phi29 dna polymerase Search Results


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  • 99
    New England Biolabs phi29 dna polymerase
    Effect of Tth SSB-255 protein on the efficiency and specificity of RCA. ( a ) Top: RCAs were performed in the absence of the SSB proteins using pUC19 <t>DNA</t> as template and <t>phi29</t> DNA polymerase for the indicated reaction times. Bottom: Signals of spot hybridization of the same samples. ( b ) Top and Bottom: same as (a), except for the absence of template DNA. ( c ) Top and Bottom: same as (a), except for the presence of the Tth SSB protein (3.0 µg/20 µl reaction volume). ( d ) Top and Bottom: same as (c), except for the absence of template DNA. ( e ) Top and Bottom: same as (a), except for the presence of the Tth SSB-255 protein (3.0 µg/20 µl reaction volume). ( f ) Top and Bottom: same as (e), except for the absence of template DNA.
    Phi29 Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 900 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phi29 dna polymerase/product/New England Biolabs
    Average 99 stars, based on 900 article reviews
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    phi29 dna polymerase - by Bioz Stars, 2020-09
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    99
    Thermo Fisher phi29 dna polymerase
    Size distribution of <t>DNA-magnesium-pyrophosphate</t> particles as a function of multiple displacement amplification reaction times, with and without the heat-inactivation step. At different time points (17, 38 and 66 h) of the MDA reaction in 3-pL droplets at 30 °C, the resulting DNA-Mg-PP i particles were released from droplets and imaged under TEM to measure their size. The first set of measurements (green) was made without heat-inactivation step. The second set of measurements (grey) was made with heat-inactivation step at 65 °C for 15 min that is typically used to inactivate <t>phi29</t> DNA polymerase and terminate the MDA reaction.
    Phi29 Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 382 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phi29 dna polymerase/product/Thermo Fisher
    Average 99 stars, based on 382 article reviews
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    phi29 dna polymerase - by Bioz Stars, 2020-09
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    90
    Monserate Biotechnology Group phi29 dna polymerase
    Effect of RNA substitutions in circular templates on rolling circle amplification with <t>phi29</t> <t>DNA</t> polymerase. ( A ) Total amount of RCA products (y-axis) generated for padlock probes with/without a terminal 3′ RNA and in the absence of synthetic RNA ligation template (template -). ( B ) Circles with 0–7 RNA substitutions in the backbone were amplified and digitally counted. The y-axis shows the number of rolling circle products (RCPs); error bars ± S.D.; n = 2. The same RCA reactions with chimeric circles were also monitored in real-time by measuring SYBR Gold incorporation on qPCR instrument ( C and E ). (C) RCA reaction curves of circles with 0, 1 and 2 RNA substitutions. ( D ) RCPs from C were imaged on microscope slides and size and intensity of individual RCPs were quantified. Black line, median; upper whisker, highest value that is within 1.5 the interquartile range of the hinge; lower whisker, lowest value within 1.5 the interquartile range of the hinge. (E) Real-time data of the same RCA reactions as in B with 0–7 RNA substitutes are displayed. Representative samples are presented from a duplicated experiment. To highlight the initial stages of RCA and to show the difference between the samples with low RCA efficiency, fluorescence intensity readout between 3000 and 6000 is presented.
    Phi29 Dna Polymerase, supplied by Monserate Biotechnology Group, used in various techniques. Bioz Stars score: 90/100, based on 53 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phi29 dna polymerase/product/Monserate Biotechnology Group
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    phi29 dna polymerase - by Bioz Stars, 2020-09
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    99
    GE Healthcare phi29 dna polymerase
    Effect of RNA substitutions in circular templates on rolling circle amplification with <t>phi29</t> <t>DNA</t> polymerase. ( A ) Total amount of RCA products (y-axis) generated for padlock probes with/without a terminal 3′ RNA and in the absence of synthetic RNA ligation template (template -). ( B ) Circles with 0–7 RNA substitutions in the backbone were amplified and digitally counted. The y-axis shows the number of rolling circle products (RCPs); error bars ± S.D.; n = 2. The same RCA reactions with chimeric circles were also monitored in real-time by measuring SYBR Gold incorporation on qPCR instrument ( C and E ). (C) RCA reaction curves of circles with 0, 1 and 2 RNA substitutions. ( D ) RCPs from C were imaged on microscope slides and size and intensity of individual RCPs were quantified. Black line, median; upper whisker, highest value that is within 1.5 the interquartile range of the hinge; lower whisker, lowest value within 1.5 the interquartile range of the hinge. (E) Real-time data of the same RCA reactions as in B with 0–7 RNA substitutes are displayed. Representative samples are presented from a duplicated experiment. To highlight the initial stages of RCA and to show the difference between the samples with low RCA efficiency, fluorescence intensity readout between 3000 and 6000 is presented.
    Phi29 Dna Polymerase, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 169 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phi29 dna polymerase/product/GE Healthcare
    Average 99 stars, based on 169 article reviews
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    phi29 dna polymerase - by Bioz Stars, 2020-09
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    93
    Illumina Inc phi29 dna polymerase
    Effect of RNA substitutions in circular templates on rolling circle amplification with <t>phi29</t> <t>DNA</t> polymerase. ( A ) Total amount of RCA products (y-axis) generated for padlock probes with/without a terminal 3′ RNA and in the absence of synthetic RNA ligation template (template -). ( B ) Circles with 0–7 RNA substitutions in the backbone were amplified and digitally counted. The y-axis shows the number of rolling circle products (RCPs); error bars ± S.D.; n = 2. The same RCA reactions with chimeric circles were also monitored in real-time by measuring SYBR Gold incorporation on qPCR instrument ( C and E ). (C) RCA reaction curves of circles with 0, 1 and 2 RNA substitutions. ( D ) RCPs from C were imaged on microscope slides and size and intensity of individual RCPs were quantified. Black line, median; upper whisker, highest value that is within 1.5 the interquartile range of the hinge; lower whisker, lowest value within 1.5 the interquartile range of the hinge. (E) Real-time data of the same RCA reactions as in B with 0–7 RNA substitutes are displayed. Representative samples are presented from a duplicated experiment. To highlight the initial stages of RCA and to show the difference between the samples with low RCA efficiency, fluorescence intensity readout between 3000 and 6000 is presented.
    Phi29 Dna Polymerase, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 93/100, based on 128 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phi29 dna polymerase/product/Illumina Inc
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    99
    Thermo Fisher reaction buffer for phi29 dna polymerase 10x
    Effect of RNA substitutions in circular templates on rolling circle amplification with <t>phi29</t> <t>DNA</t> polymerase. ( A ) Total amount of RCA products (y-axis) generated for padlock probes with/without a terminal 3′ RNA and in the absence of synthetic RNA ligation template (template -). ( B ) Circles with 0–7 RNA substitutions in the backbone were amplified and digitally counted. The y-axis shows the number of rolling circle products (RCPs); error bars ± S.D.; n = 2. The same RCA reactions with chimeric circles were also monitored in real-time by measuring SYBR Gold incorporation on qPCR instrument ( C and E ). (C) RCA reaction curves of circles with 0, 1 and 2 RNA substitutions. ( D ) RCPs from C were imaged on microscope slides and size and intensity of individual RCPs were quantified. Black line, median; upper whisker, highest value that is within 1.5 the interquartile range of the hinge; lower whisker, lowest value within 1.5 the interquartile range of the hinge. (E) Real-time data of the same RCA reactions as in B with 0–7 RNA substitutes are displayed. Representative samples are presented from a duplicated experiment. To highlight the initial stages of RCA and to show the difference between the samples with low RCA efficiency, fluorescence intensity readout between 3000 and 6000 is presented.
    Reaction Buffer For Phi29 Dna Polymerase 10x, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Kanto Chemical dna free phi29 dna polymerase
    Effect of RNA substitutions in circular templates on rolling circle amplification with <t>phi29</t> <t>DNA</t> polymerase. ( A ) Total amount of RCA products (y-axis) generated for padlock probes with/without a terminal 3′ RNA and in the absence of synthetic RNA ligation template (template -). ( B ) Circles with 0–7 RNA substitutions in the backbone were amplified and digitally counted. The y-axis shows the number of rolling circle products (RCPs); error bars ± S.D.; n = 2. The same RCA reactions with chimeric circles were also monitored in real-time by measuring SYBR Gold incorporation on qPCR instrument ( C and E ). (C) RCA reaction curves of circles with 0, 1 and 2 RNA substitutions. ( D ) RCPs from C were imaged on microscope slides and size and intensity of individual RCPs were quantified. Black line, median; upper whisker, highest value that is within 1.5 the interquartile range of the hinge; lower whisker, lowest value within 1.5 the interquartile range of the hinge. (E) Real-time data of the same RCA reactions as in B with 0–7 RNA substitutes are displayed. Representative samples are presented from a duplicated experiment. To highlight the initial stages of RCA and to show the difference between the samples with low RCA efficiency, fluorescence intensity readout between 3000 and 6000 is presented.
