bst 2 0 dna polymerase  (New England Biolabs)


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    Name:
    Bst 2 0 DNA Polymerase
    Description:
    Bst 2 0 DNA Polymerase 8 000 units
    Catalog Number:
    m0537l
    Price:
    283
    Size:
    8 000 units
    Category:
    Thermostable DNA Polymerases
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    Structured Review

    New England Biolabs bst 2 0 dna polymerase
    Bst 2 0 DNA Polymerase
    Bst 2 0 DNA Polymerase 8 000 units
    https://www.bioz.com/result/bst 2 0 dna polymerase/product/New England Biolabs
    Average 99 stars, based on 176 article reviews
    Price from $9.99 to $1999.99
    bst 2 0 dna polymerase - by Bioz Stars, 2020-08
    99/100 stars

    Images

    1) Product Images from "Genome Filtering for New DNA Biomarkers of Loa loa Infection Suitable for Loop-Mediated Isothermal Amplification"

    Article Title: Genome Filtering for New DNA Biomarkers of Loa loa Infection Suitable for Loop-Mediated Isothermal Amplification

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0139286

    Sensitivity of the L . loa RF4-based LAMP assay. Dilutions of genomic L . loa DNA were amplified with the RF4 primer set and Bst 2.0 DNA polymerase in the absence (blue) or presence (red) of the V/DEF additive. Two ul of each dilution was added to LAMP reactions. The average threshold time, defined as the time at which the change in turbidity over time (dT/dt) reaches a value of 0.1, is plotted against the concentration of L . loa DNA (ng/ml). All reactions were performed in triplicate. Error bars represent the standard deviation at each point.
    Figure Legend Snippet: Sensitivity of the L . loa RF4-based LAMP assay. Dilutions of genomic L . loa DNA were amplified with the RF4 primer set and Bst 2.0 DNA polymerase in the absence (blue) or presence (red) of the V/DEF additive. Two ul of each dilution was added to LAMP reactions. The average threshold time, defined as the time at which the change in turbidity over time (dT/dt) reaches a value of 0.1, is plotted against the concentration of L . loa DNA (ng/ml). All reactions were performed in triplicate. Error bars represent the standard deviation at each point.

    Techniques Used: Lamp Assay, Amplification, Concentration Assay, Standard Deviation

    Detection of L . loa DNA in spiked blood samples. A two-fold dilution series of genomic L . loa DNA was prepared using uninfected human whole blood. NTCs only contained uninfected human whole blood. After DNA isolation, two μl of each dilution (or NTC) was used in LAMP reactions containing the V/DEF additive with Bst 2.0 DNA polymerase. For each experiment, all samples were assayed in triplicate. Average threshold times and standard deviations are plotted against ng DNA/ml of elution buffer.
    Figure Legend Snippet: Detection of L . loa DNA in spiked blood samples. A two-fold dilution series of genomic L . loa DNA was prepared using uninfected human whole blood. NTCs only contained uninfected human whole blood. After DNA isolation, two μl of each dilution (or NTC) was used in LAMP reactions containing the V/DEF additive with Bst 2.0 DNA polymerase. For each experiment, all samples were assayed in triplicate. Average threshold times and standard deviations are plotted against ng DNA/ml of elution buffer.

    Techniques Used: DNA Extraction

    2) Product Images from "Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Ustilago maydis"

    Article Title: Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Ustilago maydis

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-13881-4

    Single factor experiment of RealAmp. The influence of each variable on the LAMP reaction was analyzed by the amplification curve ( a ) and the melt peak ( b ). For the Bst DNA polymerase optimization, Bst polymerase quantities were adjusted to 2 U, 4 U, 6
    Figure Legend Snippet: Single factor experiment of RealAmp. The influence of each variable on the LAMP reaction was analyzed by the amplification curve ( a ) and the melt peak ( b ). For the Bst DNA polymerase optimization, Bst polymerase quantities were adjusted to 2 U, 4 U, 6

    Techniques Used: Amplification

    3) Product Images from "Real-time kinetics and high-resolution melt curves in single-molecule digital LAMP to differentiate and study specific and non-specific amplification"

    Article Title: Real-time kinetics and high-resolution melt curves in single-molecule digital LAMP to differentiate and study specific and non-specific amplification

