lambda phage dna  (New England Biolabs)


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    Name:
    Lambda DNA N6 methyladenine free
    Description:
    Lambda DNA N6 methyladenine free 1 250 ug
    Catalog Number:
    N3013L
    Price:
    276
    Category:
    Genomic DNA
    Size:
    1 250 ug
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    Structured Review

    New England Biolabs lambda phage dna
    Lambda DNA N6 methyladenine free
    Lambda DNA N6 methyladenine free 1 250 ug
    https://www.bioz.com/result/lambda phage dna/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    lambda phage dna - by Bioz Stars, 2021-04
    99/100 stars

    Images

    1) Product Images from "Quantification of Trace-Level DNA by Real-Time Whole Genome Amplification"

    Article Title: Quantification of Trace-Level DNA by Real-Time Whole Genome Amplification

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0028661

    Application of the real-time DOP-PCR to diverse DNA species. Amplification profiles and their standard curves were obtained from human placental DNA (HPD; A), calf thymus DNA (CTD; B), E. coli DNA (C), and lambda phage DNA (D). Standard DNA samples from 80 fg to 80 ng and a no-template control were amplified. Six independent experiments each comprising triplicate reactions were performed, and typical results of one experiment are presented. Data for 80 ng and NTC were omitted for the plotting of standard curves.
    Figure Legend Snippet: Application of the real-time DOP-PCR to diverse DNA species. Amplification profiles and their standard curves were obtained from human placental DNA (HPD; A), calf thymus DNA (CTD; B), E. coli DNA (C), and lambda phage DNA (D). Standard DNA samples from 80 fg to 80 ng and a no-template control were amplified. Six independent experiments each comprising triplicate reactions were performed, and typical results of one experiment are presented. Data for 80 ng and NTC were omitted for the plotting of standard curves.

    Techniques Used: Degenerate Oligonucleotide–primed Polymerase Chain Reaction, Amplification

    2) Product Images from "Fabrication of long poly(dimethyl siloxane) nanochannels by replicating protein deposit from confined solution evaporation"

    Article Title: Fabrication of long poly(dimethyl siloxane) nanochannels by replicating protein deposit from confined solution evaporation

    Journal: Biomicrofluidics

    doi: 10.1063/1.4730371

    Stretching of DNA molecules (a) near the inlet (micro to nano region) and (b) at about 2 mm away from the inlet of the nanochannel. The maximum elongation of λ-DNA reaches 10 μ m, which is about 50% of its fully extended length. (Scale bar: 20 μ m)
    Figure Legend Snippet: Stretching of DNA molecules (a) near the inlet (micro to nano region) and (b) at about 2 mm away from the inlet of the nanochannel. The maximum elongation of λ-DNA reaches 10 μ m, which is about 50% of its fully extended length. (Scale bar: 20 μ m)

    Techniques Used:

    3) Product Images from "Regulation of DNA conformations and dynamics in flows with hybrid field microfluidics"

    Article Title: Regulation of DNA conformations and dynamics in flows with hybrid field microfluidics

    Journal: Biomicrofluidics

    doi: 10.1063/1.4762852

    (a) Schematic of how a DNA chain is polarized, trapped or escape, and relax in HFM; (b) and (c) Time serial snapshots of the relaxation dynamics of an intermediated-stretched (b) and an over-stretched λ-DNA (c). (d) Extension versus residence
    Figure Legend Snippet: (a) Schematic of how a DNA chain is polarized, trapped or escape, and relax in HFM; (b) and (c) Time serial snapshots of the relaxation dynamics of an intermediated-stretched (b) and an over-stretched λ-DNA (c). (d) Extension versus residence

    Techniques Used:

    The ensemble average fractional extension of ∼200 λ-DNA when switching from “flow field” alone to “hybrid field” (a) and 60 λ-DNA molecules after the sudden removal of the electric field (c). (b)
    Figure Legend Snippet: The ensemble average fractional extension of ∼200 λ-DNA when switching from “flow field” alone to “hybrid field” (a) and 60 λ-DNA molecules after the sudden removal of the electric field (c). (b)

    Techniques Used:

    (a) Schematic of the working principles of HFM, (b) a typical application scheme for an electric bias in HFM, (c) regulation the conformations and dynamics (trapping, concentration, and sudden stretching) of λ-DNA molecules in HFM, and (d) schematic
    Figure Legend Snippet: (a) Schematic of the working principles of HFM, (b) a typical application scheme for an electric bias in HFM, (c) regulation the conformations and dynamics (trapping, concentration, and sudden stretching) of λ-DNA molecules in HFM, and (d) schematic

    Techniques Used: Concentration Assay

    4) Product Images from "Conserved linear dynamics of single-molecule Brownian motion"

    Article Title: Conserved linear dynamics of single-molecule Brownian motion

    Journal: Nature Communications

    doi: 10.1038/ncomms15675

    Lattice occupancy analysis of lambda DNA. ( a ) MSD-Δ t profile of lambda DNA (Δ t =6.4 ms). The red line shows the theoretical MSD-Δ t profile. ( b ) Frequency histogram of the HE-1Δ t distribution of the experimental (top) and the S r A r simulated replicates (bottom) of lambda DNA. ( c ) Averaged MSD-Δ t profiles of the sub-trajectories captured in the same way as in Fig. 7a (red) and Fig. 7b (blue). The red and blue lines are the theoretical MSD-Δ t profiles. The green line shows the overall, theoretical MSD-Δ t profile of the experimental replicates.
    Figure Legend Snippet: Lattice occupancy analysis of lambda DNA. ( a ) MSD-Δ t profile of lambda DNA (Δ t =6.4 ms). The red line shows the theoretical MSD-Δ t profile. ( b ) Frequency histogram of the HE-1Δ t distribution of the experimental (top) and the S r A r simulated replicates (bottom) of lambda DNA. ( c ) Averaged MSD-Δ t profiles of the sub-trajectories captured in the same way as in Fig. 7a (red) and Fig. 7b (blue). The red and blue lines are the theoretical MSD-Δ t profiles. The green line shows the overall, theoretical MSD-Δ t profile of the experimental replicates.

    Techniques Used: Lambda DNA Preparation, Mass Spectrometry

    5) Product Images from "A Microneedle Functionalized with Polyethyleneimine and Nanotubes for Highly Sensitive, Label-Free Quantification of DNA"

    Article Title: A Microneedle Functionalized with Polyethyleneimine and Nanotubes for Highly Sensitive, Label-Free Quantification of DNA

    Journal: Sensors (Basel, Switzerland)

    doi: 10.3390/s17081883

    ( a ) IV curve for the SWCNTs coated microneedle with various concentration of PEI; ( b ) Cyclic voltammetry for four different types of surfaces: bare gold, 1% PEI-coated, 1% PEI/SWCNTs-coated microneedle, and 1 pM λ DNA captured needle.
    Figure Legend Snippet: ( a ) IV curve for the SWCNTs coated microneedle with various concentration of PEI; ( b ) Cyclic voltammetry for four different types of surfaces: bare gold, 1% PEI-coated, 1% PEI/SWCNTs-coated microneedle, and 1 pM λ DNA captured needle.

    Techniques Used: Concentration Assay

    6) Product Images from "Elimination of inter-domain interactions increases the cleavage fidelity of the restriction endonuclease DraIII"

    Article Title: Elimination of inter-domain interactions increases the cleavage fidelity of the restriction endonuclease DraIII

    Journal: Protein & Cell

    doi: 10.1007/s13238-014-0038-z

    Determination of Dra III Fidelity Index (FI) and star sites . (A) Determination of Dra III Fidelity Index (FI). λ DNA (1.6 nmol/L; 16 nmol/L CACNNNGTG sites) is digested by Dra III in a series of two fold dilutions. Dra III concentration: Lane 1, 3.2 μmol/L; Lane 10, 6.25 nmol/L; Lane 11, 3.125 nmol/L; Lane 21, 3.05 pmol/L; Lane 22, 1-kb DNA Ladder (NEB). The vertical arrows indicate the two critical points: HNS —the H ighest REases concentration showing N o S tar activity and LCC —the L owest REase concentration needed for C omplete C leavage on canonical sites. FI = HNS/LCC, which is 2 in this case. Asterisk represents a star band, and the hash represents a band that resulted from partial cleavage of λ DNA. The theoretical digestion pattern of Dra III to λ DNA was predicted using NEBcutter (Vincze et al., 2003 ) and was shown on the left. (B) Dra III star site in pUC19 was predicted to be the CATGTTGTG site. Lane 1: Bam HI (cut at nt 417) and Xmn I (cut at nt 2298) double digestion on pUC19 generated the 1.9-kb and 0.8-kb bands. Lane 2: Bam HI, Xmn I and Dra III triple digestion on pUC19. Asterisk indicates the star band. According to the approximate size of star bands, the CATGTTGTG site (nt 2033) was hypothesized to be the Dra III star site. Cleavage on predicted CATGTTGTG site generated the 1.6-kb and 0.3-kb star bands. Lane 3: 1-kb DNA Ladder. (C) Dra III star activity cleaves the CATGTTGTG site in pXba. Lane 1: 1-kb DNA Ladder. Lane 2: pXba was digested by Dra III. Asterisk indicates the star bands. Dra III star activity generates the expected 6.5-kb and 4.5-kb star bands on pXba. (D) Dra III star activity shows selectivity to the central “NNN” part of CATNNNGTG site. There are 11 CATNNNGTG sites in pXba and the sequences containing these sites were tested independently on oligonucleotide duplex DNAs carrying each of the sites (Table S1). The canonical CACGGCGTG site was used as positive control. Dra III shows cleavage activity to CATATGGTG, CATTACGTG, CATGTGGTG, CATAAAGTG and CATGTTGTG sites. Dra III did not cut the pseudo-palindromic CATGTTATG site
    Figure Legend Snippet: Determination of Dra III Fidelity Index (FI) and star sites . (A) Determination of Dra III Fidelity Index (FI). λ DNA (1.6 nmol/L; 16 nmol/L CACNNNGTG sites) is digested by Dra III in a series of two fold dilutions. Dra III concentration: Lane 1, 3.2 μmol/L; Lane 10, 6.25 nmol/L; Lane 11, 3.125 nmol/L; Lane 21, 3.05 pmol/L; Lane 22, 1-kb DNA Ladder (NEB). The vertical arrows indicate the two critical points: HNS —the H ighest REases concentration showing N o S tar activity and LCC —the L owest REase concentration needed for C omplete C leavage on canonical sites. FI = HNS/LCC, which is 2 in this case. Asterisk represents a star band, and the hash represents a band that resulted from partial cleavage of λ DNA. The theoretical digestion pattern of Dra III to λ DNA was predicted using NEBcutter (Vincze et al., 2003 ) and was shown on the left. (B) Dra III star site in pUC19 was predicted to be the CATGTTGTG site. Lane 1: Bam HI (cut at nt 417) and Xmn I (cut at nt 2298) double digestion on pUC19 generated the 1.9-kb and 0.8-kb bands. Lane 2: Bam HI, Xmn I and Dra III triple digestion on pUC19. Asterisk indicates the star band. According to the approximate size of star bands, the CATGTTGTG site (nt 2033) was hypothesized to be the Dra III star site. Cleavage on predicted CATGTTGTG site generated the 1.6-kb and 0.3-kb star bands. Lane 3: 1-kb DNA Ladder. (C) Dra III star activity cleaves the CATGTTGTG site in pXba. Lane 1: 1-kb DNA Ladder. Lane 2: pXba was digested by Dra III. Asterisk indicates the star bands. Dra III star activity generates the expected 6.5-kb and 4.5-kb star bands on pXba. (D) Dra III star activity shows selectivity to the central “NNN” part of CATNNNGTG site. There are 11 CATNNNGTG sites in pXba and the sequences containing these sites were tested independently on oligonucleotide duplex DNAs carrying each of the sites (Table S1). The canonical CACGGCGTG site was used as positive control. Dra III shows cleavage activity to CATATGGTG, CATTACGTG, CATGTGGTG, CATAAAGTG and CATGTTGTG sites. Dra III did not cut the pseudo-palindromic CATGTTATG site

    Techniques Used: Concentration Assay, Activity Assay, Generated, Positive Control

    The interactions between the N-terminal domain and C-terminal domain of Dra III subunit . (A) Dra III subunit structure. N-terminal domain in orange; C-terminal domain in cyan; Zinc in green; Magnesium in red. Left: Dra III subunit displayed in surface mode. Right: Dra III subunit displayed in cartoon mode. Interactions in the mouth, middle region and hydrophobic residues in the hinge region were shown in gray boxes. The locations of potential hydrogen bonds are indicated with red dotted line. (B) Determination FI of Dra III T181A. λ DNA was cleaved by diluted T181A. Lane 1: 3.2 μmol/L T181A; Lane 13: 0.78 nmol/L (LCC); Lane 21: 3.05 pmol/L; Lane 22: 1-kb DNA Ladder. FI is larger than 4000. The hash represents a partially digested band. No star band was observed. Disrupting hydrogen bond between T181 and D55 in the middle region remarkably enhanced FI
    Figure Legend Snippet: The interactions between the N-terminal domain and C-terminal domain of Dra III subunit . (A) Dra III subunit structure. N-terminal domain in orange; C-terminal domain in cyan; Zinc in green; Magnesium in red. Left: Dra III subunit displayed in surface mode. Right: Dra III subunit displayed in cartoon mode. Interactions in the mouth, middle region and hydrophobic residues in the hinge region were shown in gray boxes. The locations of potential hydrogen bonds are indicated with red dotted line. (B) Determination FI of Dra III T181A. λ DNA was cleaved by diluted T181A. Lane 1: 3.2 μmol/L T181A; Lane 13: 0.78 nmol/L (LCC); Lane 21: 3.05 pmol/L; Lane 22: 1-kb DNA Ladder. FI is larger than 4000. The hash represents a partially digested band. No star band was observed. Disrupting hydrogen bond between T181 and D55 in the middle region remarkably enhanced FI

    Techniques Used:

    7) Product Images from "Single-molecule measurements reveal that PARP1 condenses DNA by loop formation"

    Article Title: Single-molecule measurements reveal that PARP1 condenses DNA by loop formation

    Journal: bioRxiv

    doi: 10.1101/2020.09.15.297887

    TIRF imaging shows condensation of DNA by PARP1. (A) Schematic of TIRF microscopy imaging of a single λ-DNA molecule stained with Sytox Orange. A constant flow was maintained which stretches out the DNA close to its contour length. (B) Kymograph showing DNA extension over time. 400 nM PARP1 is added at the timepoint indicated by the asterisk. (C) Snapshots showing individual image frames at the indicated timepoints.
    Figure Legend Snippet: TIRF imaging shows condensation of DNA by PARP1. (A) Schematic of TIRF microscopy imaging of a single λ-DNA molecule stained with Sytox Orange. A constant flow was maintained which stretches out the DNA close to its contour length. (B) Kymograph showing DNA extension over time. 400 nM PARP1 is added at the timepoint indicated by the asterisk. (C) Snapshots showing individual image frames at the indicated timepoints.

    Techniques Used: Imaging, Microscopy, Staining

    8) Product Images from "Deconvolution of Nucleic-acid Length Distributions: A Gel Electrophoresis Analysis Tool and Applications"

    Article Title: Deconvolution of Nucleic-acid Length Distributions: A Gel Electrophoresis Analysis Tool and Applications

    Journal: bioRxiv

    doi: 10.1101/636936

    Agarose-gel analysis of tagmentation products of phage- λ DNA analyzed on (a) high-resolution and (c) low-resolution ( i.e ., mini) agarose gels. Size-distribution fits for the high-resolution gel (b) and mini gel (d) of the same tagmented λ -DNA sample subjected to increasing numbers of PCR-amplification cycles (lanes 3-5 in (a), lanes 2-4 in (c)): 8 cycles (top plots), 14 cycles (middle plots), and 20 cycles (bottom plots) in both (b) and (d). Vertical dashed lines (light red in (b), (d)) give the positions of maxima in the discrete molecular-weight ladder (blue ROI in (b), (b)).
    Figure Legend Snippet: Agarose-gel analysis of tagmentation products of phage- λ DNA analyzed on (a) high-resolution and (c) low-resolution ( i.e ., mini) agarose gels. Size-distribution fits for the high-resolution gel (b) and mini gel (d) of the same tagmented λ -DNA sample subjected to increasing numbers of PCR-amplification cycles (lanes 3-5 in (a), lanes 2-4 in (c)): 8 cycles (top plots), 14 cycles (middle plots), and 20 cycles (bottom plots) in both (b) and (d). Vertical dashed lines (light red in (b), (d)) give the positions of maxima in the discrete molecular-weight ladder (blue ROI in (b), (b)).

    Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Molecular Weight

    9) Product Images from "Force and Scale Dependence of the Elasticity of Self-Assembled DNA Bottle Brushes"

    Article Title: Force and Scale Dependence of the Elasticity of Self-Assembled DNA Bottle Brushes

    Journal: Macromolecules

    doi: 10.1021/acs.macromol.7b01795

    Effective persistence lengths of C 8 -B Sso7d -coated λ-DNA versus the mole ratio [C 8 -B Sso7d ]/[DNA(bp)] of protein to DNA basepairs, as deduced by fitting optical tweezer force extension curves with the wormlike chain model ( eq 5 ). Solution conditions: 10 mM Tris-HCl, pH 7.4, DNA concentration C DNA = 1.58 μg/mL. Note that the upper x -axis shows the protein concentration in nM. Dashed line is a guide to the eye.
    Figure Legend Snippet: Effective persistence lengths of C 8 -B Sso7d -coated λ-DNA versus the mole ratio [C 8 -B Sso7d ]/[DNA(bp)] of protein to DNA basepairs, as deduced by fitting optical tweezer force extension curves with the wormlike chain model ( eq 5 ). Solution conditions: 10 mM Tris-HCl, pH 7.4, DNA concentration C DNA = 1.58 μg/mL. Note that the upper x -axis shows the protein concentration in nM. Dashed line is a guide to the eye.

