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  • 99
    New England Biolabs monarch pcr dna cleanup kit
    (A) Assay to detect CRISPR/Cas9-mediated cleavage in vitro . A typical region of the Muc14a gene containing at least 2 binding sites for each of the gRNAs: Muc14a _3, Muc14a_4 , Muc14a_5 and Muc14a_6 (top). The <t>PCR</t> amplified <t>DNA</t> fragment was used as a digestion target for Cas9/gRNA cleavage reactions in vitro (bottom). Reactions were run on a gel to detect cleavage. A control without gRNA was included. (B) Analysis of combinations of gRNAs and Cas9 sources for X-shredding. Average male frequencies in the F1 progeny are shown for each parental genotype with a single copy of βtub85Dtub85D-cas9 transgene (1X), two copies of βtub85Dtub85D-cas9 transgene (2X) or one copy of nos-cas9 (grey bars). All lines were crossed to wild type w individuals. The reciprocal cross (female ctrl) or heterozygote βtub85Dtub85D-cas9/ + or nos-cas9/ + without gRNA (no gRNA) were used as control. The black arrow indicates gRNAs in the multiplex array and the red dotted line indicates an unbiased sex-ratio. Crosses were set as pools of males and females or as multiple male single crosses in which case error bars indicate the mean ± SD for a minimum of ten independent single crosses. For all crosses n indicates the total number of individuals (males + females) in the F1 progeny counted. (C) Developmental survival analysis of the F1 progeny of Muc14a_6/βtub85Dtub85D-cas9 males crossed to w females compared to w and βtub85Dtub85D-cas9/ + control males crossed to w females. n indicates the number of individuals recorded at every developmental stage (males + females) in the F1 progeny. Bars indicate means ± SD for at least ten independent single crosses. Statistical significance was calculated with a t test assuming unequal variance. ** p
    Monarch Pcr Dna Cleanup Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 677 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monarch pcr dna cleanup kit/product/New England Biolabs
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    99
    Thermo Fisher pcr
    (A) Assay to detect CRISPR/Cas9-mediated cleavage in vitro . A typical region of the Muc14a gene containing at least 2 binding sites for each of the gRNAs: Muc14a _3, Muc14a_4 , Muc14a_5 and Muc14a_6 (top). The <t>PCR</t> amplified <t>DNA</t> fragment was used as a digestion target for Cas9/gRNA cleavage reactions in vitro (bottom). Reactions were run on a gel to detect cleavage. A control without gRNA was included. (B) Analysis of combinations of gRNAs and Cas9 sources for X-shredding. Average male frequencies in the F1 progeny are shown for each parental genotype with a single copy of βtub85Dtub85D-cas9 transgene (1X), two copies of βtub85Dtub85D-cas9 transgene (2X) or one copy of nos-cas9 (grey bars). All lines were crossed to wild type w individuals. The reciprocal cross (female ctrl) or heterozygote βtub85Dtub85D-cas9/ + or nos-cas9/ + without gRNA (no gRNA) were used as control. The black arrow indicates gRNAs in the multiplex array and the red dotted line indicates an unbiased sex-ratio. Crosses were set as pools of males and females or as multiple male single crosses in which case error bars indicate the mean ± SD for a minimum of ten independent single crosses. For all crosses n indicates the total number of individuals (males + females) in the F1 progeny counted. (C) Developmental survival analysis of the F1 progeny of Muc14a_6/βtub85Dtub85D-cas9 males crossed to w females compared to w and βtub85Dtub85D-cas9/ + control males crossed to w females. n indicates the number of individuals recorded at every developmental stage (males + females) in the F1 progeny. Bars indicate means ± SD for at least ten independent single crosses. Statistical significance was calculated with a t test assuming unequal variance. ** p
    Pcr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 111176 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GE Healthcare illustra gfx pcr dna
    LDR in presence of multiple <t>DNA</t> templates A) LDR on a mix of <t>PCR</t> products B) LDR on a PCR product obtained from amplification of a mix of genomic DNAs. For deposition scheme, refer to fig. 3 . (Laser power and PMT gain were set to 80%).
    Illustra Gfx Pcr Dna, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 4551 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega wizard pcr preps dna purification system
    <t>UP-PCR</t> results of some patients. We have detected all the patients and finally we chose some positive bacteria and carried out the electrophoresis, so every line had PCR products. The deeper the stripe, the more the bacteria. Lines 1–9 represented PCR product of the 16S rRNA gene of Staphylococcus aureus , Staphylococcus epidermidis , Pseudomonas aeruginosa , Escherichia coli , Streptococcus viridans , Staphylococcus saprophyticus, Corynebacterium parvum , Klebsiella pneumoniae , and Enterobacter cloacae ; M for DL2000 <t>DNA</t> Marker; ten for negative control.
    Wizard Pcr Preps Dna Purification System, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 3088 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Illumina Inc truseq dna pcr free sample preparation kit
    Prdm9 Dom2 is suppressed by competition between alleles. (A) PRDM9 Cst can suppress PRDM9 Dom2 -directed recombination at Ush2a . To detect either parental (top) or recombinant (bottom) molecules, nested <t>PCR</t> was performed on <t>DNA</t> from pooled sperm samples collected from F1 hybrids from the indicated crosses (two representative biological replicates are shown for each genotype). Recombinant molecules are only detected in progeny homozygous or heterozygous null for Prdm9 Dom2 . (B) Proportions of H3K4me3 hotspots in (B6xKI)F1 hybrids due to either PRDM9 Cst or PRDM9 Dom2 . (C) PRDM9 Dom2 hotspots in (B6xKI)F1 hybrids are the same hotspots found in Prdm9 Dom2/- heterozygous mice.
    Truseq Dna Pcr Free Sample Preparation Kit, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 99/100, based on 2071 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Illumina Inc truseq dna pcr free kit
    Levels of <t>DNA</t> demethylation in the mitochondrial genome and expression levels of the mtDNA encoded genes. ( A ) Detection of nDNA contamination after mtDNA purification. Copy number for nDNA (β-globin) relative to the copy number for mtDNA was plotted. Multiple t-tests were performed between the purified mtDNA samples (gray bars) and corresponding total DNA samples (black bars) isolated from GBM cells. Bars represent the mean ± SEM ( n = 3). ( B ) Agarose gel showing <t>long-PCR</t> products from purified mtDNA samples to demonstrate the presence of mtDNA methylation. Long PCR using two pairs of primers (primer set 2) spanning across the mitochondrial genome was performed on: (i) total DNA isolated from non-treated GBM cells (Total DNA); (ii) the positive control - MeDIP performed on the long PCR products previously generated with primer set 1 and treated with the M.SssI enzyme (Pos-longPCR-5mC); (iii) purified whole mtDNA having undergone MeDIP - (mtDNA-5mC); (iv) the negative control - MeDIP performed on the long PCR products previously generated with primer set 1 (Neg-longPCR-5mC); (v) the non-antibody control for MeDIP – MeDIP was performed on the purified mtDNA without using the 5mC antibody (mtDNA-NAC); and (vi) a non-template control (H 2 O) for PCR (NTC). Fragment sizes are indicated. ( C ) Normalized levels of DNA methylation within regions of the mitochondrial genome determined on purified mtDNA samples that had undergone MeDIP with sonication. The normalized levels of DNA methylation were determined by normalizing the MeDIP results (5mC/Input) against the positive and negative controls that were used to represent 100% and 0% of DNA methylation, respectively. Statistical significance was determined between the 5Aza and VitC treated cohorts and the GBM cohort and between ND5 and all the other genes by two-way ANOVA ( n = 3). ( D ) Significant differential expression of the mitochondrial and chromosomal genes encoding the ETC subunits identified in the treated and non-treated cohorts using the Fluidigm array. Bars represent the mean of the relative quantification levels normalized to the GBM cohort ( n = 6). *, **, ***, **** indicate P values of
    Truseq Dna Pcr Free Kit, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 99/100, based on 410 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen qiaquick dna purification kit
    Levels of <t>DNA</t> demethylation in the mitochondrial genome and expression levels of the mtDNA encoded genes. ( A ) Detection of nDNA contamination after mtDNA purification. Copy number for nDNA (β-globin) relative to the copy number for mtDNA was plotted. Multiple t-tests were performed between the purified mtDNA samples (gray bars) and corresponding total DNA samples (black bars) isolated from GBM cells. Bars represent the mean ± SEM ( n = 3). ( B ) Agarose gel showing <t>long-PCR</t> products from purified mtDNA samples to demonstrate the presence of mtDNA methylation. Long PCR using two pairs of primers (primer set 2) spanning across the mitochondrial genome was performed on: (i) total DNA isolated from non-treated GBM cells (Total DNA); (ii) the positive control - MeDIP performed on the long PCR products previously generated with primer set 1 and treated with the M.SssI enzyme (Pos-longPCR-5mC); (iii) purified whole mtDNA having undergone MeDIP - (mtDNA-5mC); (iv) the negative control - MeDIP performed on the long PCR products previously generated with primer set 1 (Neg-longPCR-5mC); (v) the non-antibody control for MeDIP – MeDIP was performed on the purified mtDNA without using the 5mC antibody (mtDNA-NAC); and (vi) a non-template control (H 2 O) for PCR (NTC). Fragment sizes are indicated. ( C ) Normalized levels of DNA methylation within regions of the mitochondrial genome determined on purified mtDNA samples that had undergone MeDIP with sonication. The normalized levels of DNA methylation were determined by normalizing the MeDIP results (5mC/Input) against the positive and negative controls that were used to represent 100% and 0% of DNA methylation, respectively. Statistical significance was determined between the 5Aza and VitC treated cohorts and the GBM cohort and between ND5 and all the other genes by two-way ANOVA ( n = 3). ( D ) Significant differential expression of the mitochondrial and chromosomal genes encoding the ETC subunits identified in the treated and non-treated cohorts using the Fluidigm array. Bars represent the mean of the relative quantification levels normalized to the GBM cohort ( n = 6). *, **, ***, **** indicate P values of
    Qiaquick Dna Purification Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 859 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen epitect pcr control dna set
    <t>DNA</t> methylation status in the promoter region of ESRP2 in ovarian cancer cell lines and tissues. ( a ) ESRP2 gene expression following treatment with epigenetic drugs. Data for <t>qRT–PCR</t> are presented as the mean±s.d. of two or three experiments. ESRP2 expression following treatment with epigenetic drugs was compared with that with no treatment (NT). Student’s t -test, * P
    Epitect Pcr Control Dna Set, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 490 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs pcr
    <t>DNA</t> methylation status in the promoter region of ESRP2 in ovarian cancer cell lines and tissues. ( a ) ESRP2 gene expression following treatment with epigenetic drugs. Data for <t>qRT–PCR</t> are presented as the mean±s.d. of two or three experiments. ESRP2 expression following treatment with epigenetic drugs was compared with that with no treatment (NT). Student’s t -test, * P
    Pcr, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 18841 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Bio-Rad dna engine opticon 2 real time pcr detection system
    The conserved HoxC4-Oct- and Sp-NF-κB-binding sites are essential for full Aicda promoter activity; HoxC4, Oct1, Oct2, OcaB, Pax5, Sp1, Sp3 and NF-κB (p52 subunit) are recruited to the Aicda promoter in B cells expressing AICDA/Aicda and undergoing CSR or SHM. ( a ) pGL3-Enhancer luciferase gene reporter constructs containing WT or mutant Aicda promoter, in which the HoxC4-Oct- and/or Sp-NF-κB-binding sites were deleted or disrupted by site-directed mutagenesis, were used to transfect CH12F3-2A, 4B6 or Ramos B cells. Luciferase activity was measured after 24 h (CH12F3-2A B cells) or 16 h (Ramos and 4B6 B cells) of culture. Data are means ± s.e. (bars) of 3 independent experiments. ( b ) AICDA/Aicda expression in human spontaneously switching 4B6 and Ramos B cells, human inducible switching 2E2 B cells stimulated with nil or anti-CD40 mAb and IL-4, mouse CH12F2-2A B cells stimulated with nil or LPS, IL-4 and TGF-β1, WT C57BL/6 mouse spleen B cells stimulated nil, LPS and IL-4, or CD154 and IL-4 were analyzed by semi-quantitative <t>RT-PCR</t> using serially two-fold diluted cDNA as a template. Data are representative of 3 independent experiments. ( c ) Cross-linked chromatin was precipitated from the human or mouse B cells of panel b using a mouse mAb specific to HoxC4, rabbit Abs specific to Oct1, Oct2, OcaB, Pax5, Sp1, Sp3 or p52, or preimmune control mouse or rabbit IgG. The precipitated <t>DNA</t> was specified by PCR using AICDA or Aicda promoter primers. Data are representative of 3 independent experiments.
    Dna Engine Opticon 2 Real Time Pcr Detection System, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 89/100, based on 344 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Geneaid Biotech Ltd gel pcr dna fragments extraction kit
    Host and viral <t>DNA</t> content of differently treated ASFV samples. Quantitative dual <t>PCR</t> was executed by Virotype ASFV PCR Kit. C t values of individual and averaged samples represented by colored circles and grey box respectively. Averages were calculated from numbers of samples indicated on X axis. Gray circles indicate minimum and maximum values. C t values higher than 40 (undetectable host DNA) are represented by 41. Whole genome amplification (WGA) was executed by REPLI-g Mini Kit.
    Gel Pcr Dna Fragments Extraction Kit, supplied by Geneaid Biotech Ltd, used in various techniques. Bioz Stars score: 89/100, based on 325 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pcr  (TaKaRa)
    99
    TaKaRa pcr
    Host and viral <t>DNA</t> content of differently treated ASFV samples. Quantitative dual <t>PCR</t> was executed by Virotype ASFV PCR Kit. C t values of individual and averaged samples represented by colored circles and grey box respectively. Averages were calculated from numbers of samples indicated on X axis. Gray circles indicate minimum and maximum values. C t values higher than 40 (undetectable host DNA) are represented by 41. Whole genome amplification (WGA) was executed by REPLI-g Mini Kit.
    Pcr, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 26461 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher dnazap pcr dna degradation solutions
    Simultaneous overexpression of LPMO and CBH1 in Pf Mig1 88 . (A) Schematic diagram showing the construction cassette for dual overexpression of LPMO and CBH1 genes from P. funiculosum NCIM1228. (B) Transformants of pOAO5 after AMTM transformation in Pf Mig1 88 . Transformants were selected on 100 µg/ml hygromycin. (C) Southern blot of transformants confirmed by <t>PCR.</t> M is HindIII lambda <t>DNA</t> size marker used; Lane C indicates genomic DNA of non-transformed parental strain; Lanes 1-5 represent genomic DNA from transformants with LPMO/CBH1 cassette integration; (D) The enzymatic profile of the overexpressed enzymes in the fermentation broth of Pf Mig1 88 and six transformants.
    Dnazap Pcr Dna Degradation Solutions, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega pcr dna
    The wdr-23 promoter is induced by stress via SKN-1. (A) <t>DNA</t> microarray quantification of wdr-23 mRNA levels following a 1-h exposure to 38 μM juglone. (B) Real-time <t>RT-PCR</t> quantification of wdr-23 mRNA levels in control(RNAi) and skn-1 ( RNAi ) worms
    Pcr Dna, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 304 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    (A) Assay to detect CRISPR/Cas9-mediated cleavage in vitro . A typical region of the Muc14a gene containing at least 2 binding sites for each of the gRNAs: Muc14a _3, Muc14a_4 , Muc14a_5 and Muc14a_6 (top). The PCR amplified DNA fragment was used as a digestion target for Cas9/gRNA cleavage reactions in vitro (bottom). Reactions were run on a gel to detect cleavage. A control without gRNA was included. (B) Analysis of combinations of gRNAs and Cas9 sources for X-shredding. Average male frequencies in the F1 progeny are shown for each parental genotype with a single copy of βtub85Dtub85D-cas9 transgene (1X), two copies of βtub85Dtub85D-cas9 transgene (2X) or one copy of nos-cas9 (grey bars). All lines were crossed to wild type w individuals. The reciprocal cross (female ctrl) or heterozygote βtub85Dtub85D-cas9/ + or nos-cas9/ + without gRNA (no gRNA) were used as control. The black arrow indicates gRNAs in the multiplex array and the red dotted line indicates an unbiased sex-ratio. Crosses were set as pools of males and females or as multiple male single crosses in which case error bars indicate the mean ± SD for a minimum of ten independent single crosses. For all crosses n indicates the total number of individuals (males + females) in the F1 progeny counted. (C) Developmental survival analysis of the F1 progeny of Muc14a_6/βtub85Dtub85D-cas9 males crossed to w females compared to w and βtub85Dtub85D-cas9/ + control males crossed to w females. n indicates the number of individuals recorded at every developmental stage (males + females) in the F1 progeny. Bars indicate means ± SD for at least ten independent single crosses. Statistical significance was calculated with a t test assuming unequal variance. ** p

