exonuclease i Search Results


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  • 99
    New England Biolabs exonuclease 1 exo1
    Exonuclease activity and hybridization length affects assay sensitivity. (A) VEGF assays were designed with different lengths of the hybridization site and compared with respect to sensitivity. A 9-nt hybridization site was found to give the best signal-to-noise levels and was selected for further studies. ( B ) Different DNA polymerases were tested with regards to their ability to generate good sensitivity in an IL-8-specific assay. T4 DNA polymerase I, DNA polymerase I and Klenow fragment exo + all possess a 3′→5′ exonuclease activity and performed well in the IL-8 detection. Klenow fragment exo − , on the other hand, generated a background signal that was almost at the level of the antigen-induced signal. When exogenous <t>Exonuclease</t> I was added to the reaction, the signal-to-noise level was restored.
    Exonuclease 1 Exo1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 577 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher e coli exonuclease i
    Principle of mismatch removal. In the first step, a double-stranded polynucleotide with a mismatching base pair, e.g. resulting from ligation of oligonucleotides in a gene synthesis reaction, is cleaved by the EMC enzyme in both strands, 2–5 bp downstream of the mismatch. The short overhangs thus generated dissociate immediately at the reaction temperature of 37°C. The resulting single-stranded overhangs can be removed by different strategies: addition of a single-strand-specific 3′–5′-exonuclease, e.g. E.coli <t>exonuclease</t> I in the EMC reaction or in a subsequent exonuclease step. Alternatively, the 3′–5′-exonuclease activity of proofreading polymerases can be used in the subsequent PCR. The proposed mechanism in this case is (i) removal of single-stranded overhangs during the initial heating step from 20 to 95°C or (ii) removal of mispaired bases by ‘proofreading’ in the first elongation cycle of the overlap extension PCR.
    E Coli Exonuclease I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 84 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher exonuclease 1 exo1
    Principle of mismatch removal. In the first step, a double-stranded polynucleotide with a mismatching base pair, e.g. resulting from ligation of oligonucleotides in a gene synthesis reaction, is cleaved by the EMC enzyme in both strands, 2–5 bp downstream of the mismatch. The short overhangs thus generated dissociate immediately at the reaction temperature of 37°C. The resulting single-stranded overhangs can be removed by different strategies: addition of a single-strand-specific 3′–5′-exonuclease, e.g. E.coli <t>exonuclease</t> I in the EMC reaction or in a subsequent exonuclease step. Alternatively, the 3′–5′-exonuclease activity of proofreading polymerases can be used in the subsequent PCR. The proposed mechanism in this case is (i) removal of single-stranded overhangs during the initial heating step from 20 to 95°C or (ii) removal of mispaired bases by ‘proofreading’ in the first elongation cycle of the overlap extension PCR.
    Exonuclease 1 Exo1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore exonuclease 1 knockdown u2os cells
    Principle of mismatch removal. In the first step, a double-stranded polynucleotide with a mismatching base pair, e.g. resulting from ligation of oligonucleotides in a gene synthesis reaction, is cleaved by the EMC enzyme in both strands, 2–5 bp downstream of the mismatch. The short overhangs thus generated dissociate immediately at the reaction temperature of 37°C. The resulting single-stranded overhangs can be removed by different strategies: addition of a single-strand-specific 3′–5′-exonuclease, e.g. E.coli <t>exonuclease</t> I in the EMC reaction or in a subsequent exonuclease step. Alternatively, the 3′–5′-exonuclease activity of proofreading polymerases can be used in the subsequent PCR. The proposed mechanism in this case is (i) removal of single-stranded overhangs during the initial heating step from 20 to 95°C or (ii) removal of mispaired bases by ‘proofreading’ in the first elongation cycle of the overlap extension PCR.
    Exonuclease 1 Knockdown U2os Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 275 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    TaKaRa exonuclease i
    Amount of residual DNAs in the presence of 0% (w/v) (open circles), 5% (w/v) (closed circles), 10% (w/v) (closed triangles), 15% (w/v) (closed squares) and 20% (w/v) (closed diamonds) of PEG and ( A ) 0.1 U DNase I, ( B ) 0.15 U S1 nuclease, ( C ) 1 U exonuclease III (inset shows 10 U), ( D ) 0.5 U <t>exonuclease</t> I (inset shows 5 U). A ssDNA was used as a substrate for S1 nuclease and exonuclease I and a dsDNA was used as a substrate for DNase I and exonuclease III. PEG 4000 was used as the crowding agent for DNase I and S1 nuclease reactions, and PEG 8000 was used for exonucleases III and I. Error bars (smaller than ±2%) were omitted for clarity.
    Exonuclease I, supplied by TaKaRa, used in various techniques. Bioz Stars score: 96/100, based on 576 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    GE Healthcare exonuclease i