    Dna Free Phi29 Dna Polymerase, supplied by Kanto Chemical, used in various techniques. Bioz Stars score: 92/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    GE Healthcare phi 29 dna polymerase
    Effect of RNA substitutions in circular templates on rolling circle amplification with <t>phi29</t> <t>DNA</t> polymerase. ( A ) Total amount of RCA products (y-axis) generated for padlock probes with/without a terminal 3′ RNA and in the absence of synthetic RNA ligation template (template -). ( B ) Circles with 0–7 RNA substitutions in the backbone were amplified and digitally counted. The y-axis shows the number of rolling circle products (RCPs); error bars ± S.D.; n = 2. The same RCA reactions with chimeric circles were also monitored in real-time by measuring SYBR Gold incorporation on qPCR instrument ( C and E ). (C) RCA reaction curves of circles with 0, 1 and 2 RNA substitutions. ( D ) RCPs from C were imaged on microscope slides and size and intensity of individual RCPs were quantified. Black line, median; upper whisker, highest value that is within 1.5 the interquartile range of the hinge; lower whisker, lowest value within 1.5 the interquartile range of the hinge. (E) Real-time data of the same RCA reactions as in B with 0–7 RNA substitutes are displayed. Representative samples are presented from a duplicated experiment. To highlight the initial stages of RCA and to show the difference between the samples with low RCA efficiency, fluorescence intensity readout between 3000 and 6000 is presented.
    Phi 29 Dna Polymerase, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 94/100, based on 43 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phi 29 dna polymerase/product/GE Healthcare
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    phi 29 dna polymerase - by Bioz Stars, 2020-09
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    94
    Thermo Fisher phi29 dna polymerase 10 u µl
    Effect of RNA substitutions in circular templates on rolling circle amplification with <t>phi29</t> <t>DNA</t> polymerase. ( A ) Total amount of RCA products (y-axis) generated for padlock probes with/without a terminal 3′ RNA and in the absence of synthetic RNA ligation template (template -). ( B ) Circles with 0–7 RNA substitutions in the backbone were amplified and digitally counted. The y-axis shows the number of rolling circle products (RCPs); error bars ± S.D.; n = 2. The same RCA reactions with chimeric circles were also monitored in real-time by measuring SYBR Gold incorporation on qPCR instrument ( C and E ). (C) RCA reaction curves of circles with 0, 1 and 2 RNA substitutions. ( D ) RCPs from C were imaged on microscope slides and size and intensity of individual RCPs were quantified. Black line, median; upper whisker, highest value that is within 1.5 the interquartile range of the hinge; lower whisker, lowest value within 1.5 the interquartile range of the hinge. (E) Real-time data of the same RCA reactions as in B with 0–7 RNA substitutes are displayed. Representative samples are presented from a duplicated experiment. To highlight the initial stages of RCA and to show the difference between the samples with low RCA efficiency, fluorescence intensity readout between 3000 and 6000 is presented.
    Phi29 Dna Polymerase 10 U µl, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phi29 dna polymerase 10 u µl/product/Thermo Fisher
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    phi29 dna polymerase 10 u µl - by Bioz Stars, 2020-09
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    99
    Thermo Fisher phi29 dna polymerase reaction buffer
    Effect of RNA substitutions in circular templates on rolling circle amplification with <t>phi29</t> <t>DNA</t> polymerase. ( A ) Total amount of RCA products (y-axis) generated for padlock probes with/without a terminal 3′ RNA and in the absence of synthetic RNA ligation template (template -). ( B ) Circles with 0–7 RNA substitutions in the backbone were amplified and digitally counted. The y-axis shows the number of rolling circle products (RCPs); error bars ± S.D.; n = 2. The same RCA reactions with chimeric circles were also monitored in real-time by measuring SYBR Gold incorporation on qPCR instrument ( C and E ). (C) RCA reaction curves of circles with 0, 1 and 2 RNA substitutions. ( D ) RCPs from C were imaged on microscope slides and size and intensity of individual RCPs were quantified. Black line, median; upper whisker, highest value that is within 1.5 the interquartile range of the hinge; lower whisker, lowest value within 1.5 the interquartile range of the hinge. (E) Real-time data of the same RCA reactions as in B with 0–7 RNA substitutes are displayed. Representative samples are presented from a duplicated experiment. To highlight the initial stages of RCA and to show the difference between the samples with low RCA efficiency, fluorescence intensity readout between 3000 and 6000 is presented.
    Phi29 Dna Polymerase Reaction Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phi29 dna polymerase reaction buffer/product/Thermo Fisher
    Average 99 stars, based on 25 article reviews
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    phi29 dna polymerase reaction buffer - by Bioz Stars, 2020-09
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    92
    Enzymatics phi29 dna polymerase
    In vitro experiments demonstrating that polyacrylic acid, very similar to the polymer used in ExM, inhibits both the reverse transcription step and the rolling circle amplification step, two key enzymatic reactions of FISSEQ, but that the enzymatic activity can be restored with a passivation reaction that cancels out the charge of moieties on the polymer backbone. The passivation reaction ( 95 ), in which ethanolamine is reacted with carboxylic groups and converts them to amides with no charge (A), restores reverse transcription activity (B) and rolling circle amplification activity (C). These in vitro experiments were performed with 0.1-0.5% (w/v) polyacrylic acid, to mimic the concentration inside the expanded gel which is ∼0.2% (8.625% (w/v) sodium acrylate before expansion corresponds to 8.625/3.5^3 or ∼0.2% with expansion factor ∼3.5, and between ∼0.1% to ∼0.5% with expansion factors between 2.5 to 4). The passivation protocol was performed as in Methods section ‘Passivation’. We validated the passivation with a range of EDC concentrations, 50-150mM, that are higher than the polyacrylic acid concentration of ∼30mM (∼0.2% polyacrylic acid); however, we didn’t want the EDC concentration to be more than a few fold higher than the polyacrylic acid concentration as EDC can react with guanine in RNA ( 96 ), and therefore high concentrations of EDC may have undesired side effects. The reverse transcription reaction was performed with M-MuLV (enzymatics) according to the manufacturer’s protocol, with 1.2kb Kanamycin Positive Control RNA (Promega) as a template, and random primers. The rolling circle amplification reaction was performed with <t>phi29</t> (enzymatics) according to the manufacturer’s protocol. The template used was the 55-base long CircLigase II ssDNA Control Oligo, after circularization with CircLigase II (epicentre) according to the manufacturer’s protocol.
    Phi29 Dna Polymerase, supplied by Enzymatics, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phi29 dna polymerase/product/Enzymatics
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    phi29 dna polymerase - by Bioz Stars, 2020-09
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    92
    Epicentre Biotechnologies phi29 dna polymerase
    In vitro experiments demonstrating that polyacrylic acid, very similar to the polymer used in ExM, inhibits both the reverse transcription step and the rolling circle amplification step, two key enzymatic reactions of FISSEQ, but that the enzymatic activity can be restored with a passivation reaction that cancels out the charge of moieties on the polymer backbone. The passivation reaction ( 95 ), in which ethanolamine is reacted with carboxylic groups and converts them to amides with no charge (A), restores reverse transcription activity (B) and rolling circle amplification activity (C). These in vitro experiments were performed with 0.1-0.5% (w/v) polyacrylic acid, to mimic the concentration inside the expanded gel which is ∼0.2% (8.625% (w/v) sodium acrylate before expansion corresponds to 8.625/3.5^3 or ∼0.2% with expansion factor ∼3.5, and between ∼0.1% to ∼0.5% with expansion factors between 2.5 to 4). The passivation protocol was performed as in Methods section ‘Passivation’. We validated the passivation with a range of EDC concentrations, 50-150mM, that are higher than the polyacrylic acid concentration of ∼30mM (∼0.2% polyacrylic acid); however, we didn’t want the EDC concentration to be more than a few fold higher than the polyacrylic acid concentration as EDC can react with guanine in RNA ( 96 ), and therefore high concentrations of EDC may have undesired side effects. The reverse transcription reaction was performed with M-MuLV (enzymatics) according to the manufacturer’s protocol, with 1.2kb Kanamycin Positive Control RNA (Promega) as a template, and random primers. The rolling circle amplification reaction was performed with <t>phi29</t> (enzymatics) according to the manufacturer’s protocol. The template used was the 55-base long CircLigase II ssDNA Control Oligo, after circularization with CircLigase II (epicentre) according to the manufacturer’s protocol.
    Phi29 Dna Polymerase, supplied by Epicentre Biotechnologies, used in various techniques. Bioz Stars score: 92/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phi29 dna polymerase/product/Epicentre Biotechnologies
    Average 92 stars, based on 12 article reviews
    Price from $9.99 to $1999.99
    phi29 dna polymerase - by Bioz Stars, 2020-09
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    Image Search Results


