Counting Single DNA Molecules in a Capillary with Radial Focusing

2003 ◽  
Vol 56 (3) ◽  
pp. 149 ◽  
Author(s):  
Jinjian Zheng ◽  
Edward S. Yeung
Keyword(s):  
Single Molecule ◽  
Detection Efficiency ◽  
Signal To Noise Ratio ◽  
Sampling Rate ◽  
Ccd Camera ◽  
Counting Efficiency ◽  
Spherical Particles ◽  
Dna Molecules ◽  
Single Dna Molecules ◽  
Relative Standard

For single-molecule detection, usually a small detection volume of 10 pL or less is used to improve the signal-to-noise ratio. Detection of every molecule in a sample requires that the sample be driven through a well-defined volume to facilitate laser excitation. We report a novel approach to count single DNA molecules with nearly 100% efficiency. By applying an electric field across a 40 cm long, 75 × 75 µm2 square capillary together with hydrodynamic flow from cathode to anode, we were able to concentrate more than 95% of DNA molecules into a 10 µm region at the centre of the capillary. The YOYO-1 labelled λ-DNA molecules were imaged with an intensified CCD camera. We found that the single DNA molecule detection efficiency in a 10–17 M solution was 114 ± 21%. The mobility of the DNA molecules after radial focusing was relatively constant, with relative standard deviations ranging from 0.8% to 1.4%. This allowed us to match the sampling rate to the length of the detection window to maximize counting efficiency. Analysis of a 40.2 nL injected plug of 2 × 10–14 M λ-DNA gave a result of 492 ± 73 molecules, which agreed well with the estimated value of 484. This method should be generally useful for counting deformable molecules or non-spherical particles at extremely low concentrations.

Molecular microscopy of DNA for genome analysis: optical mapping of restriction digests

1995 ◽  
Vol 53 ◽  
pp. 50-51
Author(s):  
Edward J. Huff ◽  
Weiwen Cai ◽  
Xinghua Hu ◽  
John Huang ◽  
Junping Jing ◽  
...  
Keyword(s):  
Single Molecule ◽  
Genome Analysis ◽  
Optical Mapping ◽  
Ccd Camera ◽  
Dna Molecules ◽  
Specific Sequence ◽  
Single Dna Molecules ◽  
Single Clone ◽  
Rapid Generation ◽  
Glass Surfaces

Optical microscopy of individual DNA molecules has been an interesting technique for the past 15 years, but until recently has not been useful for genome analysis. We have developed Optical Mapping an emerging single molecule approach for the rapid generation of ordered restriction maps. Many identical individual DNA molecules from a single clone are elongated and fixed onto derivatized glass surfaces, digested with a restriction enzyme which cuts the DNA wherever a specific sequence pattern is found, stained with YOYO, and imaged with a cooled CCD camera attached to an automated epi-fluorescence microscope. Images are automatically processed to correct for non-uniform illumination, remove background, locate the DNA fragments, reject objects which do not look like single DNA molecules, recognize which fragments originate from an original uncut molecule, and calculate the relative sizes of the fragments by apparent length and fluorescence intensity. Results from many molecules are combined by clustering to recognize a consistent cutting pattern. Molecules which match the pattern are averaged to improve the sizing accuracy.

scholarly journals Somatic mutation landscapes at single-molecule resolution

2021 ◽  
Author(s):  
Stefanie V. Lensing ◽  
Peter Ellis ◽  
Federico Abascal ◽  
Iñigo Martincorena ◽  
Robert J. Osborne
Keyword(s):  
Single Molecule ◽  
Single Cells ◽  
Error Rates ◽  
Dna Molecules ◽  
Base Pairs ◽  
Single Dna Molecules ◽  
Sequencing Library ◽  
Somatic Mutagenesis ◽  
Qpcr Quantification ◽  
Duplex Sequencing

Abstract Somatic mutations drive cancer development and may contribute to ageing and other diseases. Yet, the difficulty of detecting mutations present only in single cells or small clones has limited our knowledge of somatic mutagenesis to a minority of tissues. To overcome these limitations, we introduce nanorate sequencing (NanoSeq), a new duplex sequencing protocol with error rates <5 errors per billion base pairs in single DNA molecules from cell populations. The version of the protocol described here uses clean genome fragmentation with a restriction enzyme to prevent end-repair-associated errors and ddBTPs/dATPs during A-tailing to prevent nick extension. Both changes reduce the error rate of standard duplex sequencing protocols by preventing the fixation of DNA damage into both strands of DNA molecules during library preparation. We also use qPCR quantification of the library prior to amplification to optimise the complexity of the sequencing library given the desired sequencing coverage, maximising duplex coverage. The sample preparation protocol takes between 1 and 2 days, depending on the number of samples processed. The bioinformatic protocol is described in:https://github.com/cancerit/NanoSeqhttps://github.com/fa8sanger/NanoSeq_Paper_Code

Single-molecule DNA visualization using AT-specific red and non-specific green DNA-binding fluorescent proteins

The Analyst ◽  
2019 ◽  
Vol 144 (3) ◽  
pp. 921-927 ◽  
Author(s):  
Jihyun Park ◽  
Seonghyun Lee ◽  
Nabin Won ◽  
Eunji Shin ◽  
Soo-Hyun Kim ◽  
...  
Keyword(s):  
Dna Binding ◽  
Single Molecule ◽  
Fluorescent Proteins ◽  
Physical Map ◽  
Dna Molecules ◽  
Single Dna Molecules

Two-color DNA physical map for efficient identification of single DNA molecules.