    Schematic presentation of the microsphere-based single base chain extension assays. DNA fragments containing the polymorphic site to be typed were amplified either individually or by multiplexed PCR (step 1 ). The PCR products containing a SNP site were pooled and treated with SAP and <t>exonuclease</t> I (step 2 ). After heat inactivation of the enzymes, the PCR products were used in the SBCE reactions (step 3 ) as described in Methods. For every SNP, one capture oligonucleotide probe with a unique ZipCode sequence was designed and used to assay the two alleles in each of two separate wells with a different labeled ddNTP per well. Multiplexed SNP analysis could be achieved by the employment of different ZipCode sequences for different SNPs in the presence of pooled PCR products. After the completion of the SBCE reaction, ∼1200 of each type of microsphere [with an attached oligonucleotide encoding the complement to the ZipCode sequences and a common luciferase sequence (SeqLUC)] were added to the completed SBCE reactions. The hybridization reactions were carried out at 40°C in the presence of NaCl for > 2 hr (step 4 ). The microspheres were then subjected to flow cytometric analysis (step 5 ). Minimums of 100 of each type of microsphere were read and the mean value of MESF was used for determining the genotypes. The fluorescence signal of the corresponding microsphere without SBCE reactions (microsphere alone) or SBCE reactions without AmpliTaq FS were subtracted from the MESF values.
    Exonuclease I, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 93/100, based on 1132 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Enzymatics exonuclease 1
    Improvement of single-cell methylome read mapping.  a  Schematics of snmC-seq workflow. Steps modified in snmC-seq2 are highlighted. RP-N and RP-H indicate random primers used in snmC-seq and snmC-seq2, respectively. Exo1 exonuclease 1, SAP shrimp alkaline phosphatase, SPRI solid-phase reversible immobilization.  b  Reverse read (R2) mapping is improved by reducing nucleotide concentration in random-primed DNA synthesis reaction or treatment with shrimp alkaline phosphatase, whereas forward read (R1) mapping is not affected. Each condition contained four biological replicates.  c  snmC-seq2 produces reverse reads with comparable base composition as MethylC-seq reads. The elements of all box-plots are defined as following—center line, median; box limits, first and third quartiles; whiskers, 1.5× interquartile range
    Exonuclease 1, supplied by Enzymatics, used in various techniques. Bioz Stars score: 93/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Epicentre Biotechnologies exonuclease i
    Improvement of single-cell methylome read mapping.  a  Schematics of snmC-seq workflow. Steps modified in snmC-seq2 are highlighted. RP-N and RP-H indicate random primers used in snmC-seq and snmC-seq2, respectively. Exo1 exonuclease 1, SAP shrimp alkaline phosphatase, SPRI solid-phase reversible immobilization.  b  Reverse read (R2) mapping is improved by reducing nucleotide concentration in random-primed DNA synthesis reaction or treatment with shrimp alkaline phosphatase, whereas forward read (R1) mapping is not affected. Each condition contained four biological replicates.  c  snmC-seq2 produces reverse reads with comparable base composition as MethylC-seq reads. The elements of all box-plots are defined as following—center line, median; box limits, first and third quartiles; whiskers, 1.5× interquartile range
    Exonuclease I, supplied by Epicentre Biotechnologies, used in various techniques. Bioz Stars score: 92/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Roche exonuclease i
    Improvement of single-cell methylome read mapping.  a  Schematics of snmC-seq workflow. Steps modified in snmC-seq2 are highlighted. RP-N and RP-H indicate random primers used in snmC-seq and snmC-seq2, respectively. Exo1 exonuclease 1, SAP shrimp alkaline phosphatase, SPRI solid-phase reversible immobilization.  b  Reverse read (R2) mapping is improved by reducing nucleotide concentration in random-primed DNA synthesis reaction or treatment with shrimp alkaline phosphatase, whereas forward read (R1) mapping is not affected. Each condition contained four biological replicates.  c  snmC-seq2 produces reverse reads with comparable base composition as MethylC-seq reads. The elements of all box-plots are defined as following—center line, median; box limits, first and third quartiles; whiskers, 1.5× interquartile range
    Exonuclease I, supplied by Roche, used in various techniques. Bioz Stars score: 94/100, based on 217 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Promega exonuclease i
    Improvement of single-cell methylome read mapping.  a  Schematics of snmC-seq workflow. Steps modified in snmC-seq2 are highlighted. RP-N and RP-H indicate random primers used in snmC-seq and snmC-seq2, respectively. Exo1 exonuclease 1, SAP shrimp alkaline phosphatase, SPRI solid-phase reversible immobilization.  b  Reverse read (R2) mapping is improved by reducing nucleotide concentration in random-primed DNA synthesis reaction or treatment with shrimp alkaline phosphatase, whereas forward read (R1) mapping is not affected. Each condition contained four biological replicates.  c  snmC-seq2 produces reverse reads with comparable base composition as MethylC-seq reads. The elements of all box-plots are defined as following—center line, median; box limits, first and third quartiles; whiskers, 1.5× interquartile range
