Single molecule fluorescence burst detection of DNA fragments separated by capillary electrophoresis

1995 ◽  
Vol 67 (18) ◽  
pp. 3253-3260 ◽  
Author(s):  
Brian B. Haab ◽  
Richard A. Mathies
Keyword(s):  
Capillary Electrophoresis ◽  
Single Molecule ◽  
Dna Fragments ◽  
Burst Detection ◽  
Single Molecule Fluorescence

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.

scholarly journals Selective Prespacer Processing Ensures Precise CRISPR-Cas Adaptation

2019 ◽  
Author(s):  
Sungchul Kim ◽  
Luuk Loeff ◽  
Sabina Colombo ◽  
Stan J.J. Brouns ◽  
Chirlmin Joo
Keyword(s):  
Single Molecule ◽  
Dependent Manner ◽  
Dna Fragments ◽  
Comprehensive Understanding ◽  
Single Molecule Fluorescence ◽  
Spatiotemporal Resolution ◽  
Single Stranded Dna ◽  
Correct Orientation ◽  
Crispr Array ◽  
Cellular Dna

AbstractCRISPR-Cas immunity protects prokaryotes against foreign genetic elements. CRISPR-Cas uses the highly conserved Cas1-Cas2 complex to establish inheritable memory (spacers). It remains elusive how Cas1-Cas2 acquires spacers from cellular DNA fragments (prespacers) and how it integrates them into the CRISPR array in the correct orientation. By using the high spatiotemporal resolution of single-molecule fluorescence, we reveal that Cas1-Cas2 obtains prespacers in various forms including single-stranded DNA and partial duplexes by selecting them in the DNA-length and PAM-dependent manner. Furthermore, we identify DnaQ exonucleases as enzymes that can mature the Cas1-Cas2-loaded precursor prespacers into an integration-competent size. Cas1-Cas2 protects the PAM sequence from maturation, which results in the production of asymmetrically trimmed prespacers and subsequent spacer integration in the correct orientation. This kinetic coordination in prespacer selection and PAM trimming provides comprehensive understanding of the mechanisms that underlie the integration of functional spacers in the CRISPR array.

scholarly journals Single Molecule Fluorescence Imaging and Blinking

2006 ◽  
Vol 46 (3) ◽  
pp. 164-168
Author(s):  
Hiroaki YOKOTA ◽  
Tetsuichi WAZAWA ◽  
Yoshiharu ISHII
Keyword(s):  
Fluorescence Imaging ◽  
Single Molecule ◽  
Single Molecule Fluorescence

scholarly journals Single bacterial resolvases first exploit, then constrain intrinsic dynamics of the Holliday junction to direct recombination

2021 ◽  
Author(s):  
Sujay Ray ◽  
Nibedita Pal ◽  
Nils G Walter
Keyword(s):  
Cluster Analysis ◽  
Homologous Recombination ◽  
Single Molecule ◽  
Holliday Junction ◽  
Energetic Cost ◽  
Genomic Integrity ◽  
Dna Molecules ◽  
Single Molecule Fluorescence ◽  
And Cluster Analysis ◽  
Fluorescence Observation

Abstract Homologous recombination forms and resolves an entangled DNA Holliday Junction (HJ) crucial for achieving genetic reshuffling and genome repair. To maintain genomic integrity, specialized resolvase enzymes cleave the entangled DNA into two discrete DNA molecules. However, it is unclear how two similar stacking isomers are distinguished, and how a cognate sequence is found and recognized to achieve accurate recombination. We here use single-molecule fluorescence observation and cluster analysis to examine how prototypic bacterial resolvase RuvC singles out two of the four HJ strands and achieves sequence-specific cleavage. We find that RuvC first exploits, then constrains the dynamics of intrinsic HJ isomer exchange at a sampled branch position to direct cleavage toward the catalytically competent HJ conformation and sequence, thus controlling recombination output at minimal energetic cost. Our model of rapid DNA scanning followed by ‘snap-locking’ of a cognate sequence is strikingly consistent with the conformational proofreading of other DNA-modifying enzymes.

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