alui  (New England Biolabs)


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    Name:
    AluI
    Description:
    AluI 5 000 units
    Catalog Number:
    R0137L
    Price:
    282
    Category:
    Restriction Enzymes
    Size:
    5 000 units
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    New England Biolabs alui
    AluI
    AluI 5 000 units
    https://www.bioz.com/result/alui/product/New England Biolabs
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    alui - by Bioz Stars, 2021-06
    97/100 stars

    Images

    1) Product Images from "Ser100-Phosphorylated RORα Orchestrates CAR and HNF4α to Form Active Chromatin Complex in Response to Phenobarbital to Regulate Induction of CYP2B6"

    Article Title: Ser100-Phosphorylated RORα Orchestrates CAR and HNF4α to Form Active Chromatin Complex in Response to Phenobarbital to Regulate Induction of CYP2B6

    Journal: Molecular Pharmacology

    doi: 10.1124/mol.119.118273

    Phenobarbital induced change in CYP2B6 chromatin conformations in human primary hepatocytes. 3C assay was performed using a restriction enzyme AluI and forward and reverse primers starting at −1495 and −195 upstream CYP2B6 transcription site, respectively. After digestion and ligation, the primers amplified a 207-bp DNA fragment only in PB-treated samples, as confirmed by agarose gel electrophoresis and subsequent sequencing.
    Figure Legend Snippet: Phenobarbital induced change in CYP2B6 chromatin conformations in human primary hepatocytes. 3C assay was performed using a restriction enzyme AluI and forward and reverse primers starting at −1495 and −195 upstream CYP2B6 transcription site, respectively. After digestion and ligation, the primers amplified a 207-bp DNA fragment only in PB-treated samples, as confirmed by agarose gel electrophoresis and subsequent sequencing.

    Techniques Used: Ligation, Amplification, Agarose Gel Electrophoresis, Sequencing

    2) Product Images from "Telomere damage induces internal loops that generate telomeric circles"

    Article Title: Telomere damage induces internal loops that generate telomeric circles

    Journal: Nature Communications

    doi: 10.1038/s41467-020-19139-4

    A two-step procedure for the purification of mammalian telomeres. a Top: agarose gel showing the separation of the large telomeric repeat fragments from the bulk genomic DNA in a sucrose gradient. Genomic DNA (∼2.5 mg) from SV40LT-immortalized MEFs was digested with HinfI and MspI. The digested DNA was separated by centrifugation on a sucrose gradient. Seven fractions were collected and an aliquot (∼1/500) of each fraction was loaded on an agarose gel. Bottom: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the high molecular weight (HMW) fractions. Source data are provided as a Source Data File. b Left: agarose gel showing the separation of the large telomeric repeat fragments from the remaining non-telomeric DNA, in the second purification round. The HMW DNA, contained in the last four fractions of the sucrose gradient described in (a), was recovered and digested with RsaI, AluI, MboI, HinfI, MspI, HphI, and MnlI. The digested DNA was separated on a preparative agarose gel and the DNA migrating in the area above 5 kb was extracted from the gel. The image shows an aliquot (∼1/100) of the digested DNA, separated on an agarose gel. Right: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the HMW area. Source data are provided as a Source Data File. c Dot blot analysis showing the enrichment of telomeric repeats. The indicated amounts of DNA from each enrichment step were spotted on a membrane and hybridized either with a probe recognizing the long interspersed L1 repeats or TTAGGG repeats. The amount of TTAGGG repeat signal/ng was quantified and reported relative to the signal/ng value in the initial, non-enriched DNA. Source data are provided as a Source Data File. d Single-molecule analysis showing the enrichment of the telomeric repeats. The DNA was combed onto silanized coverslips, denatured in situ and labeled sequentially with an antibody against single-stranded DNA and a Cy3-labeled (TTAGGG) 3 PNA probe.
    Figure Legend Snippet: A two-step procedure for the purification of mammalian telomeres. a Top: agarose gel showing the separation of the large telomeric repeat fragments from the bulk genomic DNA in a sucrose gradient. Genomic DNA (∼2.5 mg) from SV40LT-immortalized MEFs was digested with HinfI and MspI. The digested DNA was separated by centrifugation on a sucrose gradient. Seven fractions were collected and an aliquot (∼1/500) of each fraction was loaded on an agarose gel. Bottom: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the high molecular weight (HMW) fractions. Source data are provided as a Source Data File. b Left: agarose gel showing the separation of the large telomeric repeat fragments from the remaining non-telomeric DNA, in the second purification round. The HMW DNA, contained in the last four fractions of the sucrose gradient described in (a), was recovered and digested with RsaI, AluI, MboI, HinfI, MspI, HphI, and MnlI. The digested DNA was separated on a preparative agarose gel and the DNA migrating in the area above 5 kb was extracted from the gel. The image shows an aliquot (∼1/100) of the digested DNA, separated on an agarose gel. Right: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the HMW area. Source data are provided as a Source Data File. c Dot blot analysis showing the enrichment of telomeric repeats. The indicated amounts of DNA from each enrichment step were spotted on a membrane and hybridized either with a probe recognizing the long interspersed L1 repeats or TTAGGG repeats. The amount of TTAGGG repeat signal/ng was quantified and reported relative to the signal/ng value in the initial, non-enriched DNA. Source data are provided as a Source Data File. d Single-molecule analysis showing the enrichment of the telomeric repeats. The DNA was combed onto silanized coverslips, denatured in situ and labeled sequentially with an antibody against single-stranded DNA and a Cy3-labeled (TTAGGG) 3 PNA probe.

    Techniques Used: Purification, Agarose Gel Electrophoresis, Centrifugation, Molecular Weight, Dot Blot, In Situ, Labeling

    3) Product Images from "Regulation of Telomere Length and Suppression of Genomic Instability in Human Somatic Cells by Ku86"

    Article Title: Regulation of Telomere Length and Suppression of Genomic Instability in Human Somatic Cells by Ku86

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.24.11.5050-5059.2004

    Human Ku86-deficient cell lines have shortened telomeres. Genomic DNA was purified from the indicated cell lines, digested to completion with MboI and AluI, and then subjected to terminal restriction fragment Southern blot analysis under denaturing conditions with a (C 3 TA 2 ) 3 5′-end-radiolabeled oligonucleotide probe. Lanes: 1, HCT116 parental cell line; 2 and 3, HCT116 Ku86-heterozygous cell lines #44 and #70, respectively; 4, HCT116 p53-null cell line; 5 and 6, HCT116 p53-null Ku86-heterozygous cell lines #13 and #20, respectively. Approximate molecular size markers are shown on the far left.
    Figure Legend Snippet: Human Ku86-deficient cell lines have shortened telomeres. Genomic DNA was purified from the indicated cell lines, digested to completion with MboI and AluI, and then subjected to terminal restriction fragment Southern blot analysis under denaturing conditions with a (C 3 TA 2 ) 3 5′-end-radiolabeled oligonucleotide probe. Lanes: 1, HCT116 parental cell line; 2 and 3, HCT116 Ku86-heterozygous cell lines #44 and #70, respectively; 4, HCT116 p53-null cell line; 5 and 6, HCT116 p53-null Ku86-heterozygous cell lines #13 and #20, respectively. Approximate molecular size markers are shown on the far left.

    Techniques Used: Purification, Southern Blot

    4) Product Images from "Rhizosphere Protists Change Metabolite Profiles in Zea mays"

    Article Title: Rhizosphere Protists Change Metabolite Profiles in Zea mays

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.00857

    nMDS plots of T-RFLP profiles of bacterial communities obtained after digestion with the three different restriction enzymes MspI (A) , HhaI (B) , and AluI (C) from three soil fractions (bulk soil – circles, rhizosphere soil – triangles – adhering sand to the maize root which was gained by shaking the root, rhizosphere II – squares – remaining sand which was washed from the root). Samples not treated with protists are represented in white ( n = 10), samples treated with protists once in gray ( n = 5), and samples treated twice with protists in black ( n = 5). Stress value = 0.14.
    Figure Legend Snippet: nMDS plots of T-RFLP profiles of bacterial communities obtained after digestion with the three different restriction enzymes MspI (A) , HhaI (B) , and AluI (C) from three soil fractions (bulk soil – circles, rhizosphere soil – triangles – adhering sand to the maize root which was gained by shaking the root, rhizosphere II – squares – remaining sand which was washed from the root). Samples not treated with protists are represented in white ( n = 10), samples treated with protists once in gray ( n = 5), and samples treated twice with protists in black ( n = 5). Stress value = 0.14.

    Techniques Used:

    5) Product Images from "Limited reverse transcriptase activity of phi29 DNA polymerase"

    Article Title: Limited reverse transcriptase activity of phi29 DNA polymerase

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky190

    DNA sequencing-based analysis of rolling circle products reveals reverse transcription activity of phi29 DNA polymerase. ( A ) After RCA, short DNA oligonucleotides were hybridized to an AluI restriction site in the RCA products and RCPs were digested with AluI restriction enzyme, resulting in RCA monomers. Following digestion, monomers were PCR-amplified using primers containing Ilumina adapter sequences. PCR products were extended using IIlumina indexed primers. Finally, sequencing library was prepared using indexed primers-specific P5/7 PCR primers. The region of interest containing RNA substitutions in the original padlock probe sequence is indicated with green boxes. ( B ) Logos showing sequencing frequencies for each position within RCA monomers generated from the control DNA circle (P1 = dG), and circles containing single rG, rU, rA and rC substitutions at the RNA position (P1). Positions P1 and P2 are indicated and position P1 was additionally highlighted with the red box. ( C ) Incorporation of incorrect nucleotides for every position in the sequenced monomers from (B). Error rates, calculated as Incorporation error [%] = 1 – number of reads with expected nucleotide/total number of reads, is presented for padlock probes with single- (upper plot) and double-RNA substitutions (lower plots). P1 position for the first RNA substitution is indicated with the box.
    Figure Legend Snippet: DNA sequencing-based analysis of rolling circle products reveals reverse transcription activity of phi29 DNA polymerase. ( A ) After RCA, short DNA oligonucleotides were hybridized to an AluI restriction site in the RCA products and RCPs were digested with AluI restriction enzyme, resulting in RCA monomers. Following digestion, monomers were PCR-amplified using primers containing Ilumina adapter sequences. PCR products were extended using IIlumina indexed primers. Finally, sequencing library was prepared using indexed primers-specific P5/7 PCR primers. The region of interest containing RNA substitutions in the original padlock probe sequence is indicated with green boxes. ( B ) Logos showing sequencing frequencies for each position within RCA monomers generated from the control DNA circle (P1 = dG), and circles containing single rG, rU, rA and rC substitutions at the RNA position (P1). Positions P1 and P2 are indicated and position P1 was additionally highlighted with the red box. ( C ) Incorporation of incorrect nucleotides for every position in the sequenced monomers from (B). Error rates, calculated as Incorporation error [%] = 1 – number of reads with expected nucleotide/total number of reads, is presented for padlock probes with single- (upper plot) and double-RNA substitutions (lower plots). P1 position for the first RNA substitution is indicated with the box.