    Exonuclease I, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 65 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    New England Biolabs thermolabile exonuclease i
    Improvement of single-cell methylome read mapping.  a  Schematics of snmC-seq workflow. Steps modified in snmC-seq2 are highlighted. RP-N and RP-H indicate random primers used in snmC-seq and snmC-seq2, respectively. Exo1 exonuclease 1, SAP shrimp alkaline phosphatase, SPRI solid-phase reversible immobilization.  b  Reverse read (R2) mapping is improved by reducing nucleotide concentration in random-primed DNA synthesis reaction or treatment with shrimp alkaline phosphatase, whereas forward read (R1) mapping is not affected. Each condition contained four biological replicates.  c  snmC-seq2 produces reverse reads with comparable base composition as MethylC-seq reads. The elements of all box-plots are defined as following—center line, median; box limits, first and third quartiles; whiskers, 1.5× interquartile range
    Thermolabile Exonuclease I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Inqaba biotec exonuclease i
    Improvement of single-cell methylome read mapping.  a  Schematics of snmC-seq workflow. Steps modified in snmC-seq2 are highlighted. RP-N and RP-H indicate random primers used in snmC-seq and snmC-seq2, respectively. Exo1 exonuclease 1, SAP shrimp alkaline phosphatase, SPRI solid-phase reversible immobilization.  b  Reverse read (R2) mapping is improved by reducing nucleotide concentration in random-primed DNA synthesis reaction or treatment with shrimp alkaline phosphatase, whereas forward read (R1) mapping is not affected. Each condition contained four biological replicates.  c  snmC-seq2 produces reverse reads with comparable base composition as MethylC-seq reads. The elements of all box-plots are defined as following—center line, median; box limits, first and third quartiles; whiskers, 1.5× interquartile range
    Exonuclease I, supplied by Inqaba biotec, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    New England Biolabs exonuclease i reaction buffer
    Improvement of single-cell methylome read mapping.  a  Schematics of snmC-seq workflow. Steps modified in snmC-seq2 are highlighted. RP-N and RP-H indicate random primers used in snmC-seq and snmC-seq2, respectively. Exo1 exonuclease 1, SAP shrimp alkaline phosphatase, SPRI solid-phase reversible immobilization.  b  Reverse read (R2) mapping is improved by reducing nucleotide concentration in random-primed DNA synthesis reaction or treatment with shrimp alkaline phosphatase, whereas forward read (R1) mapping is not affected. Each condition contained four biological replicates.  c  snmC-seq2 produces reverse reads with comparable base composition as MethylC-seq reads. The elements of all box-plots are defined as following—center line, median; box limits, first and third quartiles; whiskers, 1.5× interquartile range
    Exonuclease I Reaction Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 118 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Euromedex exonuclease i
    Improvement of single-cell methylome read mapping.  a  Schematics of snmC-seq workflow. Steps modified in snmC-seq2 are highlighted. RP-N and RP-H indicate random primers used in snmC-seq and snmC-seq2, respectively. Exo1 exonuclease 1, SAP shrimp alkaline phosphatase, SPRI solid-phase reversible immobilization.  b  Reverse read (R2) mapping is improved by reducing nucleotide concentration in random-primed DNA synthesis reaction or treatment with shrimp alkaline phosphatase, whereas forward read (R1) mapping is not affected. Each condition contained four biological replicates.  c  snmC-seq2 produces reverse reads with comparable base composition as MethylC-seq reads. The elements of all box-plots are defined as following—center line, median; box limits, first and third quartiles; whiskers, 1.5× interquartile range
    Exonuclease I, supplied by Euromedex, used in various techniques. Bioz Stars score: 90/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Exonuclease activity and hybridization length affects assay sensitivity. (A) VEGF assays were designed with different lengths of the hybridization site and compared with respect to sensitivity. A 9-nt hybridization site was found to give the best signal-to-noise levels and was selected for further studies. ( B ) Different DNA polymerases were tested with regards to their ability to generate good sensitivity in an IL-8-specific assay. T4 DNA polymerase I, DNA polymerase I and Klenow fragment exo + all possess a 3′→5′ exonuclease activity and performed well in the IL-8 detection. Klenow fragment exo − , on the other hand, generated a background signal that was almost at the level of the antigen-induced signal. When exogenous Exonuclease I was added to the reaction, the signal-to-noise level was restored.