    Techniques Used: Concentration Assay, Protein Concentration

    Bottle-brush thickness from small-angle X-ray scattering. Scattering intensity (arbitrary units, a.u.) versus magnitude q of the wavevector in nm –1 . Solution conditions are 10 mM Tris-HCl, pH 7.6. Blue squares are the scattering intensities for 100 μg/mL λ-DNA coated with the C 8 -B Sso7d diblock protein polymer at a protein to DNA ratio of [C 8 -B Sso7d ]/[DNA(bp)] of 0.5 ptn/bp; black circles are the scattering intensities for 30 mg/mL free C 8 -B Sso7d protein polymer. In the figure, the scattering intensity for the free C 8 -B Sso7d protein polymer is scaled to match the high- q scattering of the of C 8 -B Sso7d /DNA bottle-brush complex. The red line is a fit to a polymer coil model with excluded volume; the orange line is the sum of a contribution due to excluded volume polymer coils (representing the excess unbound protein polymers) and a contribution due to randomly oriented rigid cylinders. Separate contributions of polymer coils and cylinders are indicated in gray.
    Figure Legend Snippet: Bottle-brush thickness from small-angle X-ray scattering. Scattering intensity (arbitrary units, a.u.) versus magnitude q of the wavevector in nm –1 . Solution conditions are 10 mM Tris-HCl, pH 7.6. Blue squares are the scattering intensities for 100 μg/mL λ-DNA coated with the C 8 -B Sso7d diblock protein polymer at a protein to DNA ratio of [C 8 -B Sso7d ]/[DNA(bp)] of 0.5 ptn/bp; black circles are the scattering intensities for 30 mg/mL free C 8 -B Sso7d protein polymer. In the figure, the scattering intensity for the free C 8 -B Sso7d protein polymer is scaled to match the high- q scattering of the of C 8 -B Sso7d /DNA bottle-brush complex. The red line is a fit to a polymer coil model with excluded volume; the orange line is the sum of a contribution due to excluded volume polymer coils (representing the excess unbound protein polymers) and a contribution due to randomly oriented rigid cylinders. Separate contributions of polymer coils and cylinders are indicated in gray.

    Techniques Used:

    10) Product Images from "High-Throughput Analysis of Global DNA Methylation Using Methyl-Sensitive Digestion"

    Article Title: High-Throughput Analysis of Global DNA Methylation Using Methyl-Sensitive Digestion

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0163184

    The workflow of the methyl-sensitive fluorescence polarization (MSFP) assay. Lambda (λ) and human genomic DNA are restricted by MspI (white) or by HpaII each alone or by a combination of HpaII and HpyCH4IV (grey) (Step 1). Subsequently, digested DNA with CpG overhangs at the 5' termini are terminally extended with fluorescence-labelled TAMRA-dCTP (C*, *C) (Step 2). TAMRA-dCTP incorporated into DNA is quantified by fluorescence polarization directly on the plate without additional purification procedures.
    Figure Legend Snippet: The workflow of the methyl-sensitive fluorescence polarization (MSFP) assay. Lambda (λ) and human genomic DNA are restricted by MspI (white) or by HpaII each alone or by a combination of HpaII and HpyCH4IV (grey) (Step 1). Subsequently, digested DNA with CpG overhangs at the 5' termini are terminally extended with fluorescence-labelled TAMRA-dCTP (C*, *C) (Step 2). TAMRA-dCTP incorporated into DNA is quantified by fluorescence polarization directly on the plate without additional purification procedures.

    Techniques Used: Fluorescence, Purification

    11) Product Images from "DNA Condensation by Partially Acetylated Poly(amido amine) Dendrimers: Effects of Dendrimer Charge Density on Complex Formation"

    Article Title: DNA Condensation by Partially Acetylated Poly(amido amine) Dendrimers: Effects of Dendrimer Charge Density on Complex Formation

    Journal: Molecules

    doi: 10.3390/molecules180910707

    ( a ) Experimental setup for imaging dendrimer binding to flow-stretched DNA with one end tethered to the surface. ( b ) Green: tethered YOYO-labeled λ-DNA in flow; red: tethered TRITC-labeled dendrimer-DNA complex in flow. ( c ) Distribution of free λ-DNA molecules lengths. ( d ) Distribution of dendrimer-DNA complex lengths.
    Figure Legend Snippet: ( a ) Experimental setup for imaging dendrimer binding to flow-stretched DNA with one end tethered to the surface. ( b ) Green: tethered YOYO-labeled λ-DNA in flow; red: tethered TRITC-labeled dendrimer-DNA complex in flow. ( c ) Distribution of free λ-DNA molecules lengths. ( d ) Distribution of dendrimer-DNA complex lengths.

    Techniques Used: Imaging, Binding Assay, Flow Cytometry, Labeling

    ( a ) Experimental setup for molecular combing. ( b ) Immobilized, aligned, YOYO-1 stained λ-DNA on PS surface. ( c ) λ-DNA/G5 PAMAM dendrimer (TRITC labeled) complexes deposited on PS coated cover glass surface (green: λ-DNA, red: PAMAM dendrimer).
    Figure Legend Snippet: ( a ) Experimental setup for molecular combing. ( b ) Immobilized, aligned, YOYO-1 stained λ-DNA on PS surface. ( c ) λ-DNA/G5 PAMAM dendrimer (TRITC labeled) complexes deposited on PS coated cover glass surface (green: λ-DNA, red: PAMAM dendrimer).

    Techniques Used: Staining, Labeling

    12) Product Images from "Single-molecule visualization reveals the damage search mechanism for the human NER protein XPC-RAD23B"

    Article Title: Single-molecule visualization reveals the damage search mechanism for the human NER protein XPC-RAD23B

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkz629

    Schematic of DNA curtain assay with quantum dot (Qdot)-conjugated XPC and initial binding position on undamaged lambda (λ) DNA. ( A ) Schematic of DNA curtains. Top left: top view of DNA curtains, bottom left: side-view of DNA curtains, and right: Qdot-conjugated XPC. The structure of XPC is adopted from yeast Rad4-Rad23 ( 10 ). ( B ) Histogram for the initial binding positions of XPC-RAD23B on undamaged λ-DNA. The error bars were obtained by bootstrapping with 70% confidence interval.
    Figure Legend Snippet: Schematic of DNA curtain assay with quantum dot (Qdot)-conjugated XPC and initial binding position on undamaged lambda (λ) DNA. ( A ) Schematic of DNA curtains. Top left: top view of DNA curtains, bottom left: side-view of DNA curtains, and right: Qdot-conjugated XPC. The structure of XPC is adopted from yeast Rad4-Rad23 ( 10 ). ( B ) Histogram for the initial binding positions of XPC-RAD23B on undamaged λ-DNA. The error bars were obtained by bootstrapping with 70% confidence interval.

    Techniques Used: Binding Assay

    Diffusion coefficients of XPC-RAD23B according to salt concentrations and collision between XPC-RAD23B and roadblock protein EcoRI E111Q at 150 mM NaCl. ( A ) Box plots of diffusion coefficients of diffusive motion ( D diff ) and constrained motion ( D cons ) at different NaCl concentrations (N: number of molecules). D diff at 40, 100 and 150 mM NaCl was 0.034 ± 0.045 μm 2 /s (∼5.4 × 10 5 bp 2 /s), 0.093 ± 0.15 μm 2 /s, (∼1.5 × 10 6 bp 2 /s) and 0.39 ± 0.23 μm 2 /s (∼6.2 × 10 6 bp 2 /s) (median ± SD), respectively. D cons was 0.010 μm 2 /s at 40 mM NaCl, 0.014 ± 0.010 μm 2 /s at 100 mM NaCl and 0.010 ± 0.020 μm 2 /s at 150 mM NaCl. ( B ) Schematic of DNA curtain experiment for the collision between XPC-RAD23B and EcoRI E111Q . EcoRI structure is adopted from protein data bank (PDB ID: 1CL8). ( C ) Kymograph for the collision between XPC-RAD23B (red) and EcoRI E111Q (green). The green arrow represents EcoRI cognate site on λ-DNA. ( D ) Quantitative analysis for the bypass events at the collision according to the collision orientation. The bypass percentage was estimated for EcoRI E111Q bound at either cognate ( N = 290) or non-specific sites ( N = 423).
    Figure Legend Snippet: Diffusion coefficients of XPC-RAD23B according to salt concentrations and collision between XPC-RAD23B and roadblock protein EcoRI E111Q at 150 mM NaCl. ( A ) Box plots of diffusion coefficients of diffusive motion ( D diff ) and constrained motion ( D cons ) at different NaCl concentrations (N: number of molecules). D diff at 40, 100 and 150 mM NaCl was 0.034 ± 0.045 μm 2 /s (∼5.4 × 10 5 bp 2 /s), 0.093 ± 0.15 μm 2 /s, (∼1.5 × 10 6 bp 2 /s) and 0.39 ± 0.23 μm 2 /s (∼6.2 × 10 6 bp 2 /s) (median ± SD), respectively. D cons was 0.010 μm 2 /s at 40 mM NaCl, 0.014 ± 0.010 μm 2 /s at 100 mM NaCl and 0.010 ± 0.020 μm 2 /s at 150 mM NaCl. ( B ) Schematic of DNA curtain experiment for the collision between XPC-RAD23B and EcoRI E111Q . EcoRI structure is adopted from protein data bank (PDB ID: 1CL8). ( C ) Kymograph for the collision between XPC-RAD23B (red) and EcoRI E111Q (green). The green arrow represents EcoRI cognate site on λ-DNA. ( D ) Quantitative analysis for the bypass events at the collision according to the collision orientation. The bypass percentage was estimated for EcoRI E111Q bound at either cognate ( N = 290) or non-specific sites ( N = 423).

    Techniques Used: Diffusion-based Assay

    XPC-RAD23B on the damaged DNA ( A ) Snap shot of single-tethered DNA curtain for the specific binding of XPC-RAD23B on CPD-modified λ-DNA. XPC-RAD23B molecules (red) are aligned at CPD sites (green arrow) on YOYO-1 stained λ-DNA (green). The black bar next to the image indicates the barrier position. The black arrow represents the flow orientation. ( B ) Histogram for binding positions of XPC-RAD23B on CPD-inserted λ-DNA. The histogram was fitted by a single Gaussian function (solid green line). The peak center is placed at 30.5 ± 3.5 kbp, which is close to the actual CPD location (33.5 kbp). The error bars were obtained by bootstrapping with 70% confidence interval. The yellow star represents the location of CPDs on the λ-DNA. ( C ) Kymographs showing the search of XPC-RAD23B for CPDs on λ-DNA. Top: CPD recognition by diffusive motion; second: CPD recognition by constrained motion; third: direct binding to CPDs; and bottom: transient binding to CPDs during diffusive motion. The green arrows next to kymographs indicate the location of CPDs. In the top panel, the yellow dashed line is an abstract line for the location of CPDs and yellow arrows represent the events that XPC-RAD23B does not identify CPDs. ( D ) Relative fraction of diffusive, constrained and direct binding to CPDs by XPC-RAD23B −22.0, 69.5, 8.5%, respectively. The total number of events (N) analyzed for relative fraction was 59. ( E ) The probability that XPC-RAD23B binds or misses CPDs when encountering the lesions. The binding probability represents CPD recognition efficiency of XPC-RAD23B in diffusive motion at CPDs. The error bar represents standard error. ( F ) Duration of binding of XPC-RAD23B to CPDs. Lifetimes were collected (blue histogram) and analyzed by fitting with a single exponential decay function (red line). The total number of molecules (N) analyzed for the lifetime was 135. The lifetime (τ) was determined to be 2.1 ± 0.2 s. (Inset) zoom-in view of lifetime histogram. The distribution of binding times, which means by stable binding of XPC-RAD23B to CPDs, is shown in the gray histogram. The total number of molecules (N) analyzed for binding times was 109.
    Figure Legend Snippet: XPC-RAD23B on the damaged DNA ( A ) Snap shot of single-tethered DNA curtain for the specific binding of XPC-RAD23B on CPD-modified λ-DNA. XPC-RAD23B molecules (red) are aligned at CPD sites (green arrow) on YOYO-1 stained λ-DNA (green). The black bar next to the image indicates the barrier position. The black arrow represents the flow orientation. ( B ) Histogram for binding positions of XPC-RAD23B on CPD-inserted λ-DNA. The histogram was fitted by a single Gaussian function (solid green line). The peak center is placed at 30.5 ± 3.5 kbp, which is close to the actual CPD location (33.5 kbp). The error bars were obtained by bootstrapping with 70% confidence interval. The yellow star represents the location of CPDs on the λ-DNA. ( C ) Kymographs showing the search of XPC-RAD23B for CPDs on λ-DNA. Top: CPD recognition by diffusive motion; second: CPD recognition by constrained motion; third: direct binding to CPDs; and bottom: transient binding to CPDs during diffusive motion. The green arrows next to kymographs indicate the location of CPDs. In the top panel, the yellow dashed line is an abstract line for the location of CPDs and yellow arrows represent the events that XPC-RAD23B does not identify CPDs. ( D ) Relative fraction of diffusive, constrained and direct binding to CPDs by XPC-RAD23B −22.0, 69.5, 8.5%, respectively. The total number of events (N) analyzed for relative fraction was 59. ( E ) The probability that XPC-RAD23B binds or misses CPDs when encountering the lesions. The binding probability represents CPD recognition efficiency of XPC-RAD23B in diffusive motion at CPDs. The error bar represents standard error. ( F ) Duration of binding of XPC-RAD23B to CPDs. Lifetimes were collected (blue histogram) and analyzed by fitting with a single exponential decay function (red line). The total number of molecules (N) analyzed for the lifetime was 135. The lifetime (τ) was determined to be 2.1 ± 0.2 s. (Inset) zoom-in view of lifetime histogram. The distribution of binding times, which means by stable binding of XPC-RAD23B to CPDs, is shown in the gray histogram. The total number of molecules (N) analyzed for binding times was 109.

    Techniques Used: Binding Assay, Modification, Staining, Flow Cytometry

    Nature of constrained motion ( A ) The position distribution of the constrained motion and immobile state and locations of consecutive AT-tracks in λ-DNA. Top: the distribution histogram of positions where both constrained and immobile species appear on undamaged λ-DNA. Middle: the distribution histogram of consecutive AT-tracks greater than 4 bp in the λ-DNA (bin size: 1 kbp). Bottom: the overlap of the above two histograms. ( B ) Correlation analysis of the positions of constrained and immobile species relative to the locations of consecutive AT-tracks. The red dashed line represents the perfect positive correlation. Pearson correlation coefficient is 0.7 with 10 −8 P -value.
    Figure Legend Snippet: Nature of constrained motion ( A ) The position distribution of the constrained motion and immobile state and locations of consecutive AT-tracks in λ-DNA. Top: the distribution histogram of positions where both constrained and immobile species appear on undamaged λ-DNA. Middle: the distribution histogram of consecutive AT-tracks greater than 4 bp in the λ-DNA (bin size: 1 kbp). Bottom: the overlap of the above two histograms. ( B ) Correlation analysis of the positions of constrained and immobile species relative to the locations of consecutive AT-tracks. The red dashed line represents the perfect positive correlation. Pearson correlation coefficient is 0.7 with 10 −8 P -value.

    Techniques Used:

    13) Product Images from "The Role of the Methyltransferase Domain of Bifunctional Restriction Enzyme RM.BpuSI in Cleavage Activity"

    Article Title: The Role of the Methyltransferase Domain of Bifunctional Restriction Enzyme RM.BpuSI in Cleavage Activity

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0080967

    Mutation of N406 in the NPPY motif abolishes MTase and cleavage activity. (A) Fluorescence spectra of WT and N406A RM.BpuSI. The high similarity of the two spectra indicated that mutation N406A does not induce significant conformation change to RM.BpuSI. Titration of SAM or SIN into mutant N406A does not show specific changes in fluorescence (data not shown). (B) Titration of SAM or SIN into WT RM .BpuSI in the presence of 400 μM ANS. The data points are fitted to a hyperbolic function and K d values are found to be 76.4 and 71.1 μM for SAM and SIN, respectively. (C) Two-fold dilutions of WT or N406A RM.BpuSI was incubated with 1 μg of λ DNA in the presence or absence of 160 μM SAM at 37°C for 1 h. While WT exhibits enhanced cleavage activity in the presence of SAM, mutant N406A does not exhibit cleavage activity in the presence or absence of SAM.
    Figure Legend Snippet: Mutation of N406 in the NPPY motif abolishes MTase and cleavage activity. (A) Fluorescence spectra of WT and N406A RM.BpuSI. The high similarity of the two spectra indicated that mutation N406A does not induce significant conformation change to RM.BpuSI. Titration of SAM or SIN into mutant N406A does not show specific changes in fluorescence (data not shown). (B) Titration of SAM or SIN into WT RM .BpuSI in the presence of 400 μM ANS. The data points are fitted to a hyperbolic function and K d values are found to be 76.4 and 71.1 μM for SAM and SIN, respectively. (C) Two-fold dilutions of WT or N406A RM.BpuSI was incubated with 1 μg of λ DNA in the presence or absence of 160 μM SAM at 37°C for 1 h. While WT exhibits enhanced cleavage activity in the presence of SAM, mutant N406A does not exhibit cleavage activity in the presence or absence of SAM.

