scholarly journals Droplet-digital Cas13a assay enables direct single-molecule microRNA quantification

2019 ◽  
Author(s):  
Tian Tian ◽  
Bowen Shu ◽  
Lei Liu ◽  
Xiaoming Zhou
Keyword(s):  
Cell Lines ◽  
Single Molecule ◽  
Absolute Quantification ◽  
Single Nucleotide ◽  
Single Molecule Level ◽  
Broad Applicability ◽  
Precise Quantification ◽  
Nucleotide Specificity ◽  
Direct Quantification

The direct quantification of microRNA at the single-molecule level is still challenging. Herein, we developed a droplet-digital Cas13a assay (ddCA) as a general approach for the amplification-free and absolute quantification of single unlabeled miRNA molecules with single-nucleotide specificity. We demonstrate its simplicity, precise quantification capability, excellent specificity and broad applicability by analyzing microRNAs from synthetic and cell lines-derived materials.

scholarly journals Single-molecule detection of deoxyribonucleoside triphosphates in microdroplets

2019 ◽  
Vol 47 (17) ◽  
pp. e101-e101 ◽  
Author(s):  
Boris Breiner ◽  
Kerr Johnson ◽  
Magdalena Stolarek ◽  
Ana-Luisa Silva ◽  
Aurel Negrea ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Single Molecule ◽  
Dna Polymerases ◽  
Single Molecule Detection ◽  
Synthesis Methods ◽  
Single Nucleotide ◽  
New Approach ◽  
Fluorescent Signal ◽  
Single Molecule Level ◽  
Current Sequence

AbstractA new approach to single-molecule DNA sequencing in which dNTPs, released by pyrophosphorolysis from the strand to be sequenced, are captured in microdroplets and read directly could have substantial advantages over current sequence-by-synthesis methods; however, there is no existing method sensitive enough to detect a single nucleotide in a microdroplet. We have developed a method for dNTP detection based on an enzymatic two-stage reaction which produces a robust fluorescent signal that is easy to detect and process. By taking advantage of the inherent specificity of DNA polymerases and ligases, coupled with volume restriction in microdroplets, this method allows us to simultaneously detect the presence of and distinguish between, the four natural dNTPs at the single-molecule level, with negligible cross-talk.

scholarly journals Bottom-up single-molecule strategy for understanding subunit function of tetrameric β-galactosidase

2018 ◽  
Vol 115 (33) ◽  
pp. 8346-8351 ◽  
Author(s):  
Xiang Li ◽  
Yu Jiang ◽  
Shaorong Chong ◽  
David R. Walt
Keyword(s):  
Single Molecule ◽  
Wild Type ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Single Molecule Level ◽  
Subunit Function ◽  
Enzyme Molecules ◽  
Individual Enzyme ◽  
Kinetics Of

In this paper, we report an example of the engineered expression of tetrameric β-galactosidase (β-gal) containing varying numbers of active monomers. Specifically, by combining wild-type and single-nucleotide polymorphism plasmids at varying ratios, tetrameric β-gal was expressed in vitro with one to four active monomers. The kinetics of individual enzyme molecules revealed four distinct populations, corresponding to the number of active monomers in the enzyme. Using single-molecule-level enzyme kinetics, we were able to measure an accurate in vitro mistranslation frequency (5.8 × 10−4 per base). In addition, we studied the kinetics of the mistranslated β-gal at the single-molecule level.

scholarly journals Dynamic single-molecule counting for the quantification and optimization of nanoparticle functionalization protocols

Nanoscale ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 4128-4136 ◽  
Author(s):  
Matěj Horáček ◽  
Dion J. Engels ◽  
Peter Zijlstra
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Medical Applications ◽  
Single Molecule Level ◽  
Microscopy Method ◽  
Super Resolution Microscopy ◽  
Direct Quantification

We provide a super-resolution microscopy method to characterize the chemical interface of nanoparticles at the single-molecule level. This provides a direct quantification and optimization of functionalization protocols for bio-medical applications.

scholarly journals Minimal detection and low biological fluctuation of mitochondrial CpG methylation at the single-molecule level

2020 ◽  
Author(s):  
Chloe Goldsmith ◽  
Jesús Rafael Rodríguez-Aguilera ◽  
Ines El-Rifai ◽  
Adrien Jarretier ◽  
Valérie Hervieu ◽  
...  
Keyword(s):  
Cell Lines ◽  
Single Molecule ◽  
Nuclear Dna ◽  
Mitochondrial Genes ◽  
Cpg Methylation ◽  
Nanopore Sequencing ◽  
Biological Processes ◽  
Single Molecule Level ◽  
Long Read ◽  
Low Levels

