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 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    alui - by Bioz Stars, 2021-03
    95/100 stars

    Images

    1) 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

    2) 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

    3) 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

    4) Product Images from "Telomeric DNA in ALT Cells Is Characterized by Free Telomeric Circles and Heterogeneous t-Loops"

    Article Title: Telomeric DNA in ALT Cells Is Characterized by Free Telomeric Circles and Heterogeneous t-Loops

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.24.22.9948-9957.2004

    Telomere measurement, telomerase activity, and telomere isolation in GM847, GM847-Tert, and VA13 cells. (A) Total DNA (10 μg) was digested with HinfI/HaeIII and separated by PFGE, and the telomeric material was detected by in-gel hybridization with a [γ- 32 P](CCCTAA) 6 probe. The signal was detected using a PhosphorImager. (B) Telomerase activity as determined by TRAP assay. IC refers to the internal PCR control. (C) Total DNA content and relative telomeric DNA abundance in GM847 psoralen photo-cross-linked fractions following genomic DNA digestion with MboI/AluI and fractionation over an A-15m Bio-Gel column. DNA content (solid line, scale on left) as determined by optical density at 260 nm (OD 260). Telomeric signal intensity (dotted line, scale on right) determined by quantitation of slot blot shown in panel D. (D) Slot blot analysis of selected fractions from the column elution shown in panel C. DNA (50 ng) was applied to a nylon membrane, probed with [γ- 32 P](CCCTAA) 6 , and detected by PhosphorImager. Controls included buffer alone (TE), the pRST5 plasmid containing 96 (TTAGGG) repeats, Bluescript (pBS) cloning vector lacking telomeric repeats, undigested GM847 genomic DNA, and MboI/AluI-digested GM847 genomic DNA.
    Figure Legend Snippet: Telomere measurement, telomerase activity, and telomere isolation in GM847, GM847-Tert, and VA13 cells. (A) Total DNA (10 μg) was digested with HinfI/HaeIII and separated by PFGE, and the telomeric material was detected by in-gel hybridization with a [γ- 32 P](CCCTAA) 6 probe. The signal was detected using a PhosphorImager. (B) Telomerase activity as determined by TRAP assay. IC refers to the internal PCR control. (C) Total DNA content and relative telomeric DNA abundance in GM847 psoralen photo-cross-linked fractions following genomic DNA digestion with MboI/AluI and fractionation over an A-15m Bio-Gel column. DNA content (solid line, scale on left) as determined by optical density at 260 nm (OD 260). Telomeric signal intensity (dotted line, scale on right) determined by quantitation of slot blot shown in panel D. (D) Slot blot analysis of selected fractions from the column elution shown in panel C. DNA (50 ng) was applied to a nylon membrane, probed with [γ- 32 P](CCCTAA) 6 , and detected by PhosphorImager. Controls included buffer alone (TE), the pRST5 plasmid containing 96 (TTAGGG) repeats, Bluescript (pBS) cloning vector lacking telomeric repeats, undigested GM847 genomic DNA, and MboI/AluI-digested GM847 genomic DNA.

    Techniques Used: Activity Assay, Isolation, Hybridization, TRAP Assay, Polymerase Chain Reaction, Fractionation, Quantitation Assay, Dot Blot, Plasmid Preparation, Clone Assay

    5) 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

    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 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

    8) 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

    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: 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

    10) 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

    11) Product Images from "Gene Expression Analysis of Zebrafish Melanocytes, Iridophores, and Retinal Pigmented Epithelium Reveals Indicators of Biological Function and Developmental Origin"

    Article Title: Gene Expression Analysis of Zebrafish Melanocytes, Iridophores, and Retinal Pigmented Epithelium Reveals Indicators of Biological Function and Developmental Origin

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0067801

    Schematic of cDNA library preparation. PolyA-selected mRNA (in red) is reverse transcribed using a polyT primer tailed with a universal primer (A). See Table S3 for primer sequences. MMLV reverse transcriptase adds cytosines to the 3′ end of the 1st strand cDNA (in black), allowing for template switching and addition of the 3′ universal primer (B). PCR amplification of the library is followed by RsaI and AluI enzymatic digestion of cDNAs (C), followed by the standard Illumina library preparation steps of end-repair, a single adenine addition, Y-adapter ligation (D), PCR enrichment, and size selection (mock gel shown in E with yellow box indicating area of gel removed for DNA extraction), prior to flowcell generation and sequencing.
    Figure Legend Snippet: Schematic of cDNA library preparation. PolyA-selected mRNA (in red) is reverse transcribed using a polyT primer tailed with a universal primer (A). See Table S3 for primer sequences. MMLV reverse transcriptase adds cytosines to the 3′ end of the 1st strand cDNA (in black), allowing for template switching and addition of the 3′ universal primer (B). PCR amplification of the library is followed by RsaI and AluI enzymatic digestion of cDNAs (C), followed by the standard Illumina library preparation steps of end-repair, a single adenine addition, Y-adapter ligation (D), PCR enrichment, and size selection (mock gel shown in E with yellow box indicating area of gel removed for DNA extraction), prior to flowcell generation and sequencing.

