alu i  (New England Biolabs)


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    Name:
    AluI
    Description:
    AluI 5 000 units
    Catalog Number:
    r0137l
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    282
    Size:
    5 000 units
    Category:
    Restriction Enzymes
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    Structured Review

    New England Biolabs alu i
    AluI
    AluI 5 000 units
    https://www.bioz.com/result/alu i/product/New England Biolabs
    Average 99 stars, based on 147 article reviews
    Price from $9.99 to $1999.99
    alu i - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Evaluation of a PCR-RFLP- ITS2 assay for discrimination of Anopheles species in northern and western Colombia"

    Article Title: Evaluation of a PCR-RFLP- ITS2 assay for discrimination of Anopheles species in northern and western Colombia

    Journal: Acta tropica

    doi: 10.1016/j.actatropica.2011.02.004

    PCR-RFLP of ITS2 region following Alu I restriction of amplimers from An. aquasalis on 2.5% agarose gel. Lanes: M: Molecular weight marker; 1–3: An. aquasalis individuals collected in Los Achiotes; 4: positive control, clone of An. aquasalis ITS2
    Figure Legend Snippet: PCR-RFLP of ITS2 region following Alu I restriction of amplimers from An. aquasalis on 2.5% agarose gel. Lanes: M: Molecular weight marker; 1–3: An. aquasalis individuals collected in Los Achiotes; 4: positive control, clone of An. aquasalis ITS2

    Techniques Used: Polymerase Chain Reaction, Agarose Gel Electrophoresis, Molecular Weight, Marker, Positive Control

    Double digest ( Alu I/ Fsp I) of ITS2 region amplified from individuals identified by the PCR-RFLP with Alu I as An. nuneztovari s.l. Lanes: M: Molecular weight marker; 1: positive control, clone of An. nuneztovari s.l. ITS2 sequence; 2–12: specimens
    Figure Legend Snippet: Double digest ( Alu I/ Fsp I) of ITS2 region amplified from individuals identified by the PCR-RFLP with Alu I as An. nuneztovari s.l. Lanes: M: Molecular weight marker; 1: positive control, clone of An. nuneztovari s.l. ITS2 sequence; 2–12: specimens

    Techniques Used: Amplification, Polymerase Chain Reaction, Molecular Weight, Marker, Positive Control, Sequencing

    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 "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 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 ▿ †

    Journal:

    doi: 10.1128/MCB.00355-08

    Telomere shortening in human DNA-PK cs +/− and DNA-PK cs −/− cell lines. (A) Genomic DNA was purified from the indicated cell lines, digested to completion with AluI and MboI, and then subjected to TRF Southern blot analysis
    Figure Legend Snippet: Telomere shortening in human DNA-PK cs +/− and DNA-PK cs −/− cell lines. (A) Genomic DNA was purified from the indicated cell lines, digested to completion with AluI and MboI, and then subjected to TRF Southern blot analysis

    Techniques Used: Purification, Southern Blot

    6) Product Images from "PCR-Restriction Fragment Length Polymorphism Method for Detection of Cyclospora cayetanensis in Environmental Waters without Microscopic Confirmation"

    Article Title: PCR-Restriction Fragment Length Polymorphism Method for Detection of Cyclospora cayetanensis in Environmental Waters without Microscopic Confirmation

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.69.8.4662-4669.2003

    RFLP analysis with Alu I of PCR (primers CYCAI2 and CYCAR1) amplicons from selected environmental samples. Lanes: M, DNA molecular weight marker VIII; +, C . cayetanensis oocysts with clear bands at 98, 88, and 50 bp and a faint band at 15 bp; 1, sample collected from the SDC on 21 April 1998; 2 to 5, individual water samples collected from the SARVB on 28 August 1998; 6 and 7, individual water samples collected from the SARYL on 28 September 1998. The term “Uncut” identifies amplicons that do not contain Alu I sites.
    Figure Legend Snippet: RFLP analysis with Alu I of PCR (primers CYCAI2 and CYCAR1) amplicons from selected environmental samples. Lanes: M, DNA molecular weight marker VIII; +, C . cayetanensis oocysts with clear bands at 98, 88, and 50 bp and a faint band at 15 bp; 1, sample collected from the SDC on 21 April 1998; 2 to 5, individual water samples collected from the SARVB on 28 August 1998; 6 and 7, individual water samples collected from the SARYL on 28 September 1998. The term “Uncut” identifies amplicons that do not contain Alu I sites.

    Techniques Used: Polymerase Chain Reaction, Environmental Sampling, Molecular Weight, Marker

    7) Product Images from "Molecular Characterization of Clinical Isolates of Aeromonas Species from Malaysia"

    Article Title: Molecular Characterization of Clinical Isolates of Aeromonas Species from Malaysia

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0030205

    Polyacrylamide gel showing 16S rDNA-RFLP patterns ( Alu I and Mbo I). L: pBR322 DNA/ BsuRI marker (Fermentas, USA), Lane 1: typical pattern of A. hydrophila (JN 686656), Lane 2: typical pattern of A. caviae (JN 686668), Lane 3: atypical pattern of A. trota (JN 686649), Lanes 4–6: atypical pattern of A. veronii (JN 686665, JN 686691, JN 686739), Lanes 7–10: A. aquariorum (JN 686662, JN 686731, JN 686725, JN 686700).
    Figure Legend Snippet: Polyacrylamide gel showing 16S rDNA-RFLP patterns ( Alu I and Mbo I). L: pBR322 DNA/ BsuRI marker (Fermentas, USA), Lane 1: typical pattern of A. hydrophila (JN 686656), Lane 2: typical pattern of A. caviae (JN 686668), Lane 3: atypical pattern of A. trota (JN 686649), Lanes 4–6: atypical pattern of A. veronii (JN 686665, JN 686691, JN 686739), Lanes 7–10: A. aquariorum (JN 686662, JN 686731, JN 686725, JN 686700).

    Techniques Used: Marker

    8) Product Images from "Molecular and epidemiological characterization of Plasmodium vivax recurrent infections in southern Mexico"

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

    Journal: Parasites & Vectors

    doi: 10.1186/1756-3305-6-109

    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.
    Figure Legend 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.