    Effect of Tth SSB-255 protein on the efficiency and specificity of RCA. ( a ) Top: RCAs were performed in the absence of the SSB proteins using pUC19 DNA as template and phi29 DNA polymerase for the indicated reaction times. Bottom: Signals of spot hybridization of the same samples. ( b ) Top and Bottom: same as (a), except for the absence of template DNA. ( c ) Top and Bottom: same as (a), except for the presence of the Tth SSB protein (3.0 µg/20 µl reaction volume). ( d ) Top and Bottom: same as (c), except for the absence of template DNA. ( e ) Top and Bottom: same as (a), except for the presence of the Tth SSB-255 protein (3.0 µg/20 µl reaction volume). ( f ) Top and Bottom: same as (e), except for the absence of template DNA.

    Journal: Nucleic Acids Research

    Article Title: Improvements of rolling circle amplification (RCA) efficiency and accuracy using Thermus thermophilus SSB mutant protein

    doi: 10.1093/nar/gkl350

    Figure Lengend Snippet: Effect of Tth SSB-255 protein on the efficiency and specificity of RCA. ( a ) Top: RCAs were performed in the absence of the SSB proteins using pUC19 DNA as template and phi29 DNA polymerase for the indicated reaction times. Bottom: Signals of spot hybridization of the same samples. ( b ) Top and Bottom: same as (a), except for the absence of template DNA. ( c ) Top and Bottom: same as (a), except for the presence of the Tth SSB protein (3.0 µg/20 µl reaction volume). ( d ) Top and Bottom: same as (c), except for the absence of template DNA. ( e ) Top and Bottom: same as (a), except for the presence of the Tth SSB-255 protein (3.0 µg/20 µl reaction volume). ( f ) Top and Bottom: same as (e), except for the absence of template DNA.