    Techniques Used: DNA Sequencing, Activity Assay, Polymerase Chain Reaction, Amplification, Sequencing, Generated

    6) Product Images from "Telomere damage induces internal loops that generate telomeric circles"

    Article Title: Telomere damage induces internal loops that generate telomeric circles

    Journal: Nature Communications

    doi: 10.1038/s41467-020-19139-4

    A two-step procedure for the purification of mammalian telomeres. a Top: agarose gel showing the separation of the large telomeric repeat fragments from the bulk genomic DNA in a sucrose gradient. Genomic DNA (∼2.5 mg) from SV40LT-immortalized MEFs was digested with HinfI and MspI. The digested DNA was separated by centrifugation on a sucrose gradient. Seven fractions were collected and an aliquot (∼1/500) of each fraction was loaded on an agarose gel. Bottom: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the high molecular weight (HMW) fractions. Source data are provided as a Source Data File. b Left: agarose gel showing the separation of the large telomeric repeat fragments from the remaining non-telomeric DNA, in the second purification round. The HMW DNA, contained in the last four fractions of the sucrose gradient described in (a), was recovered and digested with RsaI, AluI, MboI, HinfI, MspI, HphI, and MnlI. The digested DNA was separated on a preparative agarose gel and the DNA migrating in the area above 5 kb was extracted from the gel. The image shows an aliquot (∼1/100) of the digested DNA, separated on an agarose gel. Right: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the HMW area. Source data are provided as a Source Data File. c Dot blot analysis showing the enrichment of telomeric repeats. The indicated amounts of DNA from each enrichment step were spotted on a membrane and hybridized either with a probe recognizing the long interspersed L1 repeats or TTAGGG repeats. The amount of TTAGGG repeat signal/ng was quantified and reported relative to the signal/ng value in the initial, non-enriched DNA. Source data are provided as a Source Data File. d Single-molecule analysis showing the enrichment of the telomeric repeats. The DNA was combed onto silanized coverslips, denatured in situ and labeled sequentially with an antibody against single-stranded DNA and a Cy3-labeled (TTAGGG) 3 PNA probe.
    Figure Legend Snippet: A two-step procedure for the purification of mammalian telomeres. a Top: agarose gel showing the separation of the large telomeric repeat fragments from the bulk genomic DNA in a sucrose gradient. Genomic DNA (∼2.5 mg) from SV40LT-immortalized MEFs was digested with HinfI and MspI. The digested DNA was separated by centrifugation on a sucrose gradient. Seven fractions were collected and an aliquot (∼1/500) of each fraction was loaded on an agarose gel. Bottom: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the high molecular weight (HMW) fractions. Source data are provided as a Source Data File. b Left: agarose gel showing the separation of the large telomeric repeat fragments from the remaining non-telomeric DNA, in the second purification round. The HMW DNA, contained in the last four fractions of the sucrose gradient described in (a), was recovered and digested with RsaI, AluI, MboI, HinfI, MspI, HphI, and MnlI. The digested DNA was separated on a preparative agarose gel and the DNA migrating in the area above 5 kb was extracted from the gel. The image shows an aliquot (∼1/100) of the digested DNA, separated on an agarose gel. Right: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the HMW area. Source data are provided as a Source Data File. c Dot blot analysis showing the enrichment of telomeric repeats. The indicated amounts of DNA from each enrichment step were spotted on a membrane and hybridized either with a probe recognizing the long interspersed L1 repeats or TTAGGG repeats. The amount of TTAGGG repeat signal/ng was quantified and reported relative to the signal/ng value in the initial, non-enriched DNA. Source data are provided as a Source Data File. d Single-molecule analysis showing the enrichment of the telomeric repeats. The DNA was combed onto silanized coverslips, denatured in situ and labeled sequentially with an antibody against single-stranded DNA and a Cy3-labeled (TTAGGG) 3 PNA probe.

    Techniques Used: Purification, Agarose Gel Electrophoresis, Centrifugation, Molecular Weight, Dot Blot, In Situ, Labeling

    7) Product Images from "Telomere damage induces internal loops that generate telomeric circles"

    Article Title: Telomere damage induces internal loops that generate telomeric circles

    Journal: bioRxiv

    doi: 10.1101/2020.01.29.924951

    A two-step procedure for the purification of mammalian telomeres A. Top: agarose gel showing the separation of the large telomeric repeat fragments from the bulk DNA in a sucrose gradient. Genomic DNA (~2.5 mg) from SV40-MEFs was digested with HinfI and MspI. The digested DNA was separated by centrifugation on a sucrose gradient. Seven fractions were collected and an aliquot (~1/500) of each fraction was loaded on an agarose gel. Bottom: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the high molecular weight (HMW) fractions. B. Left: agarose gel showing the separation of the large telomeric repeat fragments from the remaining non-telomeric DNA, in the second purification round. The HMW DNA, contained in the last four fractions of the sucrose gradient described in (A), was recovered and digested with RsaI, AluI, MboI, HinfI, MspI, HphI and MnlI. The digested DNA was separated on a preparative agarose gel and the DNA migrating in the area above 5 kb was extracted from the gel. The image shows an aliquot (~1/100) of the digested DNA, separated on an agarose gel. Right: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the HMW area. C. Dot blot analysis showing the enrichment of telomeric repeats. The indicated amounts of DNA from each enrichment step were spotted on a membrane and hybridized either with a probe recognizing the long interspersed BamHI repeats or TTAGGG repeats. The amount of TTAGGG repeat signal/ng was quantified and reported relative to the signal/ng value in the initial, non-enriched DNA. D. Single molecule analysis showing the enrichment of the telomeric repeats. The DNA was combed onto silanized coverslips, denatured in situ and labeled sequentially with an antibody against single-stranded DNA and a Cy3-labeled (TTAGGG) 3 PNA probe.
    Figure Legend Snippet: A two-step procedure for the purification of mammalian telomeres A. Top: agarose gel showing the separation of the large telomeric repeat fragments from the bulk DNA in a sucrose gradient. Genomic DNA (~2.5 mg) from SV40-MEFs was digested with HinfI and MspI. The digested DNA was separated by centrifugation on a sucrose gradient. Seven fractions were collected and an aliquot (~1/500) of each fraction was loaded on an agarose gel. Bottom: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the high molecular weight (HMW) fractions. B. Left: agarose gel showing the separation of the large telomeric repeat fragments from the remaining non-telomeric DNA, in the second purification round. The HMW DNA, contained in the last four fractions of the sucrose gradient described in (A), was recovered and digested with RsaI, AluI, MboI, HinfI, MspI, HphI and MnlI. The digested DNA was separated on a preparative agarose gel and the DNA migrating in the area above 5 kb was extracted from the gel. The image shows an aliquot (~1/100) of the digested DNA, separated on an agarose gel. Right: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the HMW area. C. Dot blot analysis showing the enrichment of telomeric repeats. The indicated amounts of DNA from each enrichment step were spotted on a membrane and hybridized either with a probe recognizing the long interspersed BamHI repeats or TTAGGG repeats. The amount of TTAGGG repeat signal/ng was quantified and reported relative to the signal/ng value in the initial, non-enriched DNA. D. Single molecule analysis showing the enrichment of the telomeric repeats. The DNA was combed onto silanized coverslips, denatured in situ and labeled sequentially with an antibody against single-stranded DNA and a Cy3-labeled (TTAGGG) 3 PNA probe.

    Techniques Used: Purification, Agarose Gel Electrophoresis, Centrifugation, Molecular Weight, Dot Blot, In Situ, Labeling