    Techniques Used: Polymerase Chain Reaction

    9) 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

    10) 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

    11) Product Images from "The Role of RNF213 4810G > A and 4950G > A Variants in Patients with Moyamoya Disease in Korea"

    Article Title: The Role of RNF213 4810G > A and 4950G > A Variants in Patients with Moyamoya Disease in Korea

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms18112477

    Polymorphism analysis of RNF213 4448, 4810, 4863, and 4950 in moyamoya disease patients. Polymorphisms analysis of RNF213 genes amplicon by agarose gel electrophoresis after restriction endonuclease digestion. ( a ) RNF213 4448 c.13195G > A site was digested by Alu I resulting in the appearance of the GG (wild type, 135/95 bp), GA (heterozygous type, 230/135/95 bp), and AA (mutant type, 230 bp) genotypes in moyamoya disease patients; ( b ) RNF213 4810 c.14429G > A site was digested by Hpy 188I resulting in the appearance of the GG (wild type, 104/42/5 bp), GA (heterozygous type, 146/104/42/5bp), and AA (mutant type, 146 bp) genotypes in moyamoya disease patients. DNA fragments that were 5 bp or less were too small to be seen on 3% agarose gel; ( c ) RNF4863 c.14587G > A site was digested by Hpy 188I resulting in the appearance of the GG (wild type, 867 bp) and GA (hetero type, 867/719/148 bp) genotypes in moyamoya disease patients. No mutant type (AA) was found in this study. ( d ) RNF4950 c.14850G > A site was digested by Bss SαI resulting in the appearance of the GG (wild type, 162/26 bp) and GA (heterozygous type, 188/162/26 bp) genotypes. The mutant type (AA) was not found in results of restriction fragment length polymorphism (RFLP). DNA fragments that were 26 bp or less were too small to be seen on 3% agarose gel.
    Figure Legend Snippet: Polymorphism analysis of RNF213 4448, 4810, 4863, and 4950 in moyamoya disease patients. Polymorphisms analysis of RNF213 genes amplicon by agarose gel electrophoresis after restriction endonuclease digestion. ( a ) RNF213 4448 c.13195G > A site was digested by Alu I resulting in the appearance of the GG (wild type, 135/95 bp), GA (heterozygous type, 230/135/95 bp), and AA (mutant type, 230 bp) genotypes in moyamoya disease patients; ( b ) RNF213 4810 c.14429G > A site was digested by Hpy 188I resulting in the appearance of the GG (wild type, 104/42/5 bp), GA (heterozygous type, 146/104/42/5bp), and AA (mutant type, 146 bp) genotypes in moyamoya disease patients. DNA fragments that were 5 bp or less were too small to be seen on 3% agarose gel; ( c ) RNF4863 c.14587G > A site was digested by Hpy 188I resulting in the appearance of the GG (wild type, 867 bp) and GA (hetero type, 867/719/148 bp) genotypes in moyamoya disease patients. No mutant type (AA) was found in this study. ( d ) RNF4950 c.14850G > A site was digested by Bss SαI resulting in the appearance of the GG (wild type, 162/26 bp) and GA (heterozygous type, 188/162/26 bp) genotypes. The mutant type (AA) was not found in results of restriction fragment length polymorphism (RFLP). DNA fragments that were 26 bp or less were too small to be seen on 3% agarose gel.

    Techniques Used: Amplification, Agarose Gel Electrophoresis, Mutagenesis

    12) Product Images from "Winemaking and Bioprocesses Strongly Shaped the Genetic Diversity of the Ubiquitous Yeast Torulaspora delbrueckii"

    Article Title: Winemaking and Bioprocesses Strongly Shaped the Genetic Diversity of the Ubiquitous Yeast Torulaspora delbrueckii

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0094246

    Restriction patterns of D1/D2 amplicon generated by Alu I (A) or Pst I (B) for Torulaspora species. A: For Alu I restriction, four patterns were produced: 170 pb+160 pb+80 pb+70 pb+55 pb+40 pb+30 pb for T. delbrueckii and T. quercuum ; 170 pb+160 pb+120 pb+70 pb+55 pb+30 pb for T. maleeae and T. indica ; 170 pb+160 pb+95 pb+80 pb+70 pb+30 pb for T. franciscae , T. microellipsoides , T. pretoriensis ; and 330 pb+170 pb+75 pb+30 pb for T. globosa . B: For Pst I restriction, two patterns were produced: 600pb (no restriction) for T. maleeae , T. quercuum , T. indica , T. microellipsoides and T. globosa ; or 480 pb+120 pb for T. delbrueckii , T. franciscae and T. pretoriensis . Blue and pink bands represent internal upper and lower markers respectively.
    Figure Legend Snippet: Restriction patterns of D1/D2 amplicon generated by Alu I (A) or Pst I (B) for Torulaspora species. A: For Alu I restriction, four patterns were produced: 170 pb+160 pb+80 pb+70 pb+55 pb+40 pb+30 pb for T. delbrueckii and T. quercuum ; 170 pb+160 pb+120 pb+70 pb+55 pb+30 pb for T. maleeae and T. indica ; 170 pb+160 pb+95 pb+80 pb+70 pb+30 pb for T. franciscae , T. microellipsoides , T. pretoriensis ; and 330 pb+170 pb+75 pb+30 pb for T. globosa . B: For Pst I restriction, two patterns were produced: 600pb (no restriction) for T. maleeae , T. quercuum , T. indica , T. microellipsoides and T. globosa ; or 480 pb+120 pb for T. delbrueckii , T. franciscae and T. pretoriensis . Blue and pink bands represent internal upper and lower markers respectively.