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa099

    Specific amplification in digital single-molecule experiments using Bst 2.0. ( A ) Fluorescence micrographs of individual partitions are traced over time. For simplicity, we illustrate a subset of 250 of 20,000 possible partitions at three time points (0, 20 and 45 min). Of the 250 partitions in this micrograph, 30 partitions amplified. Partitions A and B are visible at 20 min; partition C becomes visible at 45 min. ( B ) Fluorescence micrographs of individual partitions are traced across temperatures during an HRM experiment. As the double-stranded DNA in each partition de-hybridizes, the intercalating dye is released and fluorescence decreases. ( C ) Plotting the fluorescence intensity as a function of time generates the standard amplification traces of individual partitions generated during a 90-min LAMP experiment. Orange curves correspond to partitions A–C from panel A. ( D ) Traces of fluorescence intensity as a function of temperature for individual partitions during melting experiments. By quantifying real-time intensity of individual partitions as temperature increases, melting traces are obtained. Temperature resolution is 1°C from 55–90°C, and 0.5°C from 90–95°C. ( E ) The derivative plot of panel D generates the standard melting curve. The temperature at which the derivative maximum occurs corresponds to the ‘melting point’ of the LAMP products in the individual partition. ( F ) The time each partition reached a fluorescence intensity of 250 RFU (TTP) as a function of temperature. ( G ) Maximum rate as a function of T m for each partition. ( H ) TTP as a function of maximum rate for each partition.
    Figure Legend Snippet: Specific amplification in digital single-molecule experiments using Bst 2.0. ( A ) Fluorescence micrographs of individual partitions are traced over time. For simplicity, we illustrate a subset of 250 of 20,000 possible partitions at three time points (0, 20 and 45 min). Of the 250 partitions in this micrograph, 30 partitions amplified. Partitions A and B are visible at 20 min; partition C becomes visible at 45 min. ( B ) Fluorescence micrographs of individual partitions are traced across temperatures during an HRM experiment. As the double-stranded DNA in each partition de-hybridizes, the intercalating dye is released and fluorescence decreases. ( C ) Plotting the fluorescence intensity as a function of time generates the standard amplification traces of individual partitions generated during a 90-min LAMP experiment. Orange curves correspond to partitions A–C from panel A. ( D ) Traces of fluorescence intensity as a function of temperature for individual partitions during melting experiments. By quantifying real-time intensity of individual partitions as temperature increases, melting traces are obtained. Temperature resolution is 1°C from 55–90°C, and 0.5°C from 90–95°C. ( E ) The derivative plot of panel D generates the standard melting curve. The temperature at which the derivative maximum occurs corresponds to the ‘melting point’ of the LAMP products in the individual partition. ( F ) The time each partition reached a fluorescence intensity of 250 RFU (TTP) as a function of temperature. ( G ) Maximum rate as a function of T m for each partition. ( H ) TTP as a function of maximum rate for each partition.

    Techniques Used: Amplification, Fluorescence, Generated

    Impacts of host (human) genomic DNA in human haploid genome equivalents (HHGE) on specific and non-specific amplification. Plots of T m as a function TTP using Bst 2.0 at ( A ) 0 HHGE per μl; ( B ) 0.01 HHGE per μL, ( C ) 1 HHGE per μl, ( D ) 100 HHGE per μl and ( E ) 5000 HHGE per μl; and using Bst 3.0 at ( F ) 0 HHGE per μl, ( G ) 0.01 HHGE per μl, ( H ) 1 HHGE per μl, ( I ) 100 HHGE per μl ( J ) 5000 HHGE per μl in the presence of template (blue) and NTC (red). N = 3 for all conditions, except Bst 3.0 at 0 and 100 HHGE per μl in the presence of template, where N = 6.
    Figure Legend Snippet: Impacts of host (human) genomic DNA in human haploid genome equivalents (HHGE) on specific and non-specific amplification. Plots of T m as a function TTP using Bst 2.0 at ( A ) 0 HHGE per μl; ( B ) 0.01 HHGE per μL, ( C ) 1 HHGE per μl, ( D ) 100 HHGE per μl and ( E ) 5000 HHGE per μl; and using Bst 3.0 at ( F ) 0 HHGE per μl, ( G ) 0.01 HHGE per μl, ( H ) 1 HHGE per μl, ( I ) 100 HHGE per μl ( J ) 5000 HHGE per μl in the presence of template (blue) and NTC (red). N = 3 for all conditions, except Bst 3.0 at 0 and 100 HHGE per μl in the presence of template, where N = 6.