    Techniques Used: Mutagenesis, Activity Assay, Fluorescence, Titration, Incubation

    14) Product Images from "DNA methylation profiling in nanochannels"

    Article Title: DNA methylation profiling in nanochannels

    Journal: Biomicrofluidics

    doi: 10.1063/1.3613671

    (a) Fluorescence images of concatenated methylated and non-methylated λ-DNA labeled with Alexa568MBD (red) and YOYO-1 (green), stretched out in nanochannels. Within each panel colors are split for clarity; (left) YOYO-1 only (DNA), (center) composite, (right) Alexa568 only (Alexa568-MBD). Schematic drawings in each panel illustrate the spatial position of the Alexa Fluor 568 MBD and the length of the λ-DNA. The scale bar in panel (b) is 5 microns.
    Figure Legend Snippet: (a) Fluorescence images of concatenated methylated and non-methylated λ-DNA labeled with Alexa568MBD (red) and YOYO-1 (green), stretched out in nanochannels. Within each panel colors are split for clarity; (left) YOYO-1 only (DNA), (center) composite, (right) Alexa568 only (Alexa568-MBD). Schematic drawings in each panel illustrate the spatial position of the Alexa Fluor 568 MBD and the length of the λ-DNA. The scale bar in panel (b) is 5 microns.

    Techniques Used: Fluorescence, Methylation, Labeling

    15) Product Images from "Archaeal Chromatin Proteins Cren7 and Sul7d Compact DNA by Bending and Bridging"

    Article Title: Archaeal Chromatin Proteins Cren7 and Sul7d Compact DNA by Bending and Bridging

    Journal: mBio

    doi: 10.1128/mBio.00804-20

    SM-TIRF visualization of the DNA compactions induced by Cren7 (A) and Sis7d (B) at different protein concentrations. Frames representing the indicated time points of a representative single λ DNA molecule from the video recorded at each protein concentration are shown plotted as a montage.
    Figure Legend Snippet: SM-TIRF visualization of the DNA compactions induced by Cren7 (A) and Sis7d (B) at different protein concentrations. Frames representing the indicated time points of a representative single λ DNA molecule from the video recorded at each protein concentration are shown plotted as a montage.

    Techniques Used: Protein Concentration

    16) Product Images from "Genome-wide epigenetic profiling of 5-hydroxymethylcytosine by long-read optical mapping"

    Article Title: Genome-wide epigenetic profiling of 5-hydroxymethylcytosine by long-read optical mapping

    Journal: bioRxiv

    doi: 10.1101/260166

    Assessment of 5-hmC labeling efficiency. Lambda DNA was nicked with Nt.BspQI (10 expected nicking sites) and labeled with either 5-hmC or fluorescent dUTP. 5-hmC was labeled according to our labeling scheme and the samples were mixed and imaged together in order to evaluate the labeling efficiency. A. representative field of view showing a mixed population of green (nicking) and red (5- hmC) labeled molecules. B. Histograms showing the number of labels per molecule for 5-hmC labeling (top) and nicking (bottom).
    Figure Legend Snippet: Assessment of 5-hmC labeling efficiency. Lambda DNA was nicked with Nt.BspQI (10 expected nicking sites) and labeled with either 5-hmC or fluorescent dUTP. 5-hmC was labeled according to our labeling scheme and the samples were mixed and imaged together in order to evaluate the labeling efficiency. A. representative field of view showing a mixed population of green (nicking) and red (5- hmC) labeled molecules. B. Histograms showing the number of labels per molecule for 5-hmC labeling (top) and nicking (bottom).

    Techniques Used: Labeling, Lambda DNA Preparation

    17) Product Images from "Trapped Chromatin Fibers Damage Flowing Red Blood Cells"

    Article Title: Trapped Chromatin Fibers Damage Flowing Red Blood Cells

    Journal: Advanced biosystems

    doi: 10.1002/adbi.201800040

    Mechanical entanglement of chromatin fibers on post-arrays in microfluidic chips. a,b) Schematics of the microfluidic device for in vitro studies of RBCs interaction with NETs. The cross-section of channels is 1066 × 35 μm. The diameter of the posts is 50 μm. The spacing between posts in different designs ranged from 10 to 80 μm. c,d) Fluorescence images show λ DNA (c), and NETs (d) trapped by posts inside the device and stained with Sytox orange dye. Both λ DNA and NETs wrap around the posts in the microfluidic device. Images were recorded under flow conditions, at 40 μL min −1 flow rate. Scale bars are 100 μm. e) SEM representative image of NETs inside a microfluidic device. Scale bar 5 μm. f) Bright field images extracted from a high-speed video (captured at 1000 frames per second) and show the breaking of a red blood cell trapped between λ DNA under flow conditions (40 μL min −1 ) in the microfluidic device over 50 ms. The λ in the Supporting Information.
    Figure Legend Snippet: Mechanical entanglement of chromatin fibers on post-arrays in microfluidic chips. a,b) Schematics of the microfluidic device for in vitro studies of RBCs interaction with NETs. The cross-section of channels is 1066 × 35 μm. The diameter of the posts is 50 μm. The spacing between posts in different designs ranged from 10 to 80 μm. c,d) Fluorescence images show λ DNA (c), and NETs (d) trapped by posts inside the device and stained with Sytox orange dye. Both λ DNA and NETs wrap around the posts in the microfluidic device. Images were recorded under flow conditions, at 40 μL min −1 flow rate. Scale bars are 100 μm. e) SEM representative image of NETs inside a microfluidic device. Scale bar 5 μm. f) Bright field images extracted from a high-speed video (captured at 1000 frames per second) and show the breaking of a red blood cell trapped between λ DNA under flow conditions (40 μL min −1 ) in the microfluidic device over 50 ms. The λ in the Supporting Information.

    Techniques Used: In Vitro, Fluorescence, Staining, Flow Cytometry, Mass Spectrometry

    Quantification of RBC fragments in blood. a) Imaging flow cytometry representative scatter plots showing the populations of RBCs and RBC fragments after passing blood through the microfluidic device with 10 μm—spaced posts without chromatin fibers (control), with λ DNA, and with NETs. b) Representative images of one RBC and two RBC fragments using fluorescence (CD235a+) and bright field. Scale bar 10 μm. c) The percentage of RBC fragments relative to the total number of CD235a+ events decreases with the increased spacing between posts. Three conditions are compared: control (blue), λ DNA (red), and NETs (green), ( N = 4 experimental repeats). Two-way ANOVA followed by Dunnett’s multiple comparisons test. *P
    Figure Legend Snippet: Quantification of RBC fragments in blood. a) Imaging flow cytometry representative scatter plots showing the populations of RBCs and RBC fragments after passing blood through the microfluidic device with 10 μm—spaced posts without chromatin fibers (control), with λ DNA, and with NETs. b) Representative images of one RBC and two RBC fragments using fluorescence (CD235a+) and bright field. Scale bar 10 μm. c) The percentage of RBC fragments relative to the total number of CD235a+ events decreases with the increased spacing between posts. Three conditions are compared: control (blue), λ DNA (red), and NETs (green), ( N = 4 experimental repeats). Two-way ANOVA followed by Dunnett’s multiple comparisons test. *P

    Techniques Used: Imaging, Flow Cytometry, Cytometry, Fluorescence

    18) Product Images from "Two glycosylase families diffusively scan DNA using a wedge residue to probe for and identify oxidatively damaged bases"

    Article Title: Two glycosylase families diffusively scan DNA using a wedge residue to probe for and identify oxidatively damaged bases

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1400386111

    Mean diffusion constants ( D traj ) and binding lifetimes of Fpg on undamaged, methylene blue (MB)-damaged, and OsO 4 -damaged λ DNA. Undamaged, MB-damaged, and OsO 4 -damaged λ DNA substrates were used in the single-molecule assay to measure
    Figure Legend Snippet: Mean diffusion constants ( D traj ) and binding lifetimes of Fpg on undamaged, methylene blue (MB)-damaged, and OsO 4 -damaged λ DNA. Undamaged, MB-damaged, and OsO 4 -damaged λ DNA substrates were used in the single-molecule assay to measure

    Techniques Used: Diffusion-based Assay, Binding Assay

    The diffusive behavior of WT and wedge mutant DNA glycosylases on undamaged λ DNA. Representative displacement vs. time traces for ( A ) Nei and ( B ) Nei Y72A. Overall diffusion constants ( D traj ) are determined by linear fits to the first 25% of
    Figure Legend Snippet: The diffusive behavior of WT and wedge mutant DNA glycosylases on undamaged λ DNA. Representative displacement vs. time traces for ( A ) Nei and ( B ) Nei Y72A. Overall diffusion constants ( D traj ) are determined by linear fits to the first 25% of

    Techniques Used: Mutagenesis, Diffusion-based Assay

    Effect of the number of damages in the λ DNA substrate on the diffusion constants and binding lifetimes of Fpg, Nei, and Nth. Undamaged (light gray), low-dose (gray), and high-dose (dark gray) treated λ DNA substrates were used in the
    Figure Legend Snippet: Effect of the number of damages in the λ DNA substrate on the diffusion constants and binding lifetimes of Fpg, Nei, and Nth. Undamaged (light gray), low-dose (gray), and high-dose (dark gray) treated λ DNA substrates were used in the

    Techniques Used: Diffusion-based Assay, Binding Assay

    19) Product Images from "Epigenetic Optical Mapping of 5-Hydroxymethylcytosine in Nanochannel Arrays"

    Article Title: Epigenetic Optical Mapping of 5-Hydroxymethylcytosine in Nanochannel Arrays

    Journal: ACS Nano

    doi: 10.1021/acsnano.8b03023

    Assessment of 5-hmC labeling efficiency. Lambda DNA was nicked with Nt.BspQI (nine expected labeling spots) and labeled with either 5-hmC or fluorescent dUTP. 5-hmC was labeled according to our labeling scheme, and the samples were mixed and imaged together in order to evaluate the labeling efficiency. (A) Representative field of view showing a mixed population of green (nicking) and red (5-hmC) labeled molecules. (B) Histograms showing the number of labels per molecule for 5-hmC labeling (top) and nicking (bottom).
    Figure Legend Snippet: Assessment of 5-hmC labeling efficiency. Lambda DNA was nicked with Nt.BspQI (nine expected labeling spots) and labeled with either 5-hmC or fluorescent dUTP. 5-hmC was labeled according to our labeling scheme, and the samples were mixed and imaged together in order to evaluate the labeling efficiency. (A) Representative field of view showing a mixed population of green (nicking) and red (5-hmC) labeled molecules. (B) Histograms showing the number of labels per molecule for 5-hmC labeling (top) and nicking (bottom).

    Techniques Used: Labeling, Lambda DNA Preparation

    20) Product Images from "Dynamic DNA binding licenses a repair factor to bypass roadblocks in search of DNA lesions"

    Article Title: Dynamic DNA binding licenses a repair factor to bypass roadblocks in search of DNA lesions

    Journal: Nature Communications

    doi: 10.1038/ncomms10607

    Visualizing protein diffusion on aligned arrays of DNA molecules. ( a ) An illustration of the DNA curtains assay ( Supplementary Information ). A quartz microscope slide is fabricated with an alternating pattern of linear chromium (Cr) diffusion barriers and oval pedestals (∼30 nm tall; 13 μm separation). The pedestals are coated with anti-digoxigenin antibodies. The flowcell surface is passivated with a fluid lipid bilayer (∼5 nm tall), and DNA (from λ-phage, 48,502 bp) is affixed to the bilayer via a biotin-streptavidin linkage. Buffer flow is used to organize DNA molecules at the linear diffusion barriers and the free DNA end is immobilized at the Cr pedestals via a digoxigenin–antibody interaction. DNA molecules that are tethered at both ends remain extended when buffer flow is turned off. ( b ) A double-tethered DNA curtain. DNA is stained with YOYO-1, a fluorescent intercalating dye (green; top). Quantum dot (QD)-conjugated Msh2–Msh3 binds specifically to the DNA molecules (magenta; bottom). We did not observe any QD signal when Msh2–Msh3 was omitted from the incubation, or when Msh2–Msh3 was incubated with an unconjugated QD. YOYO-1 was omitted from subsequent experiments because it can cause laser-induced DNA damage. Scale bar: 10 μm. ( c ) Kymograph of a single diffusing Msh2–Msh3 protein. QDs blinking (white arrows) indicates that these traces arise from single fluorescent particles.
    Figure Legend Snippet: Visualizing protein diffusion on aligned arrays of DNA molecules. ( a ) An illustration of the DNA curtains assay ( Supplementary Information ). A quartz microscope slide is fabricated with an alternating pattern of linear chromium (Cr) diffusion barriers and oval pedestals (∼30 nm tall; 13 μm separation). The pedestals are coated with anti-digoxigenin antibodies. The flowcell surface is passivated with a fluid lipid bilayer (∼5 nm tall), and DNA (from λ-phage, 48,502 bp) is affixed to the bilayer via a biotin-streptavidin linkage. Buffer flow is used to organize DNA molecules at the linear diffusion barriers and the free DNA end is immobilized at the Cr pedestals via a digoxigenin–antibody interaction. DNA molecules that are tethered at both ends remain extended when buffer flow is turned off. ( b ) A double-tethered DNA curtain. DNA is stained with YOYO-1, a fluorescent intercalating dye (green; top). Quantum dot (QD)-conjugated Msh2–Msh3 binds specifically to the DNA molecules (magenta; bottom). We did not observe any QD signal when Msh2–Msh3 was omitted from the incubation, or when Msh2–Msh3 was incubated with an unconjugated QD. YOYO-1 was omitted from subsequent experiments because it can cause laser-induced DNA damage. Scale bar: 10 μm. ( c ) Kymograph of a single diffusing Msh2–Msh3 protein. QDs blinking (white arrows) indicates that these traces arise from single fluorescent particles.

    Techniques Used: Diffusion-based Assay, Microscopy, Flow Cytometry, Staining, Incubation

    21) Product Images from "Endogenous single-strand DNA breaks at RNA polymerase II promoters in Saccharomyces cerevisiae"

    Article Title: Endogenous single-strand DNA breaks at RNA polymerase II promoters in Saccharomyces cerevisiae

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky743

    Breakage of S. cerevisiae gDNA and λ DNA at preformed ss nicks upon molecular combing. ( A-C ) Molecular combing of nick-translated gDNA from S. cerevisiae . Biotinylated nucleotides were incorporated by nick-translation conducted either in limiting (L), or non-limiting/standard conditions (N), into agarose-embedded gDNA of unperturbed, non-synchronized (A and C), or G1-synchronized (B) BY4741 cells. Biotin was detected by AlexaFluor 647-conjugated anti-biotin antibody (red) and DNA molecules were stained with YOYO-1 (green). Panel D shows examples of co-localization of nicks labeled with TdT (magenta) and R-loops labeled with the RNA:DNA hybrid specific S9.6 antibody (red), when both entities were visualized in the same sample. The percentage of co-labeled spots was estimated ∼10% of all nick-related DNA associated spots. Arrows indicate examples of co-localization. ( E–G ) Molecular combing of λ DNA. Representative images of YOYO-1 stained (green) control (F) and Nt.BbvCI nickase-treated (G) λ DNA. The size distribution histograms of combed DNA molecules before (blue) and after (orange) nickase treatment are shown in panel E. The full length intact ds λ DNA (48.5 kb) corresponds to 16.2 μm (calculated with 3 bp/nm helical repeat length), i.e. the majority of λ DNA molecules were fragmented after combing alone. Images of DNA fibers were assembled from the fields-of-view analyzed, except for panels F and G which show the original fields-of-view. For statistics see Supplementary Tables S1–S5 .
    Figure Legend Snippet: Breakage of S. cerevisiae gDNA and λ DNA at preformed ss nicks upon molecular combing. ( A-C ) Molecular combing of nick-translated gDNA from S. cerevisiae . Biotinylated nucleotides were incorporated by nick-translation conducted either in limiting (L), or non-limiting/standard conditions (N), into agarose-embedded gDNA of unperturbed, non-synchronized (A and C), or G1-synchronized (B) BY4741 cells. Biotin was detected by AlexaFluor 647-conjugated anti-biotin antibody (red) and DNA molecules were stained with YOYO-1 (green). Panel D shows examples of co-localization of nicks labeled with TdT (magenta) and R-loops labeled with the RNA:DNA hybrid specific S9.6 antibody (red), when both entities were visualized in the same sample. The percentage of co-labeled spots was estimated ∼10% of all nick-related DNA associated spots. Arrows indicate examples of co-localization. ( E–G ) Molecular combing of λ DNA. Representative images of YOYO-1 stained (green) control (F) and Nt.BbvCI nickase-treated (G) λ DNA. The size distribution histograms of combed DNA molecules before (blue) and after (orange) nickase treatment are shown in panel E. The full length intact ds λ DNA (48.5 kb) corresponds to 16.2 μm (calculated with 3 bp/nm helical repeat length), i.e. the majority of λ DNA molecules were fragmented after combing alone. Images of DNA fibers were assembled from the fields-of-view analyzed, except for panels F and G which show the original fields-of-view. For statistics see Supplementary Tables S1–S5 .

