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  • 99
    New England Biolabs λ exonuclease
    Internal modification of DNA. DNA is first tailed with either 5- E -UTP or N 6 - P -ATP, and then elongated by primer extension. The 5′-monophosphorylated template (shown in gray) is optionally digested with <t>λ-exonuclease</t> (λ-Exo) and the alkyne is reacted in CuAAC, to attach biotin to the single-stranded (ss) or double-stranded (ds) DNA. 12% denaturing PAGE, visualization by SYBR Gold staining.
    λ Exonuclease, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 647 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/λ exonuclease/product/New England Biolabs
    Average 99 stars, based on 647 article reviews
    Price from $9.99 to $1999.99
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    95
    Thermo Fisher lambda λ exonuclease
    GR binding recruits Oct-2 to octamer motifs adjacent to GR binding sites in the nucleus. (A) Nuclei prepared from CHO cells transfected with the MMTV promoter construct pHCWT, GR, and/or Oct-2 expression plasmids and treated with 10 −6 M Dex or vehicle for 15 min were restricted with Hin ) and GR and/or Oct-2 expression plasmids and treated with 10 −6 M Dex or vehicle for 15 min were restricted with Sma I and digested with <t>λ</t> exonuclease as indicated. Digestion was revealed by linear PCR extension of a T3 polymerase primer, and pause sites were positioned relative to an A sequencing track amplified with the same primer. The positions of the octamer motif sequence and GRE sequences in the MMTV LTR are summarized schematically. The Dex-, GR-, and Oct-2-specific λ pause site is indicated by the arrow. (C) Nuclei prepared from CHO cells transfected with pBluescript containing either a Gal4 binding site separated by 8 nucleotides from the octamer motif sequence from the MMTV LTR (left) or a nonspecific oligonucleotide encoding an IAP enhancer core (right) along with Gal-GR WT , Gal-GR L501P , and/or Oct-2 expression plasmids were restricted with Xho I and digested with λ exonuclease as indicated. Digestion was revealed by linear PCR extension of a T7 polymerase primer, and pause sites were positioned relative to an A sequencing track amplified with the same primer. The positions of the octamer motif-IAP sequence and of the Gal4 sequence are summarized schematically. The Gal-GR WT -, Oct-2-, and octamer motif-dependent specific λ pause site is indicated by the arrow. Western blots of cellular extracts verified that Gal-GR WT and Gal-GR L501P ).
    Lambda λ Exonuclease, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Price from $9.99 to $1999.99
    lambda λ exonuclease - by Bioz Stars, 2020-08
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    Image Search Results


    Internal modification of DNA. DNA is first tailed with either 5- E -UTP or N 6 - P -ATP, and then elongated by primer extension. The 5′-monophosphorylated template (shown in gray) is optionally digested with λ-exonuclease (λ-Exo) and the alkyne is reacted in CuAAC, to attach biotin to the single-stranded (ss) or double-stranded (ds) DNA. 12% denaturing PAGE, visualization by SYBR Gold staining.

    Journal: Nucleic Acids Research

    Article Title: Nucleotidyl transferase assisted DNA labeling with different click chemistries

    doi: 10.1093/nar/gkv544

    Figure Lengend Snippet: Internal modification of DNA. DNA is first tailed with either 5- E -UTP or N 6 - P -ATP, and then elongated by primer extension. The 5′-monophosphorylated template (shown in gray) is optionally digested with λ-exonuclease (λ-Exo) and the alkyne is reacted in CuAAC, to attach biotin to the single-stranded (ss) or double-stranded (ds) DNA. 12% denaturing PAGE, visualization by SYBR Gold staining.

    Article Snippet: Splinted ligation was performed by first annealing tailed DNA2 with DNA5/6 and DNA7 by heating to 90°C for 30 s and cooling to room temperature for 5 min, adding all other components after this step [final concentrations: 10 μM DNA2, 22.5 μM DNA5/6, 25 μM blocked and phosphorylated DNA7, 50 μM ATP, 50 mM Tris-HCl (pH 7.4), 10 mM MgCl2 , 1.5 U/μl T4 DNA ligase] incubating at 37°C for 4 h and heating to 80°C for 10 min. DNA5/6 and DNA7 were optionally removed from reaction mixtures to obtain pure, ligated/extended ssDNA by adding λ-exonuclease (0.25 U/μl for primer extension or 0.5 U/μl for ligation; New England Biolabs) directly into the reaction mixture and incubating at 37°C for 1 h, followed by 80°C for 10 min. DNA was purified by ethanol precipitation in the presence of 0.3 M sodium acetate (pH 5.5).

    Techniques: Modification, Polyacrylamide Gel Electrophoresis, Staining

    ChIP-exo 5.0 increases library yield. a Schematic of ChIP-exo 5.0. The purple triangle indicates the location of the Read_1 start site, which is also the λ exonuclease stop site. b 2% agarose gel of the electrophoresed library following 18 cycles of PCR for various S. cerevisiae transcription factors assayed by ChIP-exo 1.1 or 5.0. Following ChIP, the sample was split and libraries prepared using the indicated protocols. After splitting the sample, each reaction contained a 50 ml cell equivalent (OD 600 = 0.8) of yeast chromatin, which is five-fold less than the amount optimized for ChIP-exo 1.1. ChIP-exo 5.0 produced greater library yield for all samples. c Heatmaps comparing ChIP-exo 1.1 and 5.0 at the 975 Reb1 primary motifs in a 200 bp window. d Composite plot of data from panel ( c )

