Fluorescence Imaging of Single-Copy DNA Sequences within the Human Genome Using PNA-Directed Padlock Probe Assembly

Yaroslavsky, Anastasia; Smolina, Irina

doi:10.1016/j.chembiol.2013.02.012

Cited by 28 publications

(26 citation statements)

References 30 publications

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“…Moreover, our preliminary data indicate that, to form the PD-loop, the PNA openers can be as short as hexamers. If they again can be separated by up to 10 bp of an arbitrary sequence, then we expect to have one such site per 200 bp, on average 20 . PNA openers have already found applications in several diagnostics based on fluorescence detection 44,45 .…”

Section: Discussionmentioning

confidence: 99%

“…Cells were fixed for 15 minutes on ice and rinsed three times in a sodium-phosphate buffer (pH 6.8). Then a standard protocol used earlier for in situ detection has been applied with slight modification 20 . Specifically, at each step cells were spin down and supernatant with excess of probes and reagents was removed.…”

Section: Methodsmentioning

confidence: 99%

“…Previously, we showed that the PNA-RCA approach can be used for fluorescent in situ detection of short single-copy DNA sequences within the bacteria 18,19 and more recently in human cells 20 . Here we demonstrate that this approach can be used in a cells-in-flow format where we are able to detect single base changes.…”

Section: Introductionmentioning

confidence: 99%

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Target DNA detection and quantitation on a single cell with single base resolution

et al. 2013

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In this report, we present a new method for sensitive detection of short DNA sites in single cells with single base resolution. The method combines peptide nucleic acid (PNA) openers as the tagging probes, together with isothermal rolling circle amplification (RCA) and fluorescence-based detection, all performed in a cells-in-flow format. Bis-PNAs provide single base resolution, while RCA ensures linear signal amplification. We applied this method to detect the oncoviral DNA inserts in cancer cell lines using a flow-cytometry system. We also demonstrated quantitative detection of the selected signature sites within single cells in microfluidic nano-liter droplets. Our results show single-nucleotide polymorphism (SNP) discrimination and detection of copy-number variations (CNV) under isothermal non-denaturing conditions. This new method is ideal for many applications in which ultra-sensitive DNA characterization with single base resolution is desired on the level of single cells.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Target DNA detection and quantitation on a single cell with single base resolution

et al. 2013

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“…23 Padlock probes can distinguish single-base sequence differences, 22,24 which is not possible with the conventional FISH or in situ PCR. Moreover, in situ sequencing was also developed by combining padlock probes with nextgeneration sequencing methods.…”

Section: ·3 Operationmentioning

confidence: 99%

Microdevice in Cellular Pathology: Microfluidic Platforms for Fluorescence in situ Hybridization and Analysis of Circulating Tumor Cells

Sato

2015

ANAL. SCI.

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867 IntroductionCellular pathology techniques have progressed in recent years. Conventionally, the pathologist observes tissues with a microscope and determines whether or not the hematoxylin and eosin (H&E)-stained tissues contain cancer cells based on their morphology. The H&E stain is the traditional and most widely used staining method in medical diagnoses. 1 Recent advances in genetic analysis methods have made it possible to identify the genetic basis of human diseases, and have opened the door to individualized prevention strategies and early detection and treatment.Therefore, combinations of the traditional morphological diagnosis method and newer genetic methodologies are strongly welcomed.Fluorescence in situ hybridization (FISH) is a well-known gene-based method used to image genetic abnormalities. FISH is a microscopic technique in which specific DNA sequences tagged with fluorophores are used to detect target genes and identify their localization within a cell. FISH was developed in the early 1980s 2 and has since been improved continuously. 3 The FISH probes can only detect genes with a high degree of homology, and can visualize specific cytogenetic abnormalities such as copy number aberrations, gene mutations, and gene fusions. 4 This advanced molecular pathology technique has enabled better diagnoses of diseases, leading to more tailored therapeutic regimens.Analytes have become more multifaceted. Microfluidic devices enable the miniaturization, integration, automation, and parallelization of chemical and biochemical processes. This new technology also provides opportunity for expansion in the field of cellular pathology. Fluorescence in situ hybridization (FISH) is a well-known gene-based method to image genetic abnormalities. Development of a FISH microfluidic platform has offered the possibility of automation with significant time and cost reductions, which overcomes many drawbacks of the current protocols. Microfluidic devices are also powerful tools for single-cell analysis. Capturing the circulating tumor cells (CTCs) from blood samples is one of the most promising approaches to enable the early diagnosis of cancer. The microfluidic devices are also useful to isolate rare CTCs at high efficiency and purity. In this review, I outline recent FISH and CTC analyses using microfluidic devices, and describe their applications for the cellular diagnosis of cancers.

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“…After the binding of the PNA openers, the padlock probe efficiently hybridized the target sequence. In combination with PNA openers, padlock probes could detect a single-copy target sequence on human metaphase chromosomes by FISH (Yaroslavsky and Smolina 2013). Efficient FISH with padlock probes will contribute to cytogenetical analyses of gene repositioning of individual gene-loci in nuclei and discriminate nucleotide variation at different loci on metaphase chromosomes.…”

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confidence: 99%

FISH with Padlock Probes Can Efficiently Reveal the Genomic Position of Low or Single-Copy DNA Sequences

Matsunaga

2017

CYTOLOGIA

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A padlock probe is a circularized oligonucleotide containing complementary sequences of target regions. Padlock probes combined with rolling-circle amplification enable fluorescence in situ hybridization (FISH) to reproducibly detect the genomic position of low or single-copy DNA sequences. The FISH with padlock probes revealed that a plant gene cluster of tandemly arrayed three paralogues responsible for photosynthesis rapidly moved from the interior subnuclear region to the peripheral subnuclear region in response to light. Using a pair of peptide nucleic acids as a double-strand opener for the target region, the efficiency of FISH with padlock probes at the detection of a single-copy genomic region was much improved. FISH with padlock probes will give us reliable data of dynamics, arrangement and position of a specific single locus in both the nucleus and the condensed chromosomes.

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Fluorescence Imaging of Single-Copy DNA Sequences within the Human Genome Using PNA-Directed Padlock Probe Assembly

Cited by 28 publications

References 30 publications

Target DNA detection and quantitation on a single cell with single base resolution

Target DNA detection and quantitation on a single cell with single base resolution

Microdevice in Cellular Pathology: Microfluidic Platforms for Fluorescence in situ Hybridization and Analysis of Circulating Tumor Cells

FISH with Padlock Probes Can Efficiently Reveal the Genomic Position of Low or Single-Copy DNA Sequences

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