    Techniques Used: cDNA Library Assay, Polymerase Chain Reaction, Amplification, Ligation, Selection, DNA Extraction, Sequencing

    12) 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

    13) 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

    14) 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

    15) 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

    16) 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

    17) 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

    18) 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

    Related Articles

    Concentration Assay:

    Article Title: Limited reverse transcriptase activity of phi29 DNA polymerase
    Article Snippet: First, RCA products from the real-time RCA measurements, as described above, were diluted in PBS-Tween 0.05% to a concentration of 100 pM. .. Next, RCA products were digested with AluI restriction enzyme in a reaction mixture containing 1 × phi29 DNA polymerase buffer, 0.2 μg/μl BSA, 100 nM restriction oligonucleotide , 120 mU/μl AluI (NEB) and RCA products at a final concentration of 10 pM during 10 min incubation at 37°C. .. Subsequently, the enzyme was heat inactivated at 65°C for 2 min. After complete digestion of the 10 pM RCA products, the RCA monomer concentration is approximately 10 nM (1 h RCA of an 80 base circle yields ∼1000× amplification).

    Incubation:

    Article Title: Limited reverse transcriptase activity of phi29 DNA polymerase
    Article Snippet: First, RCA products from the real-time RCA measurements, as described above, were diluted in PBS-Tween 0.05% to a concentration of 100 pM. .. Next, RCA products were digested with AluI restriction enzyme in a reaction mixture containing 1 × phi29 DNA polymerase buffer, 0.2 μg/μl BSA, 100 nM restriction oligonucleotide , 120 mU/μl AluI (NEB) and RCA products at a final concentration of 10 pM during 10 min incubation at 37°C. .. Subsequently, the enzyme was heat inactivated at 65°C for 2 min. After complete digestion of the 10 pM RCA products, the RCA monomer concentration is approximately 10 nM (1 h RCA of an 80 base circle yields ∼1000× amplification).

    Article Title: Evaluation of a PCR-RFLP- ITS2 assay for discrimination of Anopheles species in northern and western Colombia
    Article Snippet: .. These double digests were performed in a 20 μl reaction containing 20 mM Tris-acetate, 50 mM potassium acetate, 10 mM magnesium acetate, 1 mM dithiothreitol, 1 unit Fsp I and 1 unit Alu I (New England Biolabs, Ipswich, MA, USA), and incubated at 37°C for 4 hours or overnight. ..

    other:

    Article Title: The Catalytic Subunit of DNA-Dependent Protein Kinase Regulates Proliferation, Telomere Length, and Genomic Stability in Human Somatic Cells ▿The Catalytic Subunit of DNA-Dependent Protein Kinase Regulates Proliferation, Telomere Length, and Genomic Stability in Human Somatic Cells ▿ †
    Article Snippet: Terminal restriction fragment (TRF) analysis was performed as described previously ( , ), utilizing the restriction enzymes AluI and MboI (New England Biolabs).

    Amplification:

    Article Title: Molecular Characterization of Clinical Isolates of Aeromonas Species from Malaysia
    Article Snippet: PCR and PCR-RFLP were carried out to detect the GCAT and 16S rDNA genes. .. Digestion of the amplified 16S rDNA product was carried out for 3 hours at 37°C using 2 U of Alu I (New Englands Biolabs, USA) and Mbo I (New England Biolabs, USA). .. These digested products were electrophoretically separated on 18% v/v PAGE at 160V for 5 hours.

    Polymerase Chain Reaction:

    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.

    Agarose Gel Electrophoresis:

    Article Title: Trypanosoma brucei UMSBP2 is a single-stranded telomeric DNA binding protein essential for chromosome end protection
    Article Snippet: In-gel hybridization analysis of single-stranded telomeric DNA In-gel hybridization was performed following ( ). .. Genomic DNA samples (1–1.5 μg) were digested with restriction endonucleases HinfI or HinfI and AluI (NEB Inc.) and separated on a 0.7% agarose gel. .. The samples were run in duplicates for hybridization with C-rich and G-rich telomeric probes in parallel.

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    New England Biolabs alui
    DNA sequencing-based analysis of rolling circle products reveals reverse transcription activity of phi29 DNA polymerase. ( A ) After <t>RCA,</t> short DNA oligonucleotides were hybridized to an <t>AluI</t> 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.
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    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.

    Journal: Nucleic Acids Research

    Article Title: Limited reverse transcriptase activity of phi29 DNA polymerase

    doi: 10.1093/nar/gky190

    Figure Lengend 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.

    Article Snippet: Next, RCA products were digested with AluI restriction enzyme in a reaction mixture containing 1 × phi29 DNA polymerase buffer, 0.2 μg/μl BSA, 100 nM restriction oligonucleotide , 120 mU/μl AluI (NEB) and RCA products at a final concentration of 10 pM during 10 min incubation at 37°C.

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

    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.

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gky597

    Figure Lengend 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.

    Article Snippet: Genomic DNA samples (1–1.5 μg) were digested with restriction endonucleases HinfI or HinfI and AluI (NEB Inc.) and separated on a 0.7% agarose gel.

    Techniques: Hybridization

    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.

    Journal: Molecular Pharmacology

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

    doi: 10.1124/mol.119.118273

    Figure Lengend 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.

    Article Snippet: Polynucleotide T4 kinase, T4 DNA ligase, and AluI were from New England Biolabs (Ipswich, MA).

    Techniques: Ligation, Amplification, Agarose Gel Electrophoresis, Sequencing

    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