinder that is intended for a limited type of application, making it easy to use, easy to learn and optimized for its particular task. BlobFinder can perform batch processing of image data and quantify as well as localize cells and point like source signals in fluorescence microscopy images, e.g., from FISH, in situ PLA and padlock probing, in a fast and easy way.

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Section: Introductionmentioning

confidence: 99%

BlobFinder, a tool for fluorescence microscopy image cytometry

Allalou

Wählby

2009

Computer Methods and Programs in Biomedicine

119

106

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“…Padlock/RCA was performed as previously described, 4 with minor modifications to adapt the procedure to the microchannel format. The reaction solutions (5 -60 μL) were injected into the microRCA system, and reactions were performed under stopped-flow conditions.…”

Section: Padlock/rcamentioning

confidence: 99%

“…1 The detection techniques currently used rely on fluorescence imaging; for instance, fluorescence in situ hybridization (FISH) 2 is a method that is widely used in the clinical field. Other similar detection techniques, such as, RNA scope 3 and in situ padlock probe/ rolling circle amplification (padlock/RCA) 4,5 have also been described.…”

Section: Introductionmentioning

confidence: 99%

“…7,8 In the presence of the target, the padlock probe circularizes via DNA ligation and acts as a template for RCA, which generates a long single-stranded DNA molecule; a probe conjugated with a fluorescent label can be visualized as a 1-μm-sized dot under a fluorescence microscope. This technique has been applied to the detection of specific point mutations in the mitochondrial genome of individual cells, 4 replication of porcine circovirus type 2 in cultured cells and infected tissues, 9 mRNA expression within a cell in combination with a reverse transcription reaction, 5,10 mRNA sequencing, 11 and protein localization in combination with an antigen-antibody reaction, also known as proximity ligation assay. 12 Although useful, in situ padlock/RCA has the drawback of being a labor-intensive, costly procedure.…”

Section: Introductionmentioning

confidence: 99%

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Microfluidics-based in situ Padlock/Rolling Circle Amplification System for Counting Single DNA Molecules in a Cell

et al. 2014

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“…Recently, Landegren and co-workers have introduced a highly specific in situ SNP genotyping method, which makes use of allele-specific ligation of single oligonucleotide ligation probes (padlock probes) and ROLLINGCIRCLE AMPLIFICATION (RCA) 38 . This approach can detect single nucleotide position differences between individual nucleic acids and it has been used to characterize genetic variation in mitochondrial genomes 38 .…”

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confidence: 99%

Miniaturization in functional genomics and proteomics

et al. 2005

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| Proteins are the key components of the cellular machinery responsible for processing changes that are ordered by genomic information. Analysis of most human proteins and nucleic acids is important in order to decode the complex networks that are likely to underlie many common diseases. Significant improvements in current technology are also required to dissect the regulatory processes in high-throughtput and with low cost. Miniaturization of biological assays is an important prerequisite to achieve these goals in the near future. SYSTEMS BIOLOGYThe study of the complex interactions that occur at all levels of biological information -from whole-genome sequence interactions to developmental and biochemical networks -and their functional relationship to organism-level phenotypes. NATURE REVIEWS | GENETICS VOLUME 6 | JUNE 2005 | 465 and concomitantly reducing the complexity of genomic DNA. Although PCR can be used to amplify small numbers of DNA molecules in low microlitre or nanolitre volumes in vitro, problems are associated with the dilution of heterozygous DNA. This could result in incorrect genotyping results (homozygous genotypes) because of the preferential amplification of one of the alleles. Extreme dilution indicates that there is a sufficiently high integrity of the nucleic-acid templates to allow efficient amplification. Furthermore, amplification of low concentrations of nucleic acids requires strict quality control, as it is often associated with contamination problems, which can be problematic in high-throughput applications. In contrast to nucleic-acid manipulation, it is currently impossible to amplify proteins from a biological sample. R E V I E W S Miniaturization in functional genomicsGenome research and functional genomics have catalysed the development of high-throughput and miniaturized approaches for the analysis of biomolecules [5][6][7][8] . Figure 1 | DNA micorarrays. DNA microarrays that are based on non-porous solid supports such as glass have paved the way for highly parallel miniaturized analysis of biomolecules. Using robotic workstations, ~0.25-1 nl of DNA solution are printed on a slide, creating spots that range from 100 to 150 µm in diameter. Successive samples are then spaced to avoid contact between adjacent spots, with approximately 200 µm between each spot 7 . Currently, about 50,000 cDNAs can be robotically spotted onto a microscope slide and hybridized with a fluorescently labelled probe. Using photolithographical masking techniques, arrays that have 400,000 distinct oligonucleotides have been produced, each in its own 20-µm 2 region (today, this can be as small as ~5 µm 2 ) REF. 8. After spotting, nucleic-acid samples can be hybridized on these pre-structured microarrays. Hybridization events are monitored using fluorescence scanners. A typical fluorescence read-out of highly parallel hybridization on a cDNA microarray is shown in the bottom panel. Box 1 | Technological problems in miniaturizationEvery molecular biological method can be miniaturized from microl...

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In situ genotyping individual DNA molecules by target-primed rolling-circle amplification of padlock probes

Cited by 280 publications

References 28 publications

BlobFinder, a tool for fluorescence microscopy image cytometry

BlobFinder, a tool for fluorescence microscopy image cytometry

Microfluidics-based in situ Padlock/Rolling Circle Amplification System for Counting Single DNA Molecules in a Cell

Miniaturization in functional genomics and proteomics

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