Determination of haplotypes from single DNA molecules: a method for single-molecule barcoding

2007 ◽  
Vol 28 (9) ◽  
pp. 913-921 ◽  
Author(s):  
Ming Xiao ◽  
Matthew P. Gordon ◽  
Angie Phong ◽  
Connie Ha ◽  
Ting-Fung Chan ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dna Molecules ◽  
Single Dna Molecules

Optimization of Single-Molecule Fluorescence Burst Detection of ds-DNA: Application to Capillary Electrophoresis Separations of 100–1000 Basepair Fragments

1997 ◽  
Vol 51 (10) ◽  
pp. 1579-1584 ◽  
Author(s):  
Brian B. Haab ◽  
Richard A. Mathies
Keyword(s):  
Capillary Electrophoresis ◽  
Single Molecule ◽  
Laser Power ◽  
Detection Efficiency ◽  
Fragment Size ◽  
Signal To Noise Ratio ◽  
Burst Size ◽  
Dna Fragments ◽  
Burst Detection ◽  
Single Molecule Fluorescence

Methods for optimizing the dye labeling, laser excitation, and data analysis for single-molecule fluorescence burst detection of ds-DNA have been developed and then validated through capillary electrophoresis (CE) separations of 100–1000 basepair (bp) DNA. Confocal microscopy is used to observe fluorescence bursts from individual DNA fragments labeled with the intercalation dye TO6 as they pass through the ∼ 2-μm-diameter focused laser beam. The dye concentration and laser power were optimized by studying fluorescence burst intensities from pBluescript DNA fragments. The optimal TO6 concentration was ≤100 nM, and the optimal laser power was ≤1 mW. Single-molecule counting was then used to detect CE separations of a 100–1000 bp DNA sizing ladder in 3% linear polyacrylamide. Discrete and baseline-resolved fluorescence bursts were observed in bands as small as 100 bp, and the average burst size within each band increased linearly with fragment size. By counting events using a single optimally chosen discriminator level, we achieve maximum signal-to-noise ratio (S/N) for each fragment size. If the discriminator level is ramped linearly with fragment size to achieve a constant detection efficiency, then the number of events properly reflects the relative fragment concentrations.

VISUALIZATION EX SITU OF SINGLE DNA MOLECULES INCUBATION: A FIRST STEP FOR QUANTITATIVE ANALYSIS ON MULTI-SITE DEGRADATION AND ENZYMATIC KINETICS

2009 ◽  
Vol 16 (01) ◽  
pp. 79-85
Author(s):  
XINYAN WANG ◽  
HAIJUN YANG ◽  
HUABIN WANG ◽  
PENG WANG ◽  
HAI LI
Keyword(s):  
Single Molecule ◽  
Ex Situ ◽  
In Vitro Tests ◽  
Dna Molecules ◽  
Single Molecule Level ◽  
Single Dna Molecules ◽  
Enzymatic Kinetics ◽  
Afm Imaging ◽  
Molecular Combing

Herein, we showed a different approach to directly single-molecule level visualization of the degradation of DNA in vitro tests using DNase I incubation based on high-resolution AFM imaging ex situ with fine relocation nanotechnology. A method of nanomanipulation termed as "modified dynamic molecular combing" (MDMC) was used to pattern DNA samples for further degradation and enzymatic kinetics. This strategy is potentially able to quantitatively address the mechanical-induced kinetic profiles of multi-site degradation of individual DNA molecules with very stable tension and strong immobilization on a surface and discover the mechanochemistry.

Positioning Isolation and Biochemical Analysis of Single DNA Molecules Based on Nanomanipulation and Single-Molecule PCR

2004 ◽  
Vol 126 (36) ◽  
pp. 11136-11137 ◽  
Author(s):  
Jun-hong Lü ◽  
Hai-kuo Li ◽  
Hong-jie An ◽  
Guo-hua Wang ◽  
Ying Wang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Biochemical Analysis ◽  
Dna Molecules ◽  
Single Dna Molecules

scholarly journals DNA Manipulation and Single-Molecule Imaging

Molecules ◽  
2021 ◽  
Vol 26 (4) ◽  
pp. 1050
Author(s):  
Shunsuke Takahashi ◽  
Masahiko Oshige ◽  
Shinji Katsura
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Single Molecule ◽  
Single Molecule Imaging ◽  
Dna Molecules ◽  
Dna Manipulation ◽  
Single Dna Molecules ◽  
Biological Studies ◽  
Physical Form ◽  
Almost All

DNA replication, repair, and recombination in the cell play a significant role in the regulation of the inheritance, maintenance, and transfer of genetic information. To elucidate the biomolecular mechanism in the cell, some molecular models of DNA replication, repair, and recombination have been proposed. These biological studies have been conducted using bulk assays, such as gel electrophoresis. Because in bulk assays, several millions of biomolecules are subjected to analysis, the results of the biological analysis only reveal the average behavior of a large number of biomolecules. Therefore, revealing the elementary biological processes of a protein acting on DNA (e.g., the binding of protein to DNA, DNA synthesis, the pause of DNA synthesis, and the release of protein from DNA) is difficult. Single-molecule imaging allows the analysis of the dynamic behaviors of individual biomolecules that are hidden during bulk experiments. Thus, the methods for single-molecule imaging have provided new insights into almost all of the aspects of the elementary processes of DNA replication, repair, and recombination. However, in an aqueous solution, DNA molecules are in a randomly coiled state. Thus, the manipulation of the physical form of the single DNA molecules is important. In this review, we provide an overview of the unique studies on DNA manipulation and single-molecule imaging to analyze the dynamic interaction between DNA and protein.

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