    Techniques Used: Amplification, Generated, Produced

    13) Product Images from "Heterochromatic siRNAs and DDM1 Independently Silence Aberrant 5S rDNA Transcripts in Arabidopsis"

    Article Title: Heterochromatic siRNAs and DDM1 Independently Silence Aberrant 5S rDNA Transcripts in Arabidopsis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0005932

    Effect of combined DDM1 and siRNA deficiency on 5S rDNA methylation and aberrant transcripts. A) 5S rDNA hypermethylation in ddm1 is siRNA-dependent: Southern blot comparison of Alu I and Hae III-digested genomic DNA isolated from inflorescences of wild type (WT), ddm1 , and double mutant lines nrpd1 ddm1 , rdr2 ddm1 , and dcl3 ddm1 (top panel). The probe is the same as in Figure 2E . Dilutions of the above digests were also assayed by PCR using 5S LT1 primers (bottom panel). Samples to which no restriction enzyme was added are controls (no digest). B) 5S LT1 is silenced by two overlapping processes: RNA samples from inflorescences of WT, ddm1 and the double mutant panel were analyzed by one-step RT-PCR, performed as described in Figure 1C . Control reactions were performed with ACT2 primers; reverse transcriptase was omitted from duplicate 5S LT1 and ACT2 reactions (no RT).
    Figure Legend Snippet: Effect of combined DDM1 and siRNA deficiency on 5S rDNA methylation and aberrant transcripts. A) 5S rDNA hypermethylation in ddm1 is siRNA-dependent: Southern blot comparison of Alu I and Hae III-digested genomic DNA isolated from inflorescences of wild type (WT), ddm1 , and double mutant lines nrpd1 ddm1 , rdr2 ddm1 , and dcl3 ddm1 (top panel). The probe is the same as in Figure 2E . Dilutions of the above digests were also assayed by PCR using 5S LT1 primers (bottom panel). Samples to which no restriction enzyme was added are controls (no digest). B) 5S LT1 is silenced by two overlapping processes: RNA samples from inflorescences of WT, ddm1 and the double mutant panel were analyzed by one-step RT-PCR, performed as described in Figure 1C . Control reactions were performed with ACT2 primers; reverse transcriptase was omitted from duplicate 5S LT1 and ACT2 reactions (no RT).

    Techniques Used: Methylation, Southern Blot, Isolation, Mutagenesis, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

    DDM1 limits IGS siRNA accumulation and asymmetric methylation. A) Detection of IGS siRNAs (siR1003) in dicer-like ( dcl ) mutant combinations. Blot analysis of small RNA isolated from leaves of wild type (WT); single mutants dcl2, dcl3, dcl4 ; double mutants dcl2 dcl3 ( dcl2/3 ), dcl2 dcl4 ( dcl2/4 ), dcl3 dcl4 ( dcl3/4 ); and the triple mutant dcl2 dcl3 dcl4 ( dcl2/3/4 ). B) Localization of IGS-related RNA in interphase nuclei by fluorescent in situ hybridization with an RNA probe for siR1003 (red). DNA was stained with DAPI (white). The size bar corresponds to 5 µm. C) Overaccumulation of IGS siRNAs in the ddm1 background. Blot analysis of small RNA from dcl3 and double mutant dcl3 ddm1 using probes for siR1003 and the larger 3′flanking region ( Figure 1A , diagram). WT and ddm1 signals from the same membrane are provided for comparison. D) IGS siRNA overaccumulation persists in out-crossed ddm1 . Blot analysis of small RNA isolated from WT, nrpd1 (−/−), ddm1 (−/−) and F1 heterozygotes (+/−) of each mutant crossed to the WT. E) Asymmetric cytosines in the IGS are hypermethylated in met1 and ddm1 . Southern blot analysis was performed on Alu I-digested genomic DNA isolated from WT, nrpd1 , rdr2 , two alleles of dcl3 , met1 and ddm1 . The probe corresponds to 5S LT1, shown aligned to a representative 5S rDNA repeat unit from Chromosome 5. Alu I and Hae III sites in the IGS region are indicated.
    Figure Legend Snippet: DDM1 limits IGS siRNA accumulation and asymmetric methylation. A) Detection of IGS siRNAs (siR1003) in dicer-like ( dcl ) mutant combinations. Blot analysis of small RNA isolated from leaves of wild type (WT); single mutants dcl2, dcl3, dcl4 ; double mutants dcl2 dcl3 ( dcl2/3 ), dcl2 dcl4 ( dcl2/4 ), dcl3 dcl4 ( dcl3/4 ); and the triple mutant dcl2 dcl3 dcl4 ( dcl2/3/4 ). B) Localization of IGS-related RNA in interphase nuclei by fluorescent in situ hybridization with an RNA probe for siR1003 (red). DNA was stained with DAPI (white). The size bar corresponds to 5 µm. C) Overaccumulation of IGS siRNAs in the ddm1 background. Blot analysis of small RNA from dcl3 and double mutant dcl3 ddm1 using probes for siR1003 and the larger 3′flanking region ( Figure 1A , diagram). WT and ddm1 signals from the same membrane are provided for comparison. D) IGS siRNA overaccumulation persists in out-crossed ddm1 . Blot analysis of small RNA isolated from WT, nrpd1 (−/−), ddm1 (−/−) and F1 heterozygotes (+/−) of each mutant crossed to the WT. E) Asymmetric cytosines in the IGS are hypermethylated in met1 and ddm1 . Southern blot analysis was performed on Alu I-digested genomic DNA isolated from WT, nrpd1 , rdr2 , two alleles of dcl3 , met1 and ddm1 . The probe corresponds to 5S LT1, shown aligned to a representative 5S rDNA repeat unit from Chromosome 5. Alu I and Hae III sites in the IGS region are indicated.

    Techniques Used: Methylation, Mutagenesis, Isolation, In Situ Hybridization, Staining, Southern Blot

    14) 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

    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 "Diversity of Bacteria Associated with Natural Aphid Populations"

    Article Title: Diversity of Bacteria Associated with Natural Aphid Populations

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.69.12.7216-7223.2003

    Electropherograms of the T-RFs produced by Alu I- Bse RI (AB) digestion (A, C, E, G, I, and K) and Sma I- Cla I- Xba I (SCX) digestion (B, D, F, H, J, and L) of PCR amplicons from 16S rRNA genes from the reference lines of the pea aphid A. pisum LMB95/28 (A and B), R (C and D), IS (E and F), FH (G and H), and MD (I and J) and aphid 1330 from the natural population of A. pisum (K and L). The T-RFs are shown in black, and the internal standards (50, 75, 100, 139, 150, 160, 200, 250, 300, 340, 350, 400, 450, 490, and 500 bp) are shown in gray.
    Figure Legend Snippet: Electropherograms of the T-RFs produced by Alu I- Bse RI (AB) digestion (A, C, E, G, I, and K) and Sma I- Cla I- Xba I (SCX) digestion (B, D, F, H, J, and L) of PCR amplicons from 16S rRNA genes from the reference lines of the pea aphid A. pisum LMB95/28 (A and B), R (C and D), IS (E and F), FH (G and H), and MD (I and J) and aphid 1330 from the natural population of A. pisum (K and L). The T-RFs are shown in black, and the internal standards (50, 75, 100, 139, 150, 160, 200, 250, 300, 340, 350, 400, 450, 490, and 500 bp) are shown in gray.