    I-loops are induced by single-strand damage at telomeric repeats (see also Figure S5 ). A. 2D-gel analysis showing that the t-circle arc can be strongly induced by formation of nicks and gaps at telomeres. MEFs nuclei were incubated with either 0; 1; 2.5 or 5 μg/ml of DNase I for 8 minutes at RT. The reaction was stopped and the genomic DNA was isolated. 5 μg were digested with AluI and MboI and separated on 2D-gels. The gels were blotted on a membrane and hybridized with a TTAGGG repeats probe. The ratio of the telomeric signal in the t-circle arc (yellow arrows) and in the linears (black arrows), is reported relative to the untreated sample, which was arbitrarily set to 100. B. 2D-gel analysis showing that the t-circle arc can form spontaneously, in the presence of nicks and gaps at the telomeric repeats. Isolated mouse genomic DNA was incubated with either 0; 0.1; 0.2 or 0.4 μg/ml of DNase I for 8 min at RT. The reaction was stopped, the DNA was extracted with phenol-chloroform, digested with AluI and MboI, separated on 2D-gels, blotted on a membrane and hybridized with a probe recognizing the TTAGGG repeats. The ratio of the telomeric signal in the t-circle arc and in the linears, is reported relative to the untreated sample which was arbitrarily set to 100. C. Dot blot showing the enrichment of the telomeric repeats, after the large-scale DNase I treatment. Around 500 × 10 6 SV40-MEFs nuclei were incubated either with 0 or 5 μg/ml of DNase I for 8 minutes at RT. The reaction was stopped, genomic DNA was isolated and telomeres were enriched with the procedure described in Figure 1 . The indicated amounts from each enrichment step were spotted on a membrane and hybridized with a probe recognizing the TTAGGG repeats. The telomeric signal per ng of DNA is reported relative to the non-enriched DNA. D. Accumulation of i-loops at telomeres damaged by DNase I. Telomere-enriched DNA from the experiment described in (C) was analyzed in EM. The percentage of molecules with internal loops is reported. A KpnI-digested bulk DNA control was included for the sample treated with DNase I. E. Examples of molecules with internal loops observed at telomere preparations, from DNase I-treated nuclei. Insets show 2X enlargements of the area inside the yellow rectangles.
    Figure Legend Snippet: I-loops are induced by single-strand damage at telomeric repeats (see also Figure S5 ). A. 2D-gel analysis showing that the t-circle arc can be strongly induced by formation of nicks and gaps at telomeres. MEFs nuclei were incubated with either 0; 1; 2.5 or 5 μg/ml of DNase I for 8 minutes at RT. The reaction was stopped and the genomic DNA was isolated. 5 μg were digested with AluI and MboI and separated on 2D-gels. The gels were blotted on a membrane and hybridized with a TTAGGG repeats probe. The ratio of the telomeric signal in the t-circle arc (yellow arrows) and in the linears (black arrows), is reported relative to the untreated sample, which was arbitrarily set to 100. B. 2D-gel analysis showing that the t-circle arc can form spontaneously, in the presence of nicks and gaps at the telomeric repeats. Isolated mouse genomic DNA was incubated with either 0; 0.1; 0.2 or 0.4 μg/ml of DNase I for 8 min at RT. The reaction was stopped, the DNA was extracted with phenol-chloroform, digested with AluI and MboI, separated on 2D-gels, blotted on a membrane and hybridized with a probe recognizing the TTAGGG repeats. The ratio of the telomeric signal in the t-circle arc and in the linears, is reported relative to the untreated sample which was arbitrarily set to 100. C. Dot blot showing the enrichment of the telomeric repeats, after the large-scale DNase I treatment. Around 500 × 10 6 SV40-MEFs nuclei were incubated either with 0 or 5 μg/ml of DNase I for 8 minutes at RT. The reaction was stopped, genomic DNA was isolated and telomeres were enriched with the procedure described in Figure 1 . The indicated amounts from each enrichment step were spotted on a membrane and hybridized with a probe recognizing the TTAGGG repeats. The telomeric signal per ng of DNA is reported relative to the non-enriched DNA. D. Accumulation of i-loops at telomeres damaged by DNase I. Telomere-enriched DNA from the experiment described in (C) was analyzed in EM. The percentage of molecules with internal loops is reported. A KpnI-digested bulk DNA control was included for the sample treated with DNase I. E. Examples of molecules with internal loops observed at telomere preparations, from DNase I-treated nuclei. Insets show 2X enlargements of the area inside the yellow rectangles.

    Techniques Used: Two-Dimensional Gel Electrophoresis, Incubation, Isolation, Dot Blot

    8) Product Images from "Limited reverse transcriptase activity of phi29 DNA polymerase"

    Article Title: Limited reverse transcriptase activity of phi29 DNA polymerase

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky190

    DNA sequencing-based analysis of rolling circle products reveals reverse transcription activity of phi29 DNA polymerase. ( A ) After RCA, short DNA oligonucleotides were hybridized to an AluI restriction site in the RCA products and RCPs were digested with AluI restriction enzyme, resulting in RCA monomers. Following digestion, monomers were PCR-amplified using primers containing Ilumina adapter sequences. PCR products were extended using IIlumina indexed primers. Finally, sequencing library was prepared using indexed primers-specific P5/7 PCR primers. The region of interest containing RNA substitutions in the original padlock probe sequence is indicated with green boxes. ( B ) Logos showing sequencing frequencies for each position within RCA monomers generated from the control DNA circle (P1 = dG), and circles containing single rG, rU, rA and rC substitutions at the RNA position (P1). Positions P1 and P2 are indicated and position P1 was additionally highlighted with the red box. ( C ) Incorporation of incorrect nucleotides for every position in the sequenced monomers from (B). Error rates, calculated as Incorporation error [%] = 1 – number of reads with expected nucleotide/total number of reads, is presented for padlock probes with single- (upper plot) and double-RNA substitutions (lower plots). P1 position for the first RNA substitution is indicated with the box.
    Figure Legend Snippet: DNA sequencing-based analysis of rolling circle products reveals reverse transcription activity of phi29 DNA polymerase. ( A ) After RCA, short DNA oligonucleotides were hybridized to an AluI restriction site in the RCA products and RCPs were digested with AluI restriction enzyme, resulting in RCA monomers. Following digestion, monomers were PCR-amplified using primers containing Ilumina adapter sequences. PCR products were extended using IIlumina indexed primers. Finally, sequencing library was prepared using indexed primers-specific P5/7 PCR primers. The region of interest containing RNA substitutions in the original padlock probe sequence is indicated with green boxes. ( B ) Logos showing sequencing frequencies for each position within RCA monomers generated from the control DNA circle (P1 = dG), and circles containing single rG, rU, rA and rC substitutions at the RNA position (P1). Positions P1 and P2 are indicated and position P1 was additionally highlighted with the red box. ( C ) Incorporation of incorrect nucleotides for every position in the sequenced monomers from (B). Error rates, calculated as Incorporation error [%] = 1 – number of reads with expected nucleotide/total number of reads, is presented for padlock probes with single- (upper plot) and double-RNA substitutions (lower plots). P1 position for the first RNA substitution is indicated with the box.

    Techniques Used: DNA Sequencing, Activity Assay, Polymerase Chain Reaction, Amplification, Sequencing, Generated

    9) Product Images from "Telomere damage induces internal loops that generate telomeric circles"

    Article Title: Telomere damage induces internal loops that generate telomeric circles

    Journal: Nature Communications

    doi: 10.1038/s41467-020-19139-4

    A two-step procedure for the purification of mammalian telomeres. a Top: agarose gel showing the separation of the large telomeric repeat fragments from the bulk genomic DNA in a sucrose gradient. Genomic DNA (∼2.5 mg) from SV40LT-immortalized MEFs was digested with HinfI and MspI. The digested DNA was separated by centrifugation on a sucrose gradient. Seven fractions were collected and an aliquot (∼1/500) of each fraction was loaded on an agarose gel. Bottom: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the high molecular weight (HMW) fractions. Source data are provided as a Source Data File. b Left: agarose gel showing the separation of the large telomeric repeat fragments from the remaining non-telomeric DNA, in the second purification round. The HMW DNA, contained in the last four fractions of the sucrose gradient described in (a), was recovered and digested with RsaI, AluI, MboI, HinfI, MspI, HphI, and MnlI. The digested DNA was separated on a preparative agarose gel and the DNA migrating in the area above 5 kb was extracted from the gel. The image shows an aliquot (∼1/100) of the digested DNA, separated on an agarose gel. Right: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the HMW area. Source data are provided as a Source Data File. c Dot blot analysis showing the enrichment of telomeric repeats. The indicated amounts of DNA from each enrichment step were spotted on a membrane and hybridized either with a probe recognizing the long interspersed L1 repeats or TTAGGG repeats. The amount of TTAGGG repeat signal/ng was quantified and reported relative to the signal/ng value in the initial, non-enriched DNA. Source data are provided as a Source Data File. d Single-molecule analysis showing the enrichment of the telomeric repeats. The DNA was combed onto silanized coverslips, denatured in situ and labeled sequentially with an antibody against single-stranded DNA and a Cy3-labeled (TTAGGG) 3 PNA probe.
    Figure Legend Snippet: A two-step procedure for the purification of mammalian telomeres. a Top: agarose gel showing the separation of the large telomeric repeat fragments from the bulk genomic DNA in a sucrose gradient. Genomic DNA (∼2.5 mg) from SV40LT-immortalized MEFs was digested with HinfI and MspI. The digested DNA was separated by centrifugation on a sucrose gradient. Seven fractions were collected and an aliquot (∼1/500) of each fraction was loaded on an agarose gel. Bottom: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the high molecular weight (HMW) fractions. Source data are provided as a Source Data File. b Left: agarose gel showing the separation of the large telomeric repeat fragments from the remaining non-telomeric DNA, in the second purification round. The HMW DNA, contained in the last four fractions of the sucrose gradient described in (a), was recovered and digested with RsaI, AluI, MboI, HinfI, MspI, HphI, and MnlI. The digested DNA was separated on a preparative agarose gel and the DNA migrating in the area above 5 kb was extracted from the gel. The image shows an aliquot (∼1/100) of the digested DNA, separated on an agarose gel. Right: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the HMW area. Source data are provided as a Source Data File. c Dot blot analysis showing the enrichment of telomeric repeats. The indicated amounts of DNA from each enrichment step were spotted on a membrane and hybridized either with a probe recognizing the long interspersed L1 repeats or TTAGGG repeats. The amount of TTAGGG repeat signal/ng was quantified and reported relative to the signal/ng value in the initial, non-enriched DNA. Source data are provided as a Source Data File. d Single-molecule analysis showing the enrichment of the telomeric repeats. The DNA was combed onto silanized coverslips, denatured in situ and labeled sequentially with an antibody against single-stranded DNA and a Cy3-labeled (TTAGGG) 3 PNA probe.

    Techniques Used: Purification, Agarose Gel Electrophoresis, Centrifugation, Molecular Weight, Dot Blot, In Situ, Labeling

    10) Product Images from "Telomere length regulation and transcriptional silencing in KU80-deficient Trypanosoma brucei"

    Article Title: Telomere length regulation and transcriptional silencing in KU80-deficient Trypanosoma brucei

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkh991

    Telomere shortening in Δ tb KU80 trypanosomes. Genomic DNA from wild-type cells, tb KU80 single allele knockout cells ( tb KU80 +/− ), tb KU80-deficient cells (Δ tb KU80) and from a tb KU80-deficient cell line, which expressed an ectopic copy of GFP– tb KU80 (Δ tb KU80 + GFP– tb KU80) was prepared every other week for a period of 8 weeks. The DNA was digested with AluI, HinfI and RsaI, and separated by agarose gel electrophoresis, Southern-blotted and probed with a radiolabeled telomeric (TTAGGG) 27 probe. Distinguishable bands of Δ tb KU80 telomeric DNA were used to estimate telomere shortening rates during 8 weeks.
    Figure Legend Snippet: Telomere shortening in Δ tb KU80 trypanosomes. Genomic DNA from wild-type cells, tb KU80 single allele knockout cells ( tb KU80 +/− ), tb KU80-deficient cells (Δ tb KU80) and from a tb KU80-deficient cell line, which expressed an ectopic copy of GFP– tb KU80 (Δ tb KU80 + GFP– tb KU80) was prepared every other week for a period of 8 weeks. The DNA was digested with AluI, HinfI and RsaI, and separated by agarose gel electrophoresis, Southern-blotted and probed with a radiolabeled telomeric (TTAGGG) 27 probe. Distinguishable bands of Δ tb KU80 telomeric DNA were used to estimate telomere shortening rates during 8 weeks.