    Journal: bioRxiv

    Article Title: A fly model establishes distinct mechanisms for synthetic CRISPR/Cas9 sex distorters

    doi: 10.1101/834630

    Figure Lengend Snippet: (A) Assay to detect CRISPR/Cas9-mediated cleavage in vitro . A typical region of the Muc14a gene containing at least 2 binding sites for each of the gRNAs: Muc14a _3, Muc14a_4 , Muc14a_5 and Muc14a_6 (top). The PCR amplified DNA fragment was used as a digestion target for Cas9/gRNA cleavage reactions in vitro (bottom). Reactions were run on a gel to detect cleavage. A control without gRNA was included. (B) Analysis of combinations of gRNAs and Cas9 sources for X-shredding. Average male frequencies in the F1 progeny are shown for each parental genotype with a single copy of βtub85Dtub85D-cas9 transgene (1X), two copies of βtub85Dtub85D-cas9 transgene (2X) or one copy of nos-cas9 (grey bars). All lines were crossed to wild type w individuals. The reciprocal cross (female ctrl) or heterozygote βtub85Dtub85D-cas9/ + or nos-cas9/ + without gRNA (no gRNA) were used as control. The black arrow indicates gRNAs in the multiplex array and the red dotted line indicates an unbiased sex-ratio. Crosses were set as pools of males and females or as multiple male single crosses in which case error bars indicate the mean ± SD for a minimum of ten independent single crosses. For all crosses n indicates the total number of individuals (males + females) in the F1 progeny counted. (C) Developmental survival analysis of the F1 progeny of Muc14a_6/βtub85Dtub85D-cas9 males crossed to w females compared to w and βtub85Dtub85D-cas9/ + control males crossed to w females. n indicates the number of individuals recorded at every developmental stage (males + females) in the F1 progeny. Bars indicate means ± SD for at least ten independent single crosses. Statistical significance was calculated with a t test assuming unequal variance. ** p

    Article Snippet: The amplicons were purified with NEB Monarch PCR & DNA Cleanup Kit and quantified with Nanodrop.

    Techniques: CRISPR, In Vitro, Binding Assay, Polymerase Chain Reaction, Amplification, Multiplex Assay

    Storage condition of purified ChIP DNA is important. Purified ChIP DNA was adjusted to a concentration of 1 ng/μL ( a ) or 0.1 ng/μL ( b ), aliquoted into 4 different types of microcentrifuge tubes in 15 μL volume, and stored at −20 °C. DNA was quantified using Qubit dsDNA High Sensitivity assay at the indicated time points and expressed as a percentage of the amount measured at day 0. Three independent DNA samples were used in the experiment and DNA concentration from five tubes were measured at each time point. MaxyClear, Axygen® 1.7 mL MaxyClear Snaplock Microcentrifuge Tube; LoBind, Eppendorf DNA LoBind Snap Cap PCR Tube; Siliconized, Fisherbrand™ Siliconized Low-Retention Microcentrifuge Tube; Premium, Fisherbrand™ Premium Microcentrifuge Tube

    Journal: BMC Genomics

    Article Title: Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application

    doi: 10.1186/s12864-017-4371-5

    Figure Lengend Snippet: Storage condition of purified ChIP DNA is important. Purified ChIP DNA was adjusted to a concentration of 1 ng/μL ( a ) or 0.1 ng/μL ( b ), aliquoted into 4 different types of microcentrifuge tubes in 15 μL volume, and stored at −20 °C. DNA was quantified using Qubit dsDNA High Sensitivity assay at the indicated time points and expressed as a percentage of the amount measured at day 0. Three independent DNA samples were used in the experiment and DNA concentration from five tubes were measured at each time point. MaxyClear, Axygen® 1.7 mL MaxyClear Snaplock Microcentrifuge Tube; LoBind, Eppendorf DNA LoBind Snap Cap PCR Tube; Siliconized, Fisherbrand™ Siliconized Low-Retention Microcentrifuge Tube; Premium, Fisherbrand™ Premium Microcentrifuge Tube

    Article Snippet: Similarly, DNAs were purified from de-crosslinked chromatin estimated to include 1 ng, 5 ng, 10 ng, and 50 ng of DNA after treatment of RNase A and proteinase K. The following reagents were used in the experiment: ChIP DNA Clean & Concentrator™ (Cat. # D5205) from Zymo Research (Zy) (Irvine, CA); Wizard® SV Gel and PCR Clean-Up System (Cat. # A9281) from Promega (Pr) (Fitchburg, WI); GeneJET PCR Purification Kit (Cat. # K0701) from Thermo Fisher Scientific (Th) (Waltham, MA); PureLink® PCR Purification Kit (Cat. # K310001) from Invitrogen (In) (Carlsbad, CA); Monarch® PCR & DNA Cleanup Kit (Cat. # T1030S) from New England Biolabs (Ne) (Ipswich, MA); Chromatin IP DNA Purification Kit (Cat. # 58002) from Active Motif (Am) (Carlsbad, CA); QIAquick PCR Purification Kit (Cat. # 28106) from Qiagen (Qp) (Valencia, CA), MinElute PCR Purification Kit (Cat. # 28006) from Qiagen (Qm); Agencourt AMPure XP (Cat. # A63881) from Beckman (Ba) (Indianapolis, IN), RNAClean™ XP (Cat. # A63987) from Beckman (Br), and phenol/chloroform extraction (PC) (Additional file ).