    Techniques Used: Produced, Polymerase Chain Reaction

    17) 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

    18) 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

    19) 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

    20) 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

    21) Product Images from "Spotted Fever Group and Typhus Group Rickettsioses in Humans, South Korea"

    Article Title: Spotted Fever Group and Typhus Group Rickettsioses in Humans, South Korea

    Journal: Emerging Infectious Diseases

    doi: 10.3201/eid1102.040603

    Restriction fragment length polymorphism analysis of H1 products amplified with multiplex-nested primer set from seropositive sera. Ethidium bromide–stained polyacrylamide gels of Alu I restriction endonuclease digestion of ≈420 bp rickettsial DNA amplified by using the nested primer H set WJ77/80 in the primary reactions and WJ79/83/78 in the nested reactions. Lanes: M, size marker DNA (25-bp DNA ladder); 1–18: H1–H18; 19–23: H20–24; C, Rickettsia conorii ; A, R. akari ; J, R. japonica ; F, R. felis . J–S; predicted fragments after digestion. The number on the left indicates the molecular size (in base pairs) of restriction fragments.
    Figure Legend Snippet: Restriction fragment length polymorphism analysis of H1 products amplified with multiplex-nested primer set from seropositive sera. Ethidium bromide–stained polyacrylamide gels of Alu I restriction endonuclease digestion of ≈420 bp rickettsial DNA amplified by using the nested primer H set WJ77/80 in the primary reactions and WJ79/83/78 in the nested reactions. Lanes: M, size marker DNA (25-bp DNA ladder); 1–18: H1–H18; 19–23: H20–24; C, Rickettsia conorii ; A, R. akari ; J, R. japonica ; F, R. felis . J–S; predicted fragments after digestion. The number on the left indicates the molecular size (in base pairs) of restriction fragments.

    Techniques Used: Amplification, Multiplex Assay, Staining, Marker

    Restriction fragment length polymorphism analysis of H2-products amplified with multiplex-nested primer set from seropositive sera. Ethidium bromide–stained polyacrylamide gels of Alu I restriction endonuclease digestion of ≈230-bp rickettsial DNA amplified by using the nested primer H set WJ77/80 in the primary reactions and WJ79/83/78 in the nested reactions. Lanes: M, size marker DNA (25-bp DNA ladder); 1, H3-2; 2, H7-2; 3, H8-2; 4, H13-2; 5, H14-2; 6, H15-2; 7, H18-2; 8, H19; P, Rickettsia prowazekii ; T, R. typhi . P and T; predicted fragments after digestion. The number on the left indicates the molecular size (in base pairs) of restriction fragments.
    Figure Legend Snippet: Restriction fragment length polymorphism analysis of H2-products amplified with multiplex-nested primer set from seropositive sera. Ethidium bromide–stained polyacrylamide gels of Alu I restriction endonuclease digestion of ≈230-bp rickettsial DNA amplified by using the nested primer H set WJ77/80 in the primary reactions and WJ79/83/78 in the nested reactions. Lanes: M, size marker DNA (25-bp DNA ladder); 1, H3-2; 2, H7-2; 3, H8-2; 4, H13-2; 5, H14-2; 6, H15-2; 7, H18-2; 8, H19; P, Rickettsia prowazekii ; T, R. typhi . P and T; predicted fragments after digestion. The number on the left indicates the molecular size (in base pairs) of restriction fragments.

    Techniques Used: Amplification, Multiplex Assay, Staining, Marker

    22) Product Images from "Comprehensive detection and identification of human coronaviruses, including the SARS-associated coronavirus, with a single RT-PCR assay"

    Article Title: Comprehensive detection and identification of human coronaviruses, including the SARS-associated coronavirus, with a single RT-PCR assay

    Journal: Journal of Virological Methods

    doi: 10.1016/j.jviromet.2004.07.008

    Alu I digestion of amplicons from SARS-HCoV, HCoV-229E and HCoV-OC43. Lanes 1, 3 and 5 (MW): molecular weight markers (Amplisize ladder 50–2000 bp, Bio-Rad Laboratories, Mississauga, Canada); Lane 2: SARS-HCoV (predicted fragments: 126 bp, 94 bp); Lane 4: HCoV-229E (predicted fragments: 101 bp, 86 bp, 33 bp); Lane 6: HCoV-OC43 (predicted fragment: 220 bp).
    Figure Legend Snippet: Alu I digestion of amplicons from SARS-HCoV, HCoV-229E and HCoV-OC43. Lanes 1, 3 and 5 (MW): molecular weight markers (Amplisize ladder 50–2000 bp, Bio-Rad Laboratories, Mississauga, Canada); Lane 2: SARS-HCoV (predicted fragments: 126 bp, 94 bp); Lane 4: HCoV-229E (predicted fragments: 101 bp, 86 bp, 33 bp); Lane 6: HCoV-OC43 (predicted fragment: 220 bp).

    Techniques Used: Molecular Weight

    23) Product Images from "Buccal cells DNA extraction to obtain high quality human genomic DNA suitable for polymorphism genotyping by PCR-RFLP and Real-Time PCR"

    Article Title: Buccal cells DNA extraction to obtain high quality human genomic DNA suitable for polymorphism genotyping by PCR-RFLP and Real-Time PCR

    Journal: Journal of Applied Oral Science

    doi: 10.1590/S1678-77572012000400013

    Epidermal growth factor (EGF) polymorphism (rs 4444903) genotyped by polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) using DNA extracted from different incubation times (T0, T4 and T8); A) PCR amplicons and B) digestion with Alu I
    Figure Legend Snippet: Epidermal growth factor (EGF) polymorphism (rs 4444903) genotyped by polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) using DNA extracted from different incubation times (T0, T4 and T8); A) PCR amplicons and B) digestion with Alu I

    Techniques Used: Polymerase Chain Reaction, Incubation

    24) Product Images from "Helicobactermarmotae and novel Helicobacter and Campylobacter species isolated from the livers and intestines of prairie dogs"

    Article Title: Helicobactermarmotae and novel Helicobacter and Campylobacter species isolated from the livers and intestines of prairie dogs