    Techniques Used: Knock-Out, Agarose Gel Electrophoresis

    G-overhang structure in Δ tb KU80 trypanosomes. DNA was extracted from tb KU80-deficient cells (Δ tb KU80), wild-type cells (wild type) and from HeLa cells (human), and digested with MboI and AluI restriction enzymes. Different amounts of DNA, as indicated in each lane, were separated by gel electrophoresis and analyzed with radiolabeled probes specific for G-overhangs [(CCCTAA) 4 , left panels] or C-overhangs [(TTAGGG) 4 , right panels]. To confirm equal loading the gels were denatured and re-hybridized with the same probes as above (lower panels). Different exposure times of the gels on a phosphorimager (Molecular Dynamics) are indicated on the right.
    Figure Legend Snippet: G-overhang structure in Δ tb KU80 trypanosomes. DNA was extracted from tb KU80-deficient cells (Δ tb KU80), wild-type cells (wild type) and from HeLa cells (human), and digested with MboI and AluI restriction enzymes. Different amounts of DNA, as indicated in each lane, were separated by gel electrophoresis and analyzed with radiolabeled probes specific for G-overhangs [(CCCTAA) 4 , left panels] or C-overhangs [(TTAGGG) 4 , right panels]. To confirm equal loading the gels were denatured and re-hybridized with the same probes as above (lower panels). Different exposure times of the gels on a phosphorimager (Molecular Dynamics) are indicated on the right.

    Techniques Used: Nucleic Acid Electrophoresis

    11) Product Images from "ISWI Remodelling of Physiological Chromatin Fibres Acetylated at Lysine 16 of Histone H4"

    Article Title: ISWI Remodelling of Physiological Chromatin Fibres Acetylated at Lysine 16 of Histone H4

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0088411

    ISWI and ACF remodelling activity is not inhibited by H4K16ac. (A) Scheme of the remodelling assay. Acetylated (H4K16ac) and unmodified (H4) arrays were labelled at one DNA end with the fluorescent dyes DY-682 and DY-776, respectively. Remodelling reactions contained both array types along with ATP, AluI and the remodeller. Samples of the reaction were taken at different time points (t) and the DNA fragments were analysed on an agarose gel. (B) Top: Schematic depiction of the reaction conditions of the remodelling assay. (Ac: acetylated arrays). Middle: Exemplary result of a remodelling time course with ISWI (500 nM). Nucleosome concentration was 25 nM per array type and ATP concentration was 1 µM. All samples were run on the same agarose gel and the two fluorescent labels were visualized separately by scanning the gel at the respective wave lengths. Lanes were rearranged for presentation purposes. Control reactions did not contain ISWI (–). Bottom: Quantification of remodelling progress. Based on the fluorescent signal intensity the fraction of uncut array DNA was determined for each gel lane and plotted against the remodelling time. (C) Remodelling assay as in B, but with ISWI and nucleosome concentrations of 5 nM and 100 nM, respectively. ATP concentration was 200 µM. In the plot shown in the bottom panel, the remodelling times needed to reach 50% cut array DNA were interpolated by connecting the data points by smooth lines (Excel; Microsoft). (D) Exemplary result of a remodelling time course with ACF. Reaction conditions were as in C. (kb: kilobases).
    Figure Legend Snippet: ISWI and ACF remodelling activity is not inhibited by H4K16ac. (A) Scheme of the remodelling assay. Acetylated (H4K16ac) and unmodified (H4) arrays were labelled at one DNA end with the fluorescent dyes DY-682 and DY-776, respectively. Remodelling reactions contained both array types along with ATP, AluI and the remodeller. Samples of the reaction were taken at different time points (t) and the DNA fragments were analysed on an agarose gel. (B) Top: Schematic depiction of the reaction conditions of the remodelling assay. (Ac: acetylated arrays). Middle: Exemplary result of a remodelling time course with ISWI (500 nM). Nucleosome concentration was 25 nM per array type and ATP concentration was 1 µM. All samples were run on the same agarose gel and the two fluorescent labels were visualized separately by scanning the gel at the respective wave lengths. Lanes were rearranged for presentation purposes. Control reactions did not contain ISWI (–). Bottom: Quantification of remodelling progress. Based on the fluorescent signal intensity the fraction of uncut array DNA was determined for each gel lane and plotted against the remodelling time. (C) Remodelling assay as in B, but with ISWI and nucleosome concentrations of 5 nM and 100 nM, respectively. ATP concentration was 200 µM. In the plot shown in the bottom panel, the remodelling times needed to reach 50% cut array DNA were interpolated by connecting the data points by smooth lines (Excel; Microsoft). (D) Exemplary result of a remodelling time course with ACF. Reaction conditions were as in C. (kb: kilobases).

    Techniques Used: Activity Assay, Agarose Gel Electrophoresis, Concentration Assay

    H1 inhibits ISWI activity. (A) Steady-state ATPase assay. Saturating concentrations (600 nM) of unmodified (– Ac) or acetylated (+ Ac) chromatosome arrays (+ H1) were employed to stimulate ISWI (100 nM) in presence of saturating ATP (1 mM). ATP hydrolysis rates for nucleosome arrays (– H1) were taken from Figure 2 . Error bars represent standard deviations (– H1: n = 5; + H1 n = 6). (B) Exemplary remodelling assay using nucleosome and chromatosome arrays (200 nM) as substrates for ISWI (50 nM) at 100 µM ATP. The chromatosome arrays had been reconstituted with a 1:4.5 molar ratio of nucleosomes to H1. The assay was performed as in Figure 3 except that the arrays lacked a fluorescent label and all array types were tested in separate reactions. The DNA was visualized by ethidium bromide stain. All samples of a reaction were loaded onto one gel and empty lanes were spliced out. ATP was omitted from control reactions (–). The most prominent DNA bands comprise multiples of 197 bp reflecting the distance between two AluI sites within the array ( Figure 1A ). Faint interspersed bands arose from a single AluI cut; they harbour one of the ends of the array DNA and therefore deviate in length from the internal cleavage products. Asterisks mark DNA bands originating from contaminating competitor DNA. (kb: kilobases). (C) Quantification of the experiment depicted in B. Analysis was done as in Figure 3C . (D) Summary of the remodelling activity of ISWI on chromatosome arrays reconstituted with different input amounts of H1 (see B and Figure S4 ).
    Figure Legend Snippet: H1 inhibits ISWI activity. (A) Steady-state ATPase assay. Saturating concentrations (600 nM) of unmodified (– Ac) or acetylated (+ Ac) chromatosome arrays (+ H1) were employed to stimulate ISWI (100 nM) in presence of saturating ATP (1 mM). ATP hydrolysis rates for nucleosome arrays (– H1) were taken from Figure 2 . Error bars represent standard deviations (– H1: n = 5; + H1 n = 6). (B) Exemplary remodelling assay using nucleosome and chromatosome arrays (200 nM) as substrates for ISWI (50 nM) at 100 µM ATP. The chromatosome arrays had been reconstituted with a 1:4.5 molar ratio of nucleosomes to H1. The assay was performed as in Figure 3 except that the arrays lacked a fluorescent label and all array types were tested in separate reactions. The DNA was visualized by ethidium bromide stain. All samples of a reaction were loaded onto one gel and empty lanes were spliced out. ATP was omitted from control reactions (–). The most prominent DNA bands comprise multiples of 197 bp reflecting the distance between two AluI sites within the array ( Figure 1A ). Faint interspersed bands arose from a single AluI cut; they harbour one of the ends of the array DNA and therefore deviate in length from the internal cleavage products. Asterisks mark DNA bands originating from contaminating competitor DNA. (kb: kilobases). (C) Quantification of the experiment depicted in B. Analysis was done as in Figure 3C . (D) Summary of the remodelling activity of ISWI on chromatosome arrays reconstituted with different input amounts of H1 (see B and Figure S4 ).

    Techniques Used: Activity Assay, ATPase Assay, Staining

    12) Product Images from "Telomere damage induces internal loops that generate telomeric circles"

    Article Title: Telomere damage induces internal loops that generate telomeric circles