    Article Snippet: The presence of Tth SSB-255 mutant protein shortened the elongation time required to synthesize a DNA fragment by phi29 DNA polymerase.

    Techniques: Hybridization

    Effect of Tth SSB-255 protein on RCA assays. ( a ) RCAs were carried out in the absence or presence of the indicated SSB proteins using pUC19 DNA as a template and phi29 DNA polymerase. ( b ) Same as (a), except for using linearized ( Eco RI) pUC19 DNA as the template. ( c ) Same as (a), except that the amplifications were carried out in the absence of template DNA. Lane M: molecular weight markers (100 and 12 kb).

    Journal: Nucleic Acids Research

    Article Title: Improvements of rolling circle amplification (RCA) efficiency and accuracy using Thermus thermophilus SSB mutant protein

    doi: 10.1093/nar/gkl350

    Figure Lengend Snippet: Effect of Tth SSB-255 protein on RCA assays. ( a ) RCAs were carried out in the absence or presence of the indicated SSB proteins using pUC19 DNA as a template and phi29 DNA polymerase. ( b ) Same as (a), except for using linearized ( Eco RI) pUC19 DNA as the template. ( c ) Same as (a), except that the amplifications were carried out in the absence of template DNA. Lane M: molecular weight markers (100 and 12 kb).

    Article Snippet: The presence of Tth SSB-255 mutant protein shortened the elongation time required to synthesize a DNA fragment by phi29 DNA polymerase.

    Techniques: Molecular Weight

    Plasmid DNA from the cecal sample after amplification with phi29 polymerase. 1 , 1 kb ladder and 2 , Plasmid DNA amplified with Phi29 DNA polymerase.

    Journal: Frontiers in Microbiology

    Article Title: A Comparison of Methods for the Extraction of Plasmids Capable of Conferring Antibiotic Resistance in a Human Pathogen From Complex Broiler Cecal Samples

    doi: 10.3389/fmicb.2018.01731

    Figure Lengend Snippet: Plasmid DNA from the cecal sample after amplification with phi29 polymerase. 1 , 1 kb ladder and 2 , Plasmid DNA amplified with Phi29 DNA polymerase.

    Article Snippet: Plasmid DNA was amplified by adding 1 μL of 10 μM Exo-Resistant Random Primer (Thermo Scientific), 2 μL phi29 DNA Polymerase Reaction Buffer (New England Biolabs) and 8.2 μL of MilliQ water to 5 μL of the purified treated DNA.

    Techniques: Plasmid Preparation, Amplification

    Digested plasmid DNA extracted from E. coli transformants after electroporation with the phi29 polymerase amplified DNA. 1 , 1 kb ladder; Plasmid DNA extracted from transformants selected on agar plates containing; 2 , ampicillin 32 mg/L (M_Amp_BC); 3 , ampicillin 32 mg/L (M_Amp_SC); 4 , tetracycline 16 mg/L (M_Tet_BC); 5 , tetracycline 16 mg/L (M_Tet_SC); 6 , kanamycin 25 mg/L (M_Kan); 7 , ciprofloxacin 16 mg/L (M_Cip). BC and SC refer to the two different colony morphology types, big or small colonies, on the same antibiotic plate.

    Journal: Frontiers in Microbiology

    Article Title: A Comparison of Methods for the Extraction of Plasmids Capable of Conferring Antibiotic Resistance in a Human Pathogen From Complex Broiler Cecal Samples

    doi: 10.3389/fmicb.2018.01731

    Figure Lengend Snippet: Digested plasmid DNA extracted from E. coli transformants after electroporation with the phi29 polymerase amplified DNA. 1 , 1 kb ladder; Plasmid DNA extracted from transformants selected on agar plates containing; 2 , ampicillin 32 mg/L (M_Amp_BC); 3 , ampicillin 32 mg/L (M_Amp_SC); 4 , tetracycline 16 mg/L (M_Tet_BC); 5 , tetracycline 16 mg/L (M_Tet_SC); 6 , kanamycin 25 mg/L (M_Kan); 7 , ciprofloxacin 16 mg/L (M_Cip). BC and SC refer to the two different colony morphology types, big or small colonies, on the same antibiotic plate.

    Article Snippet: Plasmid DNA was amplified by adding 1 μL of 10 μM Exo-Resistant Random Primer (Thermo Scientific), 2 μL phi29 DNA Polymerase Reaction Buffer (New England Biolabs) and 8.2 μL of MilliQ water to 5 μL of the purified treated DNA.

    Techniques: Plasmid Preparation, Electroporation, Amplification

    Effect of RNA substitutions in circular templates on rolling circle amplification with phi29 DNA polymerase. ( A ) Total amount of RCA products (y-axis) generated for padlock probes with/without a terminal 3′ RNA and in the absence of synthetic RNA ligation template (template -). ( B ) Circles with 0–7 RNA substitutions in the backbone were amplified and digitally counted. The y-axis shows the number of rolling circle products (RCPs); error bars ± S.D.; n = 2. The same RCA reactions with chimeric circles were also monitored in real-time by measuring SYBR Gold incorporation on qPCR instrument ( C and E ). (C) RCA reaction curves of circles with 0, 1 and 2 RNA substitutions. ( D ) RCPs from C were imaged on microscope slides and size and intensity of individual RCPs were quantified. Black line, median; upper whisker, highest value that is within 1.5 the interquartile range of the hinge; lower whisker, lowest value within 1.5 the interquartile range of the hinge. (E) Real-time data of the same RCA reactions as in B with 0–7 RNA substitutes are displayed. Representative samples are presented from a duplicated experiment. To highlight the initial stages of RCA and to show the difference between the samples with low RCA efficiency, fluorescence intensity readout between 3000 and 6000 is presented.