    Techniques Used: Nick Translation, Staining, Labeling

    22) Product Images from "Simple and Efficient Room-Temperature Release of Biotinylated Nucleic Acids from Streptavidin and Its Application to Selective Molecular Detection"

    Article Title: Simple and Efficient Room-Temperature Release of Biotinylated Nucleic Acids from Streptavidin and Its Application to Selective Molecular Detection

    Journal: Analytical chemistry

    doi: 10.1021/acs.analchem.9b01873

    (a) Schematic showing isolation of biotinylated DNA. A mixture of biotinylated (red) and nonbiotinylated (blue) DNA fragments are incubated with streptavidin magnetic beads (l). The beads with bound DNA are collected magnetically, and nonbiotinylated DNA is washed away (2). Biotinylated DNA is eluted with 12.5% (v/v) phenol (3). (b) Gel analysis showing: unprocessed 48.5 kbp (i) λ -phage DNA (lane l); λ -phage DNA biotinylated at one end digested with PspXI to produce fragments approximately 33.5 (ii) and 15 kbp (iii) in length (lane 2); and the fragments after isolation and phenol elution from streptavidin beads (lane 3). The red arrow indicates the biotinylated ~15 kbp fragment, (c) Normalized SS-nanopore event histograms of ECD for the initial admixture (top, n = 1158) and the product of bead isolation (bottom, n = 519). SS-nanopore diameters are 6.1 and 6.1 nm, respectively. Lower ECD corresponds to lower molecular weight (i.e., the 15 kbp biotinylated DNA). Insets show a typical conductance trace for each measurement, with initial (open pore) conductance to the left and after addition of the DNA to the right. Spikes indicate molecular translocations. Scale bars are 500 ms (horizontal) and 1 nS (vertical).
    Figure Legend Snippet: (a) Schematic showing isolation of biotinylated DNA. A mixture of biotinylated (red) and nonbiotinylated (blue) DNA fragments are incubated with streptavidin magnetic beads (l). The beads with bound DNA are collected magnetically, and nonbiotinylated DNA is washed away (2). Biotinylated DNA is eluted with 12.5% (v/v) phenol (3). (b) Gel analysis showing: unprocessed 48.5 kbp (i) λ -phage DNA (lane l); λ -phage DNA biotinylated at one end digested with PspXI to produce fragments approximately 33.5 (ii) and 15 kbp (iii) in length (lane 2); and the fragments after isolation and phenol elution from streptavidin beads (lane 3). The red arrow indicates the biotinylated ~15 kbp fragment, (c) Normalized SS-nanopore event histograms of ECD for the initial admixture (top, n = 1158) and the product of bead isolation (bottom, n = 519). SS-nanopore diameters are 6.1 and 6.1 nm, respectively. Lower ECD corresponds to lower molecular weight (i.e., the 15 kbp biotinylated DNA). Insets show a typical conductance trace for each measurement, with initial (open pore) conductance to the left and after addition of the DNA to the right. Spikes indicate molecular translocations. Scale bars are 500 ms (horizontal) and 1 nS (vertical).

    Techniques Used: Isolation, Incubation, Magnetic Beads, Molecular Weight

    23) Product Images from "Wafer-scale integration of sacrificial nanofluidic chips for detecting and manipulating single DNA molecules"

    Article Title: Wafer-scale integration of sacrificial nanofluidic chips for detecting and manipulating single DNA molecules

    Journal: Nature Communications

    doi: 10.1038/ncomms14243

    Single-molecule fluorescence imaging of DNA in sacrificial Si nanochannels. ( a ) Optical image of nanofluidic regions with nanopillars and nanochannels (40 nm deep, 200 nm wide and 500 nm pitch). ( b ) Selected fluorescence images showing λ-DNA flowing through nanopillars and nanochannels corresponding to the optical images in a . Magenta and yellow dash lines indicate the pillar interface designed for straddling and the nanochannels entry point, respectively. Here frame 1 is defined the first frame the DNA molecule enters the imaged area. The DNA flowed from the bottom to the top. ( c ) The location-dependent DNA extension due to its hydrodynamic interactions with nanostructures, with the optical graph of the nanofluidic structures added as a location reference. Here the x axis origin is set as the nanochannel entry. Each black square dot represents the DNA extension in one frame, and the data point of frame 5 is labelled. The time interval between adjacent frames was ∼18 ms. The horizontal green dash-dot line indicates the estimated dyed lambda DNA extension when it is fully stretched. The scale bar in a is 10 μm.
    Figure Legend Snippet: Single-molecule fluorescence imaging of DNA in sacrificial Si nanochannels. ( a ) Optical image of nanofluidic regions with nanopillars and nanochannels (40 nm deep, 200 nm wide and 500 nm pitch). ( b ) Selected fluorescence images showing λ-DNA flowing through nanopillars and nanochannels corresponding to the optical images in a . Magenta and yellow dash lines indicate the pillar interface designed for straddling and the nanochannels entry point, respectively. Here frame 1 is defined the first frame the DNA molecule enters the imaged area. The DNA flowed from the bottom to the top. ( c ) The location-dependent DNA extension due to its hydrodynamic interactions with nanostructures, with the optical graph of the nanofluidic structures added as a location reference. Here the x axis origin is set as the nanochannel entry. Each black square dot represents the DNA extension in one frame, and the data point of frame 5 is labelled. The time interval between adjacent frames was ∼18 ms. The horizontal green dash-dot line indicates the estimated dyed lambda DNA extension when it is fully stretched. The scale bar in a is 10 μm.

    Techniques Used: Fluorescence, Imaging, Mass Spectrometry, Lambda DNA Preparation

    24) Product Images from "Feature Tracking for High Speed AFM Imaging of Biopolymers"

    Article Title: Feature Tracking for High Speed AFM Imaging of Biopolymers

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19041044

    Amplitude signal during tracking of small features, pre- (sinusoidal, blue) and post-filtering (sinusoid removed, orange). Note that the sharp edges of the Si steps ( top plot) yields sharp edges in the filtered signal, while the rounded edges of the λ -DNA ( bottom plot) lead to a much smaller and noisier edge signal.
    Figure Legend Snippet: Amplitude signal during tracking of small features, pre- (sinusoidal, blue) and post-filtering (sinusoid removed, orange). Note that the sharp edges of the Si steps ( top plot) yields sharp edges in the filtered signal, while the rounded edges of the λ -DNA ( bottom plot) lead to a much smaller and noisier edge signal.

    Techniques Used:

    25) Product Images from "One step construction of PCR mutagenized libraries for genetic analysis by recombination cloning"

    Article Title: One step construction of PCR mutagenized libraries for genetic analysis by recombination cloning

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm583

    Cre/ lox cloning of linear DNA in vivo . ( A ) A 2255-bp lox-kanMX-lox fragment was liberated by restriction digestion of pJBN240 (fragment lane; marked by *), and recombined with a circular pAS2 lox P target plasmid (target lane) in Cre/Gam-expressing bacterial cells. The frequency of Kn R to Ap R transformants was determined. Mini-prep DNA was prepared from seven randomly chosen Kn R clones and digested with NcoI and SalI, releasing fragments of 8392, 1112 and 474 bp in correct recombinants. Std lane: lambda DNA digested with BstEII. Within the bracketed range fragments are 4822, 4324, 3675, 2323, 1929, 1371, 1264 and 702 bp. ( B ) A 1550-bp lox-tet-lox fragment (fragment lane; *) from pAK005 was recombined with pAS2 lox P (target). In this case, plasmid DNA from Tc R clones was digested with EcoRV, releasing fragments of 4746, 2554, 1394 and 1206 for correct recombinants. ( C ) The same lox-tet-lox fragment in (B) was recombined with circular pJBN240 (target), and Tc R clones were digested with EcoRV to determine if the lox-kanMX-lox region of pJBN240 had been replaced by tet sequences. Such recombinants exhibit restriction fragments of 4869 and 1752 bp following EcoRV digestion.
    Figure Legend Snippet: Cre/ lox cloning of linear DNA in vivo . ( A ) A 2255-bp lox-kanMX-lox fragment was liberated by restriction digestion of pJBN240 (fragment lane; marked by *), and recombined with a circular pAS2 lox P target plasmid (target lane) in Cre/Gam-expressing bacterial cells. The frequency of Kn R to Ap R transformants was determined. Mini-prep DNA was prepared from seven randomly chosen Kn R clones and digested with NcoI and SalI, releasing fragments of 8392, 1112 and 474 bp in correct recombinants. Std lane: lambda DNA digested with BstEII. Within the bracketed range fragments are 4822, 4324, 3675, 2323, 1929, 1371, 1264 and 702 bp. ( B ) A 1550-bp lox-tet-lox fragment (fragment lane; *) from pAK005 was recombined with pAS2 lox P (target). In this case, plasmid DNA from Tc R clones was digested with EcoRV, releasing fragments of 4746, 2554, 1394 and 1206 for correct recombinants. ( C ) The same lox-tet-lox fragment in (B) was recombined with circular pJBN240 (target), and Tc R clones were digested with EcoRV to determine if the lox-kanMX-lox region of pJBN240 had been replaced by tet sequences. Such recombinants exhibit restriction fragments of 4869 and 1752 bp following EcoRV digestion.

    Techniques Used: Clone Assay, In Vivo, Plasmid Preparation, Expressing, Lambda DNA Preparation

    26) Product Images from "Simultaneously Sizing and Quantitating Zepto-Mole DNA at High-Throughput in Free Solution"

    Article Title: Simultaneously Sizing and Quantitating Zepto-Mole DNA at High-Throughput in Free Solution

    Journal: Chemistry (Weinheim an der Bergstrasse, Germany)

    doi: 10.1002/chem.201403861

    BaNC-HDC for high-throughput DNA sizing and quantitation. Sample I: 1 kbp plus DNA ladder at the total concentration of 10 ng/µL, and the concentrations of individual fragments were presented in . Sample II: digested λ-DNA at the
    Figure Legend Snippet: BaNC-HDC for high-throughput DNA sizing and quantitation. Sample I: 1 kbp plus DNA ladder at the total concentration of 10 ng/µL, and the concentrations of individual fragments were presented in . Sample II: digested λ-DNA at the

    Techniques Used: High Throughput Screening Assay, Quantitation Assay, Concentration Assay

    27) Product Images from "Extreme mechanical diversity of human telomeric DNA revealed by fluorescence-force spectroscopy"

    Article Title: Extreme mechanical diversity of human telomeric DNA revealed by fluorescence-force spectroscopy

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1815162116

    Conformational dynamics of hTel22 under tension in 100 mM K + . ( A ) The G4 strand is annealed to a biotinylated strand and immobilized on a neutravidin-coated quartz surface. The other end is connected to a 1-μm-diameter, optically trapped bead through a λ-DNA. The G4 construct was stretched to ∼28 pN in 6.5 s. FRET was measured between Cy3 (donor) and Cy5 (acceptor) as a function of force. ( Inset ) Unfolding (black) and refolding (red). ( B ) A representative single-molecule time trace (20-ms integration time) of donor and acceptor intensities and corresponding E . Stretching and relaxation occurred at a stage speed of 455 nm/s. ( C ) f unfold and f refold in different stretching cycles from a single molecule shown in B . ( D and E ) Distributions of f unfold ( D ) and f refold ( E ). The red and black curves in D ). Only those cycles showing complete unfolding were included ( n = 188). ( F ) E vs. force example curves, one representative from each of the four peaks in f unfold distribution ( i – iv ), one representative of a ultrastable state ( v ), and one representative of gradual partial unfolding/folding without hysteresis ( vi ). ( G and H ) E vs. force curves of (dT) 22 ( G ) and hTel22 mid E population ( H ). Corresponding E histograms are shown in insets. Error bars represent SEs.
    Figure Legend Snippet: Conformational dynamics of hTel22 under tension in 100 mM K + . ( A ) The G4 strand is annealed to a biotinylated strand and immobilized on a neutravidin-coated quartz surface. The other end is connected to a 1-μm-diameter, optically trapped bead through a λ-DNA. The G4 construct was stretched to ∼28 pN in 6.5 s. FRET was measured between Cy3 (donor) and Cy5 (acceptor) as a function of force. ( Inset ) Unfolding (black) and refolding (red). ( B ) A representative single-molecule time trace (20-ms integration time) of donor and acceptor intensities and corresponding E . Stretching and relaxation occurred at a stage speed of 455 nm/s. ( C ) f unfold and f refold in different stretching cycles from a single molecule shown in B . ( D and E ) Distributions of f unfold ( D ) and f refold ( E ). The red and black curves in D ). Only those cycles showing complete unfolding were included ( n = 188). ( F ) E vs. force example curves, one representative from each of the four peaks in f unfold distribution ( i – iv ), one representative of a ultrastable state ( v ), and one representative of gradual partial unfolding/folding without hysteresis ( vi ). ( G and H ) E vs. force curves of (dT) 22 ( G ) and hTel22 mid E population ( H ). Corresponding E histograms are shown in insets. Error bars represent SEs.

    Techniques Used: Construct, Mass Spectrometry

    28) Product Images from "Fluorescent marker for direct detection of specific dsDNA sequences"

    Article Title: Fluorescent marker for direct detection of specific dsDNA sequences

    Journal: Analytical chemistry

    doi: 10.1021/ac9019895

    Histogram of measured binding positions for 204 markers bound to slide-immobilized λ-DNA molecules. The solid line is a Gaussian fit to the data. Dashed vertical lines indicate expected EcoRI target sites 1-5 on fully stretched λ-DNA.
    Figure Legend Snippet: Histogram of measured binding positions for 204 markers bound to slide-immobilized λ-DNA molecules. The solid line is a Gaussian fit to the data. Dashed vertical lines indicate expected EcoRI target sites 1-5 on fully stretched λ-DNA.

    Techniques Used: Binding Assay

    Representative fluorescence images of marker-tagged λ-DNA, stretched and immobilized on slides, with the corresponding fluorescence intensity profiles along the DNA backbone; bead positions are clearly evident as peaks in the profiles. Dashed vertical lines indicate expected EcoRI target positions 1-5 on fully stretched λ-DNA. Scale bar is 5 μm.
    Figure Legend Snippet: Representative fluorescence images of marker-tagged λ-DNA, stretched and immobilized on slides, with the corresponding fluorescence intensity profiles along the DNA backbone; bead positions are clearly evident as peaks in the profiles. Dashed vertical lines indicate expected EcoRI target positions 1-5 on fully stretched λ-DNA. Scale bar is 5 μm.

    Techniques Used: Fluorescence, Marker

    29) Product Images from "Characterization of the Type III restriction endonuclease PstII from Providencia stuartii"

    Article Title: Characterization of the Type III restriction endonuclease PstII from Providencia stuartii

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki787

    The Type III enzymes EcoP15I and PstII cannot mutually activate cleavage of T7 coliphage DNA. ( A ) Representative schematic (not to scale) of the relative orientation of EcoP15I and PstII sites in lambda (λ) and T7 phage genomic DNA. Site orientations (arrowheads) are defined as in Figure 2A . ( B ) Cleavage of λ and T7 genomic DNA by mixtures of Type III enzymes. 500 ng of λ or T7 phage DNA was mixed with 50 nM EcoP15I and/or 129 nM PstII mixture as shown in the presence of 4 mM ATP. Where indicated AdoMet was added to 100 μM. Following incubation for 1 h at 37°C, substrate and products were separated by agarose gel electrophoresis.
    Figure Legend Snippet: The Type III enzymes EcoP15I and PstII cannot mutually activate cleavage of T7 coliphage DNA. ( A ) Representative schematic (not to scale) of the relative orientation of EcoP15I and PstII sites in lambda (λ) and T7 phage genomic DNA. Site orientations (arrowheads) are defined as in Figure 2A . ( B ) Cleavage of λ and T7 genomic DNA by mixtures of Type III enzymes. 500 ng of λ or T7 phage DNA was mixed with 50 nM EcoP15I and/or 129 nM PstII mixture as shown in the presence of 4 mM ATP. Where indicated AdoMet was added to 100 μM. Following incubation for 1 h at 37°C, substrate and products were separated by agarose gel electrophoresis.

    Techniques Used: Incubation, Agarose Gel Electrophoresis

    30) Product Images from "Ionic effects on the elasticity of single DNA molecules"

    Article Title: Ionic effects on the elasticity of single DNA molecules

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi:

    Ionic effects on the elastic response of single λ DNA molecules. A portion of the overstretch transition is shown for all molecules. ( A ) Stretching λ DNA in 1 (○), 50 (▵), and 500 (◊) mM NaCl. Individual molecules often display melting hysteresis (arrows) upon relaxation of the overstretched form. ( B ) Stretching λ DNA in the presence of di- and trivalent cations with BB: 100 μM MgCl 2 (□), 100 μM putrescine 2+ (○), 100 μM spermidine 3+ (▵), or 25 μM Co(NH 3 ) 6 3+ (◊) . To compare behavior in BB alone refer to the F–x curve for 1 mM NaCl.
    Figure Legend Snippet: Ionic effects on the elastic response of single λ DNA molecules. A portion of the overstretch transition is shown for all molecules. ( A ) Stretching λ DNA in 1 (○), 50 (▵), and 500 (◊) mM NaCl. Individual molecules often display melting hysteresis (arrows) upon relaxation of the overstretched form. ( B ) Stretching λ DNA in the presence of di- and trivalent cations with BB: 100 μM MgCl 2 (□), 100 μM putrescine 2+ (○), 100 μM spermidine 3+ (▵), or 25 μM Co(NH 3 ) 6 3+ (◊) . To compare behavior in BB alone refer to the F–x curve for 1 mM NaCl.