    Journal: Nature Communications

    Article Title: Simplified ChIP-exo assays

    doi: 10.1038/s41467-018-05265-7

    Figure Lengend Snippet: ChIP-exo 5.0 increases library yield. a Schematic of ChIP-exo 5.0. The purple triangle indicates the location of the Read_1 start site, which is also the λ exonuclease stop site. b 2% agarose gel of the electrophoresed library following 18 cycles of PCR for various S. cerevisiae transcription factors assayed by ChIP-exo 1.1 or 5.0. Following ChIP, the sample was split and libraries prepared using the indicated protocols. After splitting the sample, each reaction contained a 50 ml cell equivalent (OD 600 = 0.8) of yeast chromatin, which is five-fold less than the amount optimized for ChIP-exo 1.1. ChIP-exo 5.0 produced greater library yield for all samples. c Heatmaps comparing ChIP-exo 1.1 and 5.0 at the 975 Reb1 primary motifs in a 200 bp window. d Composite plot of data from panel ( c )

    Article Snippet: The λ exonuclease digestion (100 µl) containing: 20 U λ exonuclease (NEB), 1 × λ exonuclease reaction buffer (NEB), 0.1% Triton-X 100, and 5% DMSO was incubated for 30 min at 37 °C; then washed with 10 mM Tris-HCl, pH 8.0 at 4 °C.

    Techniques: Chromatin Immunoprecipitation, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Produced

    Cdc13 protects the telomere 5′ end when bound 3 nt away from the ds-ss junction. ( a ) D10S16 contains 10 bp of telomere dsDNA and a 16 nt ssDNA 3′ overhang. The Cdc13 MBS (bold text) is located 3 nt from the ds-ss junction. The black bar represents the 14 bp guide sequence (5′-GTCACACGTCACAC-3′) used for ensuring proper annealing. “*” Indicates the radioactive label at the 3′ end of the C-strand, used for detecting the substrate and its degradation products. ( b ) Sequencing gel with the 5′DEPA reaction products. D10S16 was either pre-bound by Cdc13 or incubated with non-DNA binding BSA protein. An aliquot was taken out before addition of λ-exonuclease (-), then λ-exonuclease was added and aliquots of the reactions were stopped at different time points (20; 40; 60; 120; 240 s). ( c ) Graph showing the quantification of the gel shown in ( b ). The amount of uncleaved substrate (S) relative the reaction start point was calculated by measuring the volume of the upper two uncleaved substrate bands normalized to the volume of the loading control band (LC). Reaction products are denoted next to the gel (P). The uncropped gel is presented in Supplementary Fig. S2 .

    Journal: Scientific Reports

    Article Title: Rap1 and Cdc13 have complementary roles in preventing exonucleolytic degradation of telomere 5′ ends

    doi: 10.1038/s41598-017-08663-x

    Figure Lengend Snippet: Cdc13 protects the telomere 5′ end when bound 3 nt away from the ds-ss junction. ( a ) D10S16 contains 10 bp of telomere dsDNA and a 16 nt ssDNA 3′ overhang. The Cdc13 MBS (bold text) is located 3 nt from the ds-ss junction. The black bar represents the 14 bp guide sequence (5′-GTCACACGTCACAC-3′) used for ensuring proper annealing. “*” Indicates the radioactive label at the 3′ end of the C-strand, used for detecting the substrate and its degradation products. ( b ) Sequencing gel with the 5′DEPA reaction products. D10S16 was either pre-bound by Cdc13 or incubated with non-DNA binding BSA protein. An aliquot was taken out before addition of λ-exonuclease (-), then λ-exonuclease was added and aliquots of the reactions were stopped at different time points (20; 40; 60; 120; 240 s). ( c ) Graph showing the quantification of the gel shown in ( b ). The amount of uncleaved substrate (S) relative the reaction start point was calculated by measuring the volume of the upper two uncleaved substrate bands normalized to the volume of the loading control band (LC). Reaction products are denoted next to the gel (P). The uncropped gel is presented in Supplementary Fig. S2 .

    Article Snippet: For the binding assay, 10 fmol probe in presence of 1.5 µg competitor mix (0.5 µg each of sheared E.coli DNA (~250 bp), salmon sperm DNA and yeast t-RNA) in 1x λ-exonuclease buffer (New England Biolabs; 67 mM Glycine-KOH, pH 9.4, 2.5 MgCl2 and 50 µg/µl BSA) supplemented with 8% glycerol was mixed with varying concentrations of affinity purified Cdc13 (~0.8–4.8 μg), Rap1 (~0.07–7 μg), Rap1-DBD or DBD-mutants (~0.1–1.6 μg), in a total of 15 µl reaction.

    Techniques: Sequencing, Incubation, Binding Assay

    Schematic figure summarizing the results of this work and how it is proposed to relate to different in vivo situations. ( a ) Shows the different substrate tested with Cdc13 or Rap1 pre-bound at their respective MBS at various distances relative the ds-ss junction. “ + ” indicates protection, while “−’’ indicates no protection. ( b ) Protection by Rap1 when the 3′ overhang is very short and unable to accommodate Cdc13 binding. ( c ) Protection by Rap1 in a hypothetical situation where Cdc13 is bound very far away from the ds-ss junction (longer than tested here). ( d ) Protection may be provided by Cdc13 alone when the 3′ overhang accommodates its binding. ( e ) The wild type Rap1 DBD 337–582 is firmly attached to its MBS, and fully protects the 5′ end from degradation by λ-exonuclease. ( f ) The Rap1 wrapping loop mutant DBD 337–556 is only partly attached to the MBS, leaving the 5′ end accessible to λ-exonuclease, which cleaves off the first 3 nt of DNA before being halted at a site where the mutant DBD is more firmly attached.