AbstractCytosine DNA methylation in the CpG context (5mCpG) is associated with the transcriptional status of nuclear DNA. Due to technical limitations, it has been less clear if mitochondrial DNA (mtDNA) is methylated and whether 5mCpG has a regulatory role in this context. The main aim of this work was to develop and validate a novel tool for determining methylation of mtDNA and to corroborate its existence across different biological contexts. Using long-read nanopore sequencing we found low levels of CpG methylation (with few exceptions) and little variation across biological processes: differentiation, oxidative stress, and cancer. 5mCpG was overall higher in tissues compared to cell lines, with small additional variation between cell lines of different origin. Although we do show several significant changes in all these conditions, 5mCpG is unlikely to play a major role in defining the transcriptional status of mitochondrial genes.

scholarly journals Comparison of Multiple Displacement Amplification (MDA) and Multiple Annealing and Looping-Based Amplification Cycles (MALBAC) in Limited DNA Sequencing Based on Tube and Droplet

Micromachines ◽  
2020 ◽  
Vol 11 (7) ◽  
pp. 645
Author(s):  
Xiaoxiang Zhou ◽  
Ying Xu ◽  
Libo Zhu ◽  
Zhen Su ◽  
Xiaoming Han ◽  
...  
Keyword(s):  
Single Molecule ◽  
Multiple Displacement Amplification ◽  
Genomic Alteration ◽  
Whole Genome ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Single Molecule Level ◽  
Amplification Bias ◽  
Advantages And Disadvantages ◽  
Droplet Method

Whole genome amplification (WGA) is crucial for whole genome sequencing to investigate complex genomic alteration at the single-cell or even single-molecule level. Multiple displacement amplification (MDA) and multiple annealing and looping based amplification cycles (MALBAC) are two most widely applied WGA methods, which have different advantages and disadvantages, dependent on research objectives. Herein, we compared the MDA and MALBAC to provide more information on their performance in droplets and tubes. We observed that the droplet method could dramatically reduce the amplification bias and retain the high accuracy of replication than the conventional tube method. Furthermore, the droplet method exhibited higher efficiency and sensitivity for both homozygous and heterozygous single nucleotide variants (SNVs) at the low sequencing depth. In addition, we also found that MALBAC offered a greater uniformity and reproducibility and MDA showed a better efficiency of genomic coverage and SNV detection. Our results provided insights that will allow future decision making.

scholarly journals Low biological fluctuation of mitochondrial CpG and non-CpG methylation at the single-molecule level

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Chloe Goldsmith ◽  
Jesús Rafael Rodríguez-Aguilera ◽  
Ines El-Rifai ◽  
Adrien Jarretier-Yuste ◽  
Valérie Hervieu ◽  
...  
Keyword(s):  
Cell Lines ◽  
Single Molecule ◽  
Nuclear Dna ◽  
Pcr Amplification ◽  
Nanopore Sequencing ◽  
High Coverage ◽  
Single Molecule Level ◽  
Modified Dna ◽  
Long Read ◽  
Low Levels

AbstractMammalian cytosine DNA methylation (5mC) is associated with the integrity of the genome and the transcriptional status of nuclear DNA. Due to technical limitations, it has been less clear if mitochondrial DNA (mtDNA) is methylated and whether 5mC has a regulatory role in this context. Here, we used bisulfite-independent single-molecule sequencing of native human and mouse DNA to study mitochondrial 5mC across different biological conditions. We first validated the ability of long-read nanopore sequencing to detect 5mC in CpG (5mCpG) and non-CpG (5mCpH) context in nuclear DNA at expected genomic locations (i.e. promoters, gene bodies, enhancers, and cell type-specific transcription factor binding sites). Next, using high coverage nanopore sequencing we found low levels of mtDNA CpG and CpH methylation (with several exceptions) and little variation across biological processes: differentiation, oxidative stress, and cancer. 5mCpG and 5mCpH were overall higher in tissues compared to cell lines, with small additional variation between cell lines of different origin. Despite general low levels, global and single-base differences were found in cancer tissues compared to their adjacent counterparts, in particular for 5mCpG. In conclusion, nanopore sequencing is a useful tool for the detection of modified DNA bases on mitochondria that avoid the biases introduced by bisulfite and PCR amplification. Enhanced nanopore basecalling models will provide further resolution on the small size effects detected here, as well as rule out the presence of other DNA modifications such as oxidized forms of 5mC.

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