    Techniques: Purification, Chromatin Immunoprecipitation, Concentration Assay, Sensitive Assay, Polymerase Chain Reaction

    DNA purification reagents vary in their ability to recover low amounts of DNA from de-crosslinked chromatin. a Recovered DNA amount by different DNA purification reagents from de-crosslinked chromatin. De-crosslinked chromatin estimated to include 1 ng range DNA in ChIP elution buffer was purified following the manufacturer’s instructions. The data were generated from triplicate DNA samples derived from three independent preparations. Zy, ChIP DNA Clean Concentrator™ (Zymo Research); Pr, Wizard® SV Gel and PCR Clean-Up System (Promega); Th, GeneJET PCR Purification Kit (Thermo Fisher Scientific); In, PureLink® PCR Purification Kit (Invitrogen); Ne, Monarch® PCR DNA Cleanup Kit (New England Biolabs); Am, Chromatin IP DNA Purification Kit (Active Motif); Qp, QIAquick PCR Purification Kit (Qiagen); Qm, MinElute PCR Purification Kit (Qiagen); Ba, Agencourt AMPure XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); Br, RNAClean™ XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); PC, phenol/chloroform extraction. b Interference of PCR amplification by purified eluent of purification reagents. 9 μL eluent was mixed with 1 μL 166 bp of Drosophila probe DNA (0.0001 ng), and the resulting mixture was used as the template in 20 μl of real-time PCR reaction. The Ct value for Drosophila probe DNA from TE buffer was set as 100%. The experiment was repeated 3 times using de-crosslinked chromatin estimated to include 1 ng of DNA. c Size profiles of DNA purified by different reagents. The DNAs purified from de-crosslinked chromatin estimated to include 50 ng range DNA was analyzed by AATI Fragment Analyzer. DNA size (bp) is shown

    Journal: BMC Genomics

    Article Title: Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application

    doi: 10.1186/s12864-017-4371-5

    Figure Lengend Snippet: DNA purification reagents vary in their ability to recover low amounts of DNA from de-crosslinked chromatin. a Recovered DNA amount by different DNA purification reagents from de-crosslinked chromatin. De-crosslinked chromatin estimated to include 1 ng range DNA in ChIP elution buffer was purified following the manufacturer’s instructions. The data were generated from triplicate DNA samples derived from three independent preparations. Zy, ChIP DNA Clean Concentrator™ (Zymo Research); Pr, Wizard® SV Gel and PCR Clean-Up System (Promega); Th, GeneJET PCR Purification Kit (Thermo Fisher Scientific); In, PureLink® PCR Purification Kit (Invitrogen); Ne, Monarch® PCR DNA Cleanup Kit (New England Biolabs); Am, Chromatin IP DNA Purification Kit (Active Motif); Qp, QIAquick PCR Purification Kit (Qiagen); Qm, MinElute PCR Purification Kit (Qiagen); Ba, Agencourt AMPure XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); Br, RNAClean™ XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); PC, phenol/chloroform extraction. b Interference of PCR amplification by purified eluent of purification reagents. 9 μL eluent was mixed with 1 μL 166 bp of Drosophila probe DNA (0.0001 ng), and the resulting mixture was used as the template in 20 μl of real-time PCR reaction. The Ct value for Drosophila probe DNA from TE buffer was set as 100%. The experiment was repeated 3 times using de-crosslinked chromatin estimated to include 1 ng of DNA. c Size profiles of DNA purified by different reagents. The DNAs purified from de-crosslinked chromatin estimated to include 50 ng range DNA was analyzed by AATI Fragment Analyzer. DNA size (bp) is shown

    Article Snippet: Similarly, DNAs were purified from de-crosslinked chromatin estimated to include 1 ng, 5 ng, 10 ng, and 50 ng of DNA after treatment of RNase A and proteinase K. The following reagents were used in the experiment: ChIP DNA Clean & Concentrator™ (Cat. # D5205) from Zymo Research (Zy) (Irvine, CA); Wizard® SV Gel and PCR Clean-Up System (Cat. # A9281) from Promega (Pr) (Fitchburg, WI); GeneJET PCR Purification Kit (Cat. # K0701) from Thermo Fisher Scientific (Th) (Waltham, MA); PureLink® PCR Purification Kit (Cat. # K310001) from Invitrogen (In) (Carlsbad, CA); Monarch® PCR & DNA Cleanup Kit (Cat. # T1030S) from New England Biolabs (Ne) (Ipswich, MA); Chromatin IP DNA Purification Kit (Cat. # 58002) from Active Motif (Am) (Carlsbad, CA); QIAquick PCR Purification Kit (Cat. # 28106) from Qiagen (Qp) (Valencia, CA), MinElute PCR Purification Kit (Cat. # 28006) from Qiagen (Qm); Agencourt AMPure XP (Cat. # A63881) from Beckman (Ba) (Indianapolis, IN), RNAClean™ XP (Cat. # A63987) from Beckman (Br), and phenol/chloroform extraction (PC) (Additional file ).

    Techniques: DNA Purification, Chromatin Immunoprecipitation, Purification, Generated, Derivative Assay, Polymerase Chain Reaction, Amplification, Real-time Polymerase Chain Reaction

    Principles of Darwin Assembly. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the cut strand degraded by exonuclease III (1). Boundary and inner (mutagenic) oligonucleotides are annealed to the ssDNA plasmid (2). Key features of the oligonucleotides are highlighted: 5′-boundary oligonucleotide is 5′-biotinylated; non-complementary overhangs are shown in blue with Type IIs endonuclease recognition sites shown in white; mutations are shown as red X in the inner oligonucleotides; 3′-boundary oligonucleotide is protected at its 3′-end. After annealing, primers are extended and ligated in an isothermal assembly reaction (3). The assembled strand can be isolated by paramagnetic streptavidin-coated beads (4) and purified by alkali washing prior to PCR using outnested priming sites (5) and cloning (6) using the type IIS restriction sites (white dots). The purification step (4) is not always necessary but we found it improved PCR performance, especially for long assembly reactions ( > 1 kb).

    Journal: Nucleic Acids Research

    Article Title: Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis

    doi: 10.1093/nar/gky067

    Figure Lengend Snippet: Principles of Darwin Assembly. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the cut strand degraded by exonuclease III (1). Boundary and inner (mutagenic) oligonucleotides are annealed to the ssDNA plasmid (2). Key features of the oligonucleotides are highlighted: 5′-boundary oligonucleotide is 5′-biotinylated; non-complementary overhangs are shown in blue with Type IIs endonuclease recognition sites shown in white; mutations are shown as red X in the inner oligonucleotides; 3′-boundary oligonucleotide is protected at its 3′-end. After annealing, primers are extended and ligated in an isothermal assembly reaction (3). The assembled strand can be isolated by paramagnetic streptavidin-coated beads (4) and purified by alkali washing prior to PCR using outnested priming sites (5) and cloning (6) using the type IIS restriction sites (white dots). The purification step (4) is not always necessary but we found it improved PCR performance, especially for long assembly reactions ( > 1 kb).

    Article Snippet: PCR products were purified using GeneJET PCR Purification Kits (Thermo Fisher Scientific, Waltham MA, USA), Nucleospin Gel and PCR Clean-up (Machery-Nagel GmbH, Düren, Germany) or Monarch PCR and DNA Cleanup kits (NEB).

    Techniques: Plasmid Preparation, Isolation, Purification, Polymerase Chain Reaction, Clone Assay

    Darwin Assembly using a θ oligonucleotide. Here, a single θ oligonucleotide is used in place of the two boundary oligonucleotides allowing enzymatic cleanup after the assembly reaction. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the nicked strand degraded by exonuclease III (1). Inner oligonucleotides and a single θ oligonucleotide are annealed to the ssDNA plasmid (2). The θ oligonucleotide encodes both assembly priming and termination sequences linked by a flexible linker such that successful assembly of the mutated strand results in a closed circle (3). The template plasmid can now be linearized (e.g. at the yellow dot, by adding a targeting oligonucleotide and appropriate restriction endonuclease) and both exonuclease I and exonuclease III added to degrade any non-circular DNA (4). The mutated gene can now be amplified from the closed circle by PCR (5) and cloned into a fresh vector (6) using the type IIS restriction sites (white dots).

    Journal: Nucleic Acids Research

    Article Title: Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis

    doi: 10.1093/nar/gky067

    Figure Lengend Snippet: Darwin Assembly using a θ oligonucleotide. Here, a single θ oligonucleotide is used in place of the two boundary oligonucleotides allowing enzymatic cleanup after the assembly reaction. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the nicked strand degraded by exonuclease III (1). Inner oligonucleotides and a single θ oligonucleotide are annealed to the ssDNA plasmid (2). The θ oligonucleotide encodes both assembly priming and termination sequences linked by a flexible linker such that successful assembly of the mutated strand results in a closed circle (3). The template plasmid can now be linearized (e.g. at the yellow dot, by adding a targeting oligonucleotide and appropriate restriction endonuclease) and both exonuclease I and exonuclease III added to degrade any non-circular DNA (4). The mutated gene can now be amplified from the closed circle by PCR (5) and cloned into a fresh vector (6) using the type IIS restriction sites (white dots).

    Article Snippet: PCR products were purified using GeneJET PCR Purification Kits (Thermo Fisher Scientific, Waltham MA, USA), Nucleospin Gel and PCR Clean-up (Machery-Nagel GmbH, Düren, Germany) or Monarch PCR and DNA Cleanup kits (NEB).

    Techniques: Plasmid Preparation, Amplification, Polymerase Chain Reaction, Clone Assay

    Darwin assembled TgoT DNA polymerase library. ( A ) Five separate sequencing reactions (range and reads shown in blue) were required to sample the diversity introduced across the eight target residues (shown in red along the TgoT gene). Mutations included focused degeneracies (e.g. YWC used against Y384) or ‘small intelligent’ (S-int) diversity (NDT, VMA, ATG and TGG oligonucleotides mixed in a 12:6:1:1 ratio). Resulting incorporation is shown in box plots with outliers explicitly labelled. Wild-type contamination was determined from positions where diversity excluded those sequences (N.A.: not applicable). As with the T7 RNA polymerase library, incorporation trends and biases were analysed to identify any biases in assembly. Ranked incorporation frequencies are shown for the residues targeted with ‘small intelligent’ diversity, and the top three highest (greens) and lowest (oranges) ranked codons (based on a straight sum of ranks) are highlighted ( B ). Outnest PCR of the TgoT DNA polymerase library (expected product of 2501 bp) showing that the final PCR can be carried out with either A- (MyTaq) or B-family (Q5) polymerases ( C ). MW: 1 kb ladder (NEB). NT: no template PCR control.

    Journal: Nucleic Acids Research

    Article Title: Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis

    doi: 10.1093/nar/gky067

    Figure Lengend Snippet: Darwin assembled TgoT DNA polymerase library. ( A ) Five separate sequencing reactions (range and reads shown in blue) were required to sample the diversity introduced across the eight target residues (shown in red along the TgoT gene). Mutations included focused degeneracies (e.g. YWC used against Y384) or ‘small intelligent’ (S-int) diversity (NDT, VMA, ATG and TGG oligonucleotides mixed in a 12:6:1:1 ratio). Resulting incorporation is shown in box plots with outliers explicitly labelled. Wild-type contamination was determined from positions where diversity excluded those sequences (N.A.: not applicable). As with the T7 RNA polymerase library, incorporation trends and biases were analysed to identify any biases in assembly. Ranked incorporation frequencies are shown for the residues targeted with ‘small intelligent’ diversity, and the top three highest (greens) and lowest (oranges) ranked codons (based on a straight sum of ranks) are highlighted ( B ). Outnest PCR of the TgoT DNA polymerase library (expected product of 2501 bp) showing that the final PCR can be carried out with either A- (MyTaq) or B-family (Q5) polymerases ( C ). MW: 1 kb ladder (NEB). NT: no template PCR control.

    Article Snippet: PCR products were purified using GeneJET PCR Purification Kits (Thermo Fisher Scientific, Waltham MA, USA), Nucleospin Gel and PCR Clean-up (Machery-Nagel GmbH, Düren, Germany) or Monarch PCR and DNA Cleanup kits (NEB).