    Journal: Scientific Reports

    Article Title: Rap1 and Cdc13 have complementary roles in preventing exonucleolytic degradation of telomere 5′ ends

    doi: 10.1038/s41598-017-08663-x

    Figure Lengend Snippet: Schematic figure summarizing the results of this work and how it is proposed to relate to different in vivo situations. ( a ) Shows the different substrate tested with Cdc13 or Rap1 pre-bound at their respective MBS at various distances relative the ds-ss junction. “ + ” indicates protection, while “−’’ indicates no protection. ( b ) Protection by Rap1 when the 3′ overhang is very short and unable to accommodate Cdc13 binding. ( c ) Protection by Rap1 in a hypothetical situation where Cdc13 is bound very far away from the ds-ss junction (longer than tested here). ( d ) Protection may be provided by Cdc13 alone when the 3′ overhang accommodates its binding. ( e ) The wild type Rap1 DBD 337–582 is firmly attached to its MBS, and fully protects the 5′ end from degradation by λ-exonuclease. ( f ) The Rap1 wrapping loop mutant DBD 337–556 is only partly attached to the MBS, leaving the 5′ end accessible to λ-exonuclease, which cleaves off the first 3 nt of DNA before being halted at a site where the mutant DBD is more firmly attached.

    Article Snippet: For the binding assay, 10 fmol probe in presence of 1.5 µg competitor mix (0.5 µg each of sheared E.coli DNA (~250 bp), salmon sperm DNA and yeast t-RNA) in 1x λ-exonuclease buffer (New England Biolabs; 67 mM Glycine-KOH, pH 9.4, 2.5 MgCl2 and 50 µg/µl BSA) supplemented with 8% glycerol was mixed with varying concentrations of affinity purified Cdc13 (~0.8–4.8 μg), Rap1 (~0.07–7 μg), Rap1-DBD or DBD-mutants (~0.1–1.6 μg), in a total of 15 µl reaction.

    Techniques: In Vivo, Binding Assay, Mutagenesis

    ( a ) Schematic illustration of the 5′ DNA end protection assay (DEPA). DNA oligonucleotides are annealed to form model telomeres with a double stranded part and a single stranded 3′ overhang (I). All oligonucleotides contain a short non-telomeric guide sequence to ensure efficient annealing while the telomere part is varied to create different length overhangs and different 5′ permutations. λ-exonuclease selectively cleaves the 5′ phosphorylated end (II) of the shorter C-strand oligonucleotide which is 3′ end labelled (*). The reaction progresses in the 5′ → 3′ direction (II). To assay for 5′ end protection, Cdc13 is pre-bound to the telomere end before adding λ-exonuclease to the reaction, which will inhibit the exonuclease (III). ( b ) Schematic illustration of the assay read out. Reactions are stopped at different incubation times, de-proteinized, ethanol precipitated and run on a 10% denaturing polyacrylamide sequencing gel. A labelled oligonucleotide loading control (LC) is added before ethanol precipitation which migrates above the 3′ labelled C-strand substrate (S) on the gel. As the exonuclease reaction progresses, products of decreasing size (P) appears on the gel while the uncleaved substrate (S) diminishes. Lane I, no enzyme control (0 s); lane IIa, shorter incubation time; lane IIb, longer incubation time; lane III, a reaction where the substrate was pre-incubated with Cdc13 which gave full protection.

    Journal: Scientific Reports

    Article Title: Rap1 and Cdc13 have complementary roles in preventing exonucleolytic degradation of telomere 5′ ends

    doi: 10.1038/s41598-017-08663-x

    Figure Lengend Snippet: ( a ) Schematic illustration of the 5′ DNA end protection assay (DEPA). DNA oligonucleotides are annealed to form model telomeres with a double stranded part and a single stranded 3′ overhang (I). All oligonucleotides contain a short non-telomeric guide sequence to ensure efficient annealing while the telomere part is varied to create different length overhangs and different 5′ permutations. λ-exonuclease selectively cleaves the 5′ phosphorylated end (II) of the shorter C-strand oligonucleotide which is 3′ end labelled (*). The reaction progresses in the 5′ → 3′ direction (II). To assay for 5′ end protection, Cdc13 is pre-bound to the telomere end before adding λ-exonuclease to the reaction, which will inhibit the exonuclease (III). ( b ) Schematic illustration of the assay read out. Reactions are stopped at different incubation times, de-proteinized, ethanol precipitated and run on a 10% denaturing polyacrylamide sequencing gel. A labelled oligonucleotide loading control (LC) is added before ethanol precipitation which migrates above the 3′ labelled C-strand substrate (S) on the gel. As the exonuclease reaction progresses, products of decreasing size (P) appears on the gel while the uncleaved substrate (S) diminishes. Lane I, no enzyme control (0 s); lane IIa, shorter incubation time; lane IIb, longer incubation time; lane III, a reaction where the substrate was pre-incubated with Cdc13 which gave full protection.

    Article Snippet: For the binding assay, 10 fmol probe in presence of 1.5 µg competitor mix (0.5 µg each of sheared E.coli DNA (~250 bp), salmon sperm DNA and yeast t-RNA) in 1x λ-exonuclease buffer (New England Biolabs; 67 mM Glycine-KOH, pH 9.4, 2.5 MgCl2 and 50 µg/µl BSA) supplemented with 8% glycerol was mixed with varying concentrations of affinity purified Cdc13 (~0.8–4.8 μg), Rap1 (~0.07–7 μg), Rap1-DBD or DBD-mutants (~0.1–1.6 μg), in a total of 15 µl reaction.