    Techniques Used: Amplification

    4) Product Images from "Promoter RNA sequencing (PRSeq) for the massive and quantitative promoter analysis in vitro"

    Article Title: Promoter RNA sequencing (PRSeq) for the massive and quantitative promoter analysis in vitro

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-39892-x

    Schematic diagram of PRSeq (Promoter RNA Sequencing) method. ( A ) DNA pool preparation. Synthetic single-stranded DNA with a 3′-terminal hairpin structure is subjected to intra-strand fill-in DNA elongation to generate double-stranded recognition sequence for nicking enzyme, Nt.AlwI (green). The randomized promoter region is indicated by red color. After the nicking reaction, the DNA is subjected to DNA elongation employing Bst 2.0 DNA polymerase which has strong strand displacement activity. In this reaction, the 5′-DNA fragment generated by the nicking acts as a primer, and the random sequence in the promoter region is copied to downstream of the promoter. Finally, a primer sequence for reverse transcription is attached by ligation. Note that a part of non-randomized region of the promoter is not copied, and the copied sequence should not act as the promoter. ( B ) In vitro transcription (IVTX) of the DNA pool. The DNA pool prepared by the reactions in ( A ) is applied for IVTX. The template DNA molecules with strong and weak promoters (shown by red and purple colors) generate high and low copies of RNA, respectively, whereas most of the DNA with non-functional promoter variant (shown by blue color) do not. Since the RNA copy number is in proportion to the promoter activity linearly, activity of each promoter sequence can be evaluated by RNA sequencing of the transcript pool.
    Figure Legend Snippet: Schematic diagram of PRSeq (Promoter RNA Sequencing) method. ( A ) DNA pool preparation. Synthetic single-stranded DNA with a 3′-terminal hairpin structure is subjected to intra-strand fill-in DNA elongation to generate double-stranded recognition sequence for nicking enzyme, Nt.AlwI (green). The randomized promoter region is indicated by red color. After the nicking reaction, the DNA is subjected to DNA elongation employing Bst 2.0 DNA polymerase which has strong strand displacement activity. In this reaction, the 5′-DNA fragment generated by the nicking acts as a primer, and the random sequence in the promoter region is copied to downstream of the promoter. Finally, a primer sequence for reverse transcription is attached by ligation. Note that a part of non-randomized region of the promoter is not copied, and the copied sequence should not act as the promoter. ( B ) In vitro transcription (IVTX) of the DNA pool. The DNA pool prepared by the reactions in ( A ) is applied for IVTX. The template DNA molecules with strong and weak promoters (shown by red and purple colors) generate high and low copies of RNA, respectively, whereas most of the DNA with non-functional promoter variant (shown by blue color) do not. Since the RNA copy number is in proportion to the promoter activity linearly, activity of each promoter sequence can be evaluated by RNA sequencing of the transcript pool.

    Techniques Used: RNA Sequencing Assay, Sequencing, Activity Assay, Generated, Ligation, Activated Clotting Time Assay, In Vitro, Functional Assay, Variant Assay

    5) Product Images from "A Simple Isothermal DNA Amplification Method to Screen Black Flies for Onchocerca volvulus Infection"

    Article Title: A Simple Isothermal DNA Amplification Method to Screen Black Flies for Onchocerca volvulus Infection

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0108927

    Species-specific LAMP assay targeting OvGST1a . Genomic DNAs from O. volvulus (Ov), O. ochengi (Oo), L. loa (Lloa), Bos taurus (Bos), Simulium vitattum (Sv) and Homo sapiens (Hsa) were used as template in the LAMP assay. Detection using turbidity ( A ). Each curve represents the calculated average of triplicate turbidity curves generated with various genomic DNAs (1 ng) using Bst 2.0 DNA polymerase. Turbidity was observed only using O. volvulus genomic DNA as template. Detection using hydroxy naphthol blue ( B ). Genomic DNAs from O. volvulus (Ov), O. ochengi (Oo), L. loa (Ll), Bovine (Bt), Simulium vitattum (Sv) and human (Hs) were used as template in a PCR assay ( C ). Amplification product (∼200 bp) using LAMP primers F3 and B3 was obtained when O. volvulus genomic DNA was used (indicated by arrow). As a positive control, an actin gene fragment was PCR amplified from (Ov), (Oo), (Ll), (Bt), (Sv) and Hs DNAs using degenerate primers ( D ). Agarose gel showing amplification of a 244 bp fragment of the actin gene. Water was used in a non-template control (NTC) in all experiments. Molecular weight marker (MW) is indicated.
    Figure Legend Snippet: Species-specific LAMP assay targeting OvGST1a . Genomic DNAs from O. volvulus (Ov), O. ochengi (Oo), L. loa (Lloa), Bos taurus (Bos), Simulium vitattum (Sv) and Homo sapiens (Hsa) were used as template in the LAMP assay. Detection using turbidity ( A ). Each curve represents the calculated average of triplicate turbidity curves generated with various genomic DNAs (1 ng) using Bst 2.0 DNA polymerase. Turbidity was observed only using O. volvulus genomic DNA as template. Detection using hydroxy naphthol blue ( B ). Genomic DNAs from O. volvulus (Ov), O. ochengi (Oo), L. loa (Ll), Bovine (Bt), Simulium vitattum (Sv) and human (Hs) were used as template in a PCR assay ( C ). Amplification product (∼200 bp) using LAMP primers F3 and B3 was obtained when O. volvulus genomic DNA was used (indicated by arrow). As a positive control, an actin gene fragment was PCR amplified from (Ov), (Oo), (Ll), (Bt), (Sv) and Hs DNAs using degenerate primers ( D ). Agarose gel showing amplification of a 244 bp fragment of the actin gene. Water was used in a non-template control (NTC) in all experiments. Molecular weight marker (MW) is indicated.