    Techniques: Sequencing, Polymerase Chain Reaction

    Enzymatic Methyl-seq mechanism of action and workflow (A) Principle pathways that are important for enzymatic identification of 5mC and 5hmC using Enzymatic Methyl-seq. The actions of TET2 (blue) and T4-βGT (purple) on 5mC and its oxidation products, as well as the activity of APOBEC3A (green) on cytosine, 5gmC and 5caC are shown. The red cross represents no APOBEC3A activity. T4-βGT glucosylates 5hmC (pre-existing 5hmC and that formed by the action of TET2). TET2 converts 5mC through the intermediates 5hmC and 5fC into 5caC. APOBEC3A has limited activity on 5fC and undetectable activity on 5gmC and 5caC ( Figure 3C ). Uracil is replaced by thymine during PCR and is read as thymine during Illumina sequencing. (B) DNA is sheared to approximately 300 bp, end repaired and 3’ A-tailed. EM-seq adaptors are then ligated to the DNA. The DNA is treated with TET2 and T4-βGT before moving to the deamination reaction. The library is PCR amplified using EM-seq adaptor primers and can be sequenced on any Illumina sequencer.

    Journal: bioRxiv

    Article Title: EM-seq: Detection of DNA Methylation at Single Base Resolution from Picograms of DNA

    doi: 10.1101/2019.12.20.884692

    Figure Lengend Snippet: Enzymatic Methyl-seq mechanism of action and workflow (A) Principle pathways that are important for enzymatic identification of 5mC and 5hmC using Enzymatic Methyl-seq. The actions of TET2 (blue) and T4-βGT (purple) on 5mC and its oxidation products, as well as the activity of APOBEC3A (green) on cytosine, 5gmC and 5caC are shown. The red cross represents no APOBEC3A activity. T4-βGT glucosylates 5hmC (pre-existing 5hmC and that formed by the action of TET2). TET2 converts 5mC through the intermediates 5hmC and 5fC into 5caC. APOBEC3A has limited activity on 5fC and undetectable activity on 5gmC and 5caC ( Figure 3C ). Uracil is replaced by thymine during PCR and is read as thymine during Illumina sequencing. (B) DNA is sheared to approximately 300 bp, end repaired and 3’ A-tailed. EM-seq adaptors are then ligated to the DNA. The DNA is treated with TET2 and T4-βGT before moving to the deamination reaction. The library is PCR amplified using EM-seq adaptor primers and can be sequenced on any Illumina sequencer.

    Article Snippet: The sample was sheared using settings of 5% duty cycle, intensity 3 and 200 cycles/burst for 40 s. Sheared DNA was purified using either the Monarch PCR and DNA Cleanup kit (NEB, Ipswich, MA) or QIAquick Nucleotide Removal Kit (QIAGEN).

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

    EM-seq Illumina libraries are superior to bisulfite libraries 10 ng, 50 ng or 200 ng of NA12878 DNA were spiked with control DNA (2 ng unmethylated lambda DNA and 0.1 ng CpG methylated pUC19) and Illumina libraries were made using either EM-seq or the Zymo Gold Bisulfite Kit prepped for Illumina sequencing using NEBNext Ultra II DNA library kit reagents. Libraries were sequenced on an Illumina NovaSeq 6000 and 324 M read pairs per library were used for analysis. (A) EM-seq uses less PCR cycles but results in more PCR product than whole genome bisulfite libraries (WGBS) for all NA12878 input amounts. (B) Table of sequencing and alignment metrics for EM-seq and WGBS libraries using 324 million Illumina read pairs. Metrics were calculated using bwa-meth, Samtools, and Picard. Theoretical coverage is calculated using the number of bases sequenced/total bases in the GRCh38 reference. Percent mapped refers to reads aligned to the reference genome (grch38+controls), Percent Dups refers to reads marked as duplicate by Picard MarkDuplicates, Percent Usable refers to the set of Proper-pair, MapQ 10+, Primary, non-Duplicate reads used in methylation calling (samtools view -F 0xF00 -q 10). Effective Coverage is the Percent Usable * Theoretical coverage. (C) GC-bias plot for EM-seq and WGBS libraries. EM-seq libraries display an even GC distribution while WGBS libraries have an AT-rich and GC-poor profile.

    Journal: bioRxiv

    Article Title: EM-seq: Detection of DNA Methylation at Single Base Resolution from Picograms of DNA

    doi: 10.1101/2019.12.20.884692

    Figure Lengend Snippet: EM-seq Illumina libraries are superior to bisulfite libraries 10 ng, 50 ng or 200 ng of NA12878 DNA were spiked with control DNA (2 ng unmethylated lambda DNA and 0.1 ng CpG methylated pUC19) and Illumina libraries were made using either EM-seq or the Zymo Gold Bisulfite Kit prepped for Illumina sequencing using NEBNext Ultra II DNA library kit reagents. Libraries were sequenced on an Illumina NovaSeq 6000 and 324 M read pairs per library were used for analysis. (A) EM-seq uses less PCR cycles but results in more PCR product than whole genome bisulfite libraries (WGBS) for all NA12878 input amounts. (B) Table of sequencing and alignment metrics for EM-seq and WGBS libraries using 324 million Illumina read pairs. Metrics were calculated using bwa-meth, Samtools, and Picard. Theoretical coverage is calculated using the number of bases sequenced/total bases in the GRCh38 reference. Percent mapped refers to reads aligned to the reference genome (grch38+controls), Percent Dups refers to reads marked as duplicate by Picard MarkDuplicates, Percent Usable refers to the set of Proper-pair, MapQ 10+, Primary, non-Duplicate reads used in methylation calling (samtools view -F 0xF00 -q 10). Effective Coverage is the Percent Usable * Theoretical coverage. (C) GC-bias plot for EM-seq and WGBS libraries. EM-seq libraries display an even GC distribution while WGBS libraries have an AT-rich and GC-poor profile.

    Article Snippet: The sample was sheared using settings of 5% duty cycle, intensity 3 and 200 cycles/burst for 40 s. Sheared DNA was purified using either the Monarch PCR and DNA Cleanup kit (NEB, Ipswich, MA) or QIAquick Nucleotide Removal Kit (QIAGEN).

    Techniques: Lambda DNA Preparation, Methylation, Sequencing, Polymerase Chain Reaction

    LDR in presence of multiple DNA templates A) LDR on a mix of PCR products B) LDR on a PCR product obtained from amplification of a mix of genomic DNAs. For deposition scheme, refer to fig. 3 . (Laser power and PMT gain were set to 80%).

    Journal: BMC Microbiology

    Article Title: Bacterial discrimination by means of a universal array approach mediated by LDR (ligase detection reaction)

    doi: 10.1186/1471-2180-2-27

    Figure Lengend Snippet: LDR in presence of multiple DNA templates A) LDR on a mix of PCR products B) LDR on a PCR product obtained from amplification of a mix of genomic DNAs. For deposition scheme, refer to fig. 3 . (Laser power and PMT gain were set to 80%).

    Article Snippet: After thermal cycling was complete, 1 μl of proteinase K (1 mg/ml) was added, and the reaction heated at 70°C for 10 min and then quenched at 94°C for 15 min. After this, PCR products were purified by GFX PCR DNA purification kit (Amersham Pharmacia Biotech Inc, Piscataway-NJ), eluted in 50 μl of autoclaved water and quantified by a spectrophotometer.

    Techniques: Polymerase Chain Reaction, Amplification

    Sensitivity of LDR The reaction was performed using 100 fmol (panel A), 10 fmol (panel B), 1 fmol (panel C) of purified PCR product from P. putida DNA. All images were acquired setting both PMT gain and laser power to 85%.

    Journal: BMC Microbiology

    Article Title: Bacterial discrimination by means of a universal array approach mediated by LDR (ligase detection reaction)

    doi: 10.1186/1471-2180-2-27

    Figure Lengend Snippet: Sensitivity of LDR The reaction was performed using 100 fmol (panel A), 10 fmol (panel B), 1 fmol (panel C) of purified PCR product from P. putida DNA. All images were acquired setting both PMT gain and laser power to 85%.

    Article Snippet: After thermal cycling was complete, 1 μl of proteinase K (1 mg/ml) was added, and the reaction heated at 70°C for 10 min and then quenched at 94°C for 15 min. After this, PCR products were purified by GFX PCR DNA purification kit (Amersham Pharmacia Biotech Inc, Piscataway-NJ), eluted in 50 μl of autoclaved water and quantified by a spectrophotometer.

    Techniques: Purification, Polymerase Chain Reaction

    Schematic representation of LDR applied to microbial diversity. A) Each microbial group of interest is identified by a Common Probe and a Discriminating Oligo. The common probe is phosphorylated on its 5' end and contains a unique cZip Code affixed to its 3' end. The discriminating oligo carries a fluorescent label (Cy3) on its 5' end, and a discriminating base at its 3' terminal position. The two probes hybridize adjacently to each other on the template DNA (PCR-amplified rDNA) and the nick between the two oligos is sealed by the ligase only if there is perfect complementarity at the junction. The reaction can be thermally cycled B) The presence of a microbial group is determined by hybridizing the content of a LDR to an addressable DNA Universal Array, where unique Zip Code sequences have been spotted.

    Journal: BMC Microbiology

    Article Title: Bacterial discrimination by means of a universal array approach mediated by LDR (ligase detection reaction)

    doi: 10.1186/1471-2180-2-27

    Figure Lengend Snippet: Schematic representation of LDR applied to microbial diversity. A) Each microbial group of interest is identified by a Common Probe and a Discriminating Oligo. The common probe is phosphorylated on its 5' end and contains a unique cZip Code affixed to its 3' end. The discriminating oligo carries a fluorescent label (Cy3) on its 5' end, and a discriminating base at its 3' terminal position. The two probes hybridize adjacently to each other on the template DNA (PCR-amplified rDNA) and the nick between the two oligos is sealed by the ligase only if there is perfect complementarity at the junction. The reaction can be thermally cycled B) The presence of a microbial group is determined by hybridizing the content of a LDR to an addressable DNA Universal Array, where unique Zip Code sequences have been spotted.

    Article Snippet: After thermal cycling was complete, 1 μl of proteinase K (1 mg/ml) was added, and the reaction heated at 70°C for 10 min and then quenched at 94°C for 15 min. After this, PCR products were purified by GFX PCR DNA purification kit (Amersham Pharmacia Biotech Inc, Piscataway-NJ), eluted in 50 μl of autoclaved water and quantified by a spectrophotometer.

    Techniques: Polymerase Chain Reaction, Amplification

    UP-PCR results of some patients. We have detected all the patients and finally we chose some positive bacteria and carried out the electrophoresis, so every line had PCR products. The deeper the stripe, the more the bacteria. Lines 1–9 represented PCR product of the 16S rRNA gene of Staphylococcus aureus , Staphylococcus epidermidis , Pseudomonas aeruginosa , Escherichia coli , Streptococcus viridans , Staphylococcus saprophyticus, Corynebacterium parvum , Klebsiella pneumoniae , and Enterobacter cloacae ; M for DL2000 DNA Marker; ten for negative control.

    Journal: BioMed Research International

    Article Title: Genetic Identification and Risk Factor Analysis of Asymptomatic Bacterial Colonization on Cardiovascular Implantable Electronic Devices

    doi: 10.1155/2014/725163

    Figure Lengend Snippet: UP-PCR results of some patients. We have detected all the patients and finally we chose some positive bacteria and carried out the electrophoresis, so every line had PCR products. The deeper the stripe, the more the bacteria. Lines 1–9 represented PCR product of the 16S rRNA gene of Staphylococcus aureus , Staphylococcus epidermidis , Pseudomonas aeruginosa , Escherichia coli , Streptococcus viridans , Staphylococcus saprophyticus, Corynebacterium parvum , Klebsiella pneumoniae , and Enterobacter cloacae ; M for DL2000 DNA Marker; ten for negative control.