    Journal: Nature Communications

    doi: 10.1038/s41467-020-19139-4

    A two-step procedure for the purification of mammalian telomeres. a Top: agarose gel showing the separation of the large telomeric repeat fragments from the bulk genomic DNA in a sucrose gradient. Genomic DNA (∼2.5 mg) from SV40LT-immortalized MEFs was digested with HinfI and MspI. The digested DNA was separated by centrifugation on a sucrose gradient. Seven fractions were collected and an aliquot (∼1/500) of each fraction was loaded on an agarose gel. Bottom: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the high molecular weight (HMW) fractions. Source data are provided as a Source Data File. b Left: agarose gel showing the separation of the large telomeric repeat fragments from the remaining non-telomeric DNA, in the second purification round. The HMW DNA, contained in the last four fractions of the sucrose gradient described in (a), was recovered and digested with RsaI, AluI, MboI, HinfI, MspI, HphI, and MnlI. The digested DNA was separated on a preparative agarose gel and the DNA migrating in the area above 5 kb was extracted from the gel. The image shows an aliquot (∼1/100) of the digested DNA, separated on an agarose gel. Right: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the HMW area. Source data are provided as a Source Data File. c Dot blot analysis showing the enrichment of telomeric repeats. The indicated amounts of DNA from each enrichment step were spotted on a membrane and hybridized either with a probe recognizing the long interspersed L1 repeats or TTAGGG repeats. The amount of TTAGGG repeat signal/ng was quantified and reported relative to the signal/ng value in the initial, non-enriched DNA. Source data are provided as a Source Data File. d Single-molecule analysis showing the enrichment of the telomeric repeats. The DNA was combed onto silanized coverslips, denatured in situ and labeled sequentially with an antibody against single-stranded DNA and a Cy3-labeled (TTAGGG) 3 PNA probe.
    Figure Legend Snippet: A two-step procedure for the purification of mammalian telomeres. a Top: agarose gel showing the separation of the large telomeric repeat fragments from the bulk genomic DNA in a sucrose gradient. Genomic DNA (∼2.5 mg) from SV40LT-immortalized MEFs was digested with HinfI and MspI. The digested DNA was separated by centrifugation on a sucrose gradient. Seven fractions were collected and an aliquot (∼1/500) of each fraction was loaded on an agarose gel. Bottom: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the high molecular weight (HMW) fractions. Source data are provided as a Source Data File. b Left: agarose gel showing the separation of the large telomeric repeat fragments from the remaining non-telomeric DNA, in the second purification round. The HMW DNA, contained in the last four fractions of the sucrose gradient described in (a), was recovered and digested with RsaI, AluI, MboI, HinfI, MspI, HphI, and MnlI. The digested DNA was separated on a preparative agarose gel and the DNA migrating in the area above 5 kb was extracted from the gel. The image shows an aliquot (∼1/100) of the digested DNA, separated on an agarose gel. Right: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the HMW area. Source data are provided as a Source Data File. c Dot blot analysis showing the enrichment of telomeric repeats. The indicated amounts of DNA from each enrichment step were spotted on a membrane and hybridized either with a probe recognizing the long interspersed L1 repeats or TTAGGG repeats. The amount of TTAGGG repeat signal/ng was quantified and reported relative to the signal/ng value in the initial, non-enriched DNA. Source data are provided as a Source Data File. d Single-molecule analysis showing the enrichment of the telomeric repeats. The DNA was combed onto silanized coverslips, denatured in situ and labeled sequentially with an antibody against single-stranded DNA and a Cy3-labeled (TTAGGG) 3 PNA probe.

    Techniques Used: Purification, Agarose Gel Electrophoresis, Centrifugation, Molecular Weight, Dot Blot, In Situ, Labeling

    13) Product Images from "Trypanosoma brucei UMSBP2 is a single-stranded telomeric DNA binding protein essential for chromosome end protection"

    Article Title: Trypanosoma brucei UMSBP2 is a single-stranded telomeric DNA binding protein essential for chromosome end protection

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky597

    TbUMSBP2 knockdown altered the amount of single stranded telomeric DNA. DNA samples (1 μg) of uninduced cells (−) and cells at day 3 post TbUMSBP2 RNAi induction (+), were digested with HinfI and AluI restriction endonucleases and analyzed by in-gel hybridization to C-probe (AACCCT) 3 or G-probe (AGGGTT) 3 , first under native conditions ( A ) and then re-hybridized again to the same probes after denaturation ( B ), as described under ‘Materials and Methods’. ( C and D ) the histograms represent the relative amounts of native signal (corresponding to single-stranded telomeric DNA) normalized to the denatured (total) signals. The uninduced control samples were set as 1.
    Figure Legend Snippet: TbUMSBP2 knockdown altered the amount of single stranded telomeric DNA. DNA samples (1 μg) of uninduced cells (−) and cells at day 3 post TbUMSBP2 RNAi induction (+), were digested with HinfI and AluI restriction endonucleases and analyzed by in-gel hybridization to C-probe (AACCCT) 3 or G-probe (AGGGTT) 3 , first under native conditions ( A ) and then re-hybridized again to the same probes after denaturation ( B ), as described under ‘Materials and Methods’. ( C and D ) the histograms represent the relative amounts of native signal (corresponding to single-stranded telomeric DNA) normalized to the denatured (total) signals. The uninduced control samples were set as 1.

    Techniques Used: Hybridization

    14) Product Images from "Methylation of MdMYB1 locus mediated by RdDM pathway regulates anthocyanin biosynthesis in apple) Methylation of MdMYB1 locus mediated by RdDM pathway regulates anthocyanin biosynthesis in apple"

    Article Title: Methylation of MdMYB1 locus mediated by RdDM pathway regulates anthocyanin biosynthesis in apple) Methylation of MdMYB1 locus mediated by RdDM pathway regulates anthocyanin biosynthesis in apple

    Journal: Plant Biotechnology Journal

    doi: 10.1111/pbi.13337

    Heterologous expression of MdAGO4s or MdDRM2s rescues CHH methylation of AGO4‐binding sites in Arabidopsis ago4 and drm2 mutants. (a) Ten‐day‐old wild‐type Arabidopsis (WT), ago4 mutant, drm2 mutant and transgenic Arabidopsis (35S:: AGO4‐1/2 and 35S::DRM2‐1/2 ). DNA methylation was analysed using three methylation‐sensitive restriction endonucleases, AluI (b), HaeIII (c) and DdeI (d). Genomic DNA was extracted from 2‐week‐old seedlings, and digested genomic DNAs were amplified by PCR. Sequences lacking AluI sites (IGN5), HaeIII (AT5G27860) and DdeI (AT2G36490) were used as loading controls. Sequence and size of Arabidopsis genes are listed in Table S2 . 1–7 indicate 35S:: AGO4‐1 / ago4 , 35S:: AGO4‐2 / ago4 , ago4 , WT, drm2 , 35S:: DRM2‐1 / drm2 and 35S:: DRM2‐2 / drm2 , respectively.
    Figure Legend Snippet: Heterologous expression of MdAGO4s or MdDRM2s rescues CHH methylation of AGO4‐binding sites in Arabidopsis ago4 and drm2 mutants. (a) Ten‐day‐old wild‐type Arabidopsis (WT), ago4 mutant, drm2 mutant and transgenic Arabidopsis (35S:: AGO4‐1/2 and 35S::DRM2‐1/2 ). DNA methylation was analysed using three methylation‐sensitive restriction endonucleases, AluI (b), HaeIII (c) and DdeI (d). Genomic DNA was extracted from 2‐week‐old seedlings, and digested genomic DNAs were amplified by PCR. Sequences lacking AluI sites (IGN5), HaeIII (AT5G27860) and DdeI (AT2G36490) were used as loading controls. Sequence and size of Arabidopsis genes are listed in Table S2 . 1–7 indicate 35S:: AGO4‐1 / ago4 , 35S:: AGO4‐2 / ago4 , ago4 , WT, drm2 , 35S:: DRM2‐1 / drm2 and 35S:: DRM2‐2 / drm2 , respectively.

    Techniques Used: Expressing, Methylation, Binding Assay, Mutagenesis, Transgenic Assay, DNA Methylation Assay, Amplification, Polymerase Chain Reaction, Sequencing

    15) Product Images from "Structural and functional analysis of an OB-fold in human Ctc1 implicated in telomere maintenance and bone marrow syndromes"

    Article Title: Structural and functional analysis of an OB-fold in human Ctc1 implicated in telomere maintenance and bone marrow syndromes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx1213

    hCtc1(OB) deletion increases ExoI-resistant single-stranded telomere DNA. ( A ) Analysis of single-stranded telomeric DNA from 293T cell lines expressing full length, WT and mutant (double and deletion) hCtc1 with ExoI prior to AluI/MboI digestion. ExoI treated (+) and untreated (−) (ss)TTAGGG signal was detected with 32 P-labeled (CCCTAA) 4 probe under native condition (left) and denaturing conditions (right). The ssTTAGGG signal was normalized to the total TTAGGG signal in the same lane as indicated at the bottom. ( B ) Probing for the presence of single stranded ssCCCTAA signal in the same DNA samples as of panel (A). DNA samples were probed with 32 P-labeled (TTAGGG) 4 under native and denaturing conditions; the results show no CCCTAA signal under native conditions (left) which indicates the absence of single-stranded internal CCCTAA DNA.
    Figure Legend Snippet: hCtc1(OB) deletion increases ExoI-resistant single-stranded telomere DNA. ( A ) Analysis of single-stranded telomeric DNA from 293T cell lines expressing full length, WT and mutant (double and deletion) hCtc1 with ExoI prior to AluI/MboI digestion. ExoI treated (+) and untreated (−) (ss)TTAGGG signal was detected with 32 P-labeled (CCCTAA) 4 probe under native condition (left) and denaturing conditions (right). The ssTTAGGG signal was normalized to the total TTAGGG signal in the same lane as indicated at the bottom. ( B ) Probing for the presence of single stranded ssCCCTAA signal in the same DNA samples as of panel (A). DNA samples were probed with 32 P-labeled (TTAGGG) 4 under native and denaturing conditions; the results show no CCCTAA signal under native conditions (left) which indicates the absence of single-stranded internal CCCTAA DNA.

    Techniques Used: Expressing, Mutagenesis, Labeling

    16) Product Images from "Telomerase activity is required for the telomere G-overhang structure in Trypanosoma brucei"

    Article Title: Telomerase activity is required for the telomere G-overhang structure in Trypanosoma brucei

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-16182-y

    Most telomeres in T . brucei WT cells have a 3′ overhang that ends in 5′ TTAGGG 3′. ( a ) A diagram showing the principle of the adaptor ligation assay. Top, a natural chromosome end with a 3′ overhang (left) is ligated with an adaptor (right). The light bar of the adaptor represents the unique oligo and is radiolabeled at its 5′ end. The darker bar of the adaptor represents the common region of all guide oligos. Only when the adaptor matches the telomere end perfectly can the adaptor be ligated to the chromosome end and eventually migrate with the long telomere fragment in agarose gel electrophoresis. Bottom, the sequence of the unique oligo and seven different guide oligos are shown. The T . brucei genomic DNA from BF ( b ) and PF ( c ) WT cells (either treated with or without Exo T) was ligated to radioisotope-labeled different adaptors (TG1–6 and TGNS, as indicated on top of each lane), digested with AluI and MboI, and separated by agarose gel electrophoresis. The ethidium bromide-stained gel is shown on the left and the image of the gel exposed to a phosphorimager (exposed) is shown on the right. 1 kb DNA ladder (ThermoFisher) was used as a molecular weight marker. ( d ) Quantification of the adaptor ligation results in both BF and PF WT cells. Average values are calculated from five (BF) or three (PF) independent experiments. Error bars represent standard deviation.
    Figure Legend Snippet: Most telomeres in T . brucei WT cells have a 3′ overhang that ends in 5′ TTAGGG 3′. ( a ) A diagram showing the principle of the adaptor ligation assay. Top, a natural chromosome end with a 3′ overhang (left) is ligated with an adaptor (right). The light bar of the adaptor represents the unique oligo and is radiolabeled at its 5′ end. The darker bar of the adaptor represents the common region of all guide oligos. Only when the adaptor matches the telomere end perfectly can the adaptor be ligated to the chromosome end and eventually migrate with the long telomere fragment in agarose gel electrophoresis. Bottom, the sequence of the unique oligo and seven different guide oligos are shown. The T . brucei genomic DNA from BF ( b ) and PF ( c ) WT cells (either treated with or without Exo T) was ligated to radioisotope-labeled different adaptors (TG1–6 and TGNS, as indicated on top of each lane), digested with AluI and MboI, and separated by agarose gel electrophoresis. The ethidium bromide-stained gel is shown on the left and the image of the gel exposed to a phosphorimager (exposed) is shown on the right. 1 kb DNA ladder (ThermoFisher) was used as a molecular weight marker. ( d ) Quantification of the adaptor ligation results in both BF and PF WT cells. Average values are calculated from five (BF) or three (PF) independent experiments. Error bars represent standard deviation.