    Techniques: Sequencing, Incubation, Ethanol Precipitation

    Construction of single-strand probes. ( A ) The bacteriophage lambda DNA (gray) was used as a template, and the primers containing the common amplification primers as adaptors on the 5′ end were used to PCR amplify the spacer that is common to all of the probes. This common spacer with the amplification sequences was the further template for building the double-stranded probe precursor. The left primer with the target sequence (red) had an adaptor with a BsaI site (blue) The right primer with the target sequences (red) has an adaptor (green) with the MlyI site. ( B ) The construct was digested with BsaI (red triangle) cutting 5 bases inwards from the recognition sequence. The digestion creates a phosphorylated 5′end and a recessed 3′ end. The phosphate group is removed by shrimp alkaline phosphatase digestion. A subsequent digestion with MlyI (black triangle) cuts 5 bases inwards from the right-hand recognition sequence to create a blunt end with a 5′ phosphate. The lambda exonuclease digests the lower strand from the 5′ phosphorylated end created by the Mly I digestion leaving the upper strand intact. ( C ) The dHPLC profile of the double-stranded template has two peaks (blue) at ≈4.5 min. After exonuclease digestion (red) there is only one peak at 4.5 min, and one large early peak that represent the digested products. ( D ) The LPP is hybridized to genomic DNA (gray) and gap-filled (orange) using the genomic DNA as template. Ligation occurs when the polymerase reaches the 5′ end of the probe (yellow circle) to form a circular molecule. The amplification sequences (yellow) in the circle are then used to amplify the targeted exons. Digestion with AscI and ClaI (sites are included in the amplification primers) separate the primers from the genomic target sequences.

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

    Article Title: A comprehensive assay for targeted multiplex amplification of human DNA sequences

    doi: 10.1073/pnas.0803240105

    Figure Lengend Snippet: Construction of single-strand probes. ( A ) The bacteriophage lambda DNA (gray) was used as a template, and the primers containing the common amplification primers as adaptors on the 5′ end were used to PCR amplify the spacer that is common to all of the probes. This common spacer with the amplification sequences was the further template for building the double-stranded probe precursor. The left primer with the target sequence (red) had an adaptor with a BsaI site (blue) The right primer with the target sequences (red) has an adaptor (green) with the MlyI site. ( B ) The construct was digested with BsaI (red triangle) cutting 5 bases inwards from the recognition sequence. The digestion creates a phosphorylated 5′end and a recessed 3′ end. The phosphate group is removed by shrimp alkaline phosphatase digestion. A subsequent digestion with MlyI (black triangle) cuts 5 bases inwards from the right-hand recognition sequence to create a blunt end with a 5′ phosphate. The lambda exonuclease digests the lower strand from the 5′ phosphorylated end created by the Mly I digestion leaving the upper strand intact. ( C ) The dHPLC profile of the double-stranded template has two peaks (blue) at ≈4.5 min. After exonuclease digestion (red) there is only one peak at 4.5 min, and one large early peak that represent the digested products. ( D ) The LPP is hybridized to genomic DNA (gray) and gap-filled (orange) using the genomic DNA as template. Ligation occurs when the polymerase reaches the 5′ end of the probe (yellow circle) to form a circular molecule. The amplification sequences (yellow) in the circle are then used to amplify the targeted exons. Digestion with AscI and ClaI (sites are included in the amplification primers) separate the primers from the genomic target sequences.

    Article Snippet: The digested PCR product was treated with 0.1 units lambda exonuclease (New England Biolabs) at 37°C for 15 min in the same restriction enzyme buffer.

    Techniques: Lambda DNA Preparation, Amplification, Polymerase Chain Reaction, Sequencing, Construct, Ligation

    PFGE and Southern blot hybridization with IS 1245 -derived probe of M. avium and M. kansasii colonies. (A) PFGE with undigested DNA; (B) Southern blot hybridization with IS 1245 -derived probe. Open arrow indicates pMA100; closed arrow indicates the uncharacterized smaller hybridization band. 88.1 to 88.4 = M . avium ; 88.5 to 88.75 = PCR−IS 1245- negative M . kansasii ; 88.8 to 88.15 = PCR−IS 1245- positive M. kansasii. On the left, Lambda Ladder PFG Marker (NewEngland BioLabs) molecular size markers.

    Journal: PLoS ONE

    Article Title: First Description of Natural and Experimental Conjugation between Mycobacteria Mediated by a Linear Plasmid

    doi: 10.1371/journal.pone.0029884

    Figure Lengend Snippet: PFGE and Southern blot hybridization with IS 1245 -derived probe of M. avium and M. kansasii colonies. (A) PFGE with undigested DNA; (B) Southern blot hybridization with IS 1245 -derived probe. Open arrow indicates pMA100; closed arrow indicates the uncharacterized smaller hybridization band. 88.1 to 88.4 = M . avium ; 88.5 to 88.75 = PCR−IS 1245- negative M . kansasii ; 88.8 to 88.15 = PCR−IS 1245- positive M. kansasii. On the left, Lambda Ladder PFG Marker (NewEngland BioLabs) molecular size markers.

    Article Snippet: Besides that, the pMA100 band excised from PFGE agarose gels was treated in separate experiments with 30 U exonuclease III, 30 U exonuclease lambda or 30 U topoiosomerase I (all enzymes from New England BioLabs) for 3 h at 37°C, according to the manufacturer's protocols.

    Techniques: Southern Blot, Hybridization, Derivative Assay, Polymerase Chain Reaction, Marker

    PFGE of DNA genomic preparations. (A) PFGE with undigested DNAs from M. avium 88.3 (1) and M. kansasii 88.8 (2) under different switch times, indicated below each figure; (B) pMA100 extracted from PFGE gels and treated with exonuclease III (3) or exonuclease lambda (4); (C) pMA100 extracted from PFGE gels and treated (+) or not (-) with topoisomerase I; (D) DNA prepared with (+) or without (-) adding proteinase K to the lysis buffer; (E) same as in (D) in PFGE gels and running buffer prepared with 0.2% SDS. λ: DNA concatemers of the bacteriophage λ genome.