    Techniques Used:

    31) Product Images from "Single-molecule imaging reveals control of parental histone recycling by free histones during DNA replication"

    Article Title: Single-molecule imaging reveals control of parental histone recycling by free histones during DNA replication

    Journal: Science Advances

    doi: 10.1126/sciadv.abc0330

    Fluorescent nucleosomes on λ DNA are discretely distributed in a beads-on-a-string manner. ( A ) Crystal structure of the Xenopus nucleosome (Protein Data Bank 1AOI) illustrating the location and type of fluorescent dye (Cy5 or Alexa Fluor 647, abbreviated as A647) used to label histones. ( B ) SDS–polyacrylamide gel electrophoresis analysis of wild-type (WT) and fluorescently labeled histones and histone octamers. MW, molecular weight. ( C and D ) Native EMSA (top) and MNase protection assay (bottom) for nucleosomes labeled at H2A-K119C Cy5 (C) and H4-E63C A647 (D) reconstituted on λ DNA at increasing DNA:octamer ratios (1:0, 1:40, 1:120, and 1:200). kbp, kilobase pair. ( E ) Schematic of fluorescent λ nucleosomes immobilized in the microfluidic device for single-molecule imaging. ( F and G ) Single-molecule imaging of nucleosomes labeled at H2A-K119C Cy5 (F) and H4-E63C A647 (G) reconstituted on λ DNA at increasing DNA:octamer ratios (1:0, 1:50, 1:125, 1:200, 1:275, and 1:350). ( H and I ) Single-molecule quantification of the DNA contour length for nucleosomes labeled at H2A-K119C Cy5 (H) and H4-E63C A647 (I) reconstituted on λ DNA at increasing DNA:octamer ratios (1:0, 1:40, 1:120, and 1:200). The DNA length of individual molecules was measured on the basis of SYTOX Orange staining of the DNA (approximately 400 molecules at each histone octamer concentration).
    Figure Legend Snippet: Fluorescent nucleosomes on λ DNA are discretely distributed in a beads-on-a-string manner. ( A ) Crystal structure of the Xenopus nucleosome (Protein Data Bank 1AOI) illustrating the location and type of fluorescent dye (Cy5 or Alexa Fluor 647, abbreviated as A647) used to label histones. ( B ) SDS–polyacrylamide gel electrophoresis analysis of wild-type (WT) and fluorescently labeled histones and histone octamers. MW, molecular weight. ( C and D ) Native EMSA (top) and MNase protection assay (bottom) for nucleosomes labeled at H2A-K119C Cy5 (C) and H4-E63C A647 (D) reconstituted on λ DNA at increasing DNA:octamer ratios (1:0, 1:40, 1:120, and 1:200). kbp, kilobase pair. ( E ) Schematic of fluorescent λ nucleosomes immobilized in the microfluidic device for single-molecule imaging. ( F and G ) Single-molecule imaging of nucleosomes labeled at H2A-K119C Cy5 (F) and H4-E63C A647 (G) reconstituted on λ DNA at increasing DNA:octamer ratios (1:0, 1:50, 1:125, 1:200, 1:275, and 1:350). ( H and I ) Single-molecule quantification of the DNA contour length for nucleosomes labeled at H2A-K119C Cy5 (H) and H4-E63C A647 (I) reconstituted on λ DNA at increasing DNA:octamer ratios (1:0, 1:40, 1:120, and 1:200). The DNA length of individual molecules was measured on the basis of SYTOX Orange staining of the DNA (approximately 400 molecules at each histone octamer concentration).

    Techniques Used: Polyacrylamide Gel Electrophoresis, Labeling, Molecular Weight, Imaging, Staining, Concentration Assay

    32) Product Images from "Deconvolution of Nucleic-acid Length Distributions: A Gel Electrophoresis Analysis Tool and Applications"

    Article Title: Deconvolution of Nucleic-acid Length Distributions: A Gel Electrophoresis Analysis Tool and Applications

    Journal: bioRxiv

    doi: 10.1101/636936

    Agarose-gel analysis of tagmentation products of phage- λ DNA analyzed on (a) high-resolution and (c) low-resolution ( i.e ., mini) agarose gels. Size-distribution fits for the high-resolution gel (b) and mini gel (d) of the same tagmented λ -DNA sample subjected to increasing numbers of PCR-amplification cycles (lanes 3-5 in (a), lanes 2-4 in (c)): 8 cycles (top plots), 14 cycles (middle plots), and 20 cycles (bottom plots) in both (b) and (d). Vertical dashed lines (light red in (b), (d)) give the positions of maxima in the discrete molecular-weight ladder (blue ROI in (b), (b)).
    Figure Legend Snippet: Agarose-gel analysis of tagmentation products of phage- λ DNA analyzed on (a) high-resolution and (c) low-resolution ( i.e ., mini) agarose gels. Size-distribution fits for the high-resolution gel (b) and mini gel (d) of the same tagmented λ -DNA sample subjected to increasing numbers of PCR-amplification cycles (lanes 3-5 in (a), lanes 2-4 in (c)): 8 cycles (top plots), 14 cycles (middle plots), and 20 cycles (bottom plots) in both (b) and (d). Vertical dashed lines (light red in (b), (d)) give the positions of maxima in the discrete molecular-weight ladder (blue ROI in (b), (b)).

    Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Molecular Weight

    33) Product Images from "Inserting Extrahelical Structures into Long DNA Substrates for Single-Molecule Studies of DNA Mismatch Repair"

    Article Title: Inserting Extrahelical Structures into Long DNA Substrates for Single-Molecule Studies of DNA Mismatch Repair

    Journal: Methods in enzymology

    doi: 10.1016/bs.mie.2016.08.006

    A strategy for inserting extrahelical structures into λ-DNA. Step 1: A nicking cassette is inserted into the λ-phage genome in vivo . Step 2: Recombinant λ-DNA is purified. Step 3: Extrahelical structures are introduced via a nicking enzyme-based oligonucleotide insertion strategy. B and D represent incorporated biotinylated and digoxigenin-labeled oligonucleotides, respectively. Step 4: The resulting DNA substrates are assembled into microfluidic DNA curtains and imaged via single-molecule microscopy.
    Figure Legend Snippet: A strategy for inserting extrahelical structures into λ-DNA. Step 1: A nicking cassette is inserted into the λ-phage genome in vivo . Step 2: Recombinant λ-DNA is purified. Step 3: Extrahelical structures are introduced via a nicking enzyme-based oligonucleotide insertion strategy. B and D represent incorporated biotinylated and digoxigenin-labeled oligonucleotides, respectively. Step 4: The resulting DNA substrates are assembled into microfluidic DNA curtains and imaged via single-molecule microscopy.

    Techniques Used: In Vivo, Recombinant, Purification, Labeling, Microscopy

    Constructing λ-DNA with an internal single-stranded DNA flap. (a) Schematic of the nickase-based oligonucleotide replacement reaction. B and D represent biotinylated and digoxigenin-labeled oligonucleotides, respectively. (b) A restriction digest can be used to quantify oligonucleotide replacement rapidly. Inserting a 5′-ssDNA flap, but not a homoduplex oligonucleotide, produces a 2.7-kb fragment. Homoduplex and mock-treated λ-DNA are further digested into 2 and 0.7-kb fragments (0.7-kb band not shown). (c) A denaturing (alkaline) agarose gel confirms insertion and religation of the λ-DNA substrates. Note that the top and bottom DNA strands are separated only for the 5′-ssDNA flap substrate.
    Figure Legend Snippet: Constructing λ-DNA with an internal single-stranded DNA flap. (a) Schematic of the nickase-based oligonucleotide replacement reaction. B and D represent biotinylated and digoxigenin-labeled oligonucleotides, respectively. (b) A restriction digest can be used to quantify oligonucleotide replacement rapidly. Inserting a 5′-ssDNA flap, but not a homoduplex oligonucleotide, produces a 2.7-kb fragment. Homoduplex and mock-treated λ-DNA are further digested into 2 and 0.7-kb fragments (0.7-kb band not shown). (c) A denaturing (alkaline) agarose gel confirms insertion and religation of the λ-DNA substrates. Note that the top and bottom DNA strands are separated only for the 5′-ssDNA flap substrate.

    Techniques Used: Labeling, Agarose Gel Electrophoresis

    Visualizing Msh2–Msh3 binding to an extrahelical ssDNA flap. (A) Cartoon representation ( left ) and picture ( right ) of a microscope-mounted flowcell. The picture highlights the microfluidic connectors and the quartz prism. (B) Distribution of Msh2–Msh3 molecules on flap-containing λ-DNA. The black line is a Gaussian fit to the data ( n =503). The center of the peak corresponds to the expected location of the lesion (20 kb from the top DNA barrier, error is SD). Inset : Seven representative λ-DNA molecules ( light gray vertical lines ) with flap-bound Msh2–Msh3 ( black points ). Msh2–Msh3 recognizes lesions via (C) 1D diffusion along the DNA or (D) a direct encounter (3D collision). Each panel shows a cartoon illustration ( top ), kymograph ( middle ), and single-particle trajectory of Msh2–Msh3 ( black ) binding a DNA flap (3′-ssDNA flap; marked as 3′). Asterisk indicates transient binding by a second Msh2–Msh3. In both cases, Msh2–Msh3 releases the flap and continues to diffuse on homoduplex λ-DNA.
    Figure Legend Snippet: Visualizing Msh2–Msh3 binding to an extrahelical ssDNA flap. (A) Cartoon representation ( left ) and picture ( right ) of a microscope-mounted flowcell. The picture highlights the microfluidic connectors and the quartz prism. (B) Distribution of Msh2–Msh3 molecules on flap-containing λ-DNA. The black line is a Gaussian fit to the data ( n =503). The center of the peak corresponds to the expected location of the lesion (20 kb from the top DNA barrier, error is SD). Inset : Seven representative λ-DNA molecules ( light gray vertical lines ) with flap-bound Msh2–Msh3 ( black points ). Msh2–Msh3 recognizes lesions via (C) 1D diffusion along the DNA or (D) a direct encounter (3D collision). Each panel shows a cartoon illustration ( top ), kymograph ( middle ), and single-particle trajectory of Msh2–Msh3 ( black ) binding a DNA flap (3′-ssDNA flap; marked as 3′). Asterisk indicates transient binding by a second Msh2–Msh3. In both cases, Msh2–Msh3 releases the flap and continues to diffuse on homoduplex λ-DNA.

    Techniques Used: Binding Assay, Microscopy, Diffusion-based Assay

    34) Product Images from "Packaging of Single DNA Molecules by the Yeast Mitochondrial Protein Abf2p"

    Article Title: Packaging of Single DNA Molecules by the Yeast Mitochondrial Protein Abf2p

    Journal: Biophysical Journal

    doi:

    ( a ) Side view of the flow cell showing the trapping and excitation laser beams. ( b ) Top view of the flow cell. An individual DNA molecule held by an optical trap ( orange ) via its attached bead, and extended by flowing buffer, is moved into protein solution by translating the stage holding the flow cell perpendicular to the direction of flow. DNA was stained with YOYO-1 dye, allowing the compaction to be observed using fluorescence microscopy. The molecule was then moved back to the DNA side of the flow cell (which was protein-free), and the decompaction of the molecule was observed as protein left it, ultimately returning to its original length. ( c ) Time-lapse images of a lambda-phage DNA dimer (35 μ m contour length) undergoing compaction by Abf2p. The Abf2p concentration is 2 μ M. The time interval between successive frames is 0.5 s. The buffer flow speed is 63 μ m/s.
    Figure Legend Snippet: ( a ) Side view of the flow cell showing the trapping and excitation laser beams. ( b ) Top view of the flow cell. An individual DNA molecule held by an optical trap ( orange ) via its attached bead, and extended by flowing buffer, is moved into protein solution by translating the stage holding the flow cell perpendicular to the direction of flow. DNA was stained with YOYO-1 dye, allowing the compaction to be observed using fluorescence microscopy. The molecule was then moved back to the DNA side of the flow cell (which was protein-free), and the decompaction of the molecule was observed as protein left it, ultimately returning to its original length. ( c ) Time-lapse images of a lambda-phage DNA dimer (35 μ m contour length) undergoing compaction by Abf2p. The Abf2p concentration is 2 μ M. The time interval between successive frames is 0.5 s. The buffer flow speed is 63 μ m/s.

    Techniques Used: Flow Cytometry, Staining, Fluorescence, Microscopy, Concentration Assay

    35) Product Images from "Single-molecule analysis of 1D diffusion and transcription elongation of T7 RNA polymerase along individual stretched DNA molecules"

    Article Title: Single-molecule analysis of 1D diffusion and transcription elongation of T7 RNA polymerase along individual stretched DNA molecules

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm332

    Experimental setup for visualizing DNA–T7 RNAP interactions. ( A ) Blow-up at the bottom of the diagram shows aligned DNA molecules (green) on the surface, interacting with RNAPs (red). ( B ) A shear flow is created inside the flow cell to stretch λ-DNA molecules modified with biotin at one end. ( C ) Co-crystal structure of T7 RNAP and DNA. The epitope for AB binding is highlighted in red and DNA in green. We modified the original Chime image adopted from Zhen Huang's website (academic.brooklyn.cuny.edu/chem/zhuang). ( D ) Alexa Fluor 488-UTP labeled RNA transcripts (left) synthesized by RPAB (right) along combed T7 DNA. Scale bar = 5 μm. ( E ) Photobleaching trajectory of RPAB and (inset) free secondary antibody showing stepwise decrease in the fluorescence intensity.
    Figure Legend Snippet: Experimental setup for visualizing DNA–T7 RNAP interactions. ( A ) Blow-up at the bottom of the diagram shows aligned DNA molecules (green) on the surface, interacting with RNAPs (red). ( B ) A shear flow is created inside the flow cell to stretch λ-DNA molecules modified with biotin at one end. ( C ) Co-crystal structure of T7 RNAP and DNA. The epitope for AB binding is highlighted in red and DNA in green. We modified the original Chime image adopted from Zhen Huang's website (academic.brooklyn.cuny.edu/chem/zhuang). ( D ) Alexa Fluor 488-UTP labeled RNA transcripts (left) synthesized by RPAB (right) along combed T7 DNA. Scale bar = 5 μm. ( E ) Photobleaching trajectory of RPAB and (inset) free secondary antibody showing stepwise decrease in the fluorescence intensity.

    Techniques Used: Flow Cytometry, Modification, Binding Assay, Labeling, Synthesized, Fluorescence

    ( A ) Kymograph of T7 RNAP moving along λ-DNA stretched by shear flow. ( B ) The MSD plotted versus time for T7 RNAP moving along flow-stretched (squares) and combed DNA (circles).
    Figure Legend Snippet: ( A ) Kymograph of T7 RNAP moving along λ-DNA stretched by shear flow. ( B ) The MSD plotted versus time for T7 RNAP moving along flow-stretched (squares) and combed DNA (circles).

    Techniques Used: Flow Cytometry

    Sequence of images of T7 RNAP diffusing along ( A ) single combed unstained λ-DNA and ( B ) stained λ-DNA. Two stationary dots in the sequences are T7 RNAPs stalled at positions where the DNA is apparently attached to the substrate. The time lapse between each image is 0.2 s. Scale bar = 2.5 μm. ( C ) The displacements of proteins in the sequences shown in A and B are shown as 1 and 2, respectively, and another similarly diffusing protein as sequence 3. ( D ) Histograms of relative displacements y ( j )− y ( j −1) of protein for the sequences A and B are shown as 1 and 2, respectively, and another similarly diffusing protein as 3. The distributions are well fitted to a Gaussian centered near zero. ( E ) Histograms of the 1D diffusion coefficient ( D 1 ) and of the diffusion length, defined in the text. ( F ) The MSD (square) of protein in Figure 2 A plotted as a function of time. The error bar shown is calculated by , where is the variance in the MSD [Equation ( 3 )] and i is the measurement index going from 1 to N −1. The dotted line is a linear fit of first five MSD points.
    Figure Legend Snippet: Sequence of images of T7 RNAP diffusing along ( A ) single combed unstained λ-DNA and ( B ) stained λ-DNA. Two stationary dots in the sequences are T7 RNAPs stalled at positions where the DNA is apparently attached to the substrate. The time lapse between each image is 0.2 s. Scale bar = 2.5 μm. ( C ) The displacements of proteins in the sequences shown in A and B are shown as 1 and 2, respectively, and another similarly diffusing protein as sequence 3. ( D ) Histograms of relative displacements y ( j )− y ( j −1) of protein for the sequences A and B are shown as 1 and 2, respectively, and another similarly diffusing protein as 3. The distributions are well fitted to a Gaussian centered near zero. ( E ) Histograms of the 1D diffusion coefficient ( D 1 ) and of the diffusion length, defined in the text. ( F ) The MSD (square) of protein in Figure 2 A plotted as a function of time. The error bar shown is calculated by , where is the variance in the MSD [Equation ( 3 )] and i is the measurement index going from 1 to N −1. The dotted line is a linear fit of first five MSD points.