    Article Snippet: The PCR product was purified using Wizard PCR Preps DNA Purification System (Promega) and then ligated into the pGEM-T Easy Vector (Promega).

    Techniques: Polymerase Chain Reaction, Electrophoresis, Marker, Negative Control

    (A) Locations of primers used in PCR, as probes in dot blots, and for construction of mutY-lacZ transcriptional fusion. (B) Primer pairs (specified below gels) were used to amplify the mutY gene and surrounding region in several chemostat mutator mutants. Agarose concentrations in the gels were 1 and 0.8%, respectively. The gel lane numbered 1 corresponded to strain BW3526 carrying the mutY ::Tn 10 insertion. Lanes 2, WT strain BW2952; lanes 3, isolate 21Qa1; lanes 4, isolate L3Oa1; lanes 5, isolate 10U6; lanes 6, isolate C7F1; lanes 7, DNA markers with sizes of 23,130, 9,416, 6,557, 4,361, 2,322, and 2,027 bp. (C) Results of slot blotting of DNA extracted from mutator strains. Hybridization was at 50°C with DIG-dUTP-tailed probes mutY F2, mutY F3, and mutY F9 as well as the positive control probe for an unlinked housekeeping gene, eno .

    Journal: Journal of Bacteriology

    Article Title: Regulation of mutY and Nature of Mutator Mutations in Escherichia coli Populations under Nutrient Limitation

    doi: 10.1128/JB.184.3.739-745.2002

    Figure Lengend Snippet: (A) Locations of primers used in PCR, as probes in dot blots, and for construction of mutY-lacZ transcriptional fusion. (B) Primer pairs (specified below gels) were used to amplify the mutY gene and surrounding region in several chemostat mutator mutants. Agarose concentrations in the gels were 1 and 0.8%, respectively. The gel lane numbered 1 corresponded to strain BW3526 carrying the mutY ::Tn 10 insertion. Lanes 2, WT strain BW2952; lanes 3, isolate 21Qa1; lanes 4, isolate L3Oa1; lanes 5, isolate 10U6; lanes 6, isolate C7F1; lanes 7, DNA markers with sizes of 23,130, 9,416, 6,557, 4,361, 2,322, and 2,027 bp. (C) Results of slot blotting of DNA extracted from mutator strains. Hybridization was at 50°C with DIG-dUTP-tailed probes mutY F2, mutY F3, and mutY F9 as well as the positive control probe for an unlinked housekeeping gene, eno .

    Article Snippet: The 2,075-bp product was purified using Wizard PCR preps DNA purification kit (Promega Corp., Sydney, Australia) and electroporated into DY330 as per the method of Yu et al. ( ).

    Techniques: Polymerase Chain Reaction, Hybridization, Positive Control

    RFLP analysis of amplicons obtained with the Desulfuromonas dechlorinator-targeted primers. Amplicons were digested with the restriction endonuclease Eco RI, and fragments were separated on a 2% metaphor agarose gel. Lane 1, DNA marker V; lanes 2 to 9, digested amplicons obtained from Desulfuromonas sp. strain BB1, D. chloroethenica , the Red Cedar River (collected July 1995, May 1998, and November 1998), the Père Marquette River (locations PM 0 and PM 1, collected September 1998), and the Au Sable River, respectively; lane 10, undigested PCR product obtained from D. chloroethenica .

    Journal: Applied and Environmental Microbiology

    Article Title: 16S rRNA Gene-Based Detection of Tetrachloroethene-Dechlorinating Desulfuromonas and Dehalococcoides Species

    doi:

    Figure Lengend Snippet: RFLP analysis of amplicons obtained with the Desulfuromonas dechlorinator-targeted primers. Amplicons were digested with the restriction endonuclease Eco RI, and fragments were separated on a 2% metaphor agarose gel. Lane 1, DNA marker V; lanes 2 to 9, digested amplicons obtained from Desulfuromonas sp. strain BB1, D. chloroethenica , the Red Cedar River (collected July 1995, May 1998, and November 1998), the Père Marquette River (locations PM 0 and PM 1, collected September 1998), and the Au Sable River, respectively; lane 10, undigested PCR product obtained from D. chloroethenica .

    Article Snippet: 16S rDNA amplicons were purified (Wizard PCR Preps; Promega, Madison, Wis.) prior to sequencing by the fluorescent Dideoxy termination method at Michigan State University's sequencing facility.

    Techniques: Agarose Gel Electrophoresis, Marker, Polymerase Chain Reaction

    A comparison of the nifH constituents of the RT-PCR and PCR products derived from the gut microflora of the termite N. koshunensis . Electropherograms of the Hha I-digested nifH genes amplified from the genomic DNA by PCR (A) and from the mRNA by RT-PCR (B) are shown. The termites were fed filter paper for 5 days before nucleic acid extraction. The nifH genes were amplified with the 5′-fluorescently labeled primer IGK and the primer VCG. Numbers below the electropherograms show base lengths.

    Journal: Applied and Environmental Microbiology

    Article Title: Culture-Independent Characterization of a Gene Responsible for Nitrogen Fixation in the Symbiotic Microbial Community in the Gut of the Termite Neotermes koshunensis

    doi:

    Figure Lengend Snippet: A comparison of the nifH constituents of the RT-PCR and PCR products derived from the gut microflora of the termite N. koshunensis . Electropherograms of the Hha I-digested nifH genes amplified from the genomic DNA by PCR (A) and from the mRNA by RT-PCR (B) are shown. The termites were fed filter paper for 5 days before nucleic acid extraction. The nifH genes were amplified with the 5′-fluorescently labeled primer IGK and the primer VCG. Numbers below the electropherograms show base lengths.

    Article Snippet: The PCR products were purified on a low-melting-point agarose gel by means of the Wizard PCR prep DNA purification system (Promega).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Derivative Assay, Amplification, Labeling

    Prdm9 Dom2 is suppressed by competition between alleles. (A) PRDM9 Cst can suppress PRDM9 Dom2 -directed recombination at Ush2a . To detect either parental (top) or recombinant (bottom) molecules, nested PCR was performed on DNA from pooled sperm samples collected from F1 hybrids from the indicated crosses (two representative biological replicates are shown for each genotype). Recombinant molecules are only detected in progeny homozygous or heterozygous null for Prdm9 Dom2 . (B) Proportions of H3K4me3 hotspots in (B6xKI)F1 hybrids due to either PRDM9 Cst or PRDM9 Dom2 . (C) PRDM9 Dom2 hotspots in (B6xKI)F1 hybrids are the same hotspots found in Prdm9 Dom2/- heterozygous mice.

    Journal: PLoS Genetics

    Article Title: Multimer Formation Explains Allelic Suppression of PRDM9 Recombination Hotspots

    doi: 10.1371/journal.pgen.1005512

    Figure Lengend Snippet: Prdm9 Dom2 is suppressed by competition between alleles. (A) PRDM9 Cst can suppress PRDM9 Dom2 -directed recombination at Ush2a . To detect either parental (top) or recombinant (bottom) molecules, nested PCR was performed on DNA from pooled sperm samples collected from F1 hybrids from the indicated crosses (two representative biological replicates are shown for each genotype). Recombinant molecules are only detected in progeny homozygous or heterozygous null for Prdm9 Dom2 . (B) Proportions of H3K4me3 hotspots in (B6xKI)F1 hybrids due to either PRDM9 Cst or PRDM9 Dom2 . (C) PRDM9 Dom2 hotspots in (B6xKI)F1 hybrids are the same hotspots found in Prdm9 Dom2/- heterozygous mice.

    Article Snippet: High-throughput sequencing and data processing Pooled DNA samples from the Pbx1 recombination assay were prepared for sequencing using the TruSeq DNA PCR-Free Sample Preparation Kit (Illumina) in order to avoid PCR amplification, which could lead to template switching during amplification, in turn leading to false recombinant molecules.

    Techniques: Recombinant, Nested PCR, Mouse Assay

    Diagnostic PCR for selective editing of native glycolytic genes. Separation of PCR products resulting from outside–outside amplification to identify edited (nonwatermarked) and nonedited (watermarked) loci for PYK1 (A) and TPI1 (B) from transformants of IMX1717 (double SinLoG). (A) Lanes 1–15 show the PCR results of amplification of the PYK1 locus of randomly picked colonies. Successful editing of the locus results in a DNA fragment with a length of 670 bp. No editing of the locus results in a DNA fragment with a length of 2177 bp. Primers 11915 and 4667 were used. Lanes 1, 5, and 15 display bands of both sizes revealing selective editing. (B) Lane 1–15 show the PCR results of amplification of the TPI1 locus of randomly picked colonies. Successful editing of the locus results in a DNA fragment with a length of 378 bp. No editing of the locus results in a DNA fragment with a length of 1125 bp. Primers 3514 and 6406 were used. Lanes 9–11, 13, and 15 display bands of both sizes revealing selective editing. A negative control is indicated with “C-“ (IMX1338, SinLog). In the lanes indicated with “L”, GeneRuler DNA ladder mix was loaded. 1% (w/v) agarose in TAE.

    Journal: ACS Synthetic Biology

    Article Title: Design and Experimental Evaluation of a Minimal, Innocuous Watermarking Strategy to Distinguish Near-Identical DNA and RNA Sequences

    doi: 10.1021/acssynbio.0c00045

    Figure Lengend Snippet: Diagnostic PCR for selective editing of native glycolytic genes. Separation of PCR products resulting from outside–outside amplification to identify edited (nonwatermarked) and nonedited (watermarked) loci for PYK1 (A) and TPI1 (B) from transformants of IMX1717 (double SinLoG). (A) Lanes 1–15 show the PCR results of amplification of the PYK1 locus of randomly picked colonies. Successful editing of the locus results in a DNA fragment with a length of 670 bp. No editing of the locus results in a DNA fragment with a length of 2177 bp. Primers 11915 and 4667 were used. Lanes 1, 5, and 15 display bands of both sizes revealing selective editing. (B) Lane 1–15 show the PCR results of amplification of the TPI1 locus of randomly picked colonies. Successful editing of the locus results in a DNA fragment with a length of 378 bp. No editing of the locus results in a DNA fragment with a length of 1125 bp. Primers 3514 and 6406 were used. Lanes 9–11, 13, and 15 display bands of both sizes revealing selective editing. A negative control is indicated with “C-“ (IMX1338, SinLog). In the lanes indicated with “L”, GeneRuler DNA ladder mix was loaded. 1% (w/v) agarose in TAE.

    Article Snippet: DNA libraries were prepared using the TruSeq DNA PCR-Free Library Preparation Kit (Illumina) according to the manufacturer’s manual.

    Techniques: Diagnostic Assay, Polymerase Chain Reaction, Amplification, Negative Control

    Targeted Deletion of a Fragment in Intron 25 of the Cep290 Gene in the Mouse Retina Using a Dual AAV System (A and B) Micron IV fluorescence images for treated retina 1 (A) and retina 4 (B) on day 28 after subretinal injection. (C) PCR analysis showing targeted genomic deletion in the mouse retinas that received (1) AAV5-RK-EGFP (control) or AAV5-U11D11-RK-EGFP (treated) and (2) AAV5-SpCas9. The upper bands correspond to PCR products amplified from intron 25 of wild-type Cep290 gene, whereas the lower bands correspond to PCR products amplified from the Cep290 allele following U11D11-guided genomic deletion. (D) Percentages of wild-type and truncated DNA in the four treated retinas, as determined by next-generation sequencing.