    Journal: PLoS ONE

    Article Title: First Description of Natural and Experimental Conjugation between Mycobacteria Mediated by a Linear Plasmid

    doi: 10.1371/journal.pone.0029884

    Figure Lengend Snippet: PFGE of DNA genomic preparations. (A) PFGE with undigested DNAs from M. avium 88.3 (1) and M. kansasii 88.8 (2) under different switch times, indicated below each figure; (B) pMA100 extracted from PFGE gels and treated with exonuclease III (3) or exonuclease lambda (4); (C) pMA100 extracted from PFGE gels and treated (+) or not (-) with topoisomerase I; (D) DNA prepared with (+) or without (-) adding proteinase K to the lysis buffer; (E) same as in (D) in PFGE gels and running buffer prepared with 0.2% SDS. λ: DNA concatemers of the bacteriophage λ genome.

    Article Snippet: Besides that, the pMA100 band excised from PFGE agarose gels was treated in separate experiments with 30 U exonuclease III, 30 U exonuclease lambda or 30 U topoiosomerase I (all enzymes from New England BioLabs) for 3 h at 37°C, according to the manufacturer's protocols.

    Techniques: Lysis

    In situ detection of DNA using padlock probes and target primed rolling circle DNA synthesis . (A) The samples are cleaved with a restriction enzyme having a restriction site positioned 3' to the probe binding sequence. It is important that the enzyme does not have any other cleavage sites in close proximity to the 5'-end of the probe binding sequence to avoid degradation of the recognition sequence during exonuclease treatment. (B) The target sequence is made single stranded using the lambda exonuclease which digests duplex DNA in the 5'→3' direction in a highly processive manner, thereby making the target sequence single stranded. (C) The padlock probe is hybridized and ligated on the target sequence. Only padlock probes which are correctly hybridized at the point of ligation will be circularized. (D-E) The rolling circle reaction is initiated by using the target sequence as a primer, thereby locking the rolling circle product to the target sequence. (F) The rolling circle product is visualized by hybridizing a labeled oligonucleotide to the part of the padlock probe not recognizing the genomic hybridization target.

    Journal: BMC Molecular Biology

    Article Title: Detection of short repeated genomic sequences on metaphase chromosomes using padlock probes and target primed rolling circle DNA synthesis

    doi: 10.1186/1471-2199-8-103

    Figure Lengend Snippet: In situ detection of DNA using padlock probes and target primed rolling circle DNA synthesis . (A) The samples are cleaved with a restriction enzyme having a restriction site positioned 3' to the probe binding sequence. It is important that the enzyme does not have any other cleavage sites in close proximity to the 5'-end of the probe binding sequence to avoid degradation of the recognition sequence during exonuclease treatment. (B) The target sequence is made single stranded using the lambda exonuclease which digests duplex DNA in the 5'→3' direction in a highly processive manner, thereby making the target sequence single stranded. (C) The padlock probe is hybridized and ligated on the target sequence. Only padlock probes which are correctly hybridized at the point of ligation will be circularized. (D-E) The rolling circle reaction is initiated by using the target sequence as a primer, thereby locking the rolling circle product to the target sequence. (F) The rolling circle product is visualized by hybridizing a labeled oligonucleotide to the part of the padlock probe not recognizing the genomic hybridization target.

    Article Snippet: Lambda exonuclease Exonuclease digestion was performed in a buffer containing 1× lambda exonuclease buffer (NEB), 0.2 μg/μl BSA (NEB) and 1u/μl lambda exonuclease (NEB) for 1 min at 37°C.

    Techniques: In Situ, DNA Synthesis, Binding Assay, Sequencing, Ligation, Labeling, Hybridization

    Superior performance of ChIP-nexus in discovering relevant binding footprints for transcription factors (a) Outline of ChIP-nexus 1) The transcription factor of interest (brown) is immunoprecipitated from chromatin fragments with antibodies in the same way as during conventional ChIP-seq experiments. 2) While still bound to the antibodies, the DNA ends are repaired, dA-tailed and then ligated to a special adaptor that contains a pair of sequences for library amplification (arrows indicate the correct orientation for them to be functional), a BamHI site (black dot) for linearization, and a 9-nucleotide barcode containing 5 random bases and 4 fixed bases to remove reads resulting from over-amplification of library DNA. The barcode is part of a 5′ overhang, which reduces adaptor-adaptor ligation. 3) After the adaptor ligation step, the 5′ overhang is filled, copying the random barcode and generating blunt ends for lambda exonuclease digestion. 4) Lambda exonuclease (blue Pacman) digests until it encounters a physical barrier such as a cross-linked protein-DNA complex (‘Do not enter’ sign = ‘stop base’). 5) Single-stranded DNA is eluted and purified. 6) Self-circularization places the barcode next to the ‘stop base’. 7) An oligonucleotide (red arc) is paired with the region around the BamHI site for BamHI digestion (black scissors). 8) The digestion results in re-linearized DNA fragments with suitable Illumina sequences on both ends, ready for PCR library amplification. 9) Using single-end sequencing with the standard Illumina primer, each fragment is sequenced: first the barcode, then the genomic sequence starting with the ‘stop base’. 10) After alignment of the genomic sequences, reads with identical start positions and identical barcodes are removed. The final output is the position, number and strand orientation of the ‘stop’ bases. The frequencies of ‘stop’ bases on the positive strand are shown in red, while those on the negative strand are shown in blue. (b–e) Comparison of conventional ChIP-seq data (extended reads), ChIP-nexus data (raw stop base reads) and data generated using the original ChIP-exo protocol (raw stop base reads). (b) TBP profiles in human K562 cells at the RPS12 promoter. Although ChIP-nexus and ChIP-exo generally agree on TBP binding footprints, ChIP-nexus provides better coverage and richer details than ChIP-exo, which shows signs of over-amplification as large numbers of reads accumulate at a few discreet bases. (c) Dorsal profiles at the D. melanogaster decapentaplegic (dpp) enhancer. Five “Strong” dorsal binding sites (S1–S5) were previously mapped by in vitro DNase footprinting 12 . Note that ChIP-nexus identifies S4 as the only site with significant Dorsal binding in vivo . At the same time, ChIP-exo performed by Peconic did not detect any clear Dorsal footprint within the enhancer, in part due to the low read counts obtained. (d) Dorsal profiles at the rhomboid (rho) NEE enhancer. Four Dorsal binding sites (d1–d4) were previously mapped by in vitro DNase footprinting 14 . Note that ChIP-nexus identifies d3 as the strongest dorsal binding site in vivo , consistent with its close proximity to two Twist binding sites. Again, the original ChIP-exo protocol did not detect any clear Dorsal footprint within the enhancer. (e) Twist profiles at the same rho enhancer. Note that ChIP-nexus shows strong Twist footprints surrounding the two Twist binding sites (t1, t2) 14 . In this case, ChIP-exo performed by Peconic identified a similar Twist footprint. This shows that the Peconic experiments, which were performed with the same chromatin extracts as the Dorsal experiments, worked in principle but were less robust than our ChIP-nexus experiments.