    Journal: Nucleic Acids Research

    Article Title: Limited reverse transcriptase activity of phi29 DNA polymerase

    doi: 10.1093/nar/gky190

    Figure Lengend Snippet: Effect of RNA substitutions in circular templates on rolling circle amplification with phi29 DNA polymerase. ( A ) Total amount of RCA products (y-axis) generated for padlock probes with/without a terminal 3′ RNA and in the absence of synthetic RNA ligation template (template -). ( B ) Circles with 0–7 RNA substitutions in the backbone were amplified and digitally counted. The y-axis shows the number of rolling circle products (RCPs); error bars ± S.D.; n = 2. The same RCA reactions with chimeric circles were also monitored in real-time by measuring SYBR Gold incorporation on qPCR instrument ( C and E ). (C) RCA reaction curves of circles with 0, 1 and 2 RNA substitutions. ( D ) RCPs from C were imaged on microscope slides and size and intensity of individual RCPs were quantified. Black line, median; upper whisker, highest value that is within 1.5 the interquartile range of the hinge; lower whisker, lowest value within 1.5 the interquartile range of the hinge. (E) Real-time data of the same RCA reactions as in B with 0–7 RNA substitutes are displayed. Representative samples are presented from a duplicated experiment. To highlight the initial stages of RCA and to show the difference between the samples with low RCA efficiency, fluorescence intensity readout between 3000 and 6000 is presented.

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

    Techniques: Amplification, Generated, Ligation, Real-time Polymerase Chain Reaction, Microscopy, Whisker Assay, Fluorescence

    Phi29 DNA polymerase exhibits higher RCA rate with circles containing pyrimidine RNA substitutions. ( A ) Real-time RCA curves of circles containing 1, 2, 3 or 4 consecutive RNA substations of rG, rU, rA, rC RNA bases are displayed (number of consecutive substitutions is indicated above plots). Rate of RCA was monitored by measuring fluorescence build-up (y-axis) resulted from SYBR Gold incorporation into RCPs. Averaged fluorescence intensity for each RCA time point was calculated from a duplicated experiment. RCA was conducted in the presence of Mg 2+ and Mn 2+ (solid and dashed lines respectively). ( B ) Linear, early stage RCA velocity (y-axis) is presented for PLPs from (A) in the presence of Mg 2+ (solid lines) and Mn 2+ (dashed lines). ( C ) RCA for the control PLP (non-chimeric DNA circle, with Mg 2+ (solid) and Mn 2+ (dashed line) are displayed.

    Journal: Nucleic Acids Research

    Article Title: Limited reverse transcriptase activity of phi29 DNA polymerase

    doi: 10.1093/nar/gky190

    Figure Lengend Snippet: Phi29 DNA polymerase exhibits higher RCA rate with circles containing pyrimidine RNA substitutions. ( A ) Real-time RCA curves of circles containing 1, 2, 3 or 4 consecutive RNA substations of rG, rU, rA, rC RNA bases are displayed (number of consecutive substitutions is indicated above plots). Rate of RCA was monitored by measuring fluorescence build-up (y-axis) resulted from SYBR Gold incorporation into RCPs. Averaged fluorescence intensity for each RCA time point was calculated from a duplicated experiment. RCA was conducted in the presence of Mg 2+ and Mn 2+ (solid and dashed lines respectively). ( B ) Linear, early stage RCA velocity (y-axis) is presented for PLPs from (A) in the presence of Mg 2+ (solid lines) and Mn 2+ (dashed lines). ( C ) RCA for the control PLP (non-chimeric DNA circle, with Mg 2+ (solid) and Mn 2+ (dashed line) are displayed.

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

    Techniques: Fluorescence, Plasmid Purification

    DNA sequencing-based analysis of rolling circle products reveals reverse transcription activity of phi29 DNA polymerase. ( A ) After RCA, short DNA oligonucleotides were hybridized to an AluI restriction site in the RCA products and RCPs were digested with AluI restriction enzyme, resulting in RCA monomers. Following digestion, monomers were PCR-amplified using primers containing Ilumina adapter sequences. PCR products were extended using IIlumina indexed primers. Finally, sequencing library was prepared using indexed primers-specific P5/7 PCR primers. The region of interest containing RNA substitutions in the original padlock probe sequence is indicated with green boxes. ( B ) Logos showing sequencing frequencies for each position within RCA monomers generated from the control DNA circle (P1 = dG), and circles containing single rG, rU, rA and rC substitutions at the RNA position (P1). Positions P1 and P2 are indicated and position P1 was additionally highlighted with the red box. ( C ) Incorporation of incorrect nucleotides for every position in the sequenced monomers from (B). Error rates, calculated as Incorporation error [%] = 1 – number of reads with expected nucleotide/total number of reads, is presented for padlock probes with single- (upper plot) and double-RNA substitutions (lower plots). P1 position for the first RNA substitution is indicated with the box.

    Journal: Nucleic Acids Research

    Article Title: Limited reverse transcriptase activity of phi29 DNA polymerase

    doi: 10.1093/nar/gky190

    Figure Lengend Snippet: DNA sequencing-based analysis of rolling circle products reveals reverse transcription activity of phi29 DNA polymerase. ( A ) After RCA, short DNA oligonucleotides were hybridized to an AluI restriction site in the RCA products and RCPs were digested with AluI restriction enzyme, resulting in RCA monomers. Following digestion, monomers were PCR-amplified using primers containing Ilumina adapter sequences. PCR products were extended using IIlumina indexed primers. Finally, sequencing library was prepared using indexed primers-specific P5/7 PCR primers. The region of interest containing RNA substitutions in the original padlock probe sequence is indicated with green boxes. ( B ) Logos showing sequencing frequencies for each position within RCA monomers generated from the control DNA circle (P1 = dG), and circles containing single rG, rU, rA and rC substitutions at the RNA position (P1). Positions P1 and P2 are indicated and position P1 was additionally highlighted with the red box. ( C ) Incorporation of incorrect nucleotides for every position in the sequenced monomers from (B). Error rates, calculated as Incorporation error [%] = 1 – number of reads with expected nucleotide/total number of reads, is presented for padlock probes with single- (upper plot) and double-RNA substitutions (lower plots). P1 position for the first RNA substitution is indicated with the box.

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

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

    (A) Optimization of T4 DNA ligase concentration and time. (B) Optimization of phi29 DNA polymerase concentration and time. (C) Optimization of Nb.Mva1269I concentration and time. The assays were carried out in the reaction buffer, containing 10 nM let-7a, and 200 nM MB.

    Journal: Chemical Science

    Article Title: Target-fueled DNA walker for highly selective miRNA detection DNA walker for highly selective miRNA detection †Electronic supplementary information (ESI) available: DNA strand structure and sequences, assembly of DNA strands as noted in the text. See DOI: 10.1039/c5sc02784eClick here for additional data file.

    doi: 10.1039/c5sc02784e

    Figure Lengend Snippet: (A) Optimization of T4 DNA ligase concentration and time. (B) Optimization of phi29 DNA polymerase concentration and time. (C) Optimization of Nb.Mva1269I concentration and time. The assays were carried out in the reaction buffer, containing 10 nM let-7a, and 200 nM MB.