    Techniques Used: Ligation, Agarose Gel Electrophoresis, Sequencing, Labeling, Staining, Molecular Weight, Marker, Standard Deviation

    17) Product Images from "Covalent Modification of Bacteriophage T4 DNA Inhibits CRISPR-Cas9"

    Article Title: Covalent Modification of Bacteriophage T4 DNA Inhibits CRISPR-Cas9

    Journal: mBio

    doi: 10.1128/mBio.00648-15

    Characterization of phage T4 DNA modification. (A) Phage T4(glc-HMC), T4(HMC), and T4(C) DNA left untreated (−) or treated with (+) restriction enzymes AluI (top), which cleaves unmodified DNA; MspJI (middle), which cleaves HMC-containing DNA; or T4 glucosyltransferase (bottom), which increases the mobility of HMC-containing DNA by the addition of glucose groups. The arrows indicate the mobility shift due to glucose attachment. (B) Analysis of phage T4 DNA modification by single-molecule sequencing. Results are summarized for each genome by mapping IPD ratios at each base for each of the T4 strains studied. The coloration of each base is shown by the key at the bottom left. The T4 nucleotide sequence runs from top to bottom for each of the four genomes. The distance each colored point is displaced from the center indicates the IPD ratio (scale at bottom; leftward for the reverse strand, rightward for the forward strand). Examples of interpulse distances (indicative of modification) are shown to the right for a short segment of the T4 genome. Bars indicate the magnitude of the IPD ratio (upward for the forward strand and downward for the reverse strand). A 5′ GATC 3′ site of DAM methylation is highlighted in yellow. (C) Violin plot showing IPD ratios of A residues at 5′ GATC 3′ sequences.
    Figure Legend Snippet: Characterization of phage T4 DNA modification. (A) Phage T4(glc-HMC), T4(HMC), and T4(C) DNA left untreated (−) or treated with (+) restriction enzymes AluI (top), which cleaves unmodified DNA; MspJI (middle), which cleaves HMC-containing DNA; or T4 glucosyltransferase (bottom), which increases the mobility of HMC-containing DNA by the addition of glucose groups. The arrows indicate the mobility shift due to glucose attachment. (B) Analysis of phage T4 DNA modification by single-molecule sequencing. Results are summarized for each genome by mapping IPD ratios at each base for each of the T4 strains studied. The coloration of each base is shown by the key at the bottom left. The T4 nucleotide sequence runs from top to bottom for each of the four genomes. The distance each colored point is displaced from the center indicates the IPD ratio (scale at bottom; leftward for the reverse strand, rightward for the forward strand). Examples of interpulse distances (indicative of modification) are shown to the right for a short segment of the T4 genome. Bars indicate the magnitude of the IPD ratio (upward for the forward strand and downward for the reverse strand). A 5′ GATC 3′ site of DAM methylation is highlighted in yellow. (C) Violin plot showing IPD ratios of A residues at 5′ GATC 3′ sequences.

    Techniques Used: Modification, Mobility Shift, Sequencing, Methylation

    18) Product Images from "Genotyping of Giardia duodenalis Cysts by New Real-Time PCR Assays for Detection of Mixed Infections in Human Samples ▿"

    Article Title: Genotyping of Giardia duodenalis Cysts by New Real-Time PCR Assays for Detection of Mixed Infections in Human Samples ▿

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.02305-09

    Schematic representation of the amplification curves (left), melting curves (middle), and electrophoretic separation of products (right) obtained using the gdh , orfC4 , and tpi qPCR assays. In gel electrophoresis of gdh products, lane M is the 50-bp size ladder; lanes 1 and 6 show the assemblage A product (180 bp); lanes 2, 3, and 7 show the assemblage B product (133 bp); lanes 4, 5, and 8 show the presence of both assemblages A and B; and lane 9 shows the negative control. In gel electrophoresis of orfC4 products, lane M is the 50-bp size ladder; lanes 1 and 7 show the assemblage A product (103 bp); lanes 3, 4, 5, and 8 show the assemblage B product (171 bp); lanes 2, 6, and 9 show the presence of both assemblages A and B; and lane 10 shows the negative control. In gel electrophoresis of tpi amplicons, the product from assemblage A is not cut by AluI (lane 1) but is cut by HincII into two fragments of 47 bp and 31 bp (lane 2), whereas the B product is cut by AluI into two fragments of 45 bp and 32 bp (lane 3) but is not cut by HincII (lane 4).
    Figure Legend Snippet: Schematic representation of the amplification curves (left), melting curves (middle), and electrophoretic separation of products (right) obtained using the gdh , orfC4 , and tpi qPCR assays. In gel electrophoresis of gdh products, lane M is the 50-bp size ladder; lanes 1 and 6 show the assemblage A product (180 bp); lanes 2, 3, and 7 show the assemblage B product (133 bp); lanes 4, 5, and 8 show the presence of both assemblages A and B; and lane 9 shows the negative control. In gel electrophoresis of orfC4 products, lane M is the 50-bp size ladder; lanes 1 and 7 show the assemblage A product (103 bp); lanes 3, 4, 5, and 8 show the assemblage B product (171 bp); lanes 2, 6, and 9 show the presence of both assemblages A and B; and lane 10 shows the negative control. In gel electrophoresis of tpi amplicons, the product from assemblage A is not cut by AluI (lane 1) but is cut by HincII into two fragments of 47 bp and 31 bp (lane 2), whereas the B product is cut by AluI into two fragments of 45 bp and 32 bp (lane 3) but is not cut by HincII (lane 4).

    Techniques Used: Amplification, Real-time Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Negative Control

    19) Product Images from "Combination of methylated-DNA precipitation and methylation-sensitive restriction enzymes (COMPARE-MS) for the rapid, sensitive and quantitative detection of DNA methylation"

    Article Title: Combination of methylated-DNA precipitation and methylation-sensitive restriction enzymes (COMPARE-MS) for the rapid, sensitive and quantitative detection of DNA methylation

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gnj022

    COMPARE-MS overview and rationale. Genomic DNA is digested with AluI with or without the methylation-sensitive restriction enzyme HpaII. After digestion, either the MBD2-MBD captured methylated DNA or all digested DNA are subjected to real-time PCR at a gene-specific locus. We hypothesized that enrichment of methylated DNA by methylation-sensitive restriction enzyme digestion alone or by MBD2-MBD capture of methylated DNA alone may result in false positives associated with incomplete digestion or nonspecific capture, respectively, while the combination of the two approaches (COMPARE-MS) would maintain sensitivity while minimizing false-positive results.
    Figure Legend Snippet: COMPARE-MS overview and rationale. Genomic DNA is digested with AluI with or without the methylation-sensitive restriction enzyme HpaII. After digestion, either the MBD2-MBD captured methylated DNA or all digested DNA are subjected to real-time PCR at a gene-specific locus. We hypothesized that enrichment of methylated DNA by methylation-sensitive restriction enzyme digestion alone or by MBD2-MBD capture of methylated DNA alone may result in false positives associated with incomplete digestion or nonspecific capture, respectively, while the combination of the two approaches (COMPARE-MS) would maintain sensitivity while minimizing false-positive results.

    Techniques Used: Mass Spectrometry, Methylation, Real-time Polymerase Chain Reaction, DNA Methylation Assay

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    Article Snippet: All images were acquired with a Plan-Apochromat 63X/1.4 NO oil immersion objective, using two light-emitting diodes (365 and 470 nm) for excitation and two bandpass filters (445/450 and 530/550 nm) to collect blue and green fluorescence, respectively. .. NET digestion by nucleases In a first set of experiments, 4 μg of purified λDNA (Invitrogen) was treated with 4 U/mL DNase I (Sigma Aldrich), 4 U/mL MNase (New England BioLabs, France), or 4 U/mL Alu I (New England BioLabs) for 20 min at 37°C. .. The samples were then loaded on 0.8% agarose gels (w/v) prepared in Tris-acetate-EDTA buffer containing 0.5 μg/mL ethidium bromide (Invitrogen).

    Article Title: Genome-Wide Identification of Ampicillin Resistance Determinants in Enterococcus faecium
    Article Snippet: .. Genomic DNA from mutant libraries was isolated using the Wizard Genomic DNA Purification kit (Promega), digested with Alu I (New England Biolabs) and then purified on a Qiagen QIAquick PCR Purification column (Qiagen). .. 200 ng of the digested DNA was self-ligated by the Quick Ligation Kit (New England Biolabs) in a reaction volume of 20 µl.

    Mutagenesis:

    Article Title: Genome-Wide Identification of Ampicillin Resistance Determinants in Enterococcus faecium
    Article Snippet: .. Genomic DNA from mutant libraries was isolated using the Wizard Genomic DNA Purification kit (Promega), digested with Alu I (New England Biolabs) and then purified on a Qiagen QIAquick PCR Purification column (Qiagen). .. 200 ng of the digested DNA was self-ligated by the Quick Ligation Kit (New England Biolabs) in a reaction volume of 20 µl.