    Journal: Journal of Medical Microbiology

    doi: 10.1099/jmm.0.032144-0

    PCR products of 1.2 kb produced using Helicobacter genus-specific primers were digested by Alu I (a) or Hha I (b) and analysed by electrophoresis on a 6 % Visigel matrix. Lanes 1–4, prairie dog isolates MIT 07-5168, MIT 07-5167, MIT 04-8588
    Figure Legend Snippet: PCR products of 1.2 kb produced using Helicobacter genus-specific primers were digested by Alu I (a) or Hha I (b) and analysed by electrophoresis on a 6 % Visigel matrix. Lanes 1–4, prairie dog isolates MIT 07-5168, MIT 07-5167, MIT 04-8588

    Techniques Used: Polymerase Chain Reaction, Produced, Electrophoresis

    25) Product Images from "Re-Evaluation of a Bacterial Antifreeze Protein as an Adhesin with Ice-Binding Activity"

    Article Title: Re-Evaluation of a Bacterial Antifreeze Protein as an Adhesin with Ice-Binding Activity

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0048805

    Estimation of 312-bp repeat copy number by Southern blotting. (A) Schematic diagram of the Mp AFP coding region illustrating the relative positions of restriction enzyme cut sites. Cross-hatched lines indicate the break between the two segments of Mp AFP coding region described in Fig. 3 . Four restriction enzymes, including Mse I, Ase I, Pst I and Alu I cut only outside the 312-bp repeats. Since the cut site of Mse I (T/TAA) is present within that of Ase I (AT/TAAT), Mse I is indicated in parentheses beside Ase I. Msp I is the only restriction enzyme in this set that cuts within the 312-bp repeats in RII, and it also cuts once in RI. (B) Southern blot of the digests using a 2×312-bp repeat from RII as the probe. Undigested DNA (lane 6) remained near the well, whereas the four restriction enzymes Alu I, Pst I, Ase I and Mse I (lane 1, 2, 4 and 5) that cut outside the repeats produced a fragment of approximately 37,500 bp in length. The Msp I (lane 3) partial digest produced a ladder of bands at 312-bp intervals and those containing between 2 (624 bp) and 13 (4056 bp) repeats are marked on this blot. DNA length markers (kb) are indicated on the left of the blot.
    Figure Legend Snippet: Estimation of 312-bp repeat copy number by Southern blotting. (A) Schematic diagram of the Mp AFP coding region illustrating the relative positions of restriction enzyme cut sites. Cross-hatched lines indicate the break between the two segments of Mp AFP coding region described in Fig. 3 . Four restriction enzymes, including Mse I, Ase I, Pst I and Alu I cut only outside the 312-bp repeats. Since the cut site of Mse I (T/TAA) is present within that of Ase I (AT/TAAT), Mse I is indicated in parentheses beside Ase I. Msp I is the only restriction enzyme in this set that cuts within the 312-bp repeats in RII, and it also cuts once in RI. (B) Southern blot of the digests using a 2×312-bp repeat from RII as the probe. Undigested DNA (lane 6) remained near the well, whereas the four restriction enzymes Alu I, Pst I, Ase I and Mse I (lane 1, 2, 4 and 5) that cut outside the repeats produced a fragment of approximately 37,500 bp in length. The Msp I (lane 3) partial digest produced a ladder of bands at 312-bp intervals and those containing between 2 (624 bp) and 13 (4056 bp) repeats are marked on this blot. DNA length markers (kb) are indicated on the left of the blot.

    Techniques Used: Southern Blot, Produced

    26) 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

    27) Product Images from "Development and validation of a T7 based linear amplification for genomic DNA"

    Article Title: Development and validation of a T7 based linear amplification for genomic DNA

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-4-19

    Size distributions for starting material and IVT amplified product. Lanes 1–3 each contain 250 ng DNA run on a 2% non-denaturing agarose gel. Lanes 4–5 each contain 500 ng RNA run on a 2% denaturing agarose gel. The denaturing gel is necessary to eliminate RNA secondary structure. Lane 1: 100 bp ladder (NEB). Lane 2: starting material (yeast genomic DNA digested with Alu I and previously gel-purified to a size range of 100–700 bp). Lane 3: amplified product generated by R-PCR from 50 ng starting material. Lane 4: amplified RNA product generated by IVT from 50 ng starting material. Lane 5: 100 bp RNA Ladder (Ambion). The R-PCR amplified product appears to significantly under-represent low molecular weight species. The IVT amplified product may slightly under-represent high molecular weight species. For clarity, the denaturing gel image was rescaled to match the ladder of the non-denaturing gel.
    Figure Legend Snippet: Size distributions for starting material and IVT amplified product. Lanes 1–3 each contain 250 ng DNA run on a 2% non-denaturing agarose gel. Lanes 4–5 each contain 500 ng RNA run on a 2% denaturing agarose gel. The denaturing gel is necessary to eliminate RNA secondary structure. Lane 1: 100 bp ladder (NEB). Lane 2: starting material (yeast genomic DNA digested with Alu I and previously gel-purified to a size range of 100–700 bp). Lane 3: amplified product generated by R-PCR from 50 ng starting material. Lane 4: amplified RNA product generated by IVT from 50 ng starting material. Lane 5: 100 bp RNA Ladder (Ambion). The R-PCR amplified product appears to significantly under-represent low molecular weight species. The IVT amplified product may slightly under-represent high molecular weight species. For clarity, the denaturing gel image was rescaled to match the ladder of the non-denaturing gel.

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

    Hierarchical clustering of replicate datasets generated by direct labeling, IVT and R-PCR. Each thin bar represents a single datapoint. Red bars correspond to enrichment in the Cy5-labeled Alu I probe, while green bars correspond to enrichment in the Cy3-labeled Rsa I probe. The dendrograms (top) indicate clustering relationships among the sample replicates. The lengths of the branches represent the degree of similarity between the samples (shorter indicates higher similarity). Purple stripes to the right of the diagram highlight discordant areas (log ratios with opposite signs) in the R-PCR replicates relative to the direct labeling and IVT samples.
    Figure Legend Snippet: Hierarchical clustering of replicate datasets generated by direct labeling, IVT and R-PCR. Each thin bar represents a single datapoint. Red bars correspond to enrichment in the Cy5-labeled Alu I probe, while green bars correspond to enrichment in the Cy3-labeled Rsa I probe. The dendrograms (top) indicate clustering relationships among the sample replicates. The lengths of the branches represent the degree of similarity between the samples (shorter indicates higher similarity). Purple stripes to the right of the diagram highlight discordant areas (log ratios with opposite signs) in the R-PCR replicates relative to the direct labeling and IVT samples.