    Article Snippet: Reagents and materials T4 DNA ligase and phi29 polymerase (10 units per μL) were obtained from New England Biolabs (Beijing, China).

    Techniques: Concentration Assay

    Selective whole-genome amplification (SWGA) of Plasmodium vivax genomic DNA (gDNA) from human blood samples. (A) SWGA primers bind frequently to Plasmodium vivax gDNA and infrequently to human gDNA. (B) When phi29 encounters double-stranded gDNA, it displaces the newly synthesized strand, opening new primer binding sites on the synthesized gDNA, leading to selective amplification of templates with frequent primer binding sites. (C) Post-SWGA, the percentage of P. vivax DNA has increased relative to the percentage of host DNA.

    Journal: mBio

    Article Title: Selective Whole-Genome Amplification Is a Robust Method That Enables Scalable Whole-Genome Sequencing of Plasmodium vivax from Unprocessed Clinical Samples

    doi: 10.1128/mBio.02257-16

    Figure Lengend Snippet: Selective whole-genome amplification (SWGA) of Plasmodium vivax genomic DNA (gDNA) from human blood samples. (A) SWGA primers bind frequently to Plasmodium vivax gDNA and infrequently to human gDNA. (B) When phi29 encounters double-stranded gDNA, it displaces the newly synthesized strand, opening new primer binding sites on the synthesized gDNA, leading to selective amplification of templates with frequent primer binding sites. (C) Post-SWGA, the percentage of P. vivax DNA has increased relative to the percentage of host DNA.

    Article Snippet: Thirty to 70 ng of input DNA was added to a 50-µl reaction mixture containing 3.5 µM SWGA primers, 30 U phi29 DNA polymerase enzyme (New England Biolabs), 1× phi29 buffer (New England Biolabs), 4 mM dNTPs (Roche), 1% bovine serum albumin, and water.

    Techniques: Whole Genome Amplification, Synthesized, Binding Assay, Amplification

    Size distribution of DNA-magnesium-pyrophosphate particles as a function of multiple displacement amplification reaction times, with and without the heat-inactivation step. At different time points (17, 38 and 66 h) of the MDA reaction in 3-pL droplets at 30 °C, the resulting DNA-Mg-PP i particles were released from droplets and imaged under TEM to measure their size. The first set of measurements (green) was made without heat-inactivation step. The second set of measurements (grey) was made with heat-inactivation step at 65 °C for 15 min that is typically used to inactivate phi29 DNA polymerase and terminate the MDA reaction.

    Journal: Micromachines

    Article Title: Droplet Microfluidics Approach for Single-DNA Molecule Amplification and Condensation into DNA-Magnesium-Pyrophosphate Particles

    doi: 10.3390/mi8020062

    Figure Lengend Snippet: Size distribution of DNA-magnesium-pyrophosphate particles as a function of multiple displacement amplification reaction times, with and without the heat-inactivation step. At different time points (17, 38 and 66 h) of the MDA reaction in 3-pL droplets at 30 °C, the resulting DNA-Mg-PP i particles were released from droplets and imaged under TEM to measure their size. The first set of measurements (green) was made without heat-inactivation step. The second set of measurements (grey) was made with heat-inactivation step at 65 °C for 15 min that is typically used to inactivate phi29 DNA polymerase and terminate the MDA reaction.

    Article Snippet: In this context, DNA amplification driven by phi29 DNA polymerase provides an alternative approach to amplify long DNA molecules and because of isothermal reaction conditions [ , ], the potential problems associated with emulsion stability become irrelevant.

    Techniques: Multiple Displacement Amplification, Transmission Electron Microscopy

    Percentages of reads classified to the genus level using Kraken2 (taxonomic classification tool) from beef samples with in-house databases of mammals, archaea, bacteria, fungi, human, protozoa, and viruses. Light blue represents the proportion of “ Bos ” corresponding to beef reads. Yellow represents the presence of “ Escherichia ” in the sample. The reads that could not be classified to the genus level for mammals, archaea, bacteria, fungi, human, protozoa, or viruses are represented in gray. ( A ) Blank meat samples; Bk-0h—non-enriched blank; BK-24h—non-spiked meat sample enriched for 24 h, 1–3 biological replicates. ( B ) Extraction kits; workflow A—Nucleospin Food, workflow B—DNeasy Blood Tissue and workflow C—Zymo HostZERO. ( C ) Enrichment times; workflow A—24 h culture enrichment, workflow D—16 h culture enrichment, workflow E—16 h culture enrichment, extraction followed by DNA amplification using phi 29 DNA polymerase; all extracted with Nucleospin Food kit. ( D ) Biological and technical replicates of workflow A. Small differences in the detected species shown in panels A, C, and D can be explained by the heterogeneity of the samples and biological variation, as different replicates of the experiment were used.

    Journal: Microorganisms

    Article Title: A Practical Method to Implement Strain-Level Metagenomics-Based Foodborne Outbreak Investigation and Source Tracking in Routine

    doi: 10.3390/microorganisms8081191

    Figure Lengend Snippet: Percentages of reads classified to the genus level using Kraken2 (taxonomic classification tool) from beef samples with in-house databases of mammals, archaea, bacteria, fungi, human, protozoa, and viruses. Light blue represents the proportion of “ Bos ” corresponding to beef reads. Yellow represents the presence of “ Escherichia ” in the sample. The reads that could not be classified to the genus level for mammals, archaea, bacteria, fungi, human, protozoa, or viruses are represented in gray. ( A ) Blank meat samples; Bk-0h—non-enriched blank; BK-24h—non-spiked meat sample enriched for 24 h, 1–3 biological replicates. ( B ) Extraction kits; workflow A—Nucleospin Food, workflow B—DNeasy Blood Tissue and workflow C—Zymo HostZERO. ( C ) Enrichment times; workflow A—24 h culture enrichment, workflow D—16 h culture enrichment, workflow E—16 h culture enrichment, extraction followed by DNA amplification using phi 29 DNA polymerase; all extracted with Nucleospin Food kit. ( D ) Biological and technical replicates of workflow A. Small differences in the detected species shown in panels A, C, and D can be explained by the heterogeneity of the samples and biological variation, as different replicates of the experiment were used.

    Article Snippet: One DNA extract of the beef sample enriched for 16 h and extracted with the Nucleospin Food kit was amplified using phi 29 DNA polymerase (ThermoFisher scientific, Waltham, MA, USA) according to the manufacturer’s instructions (method E, ).