    Journal: Nucleic Acids Research

    Article Title: Homogeneous antibody-based proximity extension assays provide sensitive and specific detection of low-abundant proteins in human blood

    doi: 10.1093/nar/gkr424

    Figure Lengend Snippet: Exonuclease activity and hybridization length affects assay sensitivity. (A) VEGF assays were designed with different lengths of the hybridization site and compared with respect to sensitivity. A 9-nt hybridization site was found to give the best signal-to-noise levels and was selected for further studies. ( B ) Different DNA polymerases were tested with regards to their ability to generate good sensitivity in an IL-8-specific assay. T4 DNA polymerase I, DNA polymerase I and Klenow fragment exo + all possess a 3′→5′ exonuclease activity and performed well in the IL-8 detection. Klenow fragment exo − , on the other hand, generated a background signal that was almost at the level of the antigen-induced signal. When exogenous Exonuclease I was added to the reaction, the signal-to-noise level was restored.

    Article Snippet: After a 5-min incubation at 37°C, a 20 µl extension mix containing 66.8 mM Tris–HCl, 16.8 mM ammonium sulfate, 1 mM dithiothreitol, 33 mM magnesium chloride, 62.5 U/ml T4 DNA Polymerase [or 62.5 U/ml Klenow fragment exo(−), 125 U/ml Klenow fragment, 125 U/ml DNA Polymerase I (Fermentas), 250 U/ml Exonuclease I (New England Biolabs)] was added.

    Techniques: Activity Assay, Hybridization, Generated

    Principle of mismatch removal. In the first step, a double-stranded polynucleotide with a mismatching base pair, e.g. resulting from ligation of oligonucleotides in a gene synthesis reaction, is cleaved by the EMC enzyme in both strands, 2–5 bp downstream of the mismatch. The short overhangs thus generated dissociate immediately at the reaction temperature of 37°C. The resulting single-stranded overhangs can be removed by different strategies: addition of a single-strand-specific 3′–5′-exonuclease, e.g. E.coli exonuclease I in the EMC reaction or in a subsequent exonuclease step. Alternatively, the 3′–5′-exonuclease activity of proofreading polymerases can be used in the subsequent PCR. The proposed mechanism in this case is (i) removal of single-stranded overhangs during the initial heating step from 20 to 95°C or (ii) removal of mispaired bases by ‘proofreading’ in the first elongation cycle of the overlap extension PCR.