    Journal: Molecular Therapy

    Article Title: CRISPR/Cas9-Mediated Genome Editing as a Therapeutic Approach for Leber Congenital Amaurosis 10

    doi: 10.1016/j.ymthe.2016.12.006

    Figure Lengend Snippet: Targeted Deletion of a Fragment in Intron 25 of the Cep290 Gene in the Mouse Retina Using a Dual AAV System (A and B) Micron IV fluorescence images for treated retina 1 (A) and retina 4 (B) on day 28 after subretinal injection. (C) PCR analysis showing targeted genomic deletion in the mouse retinas that received (1) AAV5-RK-EGFP (control) or AAV5-U11D11-RK-EGFP (treated) and (2) AAV5-SpCas9. The upper bands correspond to PCR products amplified from intron 25 of wild-type Cep290 gene, whereas the lower bands correspond to PCR products amplified from the Cep290 allele following U11D11-guided genomic deletion. (D) Percentages of wild-type and truncated DNA in the four treated retinas, as determined by next-generation sequencing.

    Article Snippet: The DNA samples were fragmented to an average target fragment size of 350 bp by ultrasonication and were used to construct a sequencing library using the Illumina TruSeq DNA PCR-free sample preparation kit.

    Techniques: Fluorescence, Injection, Polymerase Chain Reaction, Amplification, Next-Generation Sequencing

    Targeted Deletion of the IVS26 Mutation with Paired sgRNAs and SpCas9 (A) Schematic diagram depicting the strategy used to remove the IVS26 mutation of CEP290 (filled star). An upstream sgRNA directs the first Cas9 cleavage to a site located upstream of the IVS26 mutation, and a downstream sgRNA directs the second Cas9 cleavage to a site downstream of the mutation. The two cleavage ends are directly ligated through the NHEJ process, resulting in the excision of the intronic fragment flanking the IVS26 mutation. The truncated intron 26 is removed during mRNA processing by the RNA splicing machinery. The locations of the upstream (U1) and downstream (D1, D2, and D3) sgRNA guide sequences are indicated in the diagram. Note that D1 sgRNA targets the sense strand, whereas the other three sgRNAs target the antisense strand. (B) PCR analysis for targeted genomic deletion. 293FT cell lines were transfected with the indicated pairs of sgRNA and SpCas9. Primers were designed to bind outside of the region to be deleted. The upper bands represent PCR products amplified from intron 26 of wild-type CEP290 , whereas the lower bands represent PCR products amplified from the CEP290 allele following genomic deletion. M, 1-kb DNA ladder. (C) Percentages of wild-type, truncated, and inverted DNA in the mutant 293FT cells transfected with paired sgRNAs and SpCas9, as determined by next-generation sequencing.

    Journal: Molecular Therapy

    Article Title: CRISPR/Cas9-Mediated Genome Editing as a Therapeutic Approach for Leber Congenital Amaurosis 10

    doi: 10.1016/j.ymthe.2016.12.006

    Figure Lengend Snippet: Targeted Deletion of the IVS26 Mutation with Paired sgRNAs and SpCas9 (A) Schematic diagram depicting the strategy used to remove the IVS26 mutation of CEP290 (filled star). An upstream sgRNA directs the first Cas9 cleavage to a site located upstream of the IVS26 mutation, and a downstream sgRNA directs the second Cas9 cleavage to a site downstream of the mutation. The two cleavage ends are directly ligated through the NHEJ process, resulting in the excision of the intronic fragment flanking the IVS26 mutation. The truncated intron 26 is removed during mRNA processing by the RNA splicing machinery. The locations of the upstream (U1) and downstream (D1, D2, and D3) sgRNA guide sequences are indicated in the diagram. Note that D1 sgRNA targets the sense strand, whereas the other three sgRNAs target the antisense strand. (B) PCR analysis for targeted genomic deletion. 293FT cell lines were transfected with the indicated pairs of sgRNA and SpCas9. Primers were designed to bind outside of the region to be deleted. The upper bands represent PCR products amplified from intron 26 of wild-type CEP290 , whereas the lower bands represent PCR products amplified from the CEP290 allele following genomic deletion. M, 1-kb DNA ladder. (C) Percentages of wild-type, truncated, and inverted DNA in the mutant 293FT cells transfected with paired sgRNAs and SpCas9, as determined by next-generation sequencing.

    Article Snippet: The DNA samples were fragmented to an average target fragment size of 350 bp by ultrasonication and were used to construct a sequencing library using the Illumina TruSeq DNA PCR-free sample preparation kit.

    Techniques: Mutagenesis, Non-Homologous End Joining, Polymerase Chain Reaction, Transfection, Amplification, Next-Generation Sequencing

    Self-Limiting CRISPR/SpCas9 System (A) Schematic diagram of the pAAV-SpCas9 plasmid. The sgRNA recognition sequences were incorporated into insertion site 1 (between the minCMV promoter and SpCas9) and/or insertion site 2 (between SpCas9-NLS and SV40 pA). ITR, inverted terminal repeat. (B) Immunoblot analysis of lysates prepared from the mutant 293FT cells transfected with the pAAV-U1D3 plasmid and the self-limiting pAAV-SpCas9 plasmids, which contained the U1 sgRNA recognition sequence (U1T) and/or the D3 sgRNA recognition sequence (D3T). The membrane was probed for SpCas9 protein (top) and β-actin (bottom; as a loading control). The ratio of SpCas9/β-actin is shown at the bottom, with the ratio for the control pAAV-SpCas9 plasmid without the sgRNA recognition sequence set to 1.0. Dashes indicate that there is no sgRNA recognition sequence at this site. (C) PCR analysis for targeted genomic deletion in the mutant cells transfected with the pAAV-U1D3 plasmid and the self-limiting pAAV-SpCas9 plasmids. Three selected samples were subjected to NGS analysis, and percentages of truncated DNA were shown below the gel image. (D and E) The levels of wild-type (D) and mutant (E) mRNAs in the mutant cells transfected with the pAAV-U1D3 plasmid and the self-limiting pAAV-SpCas9 plasmids, as measured by qRT-PCR. The data are presented as the means ± SD (n = 3). Comparisons were performed using one-way ANOVA followed by the Tukey HSD post hoc test. *p

    Journal: Molecular Therapy

    Article Title: CRISPR/Cas9-Mediated Genome Editing as a Therapeutic Approach for Leber Congenital Amaurosis 10

    doi: 10.1016/j.ymthe.2016.12.006

    Figure Lengend Snippet: Self-Limiting CRISPR/SpCas9 System (A) Schematic diagram of the pAAV-SpCas9 plasmid. The sgRNA recognition sequences were incorporated into insertion site 1 (between the minCMV promoter and SpCas9) and/or insertion site 2 (between SpCas9-NLS and SV40 pA). ITR, inverted terminal repeat. (B) Immunoblot analysis of lysates prepared from the mutant 293FT cells transfected with the pAAV-U1D3 plasmid and the self-limiting pAAV-SpCas9 plasmids, which contained the U1 sgRNA recognition sequence (U1T) and/or the D3 sgRNA recognition sequence (D3T). The membrane was probed for SpCas9 protein (top) and β-actin (bottom; as a loading control). The ratio of SpCas9/β-actin is shown at the bottom, with the ratio for the control pAAV-SpCas9 plasmid without the sgRNA recognition sequence set to 1.0. Dashes indicate that there is no sgRNA recognition sequence at this site. (C) PCR analysis for targeted genomic deletion in the mutant cells transfected with the pAAV-U1D3 plasmid and the self-limiting pAAV-SpCas9 plasmids. Three selected samples were subjected to NGS analysis, and percentages of truncated DNA were shown below the gel image. (D and E) The levels of wild-type (D) and mutant (E) mRNAs in the mutant cells transfected with the pAAV-U1D3 plasmid and the self-limiting pAAV-SpCas9 plasmids, as measured by qRT-PCR. The data are presented as the means ± SD (n = 3). Comparisons were performed using one-way ANOVA followed by the Tukey HSD post hoc test. *p

    Article Snippet: The DNA samples were fragmented to an average target fragment size of 350 bp by ultrasonication and were used to construct a sequencing library using the Illumina TruSeq DNA PCR-free sample preparation kit.

    Techniques: CRISPR, Plasmid Preparation, Mutagenesis, Transfection, Sequencing, Polymerase Chain Reaction, Next-Generation Sequencing, Quantitative RT-PCR

    Loss of Crebbp in committed lymphoid cells attenuates disease generation and CREBBP mutations occur in HSPC from a lymphoma patient. a. Analysis of the Lin- and IL7Ra+ compartments in Cd19- Crebbp-/- mice. Box plots show the median with interquartile range and whiskers represent the minimum and maximum values, n = 6 mice per genotype were quantified. Non-significance confirmed using 2-sided t-test. b. Comparison of H2AXγ foci in the IL7Ra+ progenitor populations from Cd19- Crebbp-/- mice. Data represent the mean ± SEM from n = 3 independent experiments, non-significance confirmed using 2-sided t-test. See Supplementary Table 26 for scatter plots of > 10 foci. c. KM analysis showing that no significant alterations in survival were seen for Cd19- Crebbp -/- mice (n = 27 animals) vs WT mice (n = 25 animals). P value calculated using Log-rank (Mantel-Cox) test. d. in contrast to that seen in Mx-Crebbp-/- mice. e. B-cell disease was very rare in this cohort (Cd19- Crebbp -/- LPD 4 cases, WT LPD 1 case). f. Flow sorting strategy to functionally isolate specific HSPC populations from patients with CREBBP mutated lymphomas. g. Allele specific PCR demonstrates the presence of the same mutation (183 bp band) identified from the tumour (Mut lane) within the CD34+/CD38+ HSPC population. CFU-colony forming assay, Bl- blank, WT- wild type. h . Model of mechanisms and stage specific nature of Crebbp tumour suppression in lymphoid malignancies. Following early loss of Crebbp activity in the HSPC compartment, abnormal epigenetic regulation, post-translational modifications of substrate proteins and altered transcription lead to expansion of lymphoid progenitors, blocked differentiation, increased proliferation and clonogenicity within this expanded progenitor pool. In association with an alteration of the DNA damage response mediated through suboptimal Crebbp acetylation of p53, DNA double strand breaks accumulate and accompanied by a relative decrease in apoptosis, lead to the retention of mutations within B-cell progenitors. We illustrate the epigenetic priming of specific loci at earlier time points, where H3K27ac ChIP-Seq and RNA-Seq at the critical gene X locus read out in terms of downregulation of gene expression only at later timepoints during lymphoma evolution. We speculate that the molecular aberrations cumulatively acquired interact with this primed epigenetic state and lead to the restoration of self-renewal in this abnormal pre-malignant stem cell population and to the evolution of Lymphoma.