    Journal: Nature biotechnology

    Article Title: ChIP-nexus: a novel ChIP-exo protocol for improved detection of in vivo transcription factor binding footprints

    doi: 10.1038/nbt.3121

    Figure Lengend Snippet: Superior performance of ChIP-nexus in discovering relevant binding footprints for transcription factors (a) Outline of ChIP-nexus 1) The transcription factor of interest (brown) is immunoprecipitated from chromatin fragments with antibodies in the same way as during conventional ChIP-seq experiments. 2) While still bound to the antibodies, the DNA ends are repaired, dA-tailed and then ligated to a special adaptor that contains a pair of sequences for library amplification (arrows indicate the correct orientation for them to be functional), a BamHI site (black dot) for linearization, and a 9-nucleotide barcode containing 5 random bases and 4 fixed bases to remove reads resulting from over-amplification of library DNA. The barcode is part of a 5′ overhang, which reduces adaptor-adaptor ligation. 3) After the adaptor ligation step, the 5′ overhang is filled, copying the random barcode and generating blunt ends for lambda exonuclease digestion. 4) Lambda exonuclease (blue Pacman) digests until it encounters a physical barrier such as a cross-linked protein-DNA complex (‘Do not enter’ sign = ‘stop base’). 5) Single-stranded DNA is eluted and purified. 6) Self-circularization places the barcode next to the ‘stop base’. 7) An oligonucleotide (red arc) is paired with the region around the BamHI site for BamHI digestion (black scissors). 8) The digestion results in re-linearized DNA fragments with suitable Illumina sequences on both ends, ready for PCR library amplification. 9) Using single-end sequencing with the standard Illumina primer, each fragment is sequenced: first the barcode, then the genomic sequence starting with the ‘stop base’. 10) After alignment of the genomic sequences, reads with identical start positions and identical barcodes are removed. The final output is the position, number and strand orientation of the ‘stop’ bases. The frequencies of ‘stop’ bases on the positive strand are shown in red, while those on the negative strand are shown in blue. (b–e) Comparison of conventional ChIP-seq data (extended reads), ChIP-nexus data (raw stop base reads) and data generated using the original ChIP-exo protocol (raw stop base reads). (b) TBP profiles in human K562 cells at the RPS12 promoter. Although ChIP-nexus and ChIP-exo generally agree on TBP binding footprints, ChIP-nexus provides better coverage and richer details than ChIP-exo, which shows signs of over-amplification as large numbers of reads accumulate at a few discreet bases. (c) Dorsal profiles at the D. melanogaster decapentaplegic (dpp) enhancer. Five “Strong” dorsal binding sites (S1–S5) were previously mapped by in vitro DNase footprinting 12 . Note that ChIP-nexus identifies S4 as the only site with significant Dorsal binding in vivo . At the same time, ChIP-exo performed by Peconic did not detect any clear Dorsal footprint within the enhancer, in part due to the low read counts obtained. (d) Dorsal profiles at the rhomboid (rho) NEE enhancer. Four Dorsal binding sites (d1–d4) were previously mapped by in vitro DNase footprinting 14 . Note that ChIP-nexus identifies d3 as the strongest dorsal binding site in vivo , consistent with its close proximity to two Twist binding sites. Again, the original ChIP-exo protocol did not detect any clear Dorsal footprint within the enhancer. (e) Twist profiles at the same rho enhancer. Note that ChIP-nexus shows strong Twist footprints surrounding the two Twist binding sites (t1, t2) 14 . In this case, ChIP-exo performed by Peconic identified a similar Twist footprint. This shows that the Peconic experiments, which were performed with the same chromatin extracts as the Dorsal experiments, worked in principle but were less robust than our ChIP-nexus experiments.

    Article Snippet: For lambda exonuclease digestion, each sample was incubated in 0.2 u/μl lambda exonuclease (New England Biolabs, M0262), 5% DMSO and 0.1% triton X-100 in 100 μl 1x NEB Lambda exonuclease reaction buffer at 37 °C for 60 min with constant agitation, followed by washing steps as above.