    Techniques Used: Sequencing, Staining, Diffusion-based Assay

    36) Product Images from "Quantitative single cell 5hmC sequencing reveals non-canonical gene regulation by non-CG hydroxymethylation"

    Article Title: Quantitative single cell 5hmC sequencing reveals non-canonical gene regulation by non-CG hydroxymethylation

    Journal: bioRxiv

    doi: 10.1101/2021.03.23.434325

    Quality control of unbiased clustering results of mouse cortical nuclei. a, UMAP visualization (same as Fig. 1e ) of 233 sorted cortical nuclei from wild-type mouse (brain #1 and 2) colored by biological replicates (left panel) and of 127 sorted mouse cortical nuclei from Nex-Cre transgenic mouse (brain #3) colored by cell-types defined by FANS analysis (right panel). b, UMAP visualization (same as Fig. 1e ) showing covered CG sites, number of covered CH sites, global 5hmCG level in the lambda spike-in, and number of uniquely mapped reads per nucleus.
    Figure Legend Snippet: Quality control of unbiased clustering results of mouse cortical nuclei. a, UMAP visualization (same as Fig. 1e ) of 233 sorted cortical nuclei from wild-type mouse (brain #1 and 2) colored by biological replicates (left panel) and of 127 sorted mouse cortical nuclei from Nex-Cre transgenic mouse (brain #3) colored by cell-types defined by FANS analysis (right panel). b, UMAP visualization (same as Fig. 1e ) showing covered CG sites, number of covered CH sites, global 5hmCG level in the lambda spike-in, and number of uniquely mapped reads per nucleus.

    Techniques Used: Transgenic Assay

    Development and validation of bisulfite ACE-seq for whole genome 5hmC analysis. a, Workflow comparison illustrating stepwise differences in cytosine modification status and 5hmC- protection between standard and bisulfite ACE-seq, respectively. b, Bar plots showing the cytosine modification levels in spike-in controls (methylated lambda and mutant T4 phage genomes) analyzed by BS-seq (red), bisulfite ACE-seq (dark blue) and standard ACE-seq (light blue). All methylated (5mCG) and unmodified cytosines (CHG/CHH) in the lambda phage genome should be deaminated in standard and bisulfite ACE-seq. Conversely, 5hmC should be protected at every cytosine position in the mutant T4 (5hmC only) phage genome for all three techniques. The mean values of multiple replicates are shown on the right. Bars and error bars represent the mean ± s.d. of results from multiple experiments (number of replicates are indicated on the top).
    Figure Legend Snippet: Development and validation of bisulfite ACE-seq for whole genome 5hmC analysis. a, Workflow comparison illustrating stepwise differences in cytosine modification status and 5hmC- protection between standard and bisulfite ACE-seq, respectively. b, Bar plots showing the cytosine modification levels in spike-in controls (methylated lambda and mutant T4 phage genomes) analyzed by BS-seq (red), bisulfite ACE-seq (dark blue) and standard ACE-seq (light blue). All methylated (5mCG) and unmodified cytosines (CHG/CHH) in the lambda phage genome should be deaminated in standard and bisulfite ACE-seq. Conversely, 5hmC should be protected at every cytosine position in the mutant T4 (5hmC only) phage genome for all three techniques. The mean values of multiple replicates are shown on the right. Bars and error bars represent the mean ± s.d. of results from multiple experiments (number of replicates are indicated on the top).

    Techniques Used: Modification, Methylation, Mutagenesis

    snhmC-seq workflow and buffer optimization for A3A reactions. a, Schematic comparing the library preparation steps between snhmC-seq and snmC-seq (Luo et al. 4 ). b, Box plots showing the cytosine modification states within CG (top) and CH (bottom) contexts in the spike- in methylated lambda genome (left) and mouse cortical neurons (right) between different pre-A3A elution buffer and A3A reaction buffer conditions. Single-cell BS-seq (snmC-seq) was also performed in parallel as a control. Three different A3A reaction buffer conditions (based on either MES or SPG buffers) were tested. Tris-HCl elution buffer concentrations (10 mM versus 1 mM) were also evaluated to achieve optimal A3A reaction conditions in snhmC-seq assay. MES: 2-(N-morpholino)ethanesulfonic acid; SPG: a mixture of succinic acid, sodium dihydrogen phosphate, and glycine in the molar ratios 2:7:7.
    Figure Legend Snippet: snhmC-seq workflow and buffer optimization for A3A reactions. a, Schematic comparing the library preparation steps between snhmC-seq and snmC-seq (Luo et al. 4 ). b, Box plots showing the cytosine modification states within CG (top) and CH (bottom) contexts in the spike- in methylated lambda genome (left) and mouse cortical neurons (right) between different pre-A3A elution buffer and A3A reaction buffer conditions. Single-cell BS-seq (snmC-seq) was also performed in parallel as a control. Three different A3A reaction buffer conditions (based on either MES or SPG buffers) were tested. Tris-HCl elution buffer concentrations (10 mM versus 1 mM) were also evaluated to achieve optimal A3A reaction conditions in snhmC-seq assay. MES: 2-(N-morpholino)ethanesulfonic acid; SPG: a mixture of succinic acid, sodium dihydrogen phosphate, and glycine in the molar ratios 2:7:7.

    Techniques Used: Modification, Methylation

    Evaluation of bisulfite ACE-seq in analysis of nuclei from cryo-preserved tissue samples. a, Workflow of bisulfite ACE-seq analysis of neuronal nuclei isolated from adult mouse brains (left). Bar plot in the right panel shows the global 5hmCG level in Tet1/2/3 triple KO (Tet TKO) mESCs and mouse cortical neurons analyzed by various sample preparation protocols. As a positive control, ACE-seq (light blue) was used to analyze sheared and denatured gDNA from Tet TKO mESCs or wild-type mouse cortical neurons. Bisulfite ACE-seq was applied to purified Tet TKO mESC gDNA (as a negative control) and sorted NeuN+ nuclei from mouse cortex. The black dot indicates experiments with DNA denaturation, while the grey dot indicates experiments without DNA denaturation. b, Correlation density plot comparing 5hmCG levels in 1-Mb bins between standard ACE-seq (20ng purified, excitatory neuronal DNA) and bisulfite ACE-seq (10,000 neuronal NeuN+ nuclei). c, Correlation density plot comparing global 5hmC level in 1-Mb bins between 10,000 and 100 NeuN+ nuclei with a pre-A3A denaturation step (left), between 100 NeuN+ nuclei with the pre-A3A denaturing step and without denaturing (middle), and between Tet TKO mESC DNA and 100 NeuN+ nuclei (right). d, Bar plots showing the cytosine modification levels in spike-in phage genomes between standard and bisulfite ACE-seq. The 5mCG and C deamination efficiency was assessed by the methylated lambda phage, and 5hmC protection was assessed by the mutant T4 (5hmC only) phage genome. Samples (same as in a ) were indicated by colors.
    Figure Legend Snippet: Evaluation of bisulfite ACE-seq in analysis of nuclei from cryo-preserved tissue samples. a, Workflow of bisulfite ACE-seq analysis of neuronal nuclei isolated from adult mouse brains (left). Bar plot in the right panel shows the global 5hmCG level in Tet1/2/3 triple KO (Tet TKO) mESCs and mouse cortical neurons analyzed by various sample preparation protocols. As a positive control, ACE-seq (light blue) was used to analyze sheared and denatured gDNA from Tet TKO mESCs or wild-type mouse cortical neurons. Bisulfite ACE-seq was applied to purified Tet TKO mESC gDNA (as a negative control) and sorted NeuN+ nuclei from mouse cortex. The black dot indicates experiments with DNA denaturation, while the grey dot indicates experiments without DNA denaturation. b, Correlation density plot comparing 5hmCG levels in 1-Mb bins between standard ACE-seq (20ng purified, excitatory neuronal DNA) and bisulfite ACE-seq (10,000 neuronal NeuN+ nuclei). c, Correlation density plot comparing global 5hmC level in 1-Mb bins between 10,000 and 100 NeuN+ nuclei with a pre-A3A denaturation step (left), between 100 NeuN+ nuclei with the pre-A3A denaturing step and without denaturing (middle), and between Tet TKO mESC DNA and 100 NeuN+ nuclei (right). d, Bar plots showing the cytosine modification levels in spike-in phage genomes between standard and bisulfite ACE-seq. The 5mCG and C deamination efficiency was assessed by the methylated lambda phage, and 5hmC protection was assessed by the mutant T4 (5hmC only) phage genome. Samples (same as in a ) were indicated by colors.

    Techniques Used: Isolation, Sample Prep, Positive Control, Purification, Negative Control, Modification, Methylation, Mutagenesis

    37) Product Images from "A conserved ATP- and Scc2/4-dependent activity for cohesin in tethering DNA molecules"

    Article Title: A conserved ATP- and Scc2/4-dependent activity for cohesin in tethering DNA molecules

    Journal: Science Advances

    doi: 10.1126/sciadv.aay6804

    Tentative models for permanent cohesin bridges. ( A ) Schematic representation of expected behavior of intramolecular cohesin tethers from the previously proposed ring model. The model proposes that cohesin co-entraps two DNAs within its ring structure, i.e., both DNAs occupy one physical space within cohesin. From this model, it is expected that cohesin should be fully displaced from λ-DNA molecules when tethering in cis as force is applied to separate the beads. This is not what it was observed experimentally ( Fig. 3C and fig. S5). ( B ) Schematic representation of expected behavior of intramolecular cohesin tethers from the subcompartment model. The subcompartment model is based on the assumption that DNAs are located in different physical compartments. The prediction from the model is that cohesin cannot be fully displaced from λ-DNA molecules when tethering them in cis. This is what we observed experimentally ( Fig. 3C and fig. S5). ( C ) Proposed model for a single cohesin complex with at least three subcompartments (cohesin pretzel). In this model, sister DNAs occupy two different chambers (K1 and K2) of the K (kleisin) compartment formed bet ween the SMC ATPase heads and the Scc1 subunit ( 36 ). Two possible conformations of SMC hinges are shown. Note that the experimental data are also compatible with the possibility that both DNAs jointly travel through the two chambers (K1 and K2) of the K compartment. ( D ) Schematic representation of previously proposed cohesin handcuffs models holding sister DNAs in different compartments of two separate complexes, which also fits with our experimental observations.
    Figure Legend Snippet: Tentative models for permanent cohesin bridges. ( A ) Schematic representation of expected behavior of intramolecular cohesin tethers from the previously proposed ring model. The model proposes that cohesin co-entraps two DNAs within its ring structure, i.e., both DNAs occupy one physical space within cohesin. From this model, it is expected that cohesin should be fully displaced from λ-DNA molecules when tethering in cis as force is applied to separate the beads. This is not what it was observed experimentally ( Fig. 3C and fig. S5). ( B ) Schematic representation of expected behavior of intramolecular cohesin tethers from the subcompartment model. The subcompartment model is based on the assumption that DNAs are located in different physical compartments. The prediction from the model is that cohesin cannot be fully displaced from λ-DNA molecules when tethering them in cis. This is what we observed experimentally ( Fig. 3C and fig. S5). ( C ) Proposed model for a single cohesin complex with at least three subcompartments (cohesin pretzel). In this model, sister DNAs occupy two different chambers (K1 and K2) of the K (kleisin) compartment formed bet ween the SMC ATPase heads and the Scc1 subunit ( 36 ). Two possible conformations of SMC hinges are shown. Note that the experimental data are also compatible with the possibility that both DNAs jointly travel through the two chambers (K1 and K2) of the K compartment. ( D ) Schematic representation of previously proposed cohesin handcuffs models holding sister DNAs in different compartments of two separate complexes, which also fits with our experimental observations.

    Techniques Used:

    Analysis of yeast cohesin on DNA curtains. ( A ) Schematic representation of double-tethered DNA curtains used in the study. ( B ) Image of cohesin tagged with quantum dots (magenta) bound to λ-DNA stained with YOYO-1 (green). Scale bar, 10 μm. ( C ) Survival probability plots of cohesin in the presence of ATP, ADP, ATPγS, or no nucleotide. ( D ) Lifetimes of cohesin (fast phase and slow phase) in the presence or absence of Scc2-Scc4 and different ATP analogs. Error bars are 68% confidence intervals from bootstrapping. ( E ) Image of a pair of double-tethered DNA curtains bound by cohesin. DNA molecules are in green, and cohesin is in magenta. Diagrammatic representation is shown (left). ( F ) Time-lapse images of a pair of double-tethered DNA curtains bound by cohesin as they are tethered. DNA molecules are in green, and cohesin is in magenta. Diagrammatic representation is shown (top). Pairing events were observed frequently in the DNA curtains. An average of 5 to 10 events per DNA curtain was detected.
    Figure Legend Snippet: Analysis of yeast cohesin on DNA curtains. ( A ) Schematic representation of double-tethered DNA curtains used in the study. ( B ) Image of cohesin tagged with quantum dots (magenta) bound to λ-DNA stained with YOYO-1 (green). Scale bar, 10 μm. ( C ) Survival probability plots of cohesin in the presence of ATP, ADP, ATPγS, or no nucleotide. ( D ) Lifetimes of cohesin (fast phase and slow phase) in the presence or absence of Scc2-Scc4 and different ATP analogs. Error bars are 68% confidence intervals from bootstrapping. ( E ) Image of a pair of double-tethered DNA curtains bound by cohesin. DNA molecules are in green, and cohesin is in magenta. Diagrammatic representation is shown (left). ( F ) Time-lapse images of a pair of double-tethered DNA curtains bound by cohesin as they are tethered. DNA molecules are in green, and cohesin is in magenta. Diagrammatic representation is shown (top). Pairing events were observed frequently in the DNA curtains. An average of 5 to 10 events per DNA curtain was detected.

    Techniques Used: Staining

    Cohesin bridges DNA in an ATP- and Scc2-Scc4–dependent manner. ( A ) Schematic representation of FE curve for λ-DNA exhibiting the presence (right diagram and graph) and absence (left diagram and graph) of protein DNA bridges. Dotted line is fit to worm-like chain for naked DNA. ( B ) FE curves for λ-DNAs preincubated with 1 nM cohesin and 2.5 nM Scc2-Scc4 complex and 1 mM ATP (Cohesin + Scc2/4), 1 nM cohesin and 1 mM ATP (Cohesin), 2.5 nM Scc2-Scc4 complex and 1 mM ATP (Scc2/4), or 1 nM cohesin ATPase mutant and 2.5 nM Scc2-Scc4 complex and 1 mM ATP (CohesinK38I + Scc2/4). Schematic diagram of the experimental design. After capturing a single DNA molecule between two optically trapped beads, DNA was incubated in the presence of protein in a relaxed conformation (3-μm bead distance) for 30 s in 50 mM NaCl and then moved to a buffer channel with 125 mM NaCl for extension and measurements. Only incubation with 1 nM cohesin and 2.5 nM Scc2-Scc4 complex and 1 mM ATP (Cohesin + Scc2/4) showed DNA bridging rupture events. ( C ) FE curves in the presence of increasing ionic strength. High salt favors topologically constrained and permanent DNA bridges. ( D ) Schematic representation of the experimental design to test cohesin second DNA capture. After capture of λ-DNA between the two optically trapped beads, DNA is extended and incubated for 30 s in the protein channel. DNA is moved to a buffer channel and then relaxed (3-μm bead distance) and incubated for 30 s before reextension to test for DNA bridges (E). The extended DNA is then incubated in a relaxed position in the protein channel and then moved to buffer channel and extended to confirm that bridges can be formed when protein is loaded while DNA is relaxed (F). ( E ) λ-DNA incubated with 1 nM cohesin, 2.5 nM Scc2-Scc4 complex, and 1 mM ATP in an extended conformation and then moved to a buffer channel (125 mM NaCl) in the presence of 1 mM ATP (buffer only, dark blue) or 2.5 nM Scc2-Scc4 complex and 1 mM ATP (+Scc2/4, light blue). DNAs were reextended, and the FE curves were recorded. ( F ) The λ-DNA molecules in (E) were incubated in a relaxed position (3-μm bead distance) in the presence of 1 nM cohesin, 2.5 nM Scc2-Scc4 complex, and 1 mM ATP DNAs. DNAs were moved to a buffer-only channel (125 mM NaCl containing 1 mM ATP) and reextended. FE curves show the presence of DNA bridging rupture events.
    Figure Legend Snippet: Cohesin bridges DNA in an ATP- and Scc2-Scc4–dependent manner. ( A ) Schematic representation of FE curve for λ-DNA exhibiting the presence (right diagram and graph) and absence (left diagram and graph) of protein DNA bridges. Dotted line is fit to worm-like chain for naked DNA. ( B ) FE curves for λ-DNAs preincubated with 1 nM cohesin and 2.5 nM Scc2-Scc4 complex and 1 mM ATP (Cohesin + Scc2/4), 1 nM cohesin and 1 mM ATP (Cohesin), 2.5 nM Scc2-Scc4 complex and 1 mM ATP (Scc2/4), or 1 nM cohesin ATPase mutant and 2.5 nM Scc2-Scc4 complex and 1 mM ATP (CohesinK38I + Scc2/4). Schematic diagram of the experimental design. After capturing a single DNA molecule between two optically trapped beads, DNA was incubated in the presence of protein in a relaxed conformation (3-μm bead distance) for 30 s in 50 mM NaCl and then moved to a buffer channel with 125 mM NaCl for extension and measurements. Only incubation with 1 nM cohesin and 2.5 nM Scc2-Scc4 complex and 1 mM ATP (Cohesin + Scc2/4) showed DNA bridging rupture events. ( C ) FE curves in the presence of increasing ionic strength. High salt favors topologically constrained and permanent DNA bridges. ( D ) Schematic representation of the experimental design to test cohesin second DNA capture. After capture of λ-DNA between the two optically trapped beads, DNA is extended and incubated for 30 s in the protein channel. DNA is moved to a buffer channel and then relaxed (3-μm bead distance) and incubated for 30 s before reextension to test for DNA bridges (E). The extended DNA is then incubated in a relaxed position in the protein channel and then moved to buffer channel and extended to confirm that bridges can be formed when protein is loaded while DNA is relaxed (F). ( E ) λ-DNA incubated with 1 nM cohesin, 2.5 nM Scc2-Scc4 complex, and 1 mM ATP in an extended conformation and then moved to a buffer channel (125 mM NaCl) in the presence of 1 mM ATP (buffer only, dark blue) or 2.5 nM Scc2-Scc4 complex and 1 mM ATP (+Scc2/4, light blue). DNAs were reextended, and the FE curves were recorded. ( F ) The λ-DNA molecules in (E) were incubated in a relaxed position (3-μm bead distance) in the presence of 1 nM cohesin, 2.5 nM Scc2-Scc4 complex, and 1 mM ATP DNAs. DNAs were moved to a buffer-only channel (125 mM NaCl containing 1 mM ATP) and reextended. FE curves show the presence of DNA bridging rupture events.