    Journal: Nature cell biology

    Article Title: Early loss of Crebbp confers malignant stem cell properties on lymphoid progenitors

    doi: 10.1038/ncb3597

    Figure Lengend Snippet: Loss of Crebbp in committed lymphoid cells attenuates disease generation and CREBBP mutations occur in HSPC from a lymphoma patient. a. Analysis of the Lin- and IL7Ra+ compartments in Cd19- Crebbp-/- mice. Box plots show the median with interquartile range and whiskers represent the minimum and maximum values, n = 6 mice per genotype were quantified. Non-significance confirmed using 2-sided t-test. b. Comparison of H2AXγ foci in the IL7Ra+ progenitor populations from Cd19- Crebbp-/- mice. Data represent the mean ± SEM from n = 3 independent experiments, non-significance confirmed using 2-sided t-test. See Supplementary Table 26 for scatter plots of > 10 foci. c. KM analysis showing that no significant alterations in survival were seen for Cd19- Crebbp -/- mice (n = 27 animals) vs WT mice (n = 25 animals). P value calculated using Log-rank (Mantel-Cox) test. d. in contrast to that seen in Mx-Crebbp-/- mice. e. B-cell disease was very rare in this cohort (Cd19- Crebbp -/- LPD 4 cases, WT LPD 1 case). f. Flow sorting strategy to functionally isolate specific HSPC populations from patients with CREBBP mutated lymphomas. g. Allele specific PCR demonstrates the presence of the same mutation (183 bp band) identified from the tumour (Mut lane) within the CD34+/CD38+ HSPC population. CFU-colony forming assay, Bl- blank, WT- wild type. h . Model of mechanisms and stage specific nature of Crebbp tumour suppression in lymphoid malignancies. Following early loss of Crebbp activity in the HSPC compartment, abnormal epigenetic regulation, post-translational modifications of substrate proteins and altered transcription lead to expansion of lymphoid progenitors, blocked differentiation, increased proliferation and clonogenicity within this expanded progenitor pool. In association with an alteration of the DNA damage response mediated through suboptimal Crebbp acetylation of p53, DNA double strand breaks accumulate and accompanied by a relative decrease in apoptosis, lead to the retention of mutations within B-cell progenitors. We illustrate the epigenetic priming of specific loci at earlier time points, where H3K27ac ChIP-Seq and RNA-Seq at the critical gene X locus read out in terms of downregulation of gene expression only at later timepoints during lymphoma evolution. We speculate that the molecular aberrations cumulatively acquired interact with this primed epigenetic state and lead to the restoration of self-renewal in this abnormal pre-malignant stem cell population and to the evolution of Lymphoma.

    Article Snippet: For each sample 1 ug DNA was used to prepare the sequencing library using TruSeq DNA PCR-Free Library Prep kit (Illumina) (starting from end-repairing onwards).

    Techniques: Mouse Assay, Flow Cytometry, Polymerase Chain Reaction, Mutagenesis, Activity Assay, Chromatin Immunoprecipitation, RNA Sequencing Assay, Expressing

    Levels of DNA demethylation in the mitochondrial genome and expression levels of the mtDNA encoded genes. ( A ) Detection of nDNA contamination after mtDNA purification. Copy number for nDNA (β-globin) relative to the copy number for mtDNA was plotted. Multiple t-tests were performed between the purified mtDNA samples (gray bars) and corresponding total DNA samples (black bars) isolated from GBM cells. Bars represent the mean ± SEM ( n = 3). ( B ) Agarose gel showing long-PCR products from purified mtDNA samples to demonstrate the presence of mtDNA methylation. Long PCR using two pairs of primers (primer set 2) spanning across the mitochondrial genome was performed on: (i) total DNA isolated from non-treated GBM cells (Total DNA); (ii) the positive control - MeDIP performed on the long PCR products previously generated with primer set 1 and treated with the M.SssI enzyme (Pos-longPCR-5mC); (iii) purified whole mtDNA having undergone MeDIP - (mtDNA-5mC); (iv) the negative control - MeDIP performed on the long PCR products previously generated with primer set 1 (Neg-longPCR-5mC); (v) the non-antibody control for MeDIP – MeDIP was performed on the purified mtDNA without using the 5mC antibody (mtDNA-NAC); and (vi) a non-template control (H 2 O) for PCR (NTC). Fragment sizes are indicated. ( C ) Normalized levels of DNA methylation within regions of the mitochondrial genome determined on purified mtDNA samples that had undergone MeDIP with sonication. The normalized levels of DNA methylation were determined by normalizing the MeDIP results (5mC/Input) against the positive and negative controls that were used to represent 100% and 0% of DNA methylation, respectively. Statistical significance was determined between the 5Aza and VitC treated cohorts and the GBM cohort and between ND5 and all the other genes by two-way ANOVA ( n = 3). ( D ) Significant differential expression of the mitochondrial and chromosomal genes encoding the ETC subunits identified in the treated and non-treated cohorts using the Fluidigm array. Bars represent the mean of the relative quantification levels normalized to the GBM cohort ( n = 6). *, **, ***, **** indicate P values of

    Journal: Nucleic Acids Research

    Article Title: Global DNA methylation synergistically regulates the nuclear and mitochondrial genomes in glioblastoma cells

    doi: 10.1093/nar/gky339

    Figure Lengend Snippet: Levels of DNA demethylation in the mitochondrial genome and expression levels of the mtDNA encoded genes. ( A ) Detection of nDNA contamination after mtDNA purification. Copy number for nDNA (β-globin) relative to the copy number for mtDNA was plotted. Multiple t-tests were performed between the purified mtDNA samples (gray bars) and corresponding total DNA samples (black bars) isolated from GBM cells. Bars represent the mean ± SEM ( n = 3). ( B ) Agarose gel showing long-PCR products from purified mtDNA samples to demonstrate the presence of mtDNA methylation. Long PCR using two pairs of primers (primer set 2) spanning across the mitochondrial genome was performed on: (i) total DNA isolated from non-treated GBM cells (Total DNA); (ii) the positive control - MeDIP performed on the long PCR products previously generated with primer set 1 and treated with the M.SssI enzyme (Pos-longPCR-5mC); (iii) purified whole mtDNA having undergone MeDIP - (mtDNA-5mC); (iv) the negative control - MeDIP performed on the long PCR products previously generated with primer set 1 (Neg-longPCR-5mC); (v) the non-antibody control for MeDIP – MeDIP was performed on the purified mtDNA without using the 5mC antibody (mtDNA-NAC); and (vi) a non-template control (H 2 O) for PCR (NTC). Fragment sizes are indicated. ( C ) Normalized levels of DNA methylation within regions of the mitochondrial genome determined on purified mtDNA samples that had undergone MeDIP with sonication. The normalized levels of DNA methylation were determined by normalizing the MeDIP results (5mC/Input) against the positive and negative controls that were used to represent 100% and 0% of DNA methylation, respectively. Statistical significance was determined between the 5Aza and VitC treated cohorts and the GBM cohort and between ND5 and all the other genes by two-way ANOVA ( n = 3). ( D ) Significant differential expression of the mitochondrial and chromosomal genes encoding the ETC subunits identified in the treated and non-treated cohorts using the Fluidigm array. Bars represent the mean of the relative quantification levels normalized to the GBM cohort ( n = 6). *, **, ***, **** indicate P values of

    Article Snippet: These fragments were then end repaired, A-tailed, ligated to Illumina adaptors (including the sample specific indexed) and purified without amplification to ensure conservation of methylated CpGs using the Illumina TruSeq DNA PCR-Free Kit, as described in the manufacturer's Low Sample (LS) protocol (Illumina Protocol part #15036187 Rev A Jan 2013).

    Techniques: Expressing, Purification, Isolation, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Methylation, Positive Control, Methylated DNA Immunoprecipitation, Generated, Negative Control, DNA Methylation Assay, Sonication

    DNA methylation status in the promoter region of ESRP2 in ovarian cancer cell lines and tissues. ( a ) ESRP2 gene expression following treatment with epigenetic drugs. Data for qRT–PCR are presented as the mean±s.d. of two or three experiments. ESRP2 expression following treatment with epigenetic drugs was compared with that with no treatment (NT). Student’s t -test, * P

    Journal: Oncogenesis

    Article Title: ESRP1 is overexpressed in ovarian cancer and promotes switching from mesenchymal to epithelial phenotype in ovarian cancer cells

    doi: 10.1038/oncsis.2017.87

    Figure Lengend Snippet: DNA methylation status in the promoter region of ESRP2 in ovarian cancer cell lines and tissues. ( a ) ESRP2 gene expression following treatment with epigenetic drugs. Data for qRT–PCR are presented as the mean±s.d. of two or three experiments. ESRP2 expression following treatment with epigenetic drugs was compared with that with no treatment (NT). Student’s t -test, * P

    Article Snippet: Methylated human control DNA from EpiTect PCR Control DNA Set (Qiagen) was used as positive control.

    Techniques: DNA Methylation Assay, Expressing, Quantitative RT-PCR

    DNA methylation status in the promoter region of ESRP1 in ovarian cancer cell lines and tissues. ( a ) ESRP1 gene expression in ovarian cancer cells following treatment with epigenetic drugs. Data for qRT–PCR are presented as the mean±s.d. of two or three experiments. ESRP1 expression following treatment with epigenetic drugs was compared with that with no treatment (NT). Student’s t -test; * P

    Journal: Oncogenesis

    Article Title: ESRP1 is overexpressed in ovarian cancer and promotes switching from mesenchymal to epithelial phenotype in ovarian cancer cells

    doi: 10.1038/oncsis.2017.87

    Figure Lengend Snippet: DNA methylation status in the promoter region of ESRP1 in ovarian cancer cell lines and tissues. ( a ) ESRP1 gene expression in ovarian cancer cells following treatment with epigenetic drugs. Data for qRT–PCR are presented as the mean±s.d. of two or three experiments. ESRP1 expression following treatment with epigenetic drugs was compared with that with no treatment (NT). Student’s t -test; * P

    Article Snippet: Methylated human control DNA from EpiTect PCR Control DNA Set (Qiagen) was used as positive control.

    Techniques: DNA Methylation Assay, Expressing, Quantitative RT-PCR

    The conserved HoxC4-Oct- and Sp-NF-κB-binding sites are essential for full Aicda promoter activity; HoxC4, Oct1, Oct2, OcaB, Pax5, Sp1, Sp3 and NF-κB (p52 subunit) are recruited to the Aicda promoter in B cells expressing AICDA/Aicda and undergoing CSR or SHM. ( a ) pGL3-Enhancer luciferase gene reporter constructs containing WT or mutant Aicda promoter, in which the HoxC4-Oct- and/or Sp-NF-κB-binding sites were deleted or disrupted by site-directed mutagenesis, were used to transfect CH12F3-2A, 4B6 or Ramos B cells. Luciferase activity was measured after 24 h (CH12F3-2A B cells) or 16 h (Ramos and 4B6 B cells) of culture. Data are means ± s.e. (bars) of 3 independent experiments. ( b ) AICDA/Aicda expression in human spontaneously switching 4B6 and Ramos B cells, human inducible switching 2E2 B cells stimulated with nil or anti-CD40 mAb and IL-4, mouse CH12F2-2A B cells stimulated with nil or LPS, IL-4 and TGF-β1, WT C57BL/6 mouse spleen B cells stimulated nil, LPS and IL-4, or CD154 and IL-4 were analyzed by semi-quantitative RT-PCR using serially two-fold diluted cDNA as a template. Data are representative of 3 independent experiments. ( c ) Cross-linked chromatin was precipitated from the human or mouse B cells of panel b using a mouse mAb specific to HoxC4, rabbit Abs specific to Oct1, Oct2, OcaB, Pax5, Sp1, Sp3 or p52, or preimmune control mouse or rabbit IgG. The precipitated DNA was specified by PCR using AICDA or Aicda promoter primers. Data are representative of 3 independent experiments.