    Techniques Used: Generated, Labeling, Polymerase Chain Reaction

    28) Product Images from "Association of the programmed cell death 1 (PDCD1) gene polymorphism with ankylosing spondylitis in the Korean population"

    Article Title: Association of the programmed cell death 1 (PDCD1) gene polymorphism with ankylosing spondylitis in the Korean population

    Journal: Arthritis Research & Therapy

    doi: 10.1186/ar2071

    Gel electrophoresis patterns of PD-1.5 and PD-1.9. (a) The amplified fragments of PD-1.5 were digested with Alu I: the polymerase chain reaction (PCR) product size was 333 base pairs (bp), which was digested to 264 and 69 bp. If the product was digested, the allele was identified as T; if not, it was identified as C. (b) The amplified fragments of PD-1.9 were digested with Bpu 10I: the PCR product size was 408 bp, which was digested to 260 and 145 bp. If the product was digested, the allele was identified as C; if not, it was identified as T.
    Figure Legend Snippet: Gel electrophoresis patterns of PD-1.5 and PD-1.9. (a) The amplified fragments of PD-1.5 were digested with Alu I: the polymerase chain reaction (PCR) product size was 333 base pairs (bp), which was digested to 264 and 69 bp. If the product was digested, the allele was identified as T; if not, it was identified as C. (b) The amplified fragments of PD-1.9 were digested with Bpu 10I: the PCR product size was 408 bp, which was digested to 260 and 145 bp. If the product was digested, the allele was identified as C; if not, it was identified as T.

    Techniques Used: Nucleic Acid Electrophoresis, Amplification, Polymerase Chain Reaction

    29) 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

    30) Product Images from "Helicobacter pullorum Outbreak in C57BL/6NTac and C3H/HeNTac Barrier-Maintained Mice ▿"

    Article Title: Helicobacter pullorum Outbreak in C57BL/6NTac and C3H/HeNTac Barrier-Maintained Mice ▿

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.02531-09

    Restriction fragment length polymorphism analysis patterns of the Helicobacter 16S rRNA gene with AluI and HhaI digestion. Lanes 1 to 3, Helicobacter isolates from Taconic mice; lane 4, H. pullorum human isolate MIT 98-5489; lane 5, H. typhlonius ; lane 6, H. bilis ; lane 7, H. muridarum ; lane 8, H. hepaticus ; lane 9, H. rodentium ; M, 100-bp DNA ladder.
    Figure Legend Snippet: Restriction fragment length polymorphism analysis patterns of the Helicobacter 16S rRNA gene with AluI and HhaI digestion. Lanes 1 to 3, Helicobacter isolates from Taconic mice; lane 4, H. pullorum human isolate MIT 98-5489; lane 5, H. typhlonius ; lane 6, H. bilis ; lane 7, H. muridarum ; lane 8, H. hepaticus ; lane 9, H. rodentium ; M, 100-bp DNA ladder.

    Techniques Used: Mouse Assay

    31) 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

    32) Product Images from "Flexible and scalable genotyping-by-sequencing strategies for population studies"

    Article Title: Flexible and scalable genotyping-by-sequencing strategies for population studies

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-15-979

    Effect of read count on marker dataset size and imputation. Fraction of shared post-filter, pre-imputation genetic markers and fraction of post-imputation shared genome with original sample for subsamplings of A) RsaI F 2 -44 and B) HincII F 2 -23. C) Imputed genomes for each subsample in RsaI F 2 -44 displayed in concentric rings. Sample read count declines from the outermost ring to the innermost. D) Imputed genomes for each subsample in HincII F 2 -23 displayed in concentric circles. Sample read count declines from the outermost ring to the innermost.
    Figure Legend Snippet: Effect of read count on marker dataset size and imputation. Fraction of shared post-filter, pre-imputation genetic markers and fraction of post-imputation shared genome with original sample for subsamplings of A) RsaI F 2 -44 and B) HincII F 2 -23. C) Imputed genomes for each subsample in RsaI F 2 -44 displayed in concentric rings. Sample read count declines from the outermost ring to the innermost. D) Imputed genomes for each subsample in HincII F 2 -23 displayed in concentric circles. Sample read count declines from the outermost ring to the innermost.

    Techniques Used: Marker

    Trait mapping for yellowy ( y1 ) and sugary ( su1 ) in an F 2 admixture population. A green line in the plots annotates the locations of both genes. Pre-imputation markers are shown in black and grey. Markers, post-imputation and error correction are shown in color. A) RsaI GBS dataset, su1 map. B) RsaI GBS dataset, y1 map. C) HincII GBS dataset, su1 map. D) RsaI GBS dataset, y1 map.
    Figure Legend Snippet: Trait mapping for yellowy ( y1 ) and sugary ( su1 ) in an F 2 admixture population. A green line in the plots annotates the locations of both genes. Pre-imputation markers are shown in black and grey. Markers, post-imputation and error correction are shown in color. A) RsaI GBS dataset, su1 map. B) RsaI GBS dataset, y1 map. C) HincII GBS dataset, su1 map. D) RsaI GBS dataset, y1 map.

    Techniques Used:

    Fraction of predicted sites covered in samples from a F 2 admixture population. Reads from each F 2 sample were aligned to predicted sites, then predicted sites were placed in 2 bp bins, with the fraction covered in each bin indicated by the heatmap. A) The RsaI dataset, aligned against total predicted sites. B) RsaI dataset, aligned against the subset of predicted sites with sequencing coverage in the original RsaI B73 GBS experiment. C) HincII dataset, aligned against total predicted sites. D) HincII dataset, aligned against predicted sites with at least one read coverage in the original HincII experiment. Sample order is given, left to right, in Additional file 5 : Table S1.
    Figure Legend Snippet: Fraction of predicted sites covered in samples from a F 2 admixture population. Reads from each F 2 sample were aligned to predicted sites, then predicted sites were placed in 2 bp bins, with the fraction covered in each bin indicated by the heatmap. A) The RsaI dataset, aligned against total predicted sites. B) RsaI dataset, aligned against the subset of predicted sites with sequencing coverage in the original RsaI B73 GBS experiment. C) HincII dataset, aligned against total predicted sites. D) HincII dataset, aligned against predicted sites with at least one read coverage in the original HincII experiment. Sample order is given, left to right, in Additional file 5 : Table S1.