    Techniques Used: Lamp Assay, Generated, Polymerase Chain Reaction, Amplification, Positive Control, Agarose Gel Electrophoresis, Molecular Weight, Marker

    6) Product Images from "A nuclease-polymerase chain reaction enables amplification of probes used for capture-based DNA target enrichment"

    Article Title: A nuclease-polymerase chain reaction enables amplification of probes used for capture-based DNA target enrichment

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkz870

    Diagram illustrating the BST–DSN reaction process. ( A ) When dsDNA is used as input in a BST–DSN reaction, the nuclease DSN nicks one strand of dsDNA to create a recognition site for BST polymerase which then synthesizes a complement of the opposite DNA strand while displacing the parent strand. The displaced-sense (or anti-sense) DNA strands subsequently can re-hybridize to complementary strands and form daughter dsDNA. Subsequent DSN nicking and BST amplification generated an exponential amplification of daughter dsDNA while progressively reducing the resulting DNA size. ( B ) When single stranded DNA (ssDNA) or long oligonucleotides are used as input in BST–DSN reaction, the ssDNA is first subjected to a TdT reaction in the presence of dATP to generate a poly-A tail on the 3′ end. The unpurified TdT product is then used as input in a BST–DSN reaction in the presence of an anchored-oligo-dT which is extended by BST to create dsDNA as a first step in the reaction.
    Figure Legend Snippet: Diagram illustrating the BST–DSN reaction process. ( A ) When dsDNA is used as input in a BST–DSN reaction, the nuclease DSN nicks one strand of dsDNA to create a recognition site for BST polymerase which then synthesizes a complement of the opposite DNA strand while displacing the parent strand. The displaced-sense (or anti-sense) DNA strands subsequently can re-hybridize to complementary strands and form daughter dsDNA. Subsequent DSN nicking and BST amplification generated an exponential amplification of daughter dsDNA while progressively reducing the resulting DNA size. ( B ) When single stranded DNA (ssDNA) or long oligonucleotides are used as input in BST–DSN reaction, the ssDNA is first subjected to a TdT reaction in the presence of dATP to generate a poly-A tail on the 3′ end. The unpurified TdT product is then used as input in a BST–DSN reaction in the presence of an anchored-oligo-dT which is extended by BST to create dsDNA as a first step in the reaction.

    Techniques Used: Amplification, Generated

    7) Product Images from "A novel loop-mediated isothermal amplification-based test for detecting Neospora caninum DNA"

    Article Title: A novel loop-mediated isothermal amplification-based test for detecting Neospora caninum DNA