    Journal: Nature immunology

    Article Title: HoxC4 binds to the Aicda promoter to induce AID expression, class switch DNA recombination and somatic hypermutation

    doi: 10.1038/ni.1725

    Figure Lengend Snippet: The conserved HoxC4-Oct- and Sp-NF-κB-binding sites are essential for full Aicda promoter activity; HoxC4, Oct1, Oct2, OcaB, Pax5, Sp1, Sp3 and NF-κB (p52 subunit) are recruited to the Aicda promoter in B cells expressing AICDA/Aicda and undergoing CSR or SHM. ( a ) pGL3-Enhancer luciferase gene reporter constructs containing WT or mutant Aicda promoter, in which the HoxC4-Oct- and/or Sp-NF-κB-binding sites were deleted or disrupted by site-directed mutagenesis, were used to transfect CH12F3-2A, 4B6 or Ramos B cells. Luciferase activity was measured after 24 h (CH12F3-2A B cells) or 16 h (Ramos and 4B6 B cells) of culture. Data are means ± s.e. (bars) of 3 independent experiments. ( b ) AICDA/Aicda expression in human spontaneously switching 4B6 and Ramos B cells, human inducible switching 2E2 B cells stimulated with nil or anti-CD40 mAb and IL-4, mouse CH12F2-2A B cells stimulated with nil or LPS, IL-4 and TGF-β1, WT C57BL/6 mouse spleen B cells stimulated nil, LPS and IL-4, or CD154 and IL-4 were analyzed by semi-quantitative RT-PCR using serially two-fold diluted cDNA as a template. Data are representative of 3 independent experiments. ( c ) Cross-linked chromatin was precipitated from the human or mouse B cells of panel b using a mouse mAb specific to HoxC4, rabbit Abs specific to Oct1, Oct2, OcaB, Pax5, Sp1, Sp3 or p52, or preimmune control mouse or rabbit IgG. The precipitated DNA was specified by PCR using AICDA or Aicda promoter primers. Data are representative of 3 independent experiments.

    Article Snippet: Real-time qRT-PCR analysis was performed using an DNA Engine Opticon 2 Real-Time PCR Detection System (Bio-Rad Laboratories, Inc.) to measure SYBR-green (DyNAmo HS SYBR Green, New England Biolabs, Inc.) incorporation with the following protocol: 50 °C for 2 min, 95 °C for 10 min, 40 cycles of 95 °C for 10 sec, 60 °C for 20 sec, 72 °C for 30 sec, 80 °C for 1 sec, and data acquisition at 80 °C, and 72 °C for 10 min. Melting curve analysis was performed from 72°–95 °C and samples were incubated for another 5 min at 72 °C.

    Techniques: Binding Assay, Activity Assay, Expressing, Luciferase, Construct, Mutagenesis, Quantitative RT-PCR, Polymerase Chain Reaction

    Decreased somatic mutation frequency in the Ig H chain intronic J H 4-iEµ DNA of Peyer’s patch PNA hi B220 + (GC) B cells from 3 12-week-old Hoxc4 −/− mice as compare to their Hoxc4 +/+ littermates. ( a ) Pie charts depict the proportions of sequences that carry 1, 2, 3, etc. mutations over the 720 bp J H 4-iE µ DNA analyzed. The numbers of the sequences analyzed are at the center of the pies. ( b ) HoxC4 deficiency does not alter the level of V J558 DJ H -Cµ transcripts. V J558 DJ H -C µ transcripts in Peyer’s patches B cells of these mice were measured by real-time qRT-PCR performed in triplicate samples using SYBR-green. In each sample, mRNA expression was normalized to CD79b expression. Data are means ± s.e. (bars) of triplicate samples from 3 independent pairs of Hoxc4 +/+ and Hoxc4 −/− mice. The analysis of the spectrum of mutations in Hoxc4 +/+ and Hoxc4 −/− mice is the subject of Supplementary Fig. 4 .

    Journal: Nature immunology

    Article Title: HoxC4 binds to the Aicda promoter to induce AID expression, class switch DNA recombination and somatic hypermutation

    doi: 10.1038/ni.1725

    Figure Lengend Snippet: Decreased somatic mutation frequency in the Ig H chain intronic J H 4-iEµ DNA of Peyer’s patch PNA hi B220 + (GC) B cells from 3 12-week-old Hoxc4 −/− mice as compare to their Hoxc4 +/+ littermates. ( a ) Pie charts depict the proportions of sequences that carry 1, 2, 3, etc. mutations over the 720 bp J H 4-iE µ DNA analyzed. The numbers of the sequences analyzed are at the center of the pies. ( b ) HoxC4 deficiency does not alter the level of V J558 DJ H -Cµ transcripts. V J558 DJ H -C µ transcripts in Peyer’s patches B cells of these mice were measured by real-time qRT-PCR performed in triplicate samples using SYBR-green. In each sample, mRNA expression was normalized to CD79b expression. Data are means ± s.e. (bars) of triplicate samples from 3 independent pairs of Hoxc4 +/+ and Hoxc4 −/− mice. The analysis of the spectrum of mutations in Hoxc4 +/+ and Hoxc4 −/− mice is the subject of Supplementary Fig. 4 .

    Article Snippet: Real-time qRT-PCR analysis was performed using an DNA Engine Opticon 2 Real-Time PCR Detection System (Bio-Rad Laboratories, Inc.) to measure SYBR-green (DyNAmo HS SYBR Green, New England Biolabs, Inc.) incorporation with the following protocol: 50 °C for 2 min, 95 °C for 10 min, 40 cycles of 95 °C for 10 sec, 60 °C for 20 sec, 72 °C for 30 sec, 80 °C for 1 sec, and data acquisition at 80 °C, and 72 °C for 10 min. Melting curve analysis was performed from 72°–95 °C and samples were incubated for another 5 min at 72 °C.

    Techniques: Mutagenesis, Mouse Assay, Quantitative RT-PCR, SYBR Green Assay, Expressing

    Host and viral DNA content of differently treated ASFV samples. Quantitative dual PCR was executed by Virotype ASFV PCR Kit. C t values of individual and averaged samples represented by colored circles and grey box respectively. Averages were calculated from numbers of samples indicated on X axis. Gray circles indicate minimum and maximum values. C t values higher than 40 (undetectable host DNA) are represented by 41. Whole genome amplification (WGA) was executed by REPLI-g Mini Kit.

    Journal: Viruses

    Article Title: A Simple Method for Sample Preparation to Facilitate Efficient Whole-Genome Sequencing of African Swine Fever Virus

    doi: 10.3390/v11121129

    Figure Lengend Snippet: Host and viral DNA content of differently treated ASFV samples. Quantitative dual PCR was executed by Virotype ASFV PCR Kit. C t values of individual and averaged samples represented by colored circles and grey box respectively. Averages were calculated from numbers of samples indicated on X axis. Gray circles indicate minimum and maximum values. C t values higher than 40 (undetectable host DNA) are represented by 41. Whole genome amplification (WGA) was executed by REPLI-g Mini Kit.

    Article Snippet: The PCR products were purified using Gel/PCR DNA Fragments Extraction Kit (Geneaid Biotech Ltd., Taipei, Taiwan).

    Techniques: Polymerase Chain Reaction, Whole Genome Amplification

    Simultaneous overexpression of LPMO and CBH1 in Pf Mig1 88 . (A) Schematic diagram showing the construction cassette for dual overexpression of LPMO and CBH1 genes from P. funiculosum NCIM1228. (B) Transformants of pOAO5 after AMTM transformation in Pf Mig1 88 . Transformants were selected on 100 µg/ml hygromycin. (C) Southern blot of transformants confirmed by PCR. M is HindIII lambda DNA size marker used; Lane C indicates genomic DNA of non-transformed parental strain; Lanes 1-5 represent genomic DNA from transformants with LPMO/CBH1 cassette integration; (D) The enzymatic profile of the overexpressed enzymes in the fermentation broth of Pf Mig1 88 and six transformants.

    Journal: bioRxiv

    Article Title: Insights into structure of Penicillium funiculosum LPMO and its synergistic saccharification performance with CBH1 on high substrate loading upon simultaneous overexpression

    doi: 10.1101/2020.04.16.045914

    Figure Lengend Snippet: Simultaneous overexpression of LPMO and CBH1 in Pf Mig1 88 . (A) Schematic diagram showing the construction cassette for dual overexpression of LPMO and CBH1 genes from P. funiculosum NCIM1228. (B) Transformants of pOAO5 after AMTM transformation in Pf Mig1 88 . Transformants were selected on 100 µg/ml hygromycin. (C) Southern blot of transformants confirmed by PCR. M is HindIII lambda DNA size marker used; Lane C indicates genomic DNA of non-transformed parental strain; Lanes 1-5 represent genomic DNA from transformants with LPMO/CBH1 cassette integration; (D) The enzymatic profile of the overexpressed enzymes in the fermentation broth of Pf Mig1 88 and six transformants.

    Article Snippet: Primers for transcripts to be tested were designed using boundary sequence of two exons to avoid any amplification from genomic DNA contamination. qRT-PCR was done in biological triplicates with tubulin as the endogenous control.

    Techniques: Over Expression, Transformation Assay, Southern Blot, Polymerase Chain Reaction, Lambda DNA Preparation, Marker

    Construction of LPMO expression cassette and its overexpression in Pf Mig1 88 . (A) Schematic diagram showing the assembly of LPMO cassette from P. funiculosum NCIM1228 genome. (B) Transformants of pOAO1 after AMTM transformation in Pf Mig1 88 . Transformants were selected on 100 µg/ml hygromycin. (C) Southern blot of transformants confirmed by PCR. M is Hind III lambda DNA size marker used; Lane C indicates genomic DNA of non-transformed parental strain; Lanes 1-5 represent genomic DNA from transformants with LPMO cassette integration; (D) The LPMO and FPase activities in the fermentation broth of Pf Mig1 88 and five transformants.

    Journal: bioRxiv

    Article Title: Insights into structure of Penicillium funiculosum LPMO and its synergistic saccharification performance with CBH1 on high substrate loading upon simultaneous overexpression

    doi: 10.1101/2020.04.16.045914

    Figure Lengend Snippet: Construction of LPMO expression cassette and its overexpression in Pf Mig1 88 . (A) Schematic diagram showing the assembly of LPMO cassette from P. funiculosum NCIM1228 genome. (B) Transformants of pOAO1 after AMTM transformation in Pf Mig1 88 . Transformants were selected on 100 µg/ml hygromycin. (C) Southern blot of transformants confirmed by PCR. M is Hind III lambda DNA size marker used; Lane C indicates genomic DNA of non-transformed parental strain; Lanes 1-5 represent genomic DNA from transformants with LPMO cassette integration; (D) The LPMO and FPase activities in the fermentation broth of Pf Mig1 88 and five transformants.

    Article Snippet: Primers for transcripts to be tested were designed using boundary sequence of two exons to avoid any amplification from genomic DNA contamination. qRT-PCR was done in biological triplicates with tubulin as the endogenous control.

    Techniques: Expressing, Over Expression, Transformation Assay, Southern Blot, Polymerase Chain Reaction, Lambda DNA Preparation, Marker

    The wdr-23 promoter is induced by stress via SKN-1. (A) DNA microarray quantification of wdr-23 mRNA levels following a 1-h exposure to 38 μM juglone. (B) Real-time RT-PCR quantification of wdr-23 mRNA levels in control(RNAi) and skn-1 ( RNAi ) worms

    Journal: Molecular and Cellular Biology

    Article Title: A Negative-Feedback Loop between the Detoxification/Antioxidant Response Factor SKN-1 and Its Repressor WDR-23 Matches Organism Needs with Environmental Conditions

    doi: 10.1128/MCB.00245-13

    Figure Lengend Snippet: The wdr-23 promoter is induced by stress via SKN-1. (A) DNA microarray quantification of wdr-23 mRNA levels following a 1-h exposure to 38 μM juglone. (B) Real-time RT-PCR quantification of wdr-23 mRNA levels in control(RNAi) and skn-1 ( RNAi ) worms

    Article Snippet: Full-length skn-1c cDNA with an N-terminal c-myc tag in-frame fusion was PCR amplified and expressed using the TnT T7 quick-coupled transcription/translation system for PCR DNA (Promega, Madison, WI).

    Techniques: Microarray, Quantitative RT-PCR