scholarly journals Highly specific imaging of mRNA in single cells by target RNA-initiated rolling circle amplification

2017 ◽  
Vol 8 (5) ◽  
pp. 3668-3675 ◽  
Author(s):  
Ruijie Deng ◽  
Kaixiang Zhang ◽  
Yupeng Sun ◽  
Xiaojun Ren ◽  
Jinghong Li
Keyword(s):  
Single Molecule ◽  
Single Cells ◽  
Rolling Circle Amplification ◽  
Robust Method ◽  
Single Nucleotide ◽  
Rolling Circle ◽  
Target Rna

We report a robust method for the efficient imaging of mRNA with single-nucleotide and near-single-molecule resolution in single cells.

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

TECHNOLOGY ◽  
2013 ◽  
Vol 01 (01) ◽  
pp. 88-96 ◽  
Author(s):  
Tania Konry ◽  
Adam Lerner ◽  
Martin L. Yarmush ◽  
Irina V. Smolina
Keyword(s):  
Single Cells ◽  
Rolling Circle Amplification ◽  
Copy Number Variations ◽  
New Method ◽  
Quantitative Detection ◽  
Single Nucleotide ◽  
Rolling Circle ◽  
Single Base ◽  
Target Dna ◽  
Single Base Resolution

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.

Toehold-initiated Rolling Circle Amplification for Visualizing Individual MicroRNAs In Situ in Single Cells

2014 ◽  
Vol 126 (9) ◽  
pp. 2421-2425 ◽  
Author(s):  
Ruijie Deng ◽  
Longhua Tang ◽  
Qianqian Tian ◽  
Ying Wang ◽  
Lei Lin ◽  
...  
Keyword(s):  
Single Cells ◽  
Rolling Circle Amplification ◽  
Rolling Circle

scholarly journals Clonal rolling circle amplification for on-chip DNA cluster generation

2017 ◽  
Vol 2 (1) ◽  
Author(s):  
Christian Korfhage ◽  
Evelyn Fricke ◽  
Andreas Meier ◽  
Andreas Geipel ◽  
Mark Baltes ◽  
...  
Keyword(s):  
Single Molecule ◽  
Rolling Circle Amplification ◽  
Nucleic Acid Detection ◽  
Rolling Circle ◽  
Oligonucleotide Hybridization ◽  
Dna Strands ◽  
Multiple Copies ◽  
Cluster Generation ◽  
On Chip ◽  
Micrometer Size

Abstract Generation of monoclonal DNA clusters on a surface is a useful method for digital nucleic acid detection applications (e.g. microarray or next-generation sequencing). To obtain sufficient copies per cluster for digital detection, the single molecule bound to the surface must be amplified. Here we describe ClonalRCA, a rolling-circle amplification (RCA) method for the generation of monoclonal DNA clusters based on forward and reverse primers immobilized on the surface. No primer in the reaction buffer is needed. Clusters formed by ClonalRCA comprise forward and reverse strands in multiple copies tethered to the surface within a cluster of micrometer size. Single stranded circular molecules are used as a target to create a cluster with about 10 000 forward and reverse strands. The DNA strands are available for oligonucleotide hybridization, primer extension and sequencing.

Wide diversity of PAX5 alterations in B-ALL: a Groupe Francophone de Cytogénétique Hématologique study

Blood ◽  
2010 ◽  
Vol 115 (15) ◽  
pp. 3089-3097 ◽  
Author(s):  
Etienne Coyaud ◽  
Stephanie Struski ◽  
Nais Prade ◽  
Julien Familiades ◽  
Ruth Eichner ◽  
...  
Keyword(s):  
Lymphoblastic Leukemia ◽  
Rolling Circle Amplification ◽  
High Variability ◽  
Comparative Genomic ◽  
Fusion Genes ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Rolling Circle ◽  
Rapid Amplification ◽  
Innovative Techniques

Abstract PAX5 is the main target of somatic mutations in acute B lymphoblastic leukemia (B-ALL). We analyzed 153 adult and child B-ALL harboring karyotypic abnormalities at chromosome 9p, to determine the frequency and the nature of PAX5 alterations. We found PAX5 internal rearrangements in 21% of the cases. To isolate fusion partners, we used classic and innovative techniques (rolling circle amplification-rapid amplification of cDNA ends) and single nucleotide polymorphism-comparative genomic hybridization arrays. Recurrent and novel fusion partners were identified, including NCoR1, DACH2, GOLGA6, and TAOK1 genes showing the high variability of the partners. We noted that half the fusion genes can give rise to truncated PAX5 proteins. Furthermore, malignant cells carrying PAX5 fusion genes displayed a simple karyotype. These data strongly suggest that PAX5 fusion genes are early players in leukemogenesis. In addition, PAX5 deletion was observed in 60% of B-ALL with 9p alterations. Contrary to cases with PAX5 fusions, deletions were associated with complex karyotypes and common recurrent translocations. This supports the hypothesis of the secondary nature of the deletion. Our data shed more light on the high variability of PAX5 alterations in B-ALL. Therefore, it is probable that gene fusions occur early, whereas deletions should be regarded as a late/secondary event.

scholarly journals Rolling circle amplification-driven encoding of different fluorescent molecules for simultaneous detection of multiple DNA repair enzymes at the single-molecule level

2020 ◽  
Vol 11 (22) ◽  
pp. 5724-5734
Author(s):  
Chen-chen Li ◽  
Hui-yan Chen ◽  
Juan Hu ◽  
Chun-yang Zhang
Keyword(s):  
Dna Repair ◽  
Single Molecule ◽  
Rolling Circle Amplification ◽  
Simultaneous Detection ◽  
Single Molecule Detection ◽  
Dna Repair Enzymes ◽  
Rolling Circle ◽  
Single Molecule Level ◽  
Repair Enzymes ◽  
Fluorescent Molecules

Integration of single-molecule detection with rolling circle amplification-driven encoding of different fluorescent molecules enables simultaneous detection of multiple DNA repair enzymes.

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