    Journal: Nucleic Acids Research

    Article Title: Removal of mismatched bases from synthetic genes by enzymatic mismatch cleavage

    doi: 10.1093/nar/gni058

    Figure Lengend Snippet: Principle of mismatch removal. In the first step, a double-stranded polynucleotide with a mismatching base pair, e.g. resulting from ligation of oligonucleotides in a gene synthesis reaction, is cleaved by the EMC enzyme in both strands, 2–5 bp downstream of the mismatch. The short overhangs thus generated dissociate immediately at the reaction temperature of 37°C. The resulting single-stranded overhangs can be removed by different strategies: addition of a single-strand-specific 3′–5′-exonuclease, e.g. E.coli exonuclease I in the EMC reaction or in a subsequent exonuclease step. Alternatively, the 3′–5′-exonuclease activity of proofreading polymerases can be used in the subsequent PCR. The proposed mechanism in this case is (i) removal of single-stranded overhangs during the initial heating step from 20 to 95°C or (ii) removal of mispaired bases by ‘proofreading’ in the first elongation cycle of the overlap extension PCR.

    Article Snippet: Cleavage was performed at 37°C in the buffer recommended by the manufacturer, for up to 24 h. For removal of single-stranded DNA, E.coli exonuclease I (Fermentas, St Leon-Rot, Germany) was included in some reactions.

    Techniques: Ligation, Generated, Activity Assay, Polymerase Chain Reaction

    Amount of residual DNAs in the presence of 0% (w/v) (open circles), 5% (w/v) (closed circles), 10% (w/v) (closed triangles), 15% (w/v) (closed squares) and 20% (w/v) (closed diamonds) of PEG and ( A ) 0.1 U DNase I, ( B ) 0.15 U S1 nuclease, ( C ) 1 U exonuclease III (inset shows 10 U), ( D ) 0.5 U exonuclease I (inset shows 5 U). A ssDNA was used as a substrate for S1 nuclease and exonuclease I and a dsDNA was used as a substrate for DNase I and exonuclease III. PEG 4000 was used as the crowding agent for DNase I and S1 nuclease reactions, and PEG 8000 was used for exonucleases III and I. Error bars (smaller than ±2%) were omitted for clarity.

    Journal: Nucleic Acids Research

    Article Title: Regulation of DNA nucleases by molecular crowding

    doi: 10.1093/nar/gkm445

    Figure Lengend Snippet: Amount of residual DNAs in the presence of 0% (w/v) (open circles), 5% (w/v) (closed circles), 10% (w/v) (closed triangles), 15% (w/v) (closed squares) and 20% (w/v) (closed diamonds) of PEG and ( A ) 0.1 U DNase I, ( B ) 0.15 U S1 nuclease, ( C ) 1 U exonuclease III (inset shows 10 U), ( D ) 0.5 U exonuclease I (inset shows 5 U). A ssDNA was used as a substrate for S1 nuclease and exonuclease I and a dsDNA was used as a substrate for DNase I and exonuclease III. PEG 4000 was used as the crowding agent for DNase I and S1 nuclease reactions, and PEG 8000 was used for exonucleases III and I. Error bars (smaller than ±2%) were omitted for clarity.

    Article Snippet: Exonuclease I from Escherichia coli , S1 nuclease from Asergillus oryzae and exonuclease III from E. coli were purchased from Takara Bio (Tokyo, Japan).

    Techniques:

    Initial velocities ( v ) for the DNase I ( A ) and exonuclease I ( B ) hydrolysis reactions in the presence of 0% (w/v) (open circles), 5% (w/v) (closed circles), 10% (w/v) (closed triangles), 15% (w/v) (closed squares) and 20% (w/v) PEG (closed diamonds). The DNase I reaction was carried out in a buffer containing 100 mM NaCl, 5 mM MgCl 2 and 50 mM HEPES (pH 7.2) at 25°C in the absence or presence of PEG 4000. The exonuclease I reaction was carried out in a buffer containing 100 mM NaCl, 6.7 mM MgCl 2 , 10 mM 2-mercaptoethanol and 67 mM glycine-KOH (pH 9.5) at 37°C in the absence or presence of PEG 8000. The dsDNA and ssDNA concentrations in kinetic assays were 0.1–20 μM for DNase I and 0.1–10 μM for exonuclease I. The value of v was plotted versus the concentration of substrate DNA. Error bars (smaller than ±1%) were omitted for clarity.

