Sample Preparation for Single-Molecule Enzyme Assays

2012 ◽  
pp. 323-337
Author(s):  
D.B. Craig ◽  
E.R. Nichols
Keyword(s):  
Sample Preparation ◽  
Single Molecule ◽  
Enzyme Assays

scholarly journals Sample preparation method to improve the efficiency of high-throughput single-molecule force spectroscopy

2019 ◽  
Vol 5 (4) ◽  
pp. 176-183 ◽  
Author(s):  
Lei Jin ◽  
Li Kou ◽  
Yanan Zeng ◽  
Chunguang Hu ◽  
Xiaodong Hu
Keyword(s):  
Sample Preparation ◽  
High Throughput ◽  
Single Molecule ◽  
Preparation Method ◽  
Force Spectroscopy ◽  
Experimental Result ◽  
Sample Preparation Method ◽  
Dna Stretching ◽  
Single Molecule Force Spectroscopy ◽  
Preparation Methods

Abstract Inefficient sample preparation methods hinder the performance of high-throughput single-molecule force spectroscopy (H-SMFS) for viscous damping among reactants and unstable linkage. Here, we demonstrated a sample preparation method for H-SMFS systems to achieve a higher ratio of effective target molecules per sample cell by gas-phase silanization and reactant hydrophobization. Digital holographic centrifugal force microscopy (DH-CFM) was used to verify its performance. The experimental result indicated that the DNA stretching success ratio was improved from 0.89% to 13.5%. This enhanced efficiency preparation method has potential application for force-based DNA stretching experiments and other modifying procedures.

scholarly journals Single molecule targeted sequencing for cancer gene mutation detection

2015 ◽  
Author(s):  
Yan Gao ◽  
Liwei Deng ◽  
Qin Yan ◽  
Yongqian Gao ◽  
Zengding Wu ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Gene Mutation ◽  
Single Molecule ◽  
Mutation Detection ◽  
Hla Typing ◽  
Targeted Sequencing ◽  
Rapid Decline ◽  
Cancer Gene ◽  
Cancer Genes ◽  
Targeted Capture

With the rapid decline cost of sequencing, it is now clinically affordable to examine multiple genes in a single disease-targeted test using next generation sequencing. Current targeted sequencing methods require a separate step of targeted capture enrichment during sample preparation before sequencing, and the library preparation process is labor intensive and time consuming. Here, we introduced an amplification-free Single Molecule Targeted Sequencing (SMTS) technology, which combined targeted capture and sequencing in one step. We demonstrated that this technology can detect low-frequency mutations of cancer genes. SMTS has several advantages, namely that it requires little sample preparation and avoids biases and errors introduced by PCR reaction. This technology can be applied in cancer gene mutation detection, inherited condition screening and high-resolution human leukocyte antigen (HLA) typing.

scholarly journals RT-LAMP assay for ultra-sensitive detection of SARS-CoV-2 in saliva and VTM clinical samples

2020 ◽  
Author(s):  
A. Ganguli ◽  
A. Mostafa ◽  
J. Berger ◽  
S. A. Stewart de Ramirez ◽  
A. Baltaji ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Single Molecule ◽  
State Of The Art ◽  
Rna Extraction ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Sensitive Detection ◽  
Clinical Samples ◽  
Final Reaction ◽  
Better Than

AbstractThe COVID-19 pandemic has underscored the shortcomings in the deployment of state-of-the-art diagnostic platforms. Although several PCR-based techniques have been rapidly developed to meet the growing testing needs, such techniques often need samples collected through a swab, the use of RNA extraction kits, and expensive thermocyclers in order to successfully perform the test. Isothermal amplification-based approaches have also been recently demonstrated for rapid SARS-CoV-2 detection by minimizing sample preparation while also reducing the instrumentation and reaction complexity. There are limited reports of saliva as the sample source and some of these indicate inferior sensitivity when comparing RT-LAMP with PCR-based techniques. In this paper, we demonstrate an improved sensitivity assay to test saliva using a 2-step RT-LAMP assay, where a short 10-minute RT step is performed with only B3 and BIP primers before the final reaction. We show that while the 1-step RT-LAMP demonstrate satisfactory results, the optimized 2-step approach allows for single molecule sensitivity per reaction and performs significantly better than the 1-step RT-LAMP and conventional 2-step RT-LAMP approaches with all primers included in the RT Step. Importantly, we demonstrate RNA extraction-free RT-LAMP based assays for detection of SARS-CoV-2 from VTM and saliva clinical samples.

scholarly journals Sample preparation and imaging conditions affect mEos3.2 photophysics in fission yeast cells

2020 ◽  
Author(s):  
Mengyuan Sun ◽  
Kevin Hu ◽  
Joerg Bewersdorf ◽  
Thomas D. Pollard
Keyword(s):  
Sample Preparation ◽  
Yeast Cell ◽  
Single Molecule ◽  
Fluorescent Proteins ◽  
Yeast Cells ◽  
Live Cells ◽  
Fluorescence Signal ◽  
Wide Range ◽  
Imaging Conditions ◽  
In Cells

AbstractPhotoconvertible fluorescent proteins (PCFPs) are widely used in super-resolution microscopy and studies of cellular dynamics. However, our understanding of their photophysics is still limited, hampering their quantitative application. For example, we do not know the optimal sample preparation methods or imaging conditions to count protein molecules fused to PCFPs by single-molecule localization microscopy in live and fixed cells. We also do not know how the behavior of PCFPs in live cells compares with fixed cells. Therefore, we investigated how formaldehyde fixation influences the photophysical properties of the popular green-to-red PCFP mEos3.2 in fission yeast cells under a wide range of imaging conditions. We estimated photophysical parameters by fitting a 3-state model of photoconversion and photobleaching to the time course of fluorescence signal per yeast cell expressing mEos3.2. We discovered that formaldehyde fixation makes the fluorescence signal, photoconversion rate and photobleaching rate of mEos3.2 sensitive to the buffer conditions by permeabilizing the yeast cell membrane. Under some imaging conditions, the time-integrated mEos3.2 signal per yeast cell is similar in live cells and fixed cells imaged in buffer at pH 8.5 with 1 mM DTT, indicating that light chemical fixation does not destroy mEos3.2 molecules. We also discovered that some red-state mEos3.2 molecules entered an intermediate dark state that is converted back to the red fluorescent state by 561-nm illumination. Our findings provide a guide to compare quantitatively conditions for imaging and counting of mEos3.2-tagged molecules in yeast cells. Our imaging assay and mathematical model are easy to implement and provide a simple quantitative approach to measure the time-integrated signal and the photoconversion and photobleaching rates of fluorescent proteins in cells.STATEMENT OF SIGNIFICANCEMaking quantitative measurements with single-molecule localization microscopy (SMLM) has been impeded by limited understanding of the photophysics of the fluorophores, which is very sensitive to the sample preparation and imaging conditions. We characterized the photophysics of the green-to-red photoconvertible fluorescent protein mEos3.2, which is widely used in SMLM. We combined quantitative fluorescence microscopy and mathematical modeling to measure the fluorescence signal and rate constants for photoconversion and photobleaching of mEos3.2 in live and fixed cells under a wide range of illumination intensities. Our findings provide a guide to compare conditions for imaging and counting mEos3.2-tagged proteins in cells. The presented approach is generally applicable to characterize other fluorescent proteins or dyes in cells.

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