    Techniques Used: Sequencing

    33) Product Images from "Fidelity Index Determination of DNA Methyltransferases"

    Article Title: Fidelity Index Determination of DNA Methyltransferases

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0063866

    Radioactive methylation assay using E. coli DNA-comparison between restriction enzyme digested substrate versus undigested substrate. The dilution series and presentation of data are the same as in Figure 3 . A) H 3 -methyl incorporation by M.EcoKDam. At a dilution of 1/2 4 , the substrate is fully methylated and after a dilution of 1/2 2 , the CPMs start to increase indicating star activity. This results in an FI of 4. B) H 3 -methyl incorporation by M.EcoKDam with DNA that has been digested with MboI restriction enzyme to remove all M.EcoKDam cognate sites. After a dilution of 1/2 2 , the CPMs start to increase, indicating methylation at non-cognate sites. C) H 3 -methyl incorporation by M.HhaI. At a dilution of 1/2 8 , the substrate is fully methylated and after a dilution of 1/2 3 , the CPMs start to increase indicating star activity. This results in an FI of 32. D) H 3 -methyl incorporation by M.HhaI with DNA that has been digested with HhaI restriction enzyme to remove all M.HhaI cognate sites. After a dilution of 1/2 3 , the CPMs start to increase, indicating methylation at non-cognate sites.
    Figure Legend Snippet: Radioactive methylation assay using E. coli DNA-comparison between restriction enzyme digested substrate versus undigested substrate. The dilution series and presentation of data are the same as in Figure 3 . A) H 3 -methyl incorporation by M.EcoKDam. At a dilution of 1/2 4 , the substrate is fully methylated and after a dilution of 1/2 2 , the CPMs start to increase indicating star activity. This results in an FI of 4. B) H 3 -methyl incorporation by M.EcoKDam with DNA that has been digested with MboI restriction enzyme to remove all M.EcoKDam cognate sites. After a dilution of 1/2 2 , the CPMs start to increase, indicating methylation at non-cognate sites. C) H 3 -methyl incorporation by M.HhaI. At a dilution of 1/2 8 , the substrate is fully methylated and after a dilution of 1/2 3 , the CPMs start to increase indicating star activity. This results in an FI of 32. D) H 3 -methyl incorporation by M.HhaI with DNA that has been digested with HhaI restriction enzyme to remove all M.HhaI cognate sites. After a dilution of 1/2 3 , the CPMs start to increase, indicating methylation at non-cognate sites.

    Techniques Used: Methylation, Activity Assay

    34) Product Images from "Neorickettsia sennetsu as a Neglected Cause of Fever in South-East Asia"

    Article Title: Neorickettsia sennetsu as a Neglected Cause of Fever in South-East Asia

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0003908

    Schematic alignment of N . sennetsu and related organisms showing the restrictions sites used for the RFLP. The unique RFLP pattern of the 16 sRNA gene target, after incubation with Alu I (green), Bsm FI (yellow) or Sty I (red) allow the differentiation of N . sennetsu , E . chaffeensis and A . phagocytophium as well as other potentially amplified organisms. The resulting fragment sizes are as follows; N . sennetsu – Alu I: 345bp (uncut); Bsm F1: 180bp, 80bp, 60bp, 16bp; Sty I: 215bp, 127bp; E . chaffeensis–Alu I: 345bp (uncut); Bsm F1: 328bp, 16bp; Sty I: 215bp, 127bp; A . phagocytophium–Alu I: 199bp, 145bp Bsm F1: 328bp, 16bp; Sty I: 345bp (uncut).
    Figure Legend Snippet: Schematic alignment of N . sennetsu and related organisms showing the restrictions sites used for the RFLP. The unique RFLP pattern of the 16 sRNA gene target, after incubation with Alu I (green), Bsm FI (yellow) or Sty I (red) allow the differentiation of N . sennetsu , E . chaffeensis and A . phagocytophium as well as other potentially amplified organisms. The resulting fragment sizes are as follows; N . sennetsu – Alu I: 345bp (uncut); Bsm F1: 180bp, 80bp, 60bp, 16bp; Sty I: 215bp, 127bp; E . chaffeensis–Alu I: 345bp (uncut); Bsm F1: 328bp, 16bp; Sty I: 215bp, 127bp; A . phagocytophium–Alu I: 199bp, 145bp Bsm F1: 328bp, 16bp; Sty I: 345bp (uncut).

    Techniques Used: Incubation, Amplification

    35) Product Images from "Genetic Diversity of Plasmodium vivax in Clinical Isolates from Southern Thailand using PvMSP1, PvMSP3 (PvMSP3α, PvMSP3β) Genes and Eight Microsatellite Markers"

    Article Title: Genetic Diversity of Plasmodium vivax in Clinical Isolates from Southern Thailand using PvMSP1, PvMSP3 (PvMSP3α, PvMSP3β) Genes and Eight Microsatellite Markers

    Journal: The Korean Journal of Parasitology

    doi: 10.3347/kjp.2019.57.5.469

    Restriction fragment length polymorphism patterns of Plasmodium vivax . (A) PvMSP3α after PCR/RFLP using Hha I enzyme. (B) PvMSP3β after PCR/RFLP using Pst I enzyme. (C) PvMSP1 F2 after PCR/RFLP using Alu I enzyme. M represented 100-bp marker.
    Figure Legend Snippet: Restriction fragment length polymorphism patterns of Plasmodium vivax . (A) PvMSP3α after PCR/RFLP using Hha I enzyme. (B) PvMSP3β after PCR/RFLP using Pst I enzyme. (C) PvMSP1 F2 after PCR/RFLP using Alu I enzyme. M represented 100-bp marker.

    Techniques Used: Polymerase Chain Reaction, Marker

    36) Product Images from "Genome-Wide Identification of Ampicillin Resistance Determinants in Enterococcus faecium"

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

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1002804

    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.
    Figure Legend 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.