    Journal: Nucleic Acids Research

    Article Title: Regulation of DNA nucleases by molecular crowding

    doi: 10.1093/nar/gkm445

    Figure Lengend Snippet: Initial velocities ( v ) for the DNase I ( A ) and exonuclease I ( B ) hydrolysis reactions in the presence of 0% (w/v) (open circles), 5% (w/v) (closed circles), 10% (w/v) (closed triangles), 15% (w/v) (closed squares) and 20% (w/v) PEG (closed diamonds). The DNase I reaction was carried out in a buffer containing 100 mM NaCl, 5 mM MgCl 2 and 50 mM HEPES (pH 7.2) at 25°C in the absence or presence of PEG 4000. The exonuclease I reaction was carried out in a buffer containing 100 mM NaCl, 6.7 mM MgCl 2 , 10 mM 2-mercaptoethanol and 67 mM glycine-KOH (pH 9.5) at 37°C in the absence or presence of PEG 8000. The dsDNA and ssDNA concentrations in kinetic assays were 0.1–20 μM for DNase I and 0.1–10 μM for exonuclease I. The value of v was plotted versus the concentration of substrate DNA. Error bars (smaller than ±1%) were omitted for clarity.

    Article Snippet: Exonuclease I from Escherichia coli , S1 nuclease from Asergillus oryzae and exonuclease III from E. coli were purchased from Takara Bio (Tokyo, Japan).

    Techniques: Concentration Assay

    Residual activities of DNase I and exonuclease I after incubation for 0–30 min at 60°C. Hydrolysis by DNase I in the absence (open circles) or presence (closed circles) of 20% (w/v) PEG 4000 was carried out for 10 min at 25°C. Hydrolysis by exonuclease I in the absence (open triangles) or presence (closed triangles) of 20% (w/v) PEG 8000 was carried out for 10 min at 37°C.

    Journal: Nucleic Acids Research

    Article Title: Regulation of DNA nucleases by molecular crowding

    doi: 10.1093/nar/gkm445

    Figure Lengend Snippet: Residual activities of DNase I and exonuclease I after incubation for 0–30 min at 60°C. Hydrolysis by DNase I in the absence (open circles) or presence (closed circles) of 20% (w/v) PEG 4000 was carried out for 10 min at 25°C. Hydrolysis by exonuclease I in the absence (open triangles) or presence (closed triangles) of 20% (w/v) PEG 8000 was carried out for 10 min at 37°C.

    Article Snippet: Exonuclease I from Escherichia coli , S1 nuclease from Asergillus oryzae and exonuclease III from E. coli were purchased from Takara Bio (Tokyo, Japan).

    Techniques: Incubation

    Schematic presentation of the microsphere-based single base chain extension assays. DNA fragments containing the polymorphic site to be typed were amplified either individually or by multiplexed PCR (step 1 ). The PCR products containing a SNP site were pooled and treated with SAP and exonuclease I (step 2 ). After heat inactivation of the enzymes, the PCR products were used in the SBCE reactions (step 3 ) as described in Methods. For every SNP, one capture oligonucleotide probe with a unique ZipCode sequence was designed and used to assay the two alleles in each of two separate wells with a different labeled ddNTP per well. Multiplexed SNP analysis could be achieved by the employment of different ZipCode sequences for different SNPs in the presence of pooled PCR products. After the completion of the SBCE reaction, ∼1200 of each type of microsphere [with an attached oligonucleotide encoding the complement to the ZipCode sequences and a common luciferase sequence (SeqLUC)] were added to the completed SBCE reactions. The hybridization reactions were carried out at 40°C in the presence of NaCl for > 2 hr (step 4 ). The microspheres were then subjected to flow cytometric analysis (step 5 ). Minimums of 100 of each type of microsphere were read and the mean value of MESF was used for determining the genotypes. The fluorescence signal of the corresponding microsphere without SBCE reactions (microsphere alone) or SBCE reactions without AmpliTaq FS were subtracted from the MESF values.