    Techniques: Chromatin Immunoprecipitation, Binding Assay, Immunoprecipitation, Amplification, Functional Assay, Ligation, Purification, Polymerase Chain Reaction, Sequencing, Genomic Sequencing, Generated, In Vitro, Footprinting, In Vivo

    Analysis of the Dorsal, Twist and Max in vivo footprint (a–c) For each factor, the top 200 motifs with the highest ChIP-nexus read counts were selected and are shown in descending order as heat map. The footprints show a consistent boundary on the positive strand (red) and negative strand (blue) around each motif. The zoomed-in average profile below reveals that the footprints are wider than the motif. A schematic representation of the digestion pattern is shown below using Pacman symbols for lambda exonuclease. (a) The ChIP-nexus footprint for Dorsal (NFkB) on its canonical motif (GGRWWTTCC with up to one mismatch) extends on average 5 bp away from the motif edge. Thus, the average dorsal footprint is 18 bp long (horizontal black bar). (b) The Twist ChIP-nexus footprint on the E-box motif CABATG (no mismatch) has two outside boundaries, one at 11 bp, and one at 2 bp away from the motif edge, suggesting interactions with flanking DNA sequences. Each portion of the footprint is around 8–9bp long (horizontal black bar). (c) The Max ChIP-nexus footprint on its canonical E-box motif (CACGTG, no mismatch) has an outside boundary at 8 bp away from the motif edge, as well as a boundary inside the motif (at the A/T base), suggesting two partial footprints (horizontal black bars). (d, e) Average Max and Twist ChIP-nexus footprints at the top 200 sites for all possible E-box variants (CANNTG). Each variant profile includes its reverse complement. (d) Max binds specifically to the canonical CACGTG motif and to a lesser extent to the CACATG motif. Note that the Max footprint shape looks identical between the two motifs. (e) In contrast, the Twist binding specificity and the footprint shape is more complex. Notably, the outer boundary at -11bp is stronger at the CATATG and CACATG motif, whereas the inner boundary at -2 bp is stronger at the CAGATG motif.

    Journal: Nature biotechnology

    Article Title: ChIP-nexus: a novel ChIP-exo protocol for improved detection of in vivo transcription factor binding footprints

    doi: 10.1038/nbt.3121

    Figure Lengend Snippet: Analysis of the Dorsal, Twist and Max in vivo footprint (a–c) For each factor, the top 200 motifs with the highest ChIP-nexus read counts were selected and are shown in descending order as heat map. The footprints show a consistent boundary on the positive strand (red) and negative strand (blue) around each motif. The zoomed-in average profile below reveals that the footprints are wider than the motif. A schematic representation of the digestion pattern is shown below using Pacman symbols for lambda exonuclease. (a) The ChIP-nexus footprint for Dorsal (NFkB) on its canonical motif (GGRWWTTCC with up to one mismatch) extends on average 5 bp away from the motif edge. Thus, the average dorsal footprint is 18 bp long (horizontal black bar). (b) The Twist ChIP-nexus footprint on the E-box motif CABATG (no mismatch) has two outside boundaries, one at 11 bp, and one at 2 bp away from the motif edge, suggesting interactions with flanking DNA sequences. Each portion of the footprint is around 8–9bp long (horizontal black bar). (c) The Max ChIP-nexus footprint on its canonical E-box motif (CACGTG, no mismatch) has an outside boundary at 8 bp away from the motif edge, as well as a boundary inside the motif (at the A/T base), suggesting two partial footprints (horizontal black bars). (d, e) Average Max and Twist ChIP-nexus footprints at the top 200 sites for all possible E-box variants (CANNTG). Each variant profile includes its reverse complement. (d) Max binds specifically to the canonical CACGTG motif and to a lesser extent to the CACATG motif. Note that the Max footprint shape looks identical between the two motifs. (e) In contrast, the Twist binding specificity and the footprint shape is more complex. Notably, the outer boundary at -11bp is stronger at the CATATG and CACATG motif, whereas the inner boundary at -2 bp is stronger at the CAGATG motif.

    Article Snippet: For lambda exonuclease digestion, each sample was incubated in 0.2 u/μl lambda exonuclease (New England Biolabs, M0262), 5% DMSO and 0.1% triton X-100 in 100 μl 1x NEB Lambda exonuclease reaction buffer at 37 °C for 60 min with constant agitation, followed by washing steps as above.

    Techniques: In Vivo, Chromatin Immunoprecipitation, Variant Assay, Binding Assay

    GR binding recruits Oct-2 to octamer motifs adjacent to GR binding sites in the nucleus. (A) Nuclei prepared from CHO cells transfected with the MMTV promoter construct pHCWT, GR, and/or Oct-2 expression plasmids and treated with 10 −6 M Dex or vehicle for 15 min were restricted with Hin ) and GR and/or Oct-2 expression plasmids and treated with 10 −6 M Dex or vehicle for 15 min were restricted with Sma I and digested with λ exonuclease as indicated. Digestion was revealed by linear PCR extension of a T3 polymerase primer, and pause sites were positioned relative to an A sequencing track amplified with the same primer. The positions of the octamer motif sequence and GRE sequences in the MMTV LTR are summarized schematically. The Dex-, GR-, and Oct-2-specific λ pause site is indicated by the arrow. (C) Nuclei prepared from CHO cells transfected with pBluescript containing either a Gal4 binding site separated by 8 nucleotides from the octamer motif sequence from the MMTV LTR (left) or a nonspecific oligonucleotide encoding an IAP enhancer core (right) along with Gal-GR WT , Gal-GR L501P , and/or Oct-2 expression plasmids were restricted with Xho I and digested with λ exonuclease as indicated. Digestion was revealed by linear PCR extension of a T7 polymerase primer, and pause sites were positioned relative to an A sequencing track amplified with the same primer. The positions of the octamer motif-IAP sequence and of the Gal4 sequence are summarized schematically. The Gal-GR WT -, Oct-2-, and octamer motif-dependent specific λ pause site is indicated by the arrow. Western blots of cellular extracts verified that Gal-GR WT and Gal-GR L501P ).