    Isolation:

    Article Title: Genome-Wide Identification of Ampicillin Resistance Determinants in Enterococcus faecium
    Article Snippet: .. Genomic DNA from mutant libraries was isolated using the Wizard Genomic DNA Purification kit (Promega), digested with Alu I (New England Biolabs) and then purified on a Qiagen QIAquick PCR Purification column (Qiagen). .. 200 ng of the digested DNA was self-ligated by the Quick Ligation Kit (New England Biolabs) in a reaction volume of 20 µl.

    Article Title: Development and validation of a T7 based linear amplification for genomic DNA
    Article Snippet: ChIP DNA was fragmented by sonication and isolated using antibody against di-methyl-H3 K4 as described previously [ ]. .. Restricted genomic DNA was prepared as follows: yeast genomic DNA isolated by bead lysis, phenol/chloroform extraction, and ethanol precipitation, was restricted either with Alu I or with Rsa I (New England BioLabs (NEB)). ..

    DNA Purification:

    Article Title: Genome-Wide Identification of Ampicillin Resistance Determinants in Enterococcus faecium
    Article Snippet: .. Genomic DNA from mutant libraries was isolated using the Wizard Genomic DNA Purification kit (Promega), digested with Alu I (New England Biolabs) and then purified on a Qiagen QIAquick PCR Purification column (Qiagen). .. 200 ng of the digested DNA was self-ligated by the Quick Ligation Kit (New England Biolabs) in a reaction volume of 20 µl.

    Polymerase Chain Reaction:

    Article Title: Genome-Wide Identification of Ampicillin Resistance Determinants in Enterococcus faecium
    Article Snippet: .. Genomic DNA from mutant libraries was isolated using the Wizard Genomic DNA Purification kit (Promega), digested with Alu I (New England Biolabs) and then purified on a Qiagen QIAquick PCR Purification column (Qiagen). .. 200 ng of the digested DNA was self-ligated by the Quick Ligation Kit (New England Biolabs) in a reaction volume of 20 µl.

    Article Title: Molecular and epidemiological characterization of Plasmodium vivax recurrent infections in southern Mexico
    Article Snippet: The PCR conditions were as follow: first cycle at 95°C for 3 min, followed by 35 cycles of 95°C for 30 sec, 57°C (for first PCR) or 58°C (for second PCR) for 30 sec and 72°C for 1 min, and followed by a final extension at 72°C for 10 min using a MyCycler (Biorad, Hercules). .. The PCR products were digested with Alu I ( New England Biolabs, Beverly, MA) and BstI (Promega, Madison WI) which have restriction sites in the cspr encoding Vk247 and Vk210 regions, respectively [ ]. .. DNA fragments were resolved in a 1.5% gel, visualized under a UV-transilluminator and photographed using the BioDoc-it™ digital photo-documentation system (UVP Inc, Upland , California). b) PCR-RFLP genotyping of the msp3α gene.

    Lysis:

    Article Title: Development and validation of a T7 based linear amplification for genomic DNA
    Article Snippet: ChIP DNA was fragmented by sonication and isolated using antibody against di-methyl-H3 K4 as described previously [ ]. .. Restricted genomic DNA was prepared as follows: yeast genomic DNA isolated by bead lysis, phenol/chloroform extraction, and ethanol precipitation, was restricted either with Alu I or with Rsa I (New England BioLabs (NEB)). ..

    Ethanol Precipitation:

    Article Title: Development and validation of a T7 based linear amplification for genomic DNA
    Article Snippet: ChIP DNA was fragmented by sonication and isolated using antibody against di-methyl-H3 K4 as described previously [ ]. .. Restricted genomic DNA was prepared as follows: yeast genomic DNA isolated by bead lysis, phenol/chloroform extraction, and ethanol precipitation, was restricted either with Alu I or with Rsa I (New England BioLabs (NEB)). ..

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    New England Biolabs alu i
    Schematic diagram and reproducibility of M-TraM. (A) Schematic overview of the M-TraM screening. In yellow: inverted terminal repeats (ITRs) of the himar1 transposon with outward-facing T7 promoters; in blue: the gentamicin resistance gene in the transposon. Genomic <t>DNA</t> is isolated from the E. faecium mutant library. DNA is digested with the restriction enzyme Alu I, and the DNA fragments are circularized by self-ligation. The transposon-chromosome junction together with an ITR and a T7 promoter is amplified by PCR with primers (blue arrow) that hybridize to the transposon. To eliminate foreign DNA fragments that ligated into the circularized DNA of transposon-chromosome junctions, the PCR products were re-digested with Alu I. The purified DNA fragments are used as template in the in vitro transcription reaction. The resulting RNA products are reverse transcribed into cDNA. After labelling, the cDNA is used for microarray hybridization. (B) Schematic overview of the screening strategy to identify conditionally essential genes by M-TraM. A chromosomal region encompassing three genes (A, B, and C) from three different mutants (1, 2, and 3) is shown. Each mutant carries a single transposon insertion (blue) that disrupts the function of the gene. Mutant libraries are grown in a control condition ( e.g. , BHI) and a test condition ( e.g. , in the presence of ampicillin). All the three genes are non-essential for growth in the control condition. Gene B is required only for the test condition, so mutant 2 exhibits attenuated growth or poorer survival only in the test condition, and will consequently be reduced or be entirely lost from this library (indicated by light shading). M-TraM samples are generated from the two conditions, labelled with different dyes, and hybridized to a microarray. The DNA probes of gene A and gene C on the microarray will hybridize to the samples generated from both conditions. However, the cDNA sample of gene B will be present at reduced levels only in the test condition. By comparing the signal intensity from the two conditions for each probe, genes involved in growth or survival of the test condition can be identified. (C) Reproducibility of M-TraM. Log-log plot of the microarray signal intensities from two independent experiments of mutant libraries grown under non-selective conditions in BHI broth.
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    Schematic diagram and reproducibility of M-TraM. (A) Schematic overview of the M-TraM screening. In yellow: inverted terminal repeats (ITRs) of the himar1 transposon with outward-facing T7 promoters; in blue: the gentamicin resistance gene in the transposon. Genomic DNA is isolated from the E. faecium mutant library. DNA is digested with the restriction enzyme Alu I, and the DNA fragments are circularized by self-ligation. The transposon-chromosome junction together with an ITR and a T7 promoter is amplified by PCR with primers (blue arrow) that hybridize to the transposon. To eliminate foreign DNA fragments that ligated into the circularized DNA of transposon-chromosome junctions, the PCR products were re-digested with Alu I. The purified DNA fragments are used as template in the in vitro transcription reaction. The resulting RNA products are reverse transcribed into cDNA. After labelling, the cDNA is used for microarray hybridization. (B) Schematic overview of the screening strategy to identify conditionally essential genes by M-TraM. A chromosomal region encompassing three genes (A, B, and C) from three different mutants (1, 2, and 3) is shown. Each mutant carries a single transposon insertion (blue) that disrupts the function of the gene. Mutant libraries are grown in a control condition ( e.g. , BHI) and a test condition ( e.g. , in the presence of ampicillin). All the three genes are non-essential for growth in the control condition. Gene B is required only for the test condition, so mutant 2 exhibits attenuated growth or poorer survival only in the test condition, and will consequently be reduced or be entirely lost from this library (indicated by light shading). M-TraM samples are generated from the two conditions, labelled with different dyes, and hybridized to a microarray. The DNA probes of gene A and gene C on the microarray will hybridize to the samples generated from both conditions. However, the cDNA sample of gene B will be present at reduced levels only in the test condition. By comparing the signal intensity from the two conditions for each probe, genes involved in growth or survival of the test condition can be identified. (C) Reproducibility of M-TraM. Log-log plot of the microarray signal intensities from two independent experiments of mutant libraries grown under non-selective conditions in BHI broth.

    Journal: PLoS Genetics

    Article Title: Genome-Wide Identification of Ampicillin Resistance Determinants in Enterococcus faecium

    doi: 10.1371/journal.pgen.1002804

    Figure Lengend Snippet: Schematic diagram and reproducibility of M-TraM. (A) Schematic overview of the M-TraM screening. In yellow: inverted terminal repeats (ITRs) of the himar1 transposon with outward-facing T7 promoters; in blue: the gentamicin resistance gene in the transposon. Genomic DNA is isolated from the E. faecium mutant library. DNA is digested with the restriction enzyme Alu I, and the DNA fragments are circularized by self-ligation. The transposon-chromosome junction together with an ITR and a T7 promoter is amplified by PCR with primers (blue arrow) that hybridize to the transposon. To eliminate foreign DNA fragments that ligated into the circularized DNA of transposon-chromosome junctions, the PCR products were re-digested with Alu I. The purified DNA fragments are used as template in the in vitro transcription reaction. The resulting RNA products are reverse transcribed into cDNA. After labelling, the cDNA is used for microarray hybridization. (B) Schematic overview of the screening strategy to identify conditionally essential genes by M-TraM. A chromosomal region encompassing three genes (A, B, and C) from three different mutants (1, 2, and 3) is shown. Each mutant carries a single transposon insertion (blue) that disrupts the function of the gene. Mutant libraries are grown in a control condition ( e.g. , BHI) and a test condition ( e.g. , in the presence of ampicillin). All the three genes are non-essential for growth in the control condition. Gene B is required only for the test condition, so mutant 2 exhibits attenuated growth or poorer survival only in the test condition, and will consequently be reduced or be entirely lost from this library (indicated by light shading). M-TraM samples are generated from the two conditions, labelled with different dyes, and hybridized to a microarray. The DNA probes of gene A and gene C on the microarray will hybridize to the samples generated from both conditions. However, the cDNA sample of gene B will be present at reduced levels only in the test condition. By comparing the signal intensity from the two conditions for each probe, genes involved in growth or survival of the test condition can be identified. (C) Reproducibility of M-TraM. Log-log plot of the microarray signal intensities from two independent experiments of mutant libraries grown under non-selective conditions in BHI broth.

    Article Snippet: Genomic DNA from mutant libraries was isolated using the Wizard Genomic DNA Purification kit (Promega), digested with Alu I (New England Biolabs) and then purified on a Qiagen QIAquick PCR Purification column (Qiagen).