    Journal: Genome Research

    Article Title: A Microsphere-Based Assay for Multiplexed Single Nucleotide Polymorphism Analysis Using Single Base Chain Extension

    doi:

    Figure Lengend Snippet: Schematic presentation of the microsphere-based single base chain extension assays. DNA fragments containing the polymorphic site to be typed were amplified either individually or by multiplexed PCR (step 1 ). The PCR products containing a SNP site were pooled and treated with SAP and exonuclease I (step 2 ). After heat inactivation of the enzymes, the PCR products were used in the SBCE reactions (step 3 ) as described in Methods. For every SNP, one capture oligonucleotide probe with a unique ZipCode sequence was designed and used to assay the two alleles in each of two separate wells with a different labeled ddNTP per well. Multiplexed SNP analysis could be achieved by the employment of different ZipCode sequences for different SNPs in the presence of pooled PCR products. After the completion of the SBCE reaction, ∼1200 of each type of microsphere [with an attached oligonucleotide encoding the complement to the ZipCode sequences and a common luciferase sequence (SeqLUC)] were added to the completed SBCE reactions. The hybridization reactions were carried out at 40°C in the presence of NaCl for > 2 hr (step 4 ). The microspheres were then subjected to flow cytometric analysis (step 5 ). Minimums of 100 of each type of microsphere were read and the mean value of MESF was used for determining the genotypes. The fluorescence signal of the corresponding microsphere without SBCE reactions (microsphere alone) or SBCE reactions without AmpliTaq FS were subtracted from the MESF values.

    Article Snippet: Shrimp alkaline phosphatase (SAP) and Exonuclease I (Exo I) were obtained from Amersham Pharmacia.

    Techniques: Amplification, Polymerase Chain Reaction, Sequencing, Labeling, Luciferase, Hybridization, Flow Cytometry, Fluorescence

    Improvement of single-cell methylome read mapping.  a  Schematics of snmC-seq workflow. Steps modified in snmC-seq2 are highlighted. RP-N and RP-H indicate random primers used in snmC-seq and snmC-seq2, respectively. Exo1 exonuclease 1, SAP shrimp alkaline phosphatase, SPRI solid-phase reversible immobilization.  b  Reverse read (R2) mapping is improved by reducing nucleotide concentration in random-primed DNA synthesis reaction or treatment with shrimp alkaline phosphatase, whereas forward read (R1) mapping is not affected. Each condition contained four biological replicates.  c  snmC-seq2 produces reverse reads with comparable base composition as MethylC-seq reads. The elements of all box-plots are defined as following—center line, median; box limits, first and third quartiles; whiskers, 1.5× interquartile range

    Journal: Nature Communications

    Article Title: Robust single-cell DNA methylome profiling with snmC-seq2

    doi: 10.1038/s41467-018-06355-2

    Figure Lengend Snippet: Improvement of single-cell methylome read mapping. a Schematics of snmC-seq workflow. Steps modified in snmC-seq2 are highlighted. RP-N and RP-H indicate random primers used in snmC-seq and snmC-seq2, respectively. Exo1 exonuclease 1, SAP shrimp alkaline phosphatase, SPRI solid-phase reversible immobilization. b Reverse read (R2) mapping is improved by reducing nucleotide concentration in random-primed DNA synthesis reaction or treatment with shrimp alkaline phosphatase, whereas forward read (R1) mapping is not affected. Each condition contained four biological replicates. c snmC-seq2 produces reverse reads with comparable base composition as MethylC-seq reads. The elements of all box-plots are defined as following—center line, median; box limits, first and third quartiles; whiskers, 1.5× interquartile range

    Article Snippet: A volume of 1.5 µL enzyme mix containing 1 µL Exonuclease 1 (20 U/µL, Enzymatics X8010L) and 0.5 µL rSAP (1 U/µL, NEB M0371L) was added, then incubated at 37 °C for 30 min followed by 4 °C.

    Techniques: Modification, Concentration Assay, Random Primed, DNA Synthesis