    Techniques Used: Mutagenesis, Incubation

    Cohesin and Scc2-Scc4 mediate intermolecular DNA bridges that slide on DNAs. ( A ) Schematic representation of the experimental design for the dual-trap optical tweezer to generate permanent intermolecular cohesin bridges. Two λ-DNA molecules are tethered between the two beads and incubated in a relaxed position (3-μm bead distance) in the presence or absence of protein in buffer containing 50 mM NaCl. The relaxed molecules are then moved to a different channel containing 300 mM NaCl and reextended. Imaging is done before incubations and after reextension in a buffer containing 300 mM NaCl and 50 nM SYTOX Orange to visualize DNA. ( B ) Two λ-DNA molecules were tethered and treated as described in (A) and incubated with either (i) 1 nM cohesin, 2.5 nM Scc2-Scc4, and no ATP (Cohesin + Scc2/4, left); (ii) 1 nM cohesin, 2.5 nM Scc2-Scc4, and 1 mM ATP (Cohesin + Scc2/4, middle); or (iii) 1 nM cohesin ATPase mutant K38I, 2.5 nM Scc2-Scc4, and 1 mM ATP (K38I + Scc2/4, right). Imaging was performed before incubation and after DNA reextension in a buffer containing 300 mM NaCl to minimize DNA entanglement and 50 nM SYTOX Orange to visualize DNA. Images from three independent experiments are shown. Three independent experiments are shown for each category. ( C ) Schematic representation of the experimental design to test for sliding of permanent cohesin bridges (top diagram). Following the formation of an intermolecular cohesin bridge (see fig. S8 for details in bridge formation protocol), beads 3 and 4 were moved together in the x axis to slide the bridge along DNA1. Images showing two representative sliding experiments are shown. Experiments were performed in a buffer containing 300 mM NaCl and 50 nM SYTOX Orange. Movies of the experiments are shown in movies S4 and S5. The experiment was performed three times, and sliding was observed in all cases. ( D ) Schematic representation of the experimental design to disrupt intermolecular cohesin bridges. Following the formation of an intermolecular cohesin bridge, bead 3 is moved down in the y axis until one of the DNA ends loses contact with the bead. Imaging was performed before and after the pull in a buffer containing 300 mM NaCl and 50 nM SYTOX Orange. Representative experiment is shown. A movie of the experiment is shown in movie S6.
    Figure Legend Snippet: Cohesin and Scc2-Scc4 mediate intermolecular DNA bridges that slide on DNAs. ( A ) Schematic representation of the experimental design for the dual-trap optical tweezer to generate permanent intermolecular cohesin bridges. Two λ-DNA molecules are tethered between the two beads and incubated in a relaxed position (3-μm bead distance) in the presence or absence of protein in buffer containing 50 mM NaCl. The relaxed molecules are then moved to a different channel containing 300 mM NaCl and reextended. Imaging is done before incubations and after reextension in a buffer containing 300 mM NaCl and 50 nM SYTOX Orange to visualize DNA. ( B ) Two λ-DNA molecules were tethered and treated as described in (A) and incubated with either (i) 1 nM cohesin, 2.5 nM Scc2-Scc4, and no ATP (Cohesin + Scc2/4, left); (ii) 1 nM cohesin, 2.5 nM Scc2-Scc4, and 1 mM ATP (Cohesin + Scc2/4, middle); or (iii) 1 nM cohesin ATPase mutant K38I, 2.5 nM Scc2-Scc4, and 1 mM ATP (K38I + Scc2/4, right). Imaging was performed before incubation and after DNA reextension in a buffer containing 300 mM NaCl to minimize DNA entanglement and 50 nM SYTOX Orange to visualize DNA. Images from three independent experiments are shown. Three independent experiments are shown for each category. ( C ) Schematic representation of the experimental design to test for sliding of permanent cohesin bridges (top diagram). Following the formation of an intermolecular cohesin bridge (see fig. S8 for details in bridge formation protocol), beads 3 and 4 were moved together in the x axis to slide the bridge along DNA1. Images showing two representative sliding experiments are shown. Experiments were performed in a buffer containing 300 mM NaCl and 50 nM SYTOX Orange. Movies of the experiments are shown in movies S4 and S5. The experiment was performed three times, and sliding was observed in all cases. ( D ) Schematic representation of the experimental design to disrupt intermolecular cohesin bridges. Following the formation of an intermolecular cohesin bridge, bead 3 is moved down in the y axis until one of the DNA ends loses contact with the bead. Imaging was performed before and after the pull in a buffer containing 300 mM NaCl and 50 nM SYTOX Orange. Representative experiment is shown. A movie of the experiment is shown in movie S6.

    Techniques Used: Incubation, Imaging, Mutagenesis

    Cohesin does not compact linear DNA molecules stretched under low force. ( A ) FE curve for λ-DNA preincubated with 1 nM human cohesin and 1 mM ATP in 125 mM NaCl buffer (hCohesin). Dotted line is fit to worm-like chain model. After capturing a single DNA molecule between two optically trapped beads, DNA was incubated in the presence of protein in 50 mM NaCl buffer in a relaxed conformation (3-μm bead distance) for 30 s and then moved to the 125 mM NaCl buffer channel for extension and measurements. No evidence of DNA bridges was observed under this condition. ( B ) FE curve for λ-DNA preincubated with 1 nM human cohesin, 2.5 nM yeast Scc2-Scc4, and 1 mM ATP in 125 mM NaCl buffer (hCohesin + Scc2/4). Experimental procedure as in (A). FE curves exhibited multiple rupture events indicating the presence of reversible and permanent DNA bridges. ( C ) DNA compaction trace for λ-DNA molecule extended using a force of 1 pN (top). The DNA was tethered between two beads. One bead was clamped (fixed), while a 1-pN force was applied to the second bead to maintain the molecule extended. The DNA was then incubated in the presence of 1 nM condensin (1 mM ATP in 50 mM NaCl) (left, magenta trace). The FE curve for the λ-DNA full extension after incubation is shown (bottom). Additional examples can be found in fig. S10. ( D ) DNA compaction trace for λ-DNA molecule extended using a force of 1 pN (top) in the presence of 1 nM cohesin and 2.5 nM Scc2-Scc4 complex (1 mM ATP in 50 mM NaCl) (right, yellow trace). The distance between the beads was recorded over time. The FE curve for the λ-DNA full extension after incubation is shown (bottom). Additional examples can be found in fig. S10. ( E ) Kymograms of single-tethered λ-DNA stained with (YOYO-1) during the incubation with yeast cohesin and Scc2-Scc4 in the presence of ATP in 50 mM NaCl buffer at a flow rate of 20 μl/min. HF, high flow. The free end of DNA is marked with orange arrowheads. No compaction of single-tethered λ-DNAs was observed. ( F ) Kymograms of single-tethered λ-DNA stained with (YOYO-1) during the incubation with yeast cohesin and Scc2-Scc4 in the presence of ATP in 50 mM NaCl buffer at a flow rate of 10 μl/min. The conditions are as in (E) except for the reduced flow rate. Slow compaction of single-tethered λ-DNAs was observed over time (orange arrowheads mark the free end of DNA). ( G ) Kymograms of single-tethered λ-DNA stained with (YOYO-1) during the incubation with yeast cohesin and Scc2-Scc4 in the presence of ATP in 50 mM NaCl buffer at stopped flow. The free end of DNA is marked with orange arrowheads. The HF phase at the end of the experiment shows that the DNA was compacted during the stopped flow phase. Note that under stopped flow conditions, DNA molecules that diffuse laterally on the flow chip can transiently cross the field of view and also appear in a kymogram representation. Examples are marked with asterisks (*). These events bear no relevance for the interpretations of the assay.
    Figure Legend Snippet: Cohesin does not compact linear DNA molecules stretched under low force. ( A ) FE curve for λ-DNA preincubated with 1 nM human cohesin and 1 mM ATP in 125 mM NaCl buffer (hCohesin). Dotted line is fit to worm-like chain model. After capturing a single DNA molecule between two optically trapped beads, DNA was incubated in the presence of protein in 50 mM NaCl buffer in a relaxed conformation (3-μm bead distance) for 30 s and then moved to the 125 mM NaCl buffer channel for extension and measurements. No evidence of DNA bridges was observed under this condition. ( B ) FE curve for λ-DNA preincubated with 1 nM human cohesin, 2.5 nM yeast Scc2-Scc4, and 1 mM ATP in 125 mM NaCl buffer (hCohesin + Scc2/4). Experimental procedure as in (A). FE curves exhibited multiple rupture events indicating the presence of reversible and permanent DNA bridges. ( C ) DNA compaction trace for λ-DNA molecule extended using a force of 1 pN (top). The DNA was tethered between two beads. One bead was clamped (fixed), while a 1-pN force was applied to the second bead to maintain the molecule extended. The DNA was then incubated in the presence of 1 nM condensin (1 mM ATP in 50 mM NaCl) (left, magenta trace). The FE curve for the λ-DNA full extension after incubation is shown (bottom). Additional examples can be found in fig. S10. ( D ) DNA compaction trace for λ-DNA molecule extended using a force of 1 pN (top) in the presence of 1 nM cohesin and 2.5 nM Scc2-Scc4 complex (1 mM ATP in 50 mM NaCl) (right, yellow trace). The distance between the beads was recorded over time. The FE curve for the λ-DNA full extension after incubation is shown (bottom). Additional examples can be found in fig. S10. ( E ) Kymograms of single-tethered λ-DNA stained with (YOYO-1) during the incubation with yeast cohesin and Scc2-Scc4 in the presence of ATP in 50 mM NaCl buffer at a flow rate of 20 μl/min. HF, high flow. The free end of DNA is marked with orange arrowheads. No compaction of single-tethered λ-DNAs was observed. ( F ) Kymograms of single-tethered λ-DNA stained with (YOYO-1) during the incubation with yeast cohesin and Scc2-Scc4 in the presence of ATP in 50 mM NaCl buffer at a flow rate of 10 μl/min. The conditions are as in (E) except for the reduced flow rate. Slow compaction of single-tethered λ-DNAs was observed over time (orange arrowheads mark the free end of DNA). ( G ) Kymograms of single-tethered λ-DNA stained with (YOYO-1) during the incubation with yeast cohesin and Scc2-Scc4 in the presence of ATP in 50 mM NaCl buffer at stopped flow. The free end of DNA is marked with orange arrowheads. The HF phase at the end of the experiment shows that the DNA was compacted during the stopped flow phase. Note that under stopped flow conditions, DNA molecules that diffuse laterally on the flow chip can transiently cross the field of view and also appear in a kymogram representation. Examples are marked with asterisks (*). These events bear no relevance for the interpretations of the assay.

    Techniques Used: Incubation, Staining, Flow Cytometry, Chromatin Immunoprecipitation

    38) Product Images from "TAMRA-polypyrrole for A/T sequence visualization on DNA molecules"

    Article Title: TAMRA-polypyrrole for A/T sequence visualization on DNA molecules

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky531

    TAMRA-pPy-stained λ DNA tethered on the surface of a flow cell. ( A ) Free-floating λ DNA (48.5 kb) molecules with a mushroom-like conformation in the flow off condition. ( B ) Fully elongated λ DNA molecules with a flow of 100 μl/min. Arrows indicate flow direction. Scale bar 10 μm. ( C ) Comparison of experimentally measured fluorescence intensity (red) with in silico sequence frequencies from the λ genome sequence. The black solid line represents A/T (W), the gray dotted line indicates four consecutive A/T ( W 4 ), and the grey solid line is W 9 . ( D ) Cross-correlation ( cc ) coefficient values calculated from the alignment of 20 molecular images with the genome using three kinds of binding sequences ( W, W 4 and W 9 ). The control cc was obtained from 100 computer-generated random sequences (*** P
    Figure Legend Snippet: TAMRA-pPy-stained λ DNA tethered on the surface of a flow cell. ( A ) Free-floating λ DNA (48.5 kb) molecules with a mushroom-like conformation in the flow off condition. ( B ) Fully elongated λ DNA molecules with a flow of 100 μl/min. Arrows indicate flow direction. Scale bar 10 μm. ( C ) Comparison of experimentally measured fluorescence intensity (red) with in silico sequence frequencies from the λ genome sequence. The black solid line represents A/T (W), the gray dotted line indicates four consecutive A/T ( W 4 ), and the grey solid line is W 9 . ( D ) Cross-correlation ( cc ) coefficient values calculated from the alignment of 20 molecular images with the genome using three kinds of binding sequences ( W, W 4 and W 9 ). The control cc was obtained from 100 computer-generated random sequences (*** P

    Techniques Used: Staining, Flow Cytometry, Fluorescence, In Silico, Sequencing, Binding Assay, Generated

    ( A ) TAMRA-pPy-stained polytene chromosomes from D. melanogaster . Polytene chromosome samples were prepared via a conventional acid-wash protocol and exhibit bands and interbands in chromosomes on a fluorescence microscope. ( B ) Representative fluorescent λ DNA images stained with DAPI and TAMRA-polypyrrole. ( C ) Intensity profiles of DAPI and TAMRA-polypyrrole-stained λ DNA. Red line is TAMRA-polypyrrole, and blue line is DAPI staining. ( D ) Intensity contrast ratios for DAPI and TAMRA-polypyrrole. Bars represent the highest values over the lowest values for each DNA molecule. Error bars represent the minimum and maximum values for the images in (B).
    Figure Legend Snippet: ( A ) TAMRA-pPy-stained polytene chromosomes from D. melanogaster . Polytene chromosome samples were prepared via a conventional acid-wash protocol and exhibit bands and interbands in chromosomes on a fluorescence microscope. ( B ) Representative fluorescent λ DNA images stained with DAPI and TAMRA-polypyrrole. ( C ) Intensity profiles of DAPI and TAMRA-polypyrrole-stained λ DNA. Red line is TAMRA-polypyrrole, and blue line is DAPI staining. ( D ) Intensity contrast ratios for DAPI and TAMRA-polypyrrole. Bars represent the highest values over the lowest values for each DNA molecule. Error bars represent the minimum and maximum values for the images in (B).

    Techniques Used: Staining, Fluorescence, Microscopy

    TAMRA-pPy-stained λ DNA electrostatically immobilized on a surface ( A ) λ DNA on a positively charged surface; yellow arrows indicate the genome direction 5′→3′. ( B ) λ concatemers from monomer to heptamer. Gray bars represent the A/T frequency. ( C ) S. cerevisiae chromosome I DNA fragments aligned with the in silico A/T frequency map. Averaged cc value was 0.64 ± 0.14, whereas cc for computer-generated random images was 0.41 ± 0.06 ( P
    Figure Legend Snippet: TAMRA-pPy-stained λ DNA electrostatically immobilized on a surface ( A ) λ DNA on a positively charged surface; yellow arrows indicate the genome direction 5′→3′. ( B ) λ concatemers from monomer to heptamer. Gray bars represent the A/T frequency. ( C ) S. cerevisiae chromosome I DNA fragments aligned with the in silico A/T frequency map. Averaged cc value was 0.64 ± 0.14, whereas cc for computer-generated random images was 0.41 ± 0.06 ( P

    Techniques Used: Staining, In Silico, Generated

    39) Product Images from "Comparative analysis of the end-joining activity of several DNA ligases"

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0190062

    Wild type DNA ligase λ DNA digest ligation assay. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1 ), NruI (G/C Blunt, 2 ), BstNI (5′ SBO, 3 ), Hpy188I (3′SBO, 4 ), NdeI (2 BO, 5 ) and BamHI (4 BO, 6 ), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in the presence of T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 6% polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and, hLig3 (D), respectively. E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.
    Figure Legend Snippet: Wild type DNA ligase λ DNA digest ligation assay. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1 ), NruI (G/C Blunt, 2 ), BstNI (5′ SBO, 3 ), Hpy188I (3′SBO, 4 ), NdeI (2 BO, 5 ) and BamHI (4 BO, 6 ), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in the presence of T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 6% polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and, hLig3 (D), respectively. E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.