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

    37) Product Images from "Direct Quantitative Monitoring of Homology-Directed DNA Repair of Damaged Telomeres"

    Article Title: Direct Quantitative Monitoring of Homology-Directed DNA Repair of Damaged Telomeres

    Journal: Methods in enzymology

    doi: 10.1016/bs.mie.2017.11.010

    Overview of the SMARD assay to study break-induced telomere synthesis. (A) Induction of DSBs at the telomeres and labeling of nascent DNA by thymidine analogues: IdU and CIdU. (B) Cell lysis and digestion of genomic DNA by Alu I and Mbo I. The × sign represents multiple restriction sites for the two enzymes in nontelomeric DNA. However, due to the GC-rich nature of telomeric DNA, most of the telomeres remain uncut by these two frequent cutters. (C) Agarose gel electrophoresis to separate the digested nontelomeric genomic DNA from the telomeres. (D) The isolated DNA is YOYO-1 stained and stretched onto silanized cover glasses. (E) Immunofluorescence-FISH to detect nascent telomeres. (F) Image acquisition and analysis.
    Figure Legend Snippet: Overview of the SMARD assay to study break-induced telomere synthesis. (A) Induction of DSBs at the telomeres and labeling of nascent DNA by thymidine analogues: IdU and CIdU. (B) Cell lysis and digestion of genomic DNA by Alu I and Mbo I. The × sign represents multiple restriction sites for the two enzymes in nontelomeric DNA. However, due to the GC-rich nature of telomeric DNA, most of the telomeres remain uncut by these two frequent cutters. (C) Agarose gel electrophoresis to separate the digested nontelomeric genomic DNA from the telomeres. (D) The isolated DNA is YOYO-1 stained and stretched onto silanized cover glasses. (E) Immunofluorescence-FISH to detect nascent telomeres. (F) Image acquisition and analysis.

    Techniques Used: Labeling, Lysis, Agarose Gel Electrophoresis, Isolation, Staining, Immunofluorescence, Fluorescence In Situ Hybridization

    Autoradiogram of an agarose gel hybridized with a 32 P-telomeric probe. Lane 1: Undigested DNA from a U2OS cell line. Lane 2: DNA from U2OS cells digested with Alu I and Mbo I. Lane 3: DNA from U2OS cells labeled with 30μ M IdU and 30μ M CIdU and digested with Alu I and Mbo I. This lane shows that the incorporation of the thymidine analogue does not affect the digestion efficiency of the restriction enzymes.
    Figure Legend Snippet: Autoradiogram of an agarose gel hybridized with a 32 P-telomeric probe. Lane 1: Undigested DNA from a U2OS cell line. Lane 2: DNA from U2OS cells digested with Alu I and Mbo I. Lane 3: DNA from U2OS cells labeled with 30μ M IdU and 30μ M CIdU and digested with Alu I and Mbo I. This lane shows that the incorporation of the thymidine analogue does not affect the digestion efficiency of the restriction enzymes.

    Techniques Used: Agarose Gel Electrophoresis, Labeling

    38) 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

    39) 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

    40) 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

    Related Articles

    Polymerase Chain Reaction:

    Article Title: PCR-Restriction Fragment Length Polymorphism Method for Detection of Cyclospora cayetanensis in Environmental Waters without Microscopic Confirmation
    Article Snippet: .. The positive PCR products from the second reaction (10 μl) were digested with 1 U of Alu I (New England Biolabs) at 37°C for 2 h. Fragments were separated, visualized, and photographed as described above. .. During our initial SAR monitoring study, we detected five samples that were PCR-RFLP positive for C . cayetanensis as described by Relman et al. ( ) and Jinneman et al. ( ).

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

    Incubation:

    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. ..

    Amplification:

    Article Title: Molecular Characterization of Clinical Isolates of Aeromonas Species from Malaysia
    Article Snippet: .. 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.

    Agarose Gel Electrophoresis:

    Article Title: Trypanosoma brucei UMSBP2 is a single-stranded telomeric DNA binding protein essential for chromosome end protection
    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. .. The samples were run in duplicates for hybridization with C-rich and G-rich telomeric probes in parallel.

    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).

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    New England Biolabs alu i
    PCR-RFLP of ITS2 region following <t>Alu</t> I restriction of amplimers from An. aquasalis on 2.5% agarose gel. Lanes: M: Molecular weight marker; 1–3: An. aquasalis individuals collected in Los Achiotes; 4: positive control, clone of An. aquasalis ITS2
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    PCR-RFLP of ITS2 region following Alu I restriction of amplimers from An. aquasalis on 2.5% agarose gel. Lanes: M: Molecular weight marker; 1–3: An. aquasalis individuals collected in Los Achiotes; 4: positive control, clone of An. aquasalis ITS2

    Journal: Acta tropica

    Article Title: Evaluation of a PCR-RFLP- ITS2 assay for discrimination of Anopheles species in northern and western Colombia

    doi: 10.1016/j.actatropica.2011.02.004

    Figure Lengend Snippet: PCR-RFLP of ITS2 region following Alu I restriction of amplimers from An. aquasalis on 2.5% agarose gel. Lanes: M: Molecular weight marker; 1–3: An. aquasalis individuals collected in Los Achiotes; 4: positive control, clone of An. aquasalis ITS2

    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.

    Techniques: Polymerase Chain Reaction, Agarose Gel Electrophoresis, Molecular Weight, Marker, Positive Control

    Double digest ( Alu I/ Fsp I) of ITS2 region amplified from individuals identified by the PCR-RFLP with Alu I as An. nuneztovari s.l. Lanes: M: Molecular weight marker; 1: positive control, clone of An. nuneztovari s.l. ITS2 sequence; 2–12: specimens

    Journal: Acta tropica

    Article Title: Evaluation of a PCR-RFLP- ITS2 assay for discrimination of Anopheles species in northern and western Colombia

    doi: 10.1016/j.actatropica.2011.02.004

    Figure Lengend Snippet: Double digest ( Alu I/ Fsp I) of ITS2 region amplified from individuals identified by the PCR-RFLP with Alu I as An. nuneztovari s.l. Lanes: M: Molecular weight marker; 1: positive control, clone of An. nuneztovari s.l. ITS2 sequence; 2–12: specimens

    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.

    Techniques: Amplification, Polymerase Chain Reaction, Molecular Weight, Marker, Positive Control, Sequencing

    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

    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.

    Journal: Molecular and Cellular Biology

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

    doi: 10.1128/MCB.24.22.9948-9957.2004

    Figure Lengend 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.

    Article Snippet: Restriction digestion with MboI and AluI (New England Biolabs) was done with enzyme concentrations of 3 U/μg of DNA.

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