    Journal: Molecular and Cellular Biology

    Article Title: Recruitment of Octamer Transcription Factors to DNA by Glucocorticoid Receptor

    doi:

    Figure Lengend Snippet: GR binding recruits Oct-2 to octamer motifs adjacent to GR binding sites in the nucleus. (A) Nuclei prepared from CHO cells transfected with the MMTV promoter construct pHCWT, GR, and/or Oct-2 expression plasmids and treated with 10 −6 M Dex or vehicle for 15 min were restricted with Hin ) and GR and/or Oct-2 expression plasmids and treated with 10 −6 M Dex or vehicle for 15 min were restricted with Sma I and digested with λ exonuclease as indicated. Digestion was revealed by linear PCR extension of a T3 polymerase primer, and pause sites were positioned relative to an A sequencing track amplified with the same primer. The positions of the octamer motif sequence and GRE sequences in the MMTV LTR are summarized schematically. The Dex-, GR-, and Oct-2-specific λ pause site is indicated by the arrow. (C) Nuclei prepared from CHO cells transfected with pBluescript containing either a Gal4 binding site separated by 8 nucleotides from the octamer motif sequence from the MMTV LTR (left) or a nonspecific oligonucleotide encoding an IAP enhancer core (right) along with Gal-GR WT , Gal-GR L501P , and/or Oct-2 expression plasmids were restricted with Xho I and digested with λ exonuclease as indicated. Digestion was revealed by linear PCR extension of a T7 polymerase primer, and pause sites were positioned relative to an A sequencing track amplified with the same primer. The positions of the octamer motif-IAP sequence and of the Gal4 sequence are summarized schematically. The Gal-GR WT -, Oct-2-, and octamer motif-dependent specific λ pause site is indicated by the arrow. Western blots of cellular extracts verified that Gal-GR WT and Gal-GR L501P ).

    Article Snippet: Each sample was simultaneously digested with 100 U of restriction enzyme and 15 U of λ exonuclease (Life Technologies) for 15 min at 30°C.

    Techniques: Binding Assay, Transfection, Construct, Expressing, Polymerase Chain Reaction, Sequencing, Amplification, Western Blot

    Copper(I)-oxygen efficiently reveals incorporated BrdU; the revelation can be further increased by means of exonucleases. A ) The results of the detection of the BrdU labeling of replicated DNA using acid (4 N HCl) or hydroxide (0.07 M NaOH) or DNase I treatment or the one-step or the two-step procedure are shown. All of the images were taken using 99-ms time to be able to compare the signal intensity. In the one-step procedure (the image labeled as Cu), the 30-minute treatment with copper(I)-oxygen was used exclusively. In the two-step protocol, a 10-minute treatment of the samples with copper(I)-oxygen was followed by incubation with exonuclease III or exonuclease λ. The model shows the situation for both one-step and two-step procedures. Note that exonuclease λ reveals BrdU-labeled parts in the proximity of close single gaps as it has no activity at nicks and limited activity at gaps. Only close single gaps can result into the formation of double-strand break. Although only one strand is usually labeled by BrdU, the situation is shown as if both strands were labeled in the schematic picture. The revealed parts of distinct strands are distinguished by colors. Bar: 20 µm. B ) Relative signal intensity is shown in the graph.

    Journal: PLoS ONE

    Article Title: Atomic Scissors: A New Method of Tracking the 5-Bromo-2?-Deoxyuridine-Labeled DNA In Situ

    doi: 10.1371/journal.pone.0052584

    Figure Lengend Snippet: Copper(I)-oxygen efficiently reveals incorporated BrdU; the revelation can be further increased by means of exonucleases. A ) The results of the detection of the BrdU labeling of replicated DNA using acid (4 N HCl) or hydroxide (0.07 M NaOH) or DNase I treatment or the one-step or the two-step procedure are shown. All of the images were taken using 99-ms time to be able to compare the signal intensity. In the one-step procedure (the image labeled as Cu), the 30-minute treatment with copper(I)-oxygen was used exclusively. In the two-step protocol, a 10-minute treatment of the samples with copper(I)-oxygen was followed by incubation with exonuclease III or exonuclease λ. The model shows the situation for both one-step and two-step procedures. Note that exonuclease λ reveals BrdU-labeled parts in the proximity of close single gaps as it has no activity at nicks and limited activity at gaps. Only close single gaps can result into the formation of double-strand break. Although only one strand is usually labeled by BrdU, the situation is shown as if both strands were labeled in the schematic picture. The revealed parts of distinct strands are distinguished by colors. Bar: 20 µm. B ) Relative signal intensity is shown in the graph.

    Article Snippet: Enzymes used These enzymes and condition were used: Terminal deoxynucleotidyl transferase (TdT; 2 U/µl, 10 minutes, 37°C, Fermentas), buffer for TdT, 0.05 mM dATP, dGTP, dCTP and 0.05 mM Alexa Fluor® 555-aha-2′-deoxyuridine-5′-triphosphate (Alexa-dUTP); DNA polymerase I (0.2 U/µl, 10 minutes, RT, Fermentas), buffer for DNA polymerase I, 0.05 mM dATP, dGTP, dCTP and 0.05 mM Alexa-dUTP; Klenow fragment (0.2 U/µl, 10 minutes, RT, Fermentas), buffer for the Klenow fragment, 0.05 mM dATP, dGTP, dCTP and 0.05 mM Alexa-dUTP; Klenow fragment Exo- (0.2 U/µl, 10 minutes, RT, Fermentas), buffer for the Klenow fragment Exo-, 0.05 mM dATP, dGTP, dCTP and 0.05 mM Alexa-dUTP; Exonuclease III (1 U/µl, 30 minutes, RT, Fermentas), buffer for exonuclease III; Exonuclease λ (0.1 U/µl, 30 minutes, RT, Fermentas), buffer for exonuclease λ; Shrimp alkaline phosphomonoesterase (phosphatase; SAP; 1 U/µl, 20 minutes, 37°C, Fermentas), buffer for SAP.

    Techniques: Labeling, Mass Spectrometry, Incubation, Activity Assay