    Techniques Used: Ligation, Agarose Gel Electrophoresis, Staining

    Effect of DBDs on blunt/cohesive end λ DNA Re-ligation. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1), NruI (G/C Blunt, 2), BstNI (5′ SBO, 3), Hpy188I (3′SBO, 4), NdeI (2 BO, 5) and BamHI (4 BO, 6), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). (E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.
    Figure Legend Snippet: Effect of DBDs on blunt/cohesive end λ DNA Re-ligation. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1), NruI (G/C Blunt, 2), BstNI (5′ SBO, 3), Hpy188I (3′SBO, 4), NdeI (2 BO, 5) and BamHI (4 BO, 6), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). (E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.

    Techniques Used: Ligation, Agarose Gel Electrophoresis, Staining

    40) Product Images from "Uncoupling of sister replisomes during eukaryotic DNA replication"

    Article Title: Uncoupling of sister replisomes during eukaryotic DNA replication

    Journal: Molecular cell

    doi: 10.1016/j.molcel.2010.11.027

    Replication kinetics of immobilized λ DNA Quantification of replication on singly-tethered (black) and doubly-tethered (grey) DNA after 15 minutes incubation in NPE. (A) Number of replication bubbles (anti-dig tracts) per immobilized λ DNA. (B) Length distributions of replication bubbles. (C) Percent replication of individual λ DNA molecules. Error bars indicate standard deviations.
    Figure Legend Snippet: Replication kinetics of immobilized λ DNA Quantification of replication on singly-tethered (black) and doubly-tethered (grey) DNA after 15 minutes incubation in NPE. (A) Number of replication bubbles (anti-dig tracts) per immobilized λ DNA. (B) Length distributions of replication bubbles. (C) Percent replication of individual λ DNA molecules. Error bars indicate standard deviations.

    Techniques Used: Incubation

    Single-molecule visualization of eukaryotic replication (A) Possible configurations of sister replisomes. The sister replisomes assembled at each origin (i) travel away from each other (ii) or remain physically coupled (iii). Doubly-tethered DNA is replicated efficiently by independently functioning replisomes (iv) but not physically coupled replisomes, which stall after available slack in the DNA is consumed (v). (B) Protocol to induce replication of λ DNA immobilized at one end in a flow cell. (C) Visualization of replicated DNA by TIRF microscopy. λ DNA was incubated with extracts lacking (i, ii) or containing (iii, iv) Geminin and stained with anti-digoxigenin or SYTOX, as indicated.
    Figure Legend Snippet: Single-molecule visualization of eukaryotic replication (A) Possible configurations of sister replisomes. The sister replisomes assembled at each origin (i) travel away from each other (ii) or remain physically coupled (iii). Doubly-tethered DNA is replicated efficiently by independently functioning replisomes (iv) but not physically coupled replisomes, which stall after available slack in the DNA is consumed (v). (B) Protocol to induce replication of λ DNA immobilized at one end in a flow cell. (C) Visualization of replicated DNA by TIRF microscopy. λ DNA was incubated with extracts lacking (i, ii) or containing (iii, iv) Geminin and stained with anti-digoxigenin or SYTOX, as indicated.

    Techniques Used: Flow Cytometry, Microscopy, Incubation, Staining

    Replication of stretched DNA by a single pair of diverging forks (A) Scheme used to limit replication initiation to a single event on each λ DNA molecule and to visualize bidirectional replication. (B) SYTOX (top), anti-dig (middle), and merged (bottom) images of three mechanically stretched λ DNA molecules containing a single replication bubble. Extent of slack and replication are indicated. (C) Number of replication bubbles per monomeric λ DNAs (n=39). (D) Extent of replication versus the amount of slack on individual λ DNA molecules that underwent single initiations. The amount of slack was calculated by comparing the end-to-end distance of doubly-tethered DNA molecule to the B-form contour length of λ DNA (16.5 μm). The solid line depicts the extent of replication expected if replication stops when the slack initially present in the λ DNA is used up, as expected for physically coupled forks.
    Figure Legend Snippet: Replication of stretched DNA by a single pair of diverging forks (A) Scheme used to limit replication initiation to a single event on each λ DNA molecule and to visualize bidirectional replication. (B) SYTOX (top), anti-dig (middle), and merged (bottom) images of three mechanically stretched λ DNA molecules containing a single replication bubble. Extent of slack and replication are indicated. (C) Number of replication bubbles per monomeric λ DNAs (n=39). (D) Extent of replication versus the amount of slack on individual λ DNA molecules that underwent single initiations. The amount of slack was calculated by comparing the end-to-end distance of doubly-tethered DNA molecule to the B-form contour length of λ DNA (16.5 μm). The solid line depicts the extent of replication expected if replication stops when the slack initially present in the λ DNA is used up, as expected for physically coupled forks.

    Techniques Used:

    Analysis of fork-rates (A) Length of anti-dig tracts under single-initiation conditions on singly-tethered (black) and doubly-tethered (grey) λ DNA molecules. Error bars indicate standard deviations. (B) Lengths of sister anti-dig tracts of the rightward fork versus the leftward fork on singly (black) and doubly-tethered (grey) DNA molecules. The dashed line represents perfectly correlated sister forks.
    Figure Legend Snippet: Analysis of fork-rates (A) Length of anti-dig tracts under single-initiation conditions on singly-tethered (black) and doubly-tethered (grey) λ DNA molecules. Error bars indicate standard deviations. (B) Lengths of sister anti-dig tracts of the rightward fork versus the leftward fork on singly (black) and doubly-tethered (grey) DNA molecules. The dashed line represents perfectly correlated sister forks.

    Techniques Used:

    Related Articles

    other:

    Article Title: Quantification of Trace-Level DNA by Real-Time Whole Genome Amplification
    Article Snippet: Concentrations of CTD, E. coli DNA, and lambda phage DNA were determined by measuring UV absorbance at 260 nm.

    Article Title: Conserved linear dynamics of single-molecule Brownian motion
    Article Snippet: Materials Supercoiled ColE1 (6.6 kbp) DNA was obtained from Nippon Gene (Toyama, Japan) whereas the lambda phage DNA was obtained from New England Biolabs (Hitchin, UK).

    Concentration Assay:

    Article Title: Regulation of DNA conformations and dynamics in flows with hybrid field microfluidics
    Article Snippet: .. A dilute solution (∼0.03 μ g/ml, about 10−4 of the overlapping concentration, at which the macromolecules completely fill the space but not overlapping) of λ-DNA (New England Biolab, Inc) in 10 mM TE buffer ( p H = 8.0) with 4% β-mercaptoethanol was used as the experimental fluid. .. DNA molecules were labeled with YOYO-1 dyes at a dye-to-base-pair molar ratio of 1:5.

    Construct:

    Article Title: Single-molecule measurements reveal that PARP1 condenses DNA by loop formation
    Article Snippet: .. Magnetic tweezers DNA construct The central part of the DNA construct used for magnetic tweezers was a 7.9 kbp fragment produced by restriction digestion of λ-DNA using SapI and BsaI (NEB) and subsequent agarose gel purification. .. A 478 bp fragment of biotin labelled DNA was produced by PCR with Taq Polymerase (NEB) of λ-DNA using the following primers: 5’-CGAACTCTTCAAATTCTTCTTCCA-3’ and 5’-GATTGCTCTTCTGTAAGGTTTTG-3’ with a 5:1 ratio of dTTP:biotin-11-dUTP (Jena Bioscience).

    Produced:

    Article Title: Single-molecule measurements reveal that PARP1 condenses DNA by loop formation
    Article Snippet: .. Magnetic tweezers DNA construct The central part of the DNA construct used for magnetic tweezers was a 7.9 kbp fragment produced by restriction digestion of λ-DNA using SapI and BsaI (NEB) and subsequent agarose gel purification. .. A 478 bp fragment of biotin labelled DNA was produced by PCR with Taq Polymerase (NEB) of λ-DNA using the following primers: 5’-CGAACTCTTCAAATTCTTCTTCCA-3’ and 5’-GATTGCTCTTCTGTAAGGTTTTG-3’ with a 5:1 ratio of dTTP:biotin-11-dUTP (Jena Bioscience).

    Agarose Gel Electrophoresis:

    Article Title: Single-molecule measurements reveal that PARP1 condenses DNA by loop formation
    Article Snippet: .. Magnetic tweezers DNA construct The central part of the DNA construct used for magnetic tweezers was a 7.9 kbp fragment produced by restriction digestion of λ-DNA using SapI and BsaI (NEB) and subsequent agarose gel purification. .. A 478 bp fragment of biotin labelled DNA was produced by PCR with Taq Polymerase (NEB) of λ-DNA using the following primers: 5’-CGAACTCTTCAAATTCTTCTTCCA-3’ and 5’-GATTGCTCTTCTGTAAGGTTTTG-3’ with a 5:1 ratio of dTTP:biotin-11-dUTP (Jena Bioscience).

    Purification:

    Article Title: Single-molecule measurements reveal that PARP1 condenses DNA by loop formation
    Article Snippet: .. Magnetic tweezers DNA construct The central part of the DNA construct used for magnetic tweezers was a 7.9 kbp fragment produced by restriction digestion of λ-DNA using SapI and BsaI (NEB) and subsequent agarose gel purification. .. A 478 bp fragment of biotin labelled DNA was produced by PCR with Taq Polymerase (NEB) of λ-DNA using the following primers: 5’-CGAACTCTTCAAATTCTTCTTCCA-3’ and 5’-GATTGCTCTTCTGTAAGGTTTTG-3’ with a 5:1 ratio of dTTP:biotin-11-dUTP (Jena Bioscience).

    Labeling:

    Article Title: Fabrication of long poly(dimethyl siloxane) nanochannels by replicating protein deposit from confined solution evaporation
    Article Snippet: .. Bovine serum albumin (BSA) was purchased from Sigma-Aldrich. λ-DNA was purchased from New England BioLab and labeled with YOYO-1 (Y3601, purchased from Invitrogen) for observation. ..

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    New England Biolabs lambda phage dna
    Application of the real-time DOP-PCR to diverse <t>DNA</t> species. Amplification profiles and their standard curves were obtained from human placental DNA (HPD; A), calf thymus DNA (CTD; B), E. coli DNA (C), and <t>lambda</t> phage DNA (D). Standard DNA samples from 80 fg to 80 ng and a no-template control were amplified. Six independent experiments each comprising triplicate reactions were performed, and typical results of one experiment are presented. Data for 80 ng and NTC were omitted for the plotting of standard curves.
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    Application of the real-time DOP-PCR to diverse DNA species. Amplification profiles and their standard curves were obtained from human placental DNA (HPD; A), calf thymus DNA (CTD; B), E. coli DNA (C), and lambda phage DNA (D). Standard DNA samples from 80 fg to 80 ng and a no-template control were amplified. Six independent experiments each comprising triplicate reactions were performed, and typical results of one experiment are presented. Data for 80 ng and NTC were omitted for the plotting of standard curves.

    Journal: PLoS ONE

    Article Title: Quantification of Trace-Level DNA by Real-Time Whole Genome Amplification

    doi: 10.1371/journal.pone.0028661

    Figure Lengend Snippet: Application of the real-time DOP-PCR to diverse DNA species. Amplification profiles and their standard curves were obtained from human placental DNA (HPD; A), calf thymus DNA (CTD; B), E. coli DNA (C), and lambda phage DNA (D). Standard DNA samples from 80 fg to 80 ng and a no-template control were amplified. Six independent experiments each comprising triplicate reactions were performed, and typical results of one experiment are presented. Data for 80 ng and NTC were omitted for the plotting of standard curves.

    Article Snippet: Concentrations of CTD, E. coli DNA, and lambda phage DNA were determined by measuring UV absorbance at 260 nm.

    Techniques: Degenerate Oligonucleotide–primed Polymerase Chain Reaction, Amplification

    Stretching of DNA molecules (a) near the inlet (micro to nano region) and (b) at about 2 mm away from the inlet of the nanochannel. The maximum elongation of λ-DNA reaches 10 μ m, which is about 50% of its fully extended length. (Scale bar: 20 μ m)

    Journal: Biomicrofluidics

    Article Title: Fabrication of long poly(dimethyl siloxane) nanochannels by replicating protein deposit from confined solution evaporation

    doi: 10.1063/1.4730371

    Figure Lengend Snippet: Stretching of DNA molecules (a) near the inlet (micro to nano region) and (b) at about 2 mm away from the inlet of the nanochannel. The maximum elongation of λ-DNA reaches 10 μ m, which is about 50% of its fully extended length. (Scale bar: 20 μ m)

    Article Snippet: Bovine serum albumin (BSA) was purchased from Sigma-Aldrich. λ-DNA was purchased from New England BioLab and labeled with YOYO-1 (Y3601, purchased from Invitrogen) for observation.

    Techniques:

    (a) Schematic of how a DNA chain is polarized, trapped or escape, and relax in HFM; (b) and (c) Time serial snapshots of the relaxation dynamics of an intermediated-stretched (b) and an over-stretched λ-DNA (c). (d) Extension versus residence

    Journal: Biomicrofluidics

    Article Title: Regulation of DNA conformations and dynamics in flows with hybrid field microfluidics

    doi: 10.1063/1.4762852

    Figure Lengend Snippet: (a) Schematic of how a DNA chain is polarized, trapped or escape, and relax in HFM; (b) and (c) Time serial snapshots of the relaxation dynamics of an intermediated-stretched (b) and an over-stretched λ-DNA (c). (d) Extension versus residence

    Article Snippet: A dilute solution (∼0.03 μ g/ml, about 10−4 of the overlapping concentration, at which the macromolecules completely fill the space but not overlapping) of λ-DNA (New England Biolab, Inc) in 10 mM TE buffer ( p H = 8.0) with 4% β-mercaptoethanol was used as the experimental fluid.

    Techniques:

    The ensemble average fractional extension of ∼200 λ-DNA when switching from “flow field” alone to “hybrid field” (a) and 60 λ-DNA molecules after the sudden removal of the electric field (c). (b)

    Journal: Biomicrofluidics

    Article Title: Regulation of DNA conformations and dynamics in flows with hybrid field microfluidics

    doi: 10.1063/1.4762852

    Figure Lengend Snippet: The ensemble average fractional extension of ∼200 λ-DNA when switching from “flow field” alone to “hybrid field” (a) and 60 λ-DNA molecules after the sudden removal of the electric field (c). (b)

    Article Snippet: A dilute solution (∼0.03 μ g/ml, about 10−4 of the overlapping concentration, at which the macromolecules completely fill the space but not overlapping) of λ-DNA (New England Biolab, Inc) in 10 mM TE buffer ( p H = 8.0) with 4% β-mercaptoethanol was used as the experimental fluid.

    Techniques:

    (a) Schematic of the working principles of HFM, (b) a typical application scheme for an electric bias in HFM, (c) regulation the conformations and dynamics (trapping, concentration, and sudden stretching) of λ-DNA molecules in HFM, and (d) schematic

    Journal: Biomicrofluidics

    Article Title: Regulation of DNA conformations and dynamics in flows with hybrid field microfluidics

    doi: 10.1063/1.4762852

    Figure Lengend Snippet: (a) Schematic of the working principles of HFM, (b) a typical application scheme for an electric bias in HFM, (c) regulation the conformations and dynamics (trapping, concentration, and sudden stretching) of λ-DNA molecules in HFM, and (d) schematic

    Article Snippet: A dilute solution (∼0.03 μ g/ml, about 10−4 of the overlapping concentration, at which the macromolecules completely fill the space but not overlapping) of λ-DNA (New England Biolab, Inc) in 10 mM TE buffer ( p H = 8.0) with 4% β-mercaptoethanol was used as the experimental fluid.

    Techniques: Concentration Assay

    Lattice occupancy analysis of lambda DNA. ( a ) MSD-Δ t profile of lambda DNA (Δ t =6.4 ms). The red line shows the theoretical MSD-Δ t profile. ( b ) Frequency histogram of the HE-1Δ t distribution of the experimental (top) and the S r A r simulated replicates (bottom) of lambda DNA. ( c ) Averaged MSD-Δ t profiles of the sub-trajectories captured in the same way as in Fig. 7a (red) and Fig. 7b (blue). The red and blue lines are the theoretical MSD-Δ t profiles. The green line shows the overall, theoretical MSD-Δ t profile of the experimental replicates.

    Journal: Nature Communications

    Article Title: Conserved linear dynamics of single-molecule Brownian motion

    doi: 10.1038/ncomms15675

    Figure Lengend Snippet: Lattice occupancy analysis of lambda DNA. ( a ) MSD-Δ t profile of lambda DNA (Δ t =6.4 ms). The red line shows the theoretical MSD-Δ t profile. ( b ) Frequency histogram of the HE-1Δ t distribution of the experimental (top) and the S r A r simulated replicates (bottom) of lambda DNA. ( c ) Averaged MSD-Δ t profiles of the sub-trajectories captured in the same way as in Fig. 7a (red) and Fig. 7b (blue). The red and blue lines are the theoretical MSD-Δ t profiles. The green line shows the overall, theoretical MSD-Δ t profile of the experimental replicates.

    Article Snippet: Materials Supercoiled ColE1 (6.6 kbp) DNA was obtained from Nippon Gene (Toyama, Japan) whereas the lambda phage DNA was obtained from New England Biolabs (Hitchin, UK).

    Techniques: Lambda DNA Preparation, Mass Spectrometry