    Techniques: Isolation, Mutagenesis, Ligation, Amplification, Polymerase Chain Reaction, Purification, In Vitro, Microarray, Hybridization, Generated

    Plasmodium vivax msp3α genotypes. PCR products were digested with Alu I, and the digested products are separated side-by-side with their undigested products on agarose gels. a) Shows different genotypes indicated by capital letters underneath. b) Lanes 1, 3, 5, 7, 9, and 11 are undigested products of ~2.0 kb, and lanes 2, 4, 6, 8, 10, and 12 were the same products digested with Alu I. Lane 2 presents genotype C; lane 4, 10, and 12 present genotype B; lane 6 and 8 are mixed genotypes of A+C; c) genotype G; d) genotype D; and e) genotype H. m, 100 bp DNA ladder.

    Journal: Parasites & Vectors

    Article Title: Molecular and epidemiological characterization of Plasmodium vivax recurrent infections in southern Mexico

    doi: 10.1186/1756-3305-6-109

    Figure Lengend Snippet: Plasmodium vivax msp3α genotypes. PCR products were digested with Alu I, and the digested products are separated side-by-side with their undigested products on agarose gels. a) Shows different genotypes indicated by capital letters underneath. b) Lanes 1, 3, 5, 7, 9, and 11 are undigested products of ~2.0 kb, and lanes 2, 4, 6, 8, 10, and 12 were the same products digested with Alu I. Lane 2 presents genotype C; lane 4, 10, and 12 present genotype B; lane 6 and 8 are mixed genotypes of A+C; c) genotype G; d) genotype D; and e) genotype H. m, 100 bp DNA ladder.

    Article Snippet: The PCR products were digested with Alu I ( New England Biolabs, Beverly, MA) and BstI (Promega, Madison WI) which have restriction sites in the cspr encoding Vk247 and Vk210 regions, respectively [ ].

    Techniques: Polymerase Chain Reaction

    Diversity of NRPS studied by amplified fragment restriction fingerprinting method. UPGMA dendrogram ( a ) was inferred by restriction pattern of NRPS amplicons digested by Hae III ( b ) and Alu I ( c ). Lanes in the gels are in original order; irrespective of isolates appear in dendrogram. The gels were analyzed by densitometry and the bands whose areas were greater than 5% of the whole lane area were used for generating binary matrix. The similarities were calculated using the Jaccard’s coefficient, and the clustering was done by using the unweighted pair group method. Genus level affiliation of the isolates is also shown ( d ). Original gels are presented in supplementary Figure S5 .

    Journal: Scientific Reports

    Article Title: Intertidal marine sediment harbours Actinobacteria with promising bioactive and biosynthetic potential

    doi: 10.1038/s41598-017-09672-6

    Figure Lengend Snippet: Diversity of NRPS studied by amplified fragment restriction fingerprinting method. UPGMA dendrogram ( a ) was inferred by restriction pattern of NRPS amplicons digested by Hae III ( b ) and Alu I ( c ). Lanes in the gels are in original order; irrespective of isolates appear in dendrogram. The gels were analyzed by densitometry and the bands whose areas were greater than 5% of the whole lane area were used for generating binary matrix. The similarities were calculated using the Jaccard’s coefficient, and the clustering was done by using the unweighted pair group method. Genus level affiliation of the isolates is also shown ( d ). Original gels are presented in supplementary Figure S5 .

    Article Snippet: The amplified PKS-II and NRPS fragments were independently digested by Alu I and Hae III (New England Biolabs, Inc.), following the protocol suggested by manufacturer.

    Techniques: Amplification

    Diversity of PKS-II studied by amplified fragment restriction fingerprinting method. UPGMA dendrogram ( a ) was inferred by restriction pattern of PKS-II amplicons digested by Hae III ( b ) and Alu I ( c ). Lanes in the gels are in original order; irrespective of isolates appear in dendrogram. The gels were analyzed by densitometry and the bands whose areas were greater than 5% of the whole lane area were used for generating binary matrix. The similarities were calculated using the Jaccard’s coefficient, and the clustering was done by using the unweighted pair group method. Genus level affiliation of the isolates is also shown ( d ). Original gels are presented in supplementary Figure S6 .

    Journal: Scientific Reports

    Article Title: Intertidal marine sediment harbours Actinobacteria with promising bioactive and biosynthetic potential

    doi: 10.1038/s41598-017-09672-6

    Figure Lengend Snippet: Diversity of PKS-II studied by amplified fragment restriction fingerprinting method. UPGMA dendrogram ( a ) was inferred by restriction pattern of PKS-II amplicons digested by Hae III ( b ) and Alu I ( c ). Lanes in the gels are in original order; irrespective of isolates appear in dendrogram. The gels were analyzed by densitometry and the bands whose areas were greater than 5% of the whole lane area were used for generating binary matrix. The similarities were calculated using the Jaccard’s coefficient, and the clustering was done by using the unweighted pair group method. Genus level affiliation of the isolates is also shown ( d ). Original gels are presented in supplementary Figure S6 .

    Article Snippet: The amplified PKS-II and NRPS fragments were independently digested by Alu I and Hae III (New England Biolabs, Inc.), following the protocol suggested by manufacturer.

    Techniques: Amplification

    Alu-I-derived NETs retain microbicidal activity partially dependent on DNA integrity . S. aureus, E. coli C1845, S. flexneri , and S. enterica serovar Typhimurium SL1344 were incubated for 45 min in the presence of isolated NETs obtained after Alu I treatment of A23187- or PMA-activated PMN. In some experiments, NET samples were pretreated with DNase to dismantle NETs. Bacterial viability was measured by a colony count assay (CFU/mL). Results are expressed as percentage bacterial viability, calculated from CFU/mL values of bacteria exposed to NETs relative to bacteria not exposed to NETs (control tube = C). * p

    Journal: Frontiers in Immunology

    Article Title: An Improved Strategy to Recover Large Fragments of Functional Human Neutrophil Extracellular Traps

    doi: 10.3389/fimmu.2013.00166

    Figure Lengend Snippet: Alu-I-derived NETs retain microbicidal activity partially dependent on DNA integrity . S. aureus, E. coli C1845, S. flexneri , and S. enterica serovar Typhimurium SL1344 were incubated for 45 min in the presence of isolated NETs obtained after Alu I treatment of A23187- or PMA-activated PMN. In some experiments, NET samples were pretreated with DNase to dismantle NETs. Bacterial viability was measured by a colony count assay (CFU/mL). Results are expressed as percentage bacterial viability, calculated from CFU/mL values of bacteria exposed to NETs relative to bacteria not exposed to NETs (control tube = C). * p

    Article Snippet: NET digestion by nucleases In a first set of experiments, 4 μg of purified λDNA (Invitrogen) was treated with 4 U/mL DNase I (Sigma Aldrich), 4 U/mL MNase (New England BioLabs, France), or 4 U/mL Alu I (New England BioLabs) for 20 min at 37°C.

    Techniques: Derivative Assay, Activity Assay, Incubation, Isolation

    DNA nucleases induce NET digestion . (A) Migration profile of pure λDNA after digestion with 4 U/mL DNase, MNase, or Alu-I. (B) Alu-I, DNase, and MNase dose-effects on NET dsDNA obtained after A23187 stimulation of PMN. Incubation with the restriction enzymes lasted 20 min at 37°C. DNA migration took place in 0.8% agarose gel containing ethidium bromide.

    Journal: Frontiers in Immunology

    Article Title: An Improved Strategy to Recover Large Fragments of Functional Human Neutrophil Extracellular Traps

    doi: 10.3389/fimmu.2013.00166

    Figure Lengend Snippet: DNA nucleases induce NET digestion . (A) Migration profile of pure λDNA after digestion with 4 U/mL DNase, MNase, or Alu-I. (B) Alu-I, DNase, and MNase dose-effects on NET dsDNA obtained after A23187 stimulation of PMN. Incubation with the restriction enzymes lasted 20 min at 37°C. DNA migration took place in 0.8% agarose gel containing ethidium bromide.

    Article Snippet: NET digestion by nucleases In a first set of experiments, 4 μg of purified λDNA (Invitrogen) was treated with 4 U/mL DNase I (Sigma Aldrich), 4 U/mL MNase (New England BioLabs, France), or 4 U/mL Alu I (New England BioLabs) for 20 min at 37°C.

    Techniques: Migration, Incubation, Agarose Gel Electrophoresis

    NETs are recovered after AluI treatment of activated PMN . Unstimulated PMN and PMA- or A23187-stimulated PMN were treated with the restriction enzyme Alu-I. (A) NET samples migrated on agarose gel were stained with ethidium bromide: dsDNA fragments were revealed as a smearing pattern along the gel. (B) Visualization of NET protein content by silver staining. Few proteins were observed in the untreated sample, whereas numerous proteins were observed in PMA- and A23187-NET samples, with similar profiles. (C) Identification of three specific NET proteins (LF, H3, and cit-H3) by immunoblotting. Cit-H3 is a signature of netosis. These experiments were repeated at least six times with PMN from different healthy controls.

    Journal: Frontiers in Immunology

    Article Title: An Improved Strategy to Recover Large Fragments of Functional Human Neutrophil Extracellular Traps

    doi: 10.3389/fimmu.2013.00166

    Figure Lengend Snippet: NETs are recovered after AluI treatment of activated PMN . Unstimulated PMN and PMA- or A23187-stimulated PMN were treated with the restriction enzyme Alu-I. (A) NET samples migrated on agarose gel were stained with ethidium bromide: dsDNA fragments were revealed as a smearing pattern along the gel. (B) Visualization of NET protein content by silver staining. Few proteins were observed in the untreated sample, whereas numerous proteins were observed in PMA- and A23187-NET samples, with similar profiles. (C) Identification of three specific NET proteins (LF, H3, and cit-H3) by immunoblotting. Cit-H3 is a signature of netosis. These experiments were repeated at least six times with PMN from different healthy controls.

    Article Snippet: NET digestion by nucleases In a first set of experiments, 4 μg of purified λDNA (Invitrogen) was treated with 4 U/mL DNase I (Sigma Aldrich), 4 U/mL MNase (New England BioLabs, France), or 4 U/mL Alu I (New England BioLabs) for 20 min at 37°C.

    Techniques: Agarose Gel Electrophoresis, Staining, Silver Staining