    Techniques: Amplification

    Effect of RNA substitutions in circular templates on rolling circle amplification with phi29 DNA polymerase. ( A ) Total amount of RCA products (y-axis) generated for padlock probes with/without a terminal 3′ RNA and in the absence of synthetic RNA ligation template (template -). ( B ) Circles with 0–7 RNA substitutions in the backbone were amplified and digitally counted. The y-axis shows the number of rolling circle products (RCPs); error bars ± S.D.; n = 2. The same RCA reactions with chimeric circles were also monitored in real-time by measuring SYBR Gold incorporation on qPCR instrument ( C and E ). (C) RCA reaction curves of circles with 0, 1 and 2 RNA substitutions. ( D ) RCPs from C were imaged on microscope slides and size and intensity of individual RCPs were quantified. Black line, median; upper whisker, highest value that is within 1.5 the interquartile range of the hinge; lower whisker, lowest value within 1.5 the interquartile range of the hinge. (E) Real-time data of the same RCA reactions as in B with 0–7 RNA substitutes are displayed. Representative samples are presented from a duplicated experiment. To highlight the initial stages of RCA and to show the difference between the samples with low RCA efficiency, fluorescence intensity readout between 3000 and 6000 is presented.

    Journal: Nucleic Acids Research

    Article Title: Limited reverse transcriptase activity of phi29 DNA polymerase

    doi: 10.1093/nar/gky190

    Figure Lengend Snippet: Effect of RNA substitutions in circular templates on rolling circle amplification with phi29 DNA polymerase. ( A ) Total amount of RCA products (y-axis) generated for padlock probes with/without a terminal 3′ RNA and in the absence of synthetic RNA ligation template (template -). ( B ) Circles with 0–7 RNA substitutions in the backbone were amplified and digitally counted. The y-axis shows the number of rolling circle products (RCPs); error bars ± S.D.; n = 2. The same RCA reactions with chimeric circles were also monitored in real-time by measuring SYBR Gold incorporation on qPCR instrument ( C and E ). (C) RCA reaction curves of circles with 0, 1 and 2 RNA substitutions. ( D ) RCPs from C were imaged on microscope slides and size and intensity of individual RCPs were quantified. Black line, median; upper whisker, highest value that is within 1.5 the interquartile range of the hinge; lower whisker, lowest value within 1.5 the interquartile range of the hinge. (E) Real-time data of the same RCA reactions as in B with 0–7 RNA substitutes are displayed. Representative samples are presented from a duplicated experiment. To highlight the initial stages of RCA and to show the difference between the samples with low RCA efficiency, fluorescence intensity readout between 3000 and 6000 is presented.

    Article Snippet: Following the ligation, 2 μl ligation volume (circles) was added to 18 μl RCA reaction mix containing 0.1 U/μl phi29 DNA polymerase (Monserate Biotechnology Group) 1 × phi29 reaction buffer (Thermo Fisher), 125 μM dNTP (DNA Gdansk), 0.2 μg/μl BSA (NEB) and 1 × SYBR Gold (S11194, Invitrogen) to a final circles’ concentration of 1 nM.

    Techniques: Amplification, Generated, Ligation, Real-time Polymerase Chain Reaction, Microscopy, Whisker Assay, Fluorescence

    Phi29 DNA polymerase exhibits higher RCA rate with circles containing pyrimidine RNA substitutions. ( A ) Real-time RCA curves of circles containing 1, 2, 3 or 4 consecutive RNA substations of rG, rU, rA, rC RNA bases are displayed (number of consecutive substitutions is indicated above plots). Rate of RCA was monitored by measuring fluorescence build-up (y-axis) resulted from SYBR Gold incorporation into RCPs. Averaged fluorescence intensity for each RCA time point was calculated from a duplicated experiment. RCA was conducted in the presence of Mg 2+ and Mn 2+ (solid and dashed lines respectively). ( B ) Linear, early stage RCA velocity (y-axis) is presented for PLPs from (A) in the presence of Mg 2+ (solid lines) and Mn 2+ (dashed lines). ( C ) RCA for the control PLP (non-chimeric DNA circle, with Mg 2+ (solid) and Mn 2+ (dashed line) are displayed.

    Journal: Nucleic Acids Research

    Article Title: Limited reverse transcriptase activity of phi29 DNA polymerase

    doi: 10.1093/nar/gky190

    Figure Lengend Snippet: Phi29 DNA polymerase exhibits higher RCA rate with circles containing pyrimidine RNA substitutions. ( A ) Real-time RCA curves of circles containing 1, 2, 3 or 4 consecutive RNA substations of rG, rU, rA, rC RNA bases are displayed (number of consecutive substitutions is indicated above plots). Rate of RCA was monitored by measuring fluorescence build-up (y-axis) resulted from SYBR Gold incorporation into RCPs. Averaged fluorescence intensity for each RCA time point was calculated from a duplicated experiment. RCA was conducted in the presence of Mg 2+ and Mn 2+ (solid and dashed lines respectively). ( B ) Linear, early stage RCA velocity (y-axis) is presented for PLPs from (A) in the presence of Mg 2+ (solid lines) and Mn 2+ (dashed lines). ( C ) RCA for the control PLP (non-chimeric DNA circle, with Mg 2+ (solid) and Mn 2+ (dashed line) are displayed.

    Article Snippet: Following the ligation, 2 μl ligation volume (circles) was added to 18 μl RCA reaction mix containing 0.1 U/μl phi29 DNA polymerase (Monserate Biotechnology Group) 1 × phi29 reaction buffer (Thermo Fisher), 125 μM dNTP (DNA Gdansk), 0.2 μg/μl BSA (NEB) and 1 × SYBR Gold (S11194, Invitrogen) to a final circles’ concentration of 1 nM.

    Techniques: Fluorescence, Plasmid Purification

    DNA sequencing-based analysis of rolling circle products reveals reverse transcription activity of phi29 DNA polymerase. ( A ) After RCA, short DNA oligonucleotides were hybridized to an AluI restriction site in the RCA products and RCPs were digested with AluI restriction enzyme, resulting in RCA monomers. Following digestion, monomers were PCR-amplified using primers containing Ilumina adapter sequences. PCR products were extended using IIlumina indexed primers. Finally, sequencing library was prepared using indexed primers-specific P5/7 PCR primers. The region of interest containing RNA substitutions in the original padlock probe sequence is indicated with green boxes. ( B ) Logos showing sequencing frequencies for each position within RCA monomers generated from the control DNA circle (P1 = dG), and circles containing single rG, rU, rA and rC substitutions at the RNA position (P1). Positions P1 and P2 are indicated and position P1 was additionally highlighted with the red box. ( C ) Incorporation of incorrect nucleotides for every position in the sequenced monomers from (B). Error rates, calculated as Incorporation error [%] = 1 – number of reads with expected nucleotide/total number of reads, is presented for padlock probes with single- (upper plot) and double-RNA substitutions (lower plots). P1 position for the first RNA substitution is indicated with the box.

    Journal: Nucleic Acids Research

    Article Title: Limited reverse transcriptase activity of phi29 DNA polymerase

    doi: 10.1093/nar/gky190

    Figure Lengend Snippet: DNA sequencing-based analysis of rolling circle products reveals reverse transcription activity of phi29 DNA polymerase. ( A ) After RCA, short DNA oligonucleotides were hybridized to an AluI restriction site in the RCA products and RCPs were digested with AluI restriction enzyme, resulting in RCA monomers. Following digestion, monomers were PCR-amplified using primers containing Ilumina adapter sequences. PCR products were extended using IIlumina indexed primers. Finally, sequencing library was prepared using indexed primers-specific P5/7 PCR primers. The region of interest containing RNA substitutions in the original padlock probe sequence is indicated with green boxes. ( B ) Logos showing sequencing frequencies for each position within RCA monomers generated from the control DNA circle (P1 = dG), and circles containing single rG, rU, rA and rC substitutions at the RNA position (P1). Positions P1 and P2 are indicated and position P1 was additionally highlighted with the red box. ( C ) Incorporation of incorrect nucleotides for every position in the sequenced monomers from (B). Error rates, calculated as Incorporation error [%] = 1 – number of reads with expected nucleotide/total number of reads, is presented for padlock probes with single- (upper plot) and double-RNA substitutions (lower plots). P1 position for the first RNA substitution is indicated with the box.

    Article Snippet: Following the ligation, 2 μl ligation volume (circles) was added to 18 μl RCA reaction mix containing 0.1 U/μl phi29 DNA polymerase (Monserate Biotechnology Group) 1 × phi29 reaction buffer (Thermo Fisher), 125 μM dNTP (DNA Gdansk), 0.2 μg/μl BSA (NEB) and 1 × SYBR Gold (S11194, Invitrogen) to a final circles’ concentration of 1 nM.

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

    In vitro experiments demonstrating that polyacrylic acid, very similar to the polymer used in ExM, inhibits both the reverse transcription step and the rolling circle amplification step, two key enzymatic reactions of FISSEQ, but that the enzymatic activity can be restored with a passivation reaction that cancels out the charge of moieties on the polymer backbone. The passivation reaction ( 95 ), in which ethanolamine is reacted with carboxylic groups and converts them to amides with no charge (A), restores reverse transcription activity (B) and rolling circle amplification activity (C). These in vitro experiments were performed with 0.1-0.5% (w/v) polyacrylic acid, to mimic the concentration inside the expanded gel which is ∼0.2% (8.625% (w/v) sodium acrylate before expansion corresponds to 8.625/3.5^3 or ∼0.2% with expansion factor ∼3.5, and between ∼0.1% to ∼0.5% with expansion factors between 2.5 to 4). The passivation protocol was performed as in Methods section ‘Passivation’. We validated the passivation with a range of EDC concentrations, 50-150mM, that are higher than the polyacrylic acid concentration of ∼30mM (∼0.2% polyacrylic acid); however, we didn’t want the EDC concentration to be more than a few fold higher than the polyacrylic acid concentration as EDC can react with guanine in RNA ( 96 ), and therefore high concentrations of EDC may have undesired side effects. The reverse transcription reaction was performed with M-MuLV (enzymatics) according to the manufacturer’s protocol, with 1.2kb Kanamycin Positive Control RNA (Promega) as a template, and random primers. The rolling circle amplification reaction was performed with phi29 (enzymatics) according to the manufacturer’s protocol. The template used was the 55-base long CircLigase II ssDNA Control Oligo, after circularization with CircLigase II (epicentre) according to the manufacturer’s protocol.

    Journal: bioRxiv

    Article Title: Expansion Sequencing: Spatially Precise In Situ Transcriptomics in Intact Biological Systems

    doi: 10.1101/2020.05.13.094268

    Figure Lengend Snippet: In vitro experiments demonstrating that polyacrylic acid, very similar to the polymer used in ExM, inhibits both the reverse transcription step and the rolling circle amplification step, two key enzymatic reactions of FISSEQ, but that the enzymatic activity can be restored with a passivation reaction that cancels out the charge of moieties on the polymer backbone. The passivation reaction ( 95 ), in which ethanolamine is reacted with carboxylic groups and converts them to amides with no charge (A), restores reverse transcription activity (B) and rolling circle amplification activity (C). These in vitro experiments were performed with 0.1-0.5% (w/v) polyacrylic acid, to mimic the concentration inside the expanded gel which is ∼0.2% (8.625% (w/v) sodium acrylate before expansion corresponds to 8.625/3.5^3 or ∼0.2% with expansion factor ∼3.5, and between ∼0.1% to ∼0.5% with expansion factors between 2.5 to 4). The passivation protocol was performed as in Methods section ‘Passivation’. We validated the passivation with a range of EDC concentrations, 50-150mM, that are higher than the polyacrylic acid concentration of ∼30mM (∼0.2% polyacrylic acid); however, we didn’t want the EDC concentration to be more than a few fold higher than the polyacrylic acid concentration as EDC can react with guanine in RNA ( 96 ), and therefore high concentrations of EDC may have undesired side effects. The reverse transcription reaction was performed with M-MuLV (enzymatics) according to the manufacturer’s protocol, with 1.2kb Kanamycin Positive Control RNA (Promega) as a template, and random primers. The rolling circle amplification reaction was performed with phi29 (enzymatics) according to the manufacturer’s protocol. The template used was the 55-base long CircLigase II ssDNA Control Oligo, after circularization with CircLigase II (epicentre) according to the manufacturer’s protocol.

    Article Snippet: Next, rolling circle amplification was performed overnight at 30°C with 1 U/µl phi29 DNA polymerase (Enzymatics, cat. no. P7020-HC-L), 250 µM dNTP, 40 µM aminoallyl dUTP and 1X Phi29 buffer.

    Techniques: In Vitro, Amplification, Activity Assay, Concentration Assay, Positive Control