    Journal: Parasites & Vectors

    doi: 10.1186/s13071-017-2549-y

    Using the LAMP assay for detecting N. caninum plasmid DNA containing the Nc-5 region on a SYBRsafe stained agarose gel. a The effects of reaction time. Lanes 1 and 2: 60 min; Lanes 3 and 4: 50 min; Lanes 5 and 6: 40 min. Lanes 2, 4 and 6 represent negative controls for each reaction time. b Optimizing LAMP assay conditions. Lanes 1 and 2: 30 min; Lanes 3 and 4: 20 min. Lanes 2 and 4 are the negative control for each reaction time. c Restriction analysis of N. caninum LAMP products amplified from plasmid DNA containing the Nc-5 region. The digestion products were run on a 3% agarose gel. Lane 1: N. caninum LAMP product; Lane 2: Msp I digestion of N. caninum product (132–218 bp bands, according to predicted size). Lane MM in a , b and c is the HyperLadder II (Bioline) DNA molecular marker
    Figure Legend Snippet: Using the LAMP assay for detecting N. caninum plasmid DNA containing the Nc-5 region on a SYBRsafe stained agarose gel. a The effects of reaction time. Lanes 1 and 2: 60 min; Lanes 3 and 4: 50 min; Lanes 5 and 6: 40 min. Lanes 2, 4 and 6 represent negative controls for each reaction time. b Optimizing LAMP assay conditions. Lanes 1 and 2: 30 min; Lanes 3 and 4: 20 min. Lanes 2 and 4 are the negative control for each reaction time. c Restriction analysis of N. caninum LAMP products amplified from plasmid DNA containing the Nc-5 region. The digestion products were run on a 3% agarose gel. Lane 1: N. caninum LAMP product; Lane 2: Msp I digestion of N. caninum product (132–218 bp bands, according to predicted size). Lane MM in a , b and c is the HyperLadder II (Bioline) DNA molecular marker

    Techniques Used: Lamp Assay, Plasmid Preparation, Staining, Agarose Gel Electrophoresis, Negative Control, Amplification, Marker

    LAMP and semi-nested PCR Limit of Detection (LoD). a LAMP reaction. b Semi-nested PCR. Ten-fold serial dilutions of plasmid DNA were used in both a and b ; they contained the Nc-5 region for detecting N. caninum by agarose gel electrophoresis analysis. From left to right: Lane MM: HyperLadder II (Bioline) DNA molecular marker; Lane 1: amplification of 1 ng plasmid DNA containing the cloned N. caninum fragment; Lanes 2–7: 10-fold serial dilutions of N. caninum plasmid DNA (10 −1 to 10 −6 ng); Lane 8: negative control without target DNA. Semi-nested PCR products showed specific amplification of N. caninum , having 10 −5 ng LoD whereas LAMP LoD was 10 −6 ng. LAMP and semi-nested PCR LoD in both c and d using serial dilutions of N.caninum genomic DNA (NC-1 strain) by visualization on an agarose gel. c LAMP reaction. Lane MM: HyperLadder II (Bioline) DNA molecular marker; Lane 1: amplification of N. caninum genomic DNA (50 ng); Lanes 2–7: 10-fold serial dilutions (10 −1 to 10 −6 ng); Lane 8: positive control (plasmid DNA); Lane 9: negative control (no DNA template). d Semi-nested PCR. Lane MM: HyperLadder II (Bioline) DNA molecular marker; Lane 1: negative control (no DNA template); Lane 2: amplification of N. caninum genomic DNA (50 ng); Lanes 3–8: 10-fold serial dilutions (10 −1 to 10 −6 ng); Lanes 9, 10: positive controls (plasmid DNA)
    Figure Legend Snippet: LAMP and semi-nested PCR Limit of Detection (LoD). a LAMP reaction. b Semi-nested PCR. Ten-fold serial dilutions of plasmid DNA were used in both a and b ; they contained the Nc-5 region for detecting N. caninum by agarose gel electrophoresis analysis. From left to right: Lane MM: HyperLadder II (Bioline) DNA molecular marker; Lane 1: amplification of 1 ng plasmid DNA containing the cloned N. caninum fragment; Lanes 2–7: 10-fold serial dilutions of N. caninum plasmid DNA (10 −1 to 10 −6 ng); Lane 8: negative control without target DNA. Semi-nested PCR products showed specific amplification of N. caninum , having 10 −5 ng LoD whereas LAMP LoD was 10 −6 ng. LAMP and semi-nested PCR LoD in both c and d using serial dilutions of N.caninum genomic DNA (NC-1 strain) by visualization on an agarose gel. c LAMP reaction. Lane MM: HyperLadder II (Bioline) DNA molecular marker; Lane 1: amplification of N. caninum genomic DNA (50 ng); Lanes 2–7: 10-fold serial dilutions (10 −1 to 10 −6 ng); Lane 8: positive control (plasmid DNA); Lane 9: negative control (no DNA template). d Semi-nested PCR. Lane MM: HyperLadder II (Bioline) DNA molecular marker; Lane 1: negative control (no DNA template); Lane 2: amplification of N. caninum genomic DNA (50 ng); Lanes 3–8: 10-fold serial dilutions (10 −1 to 10 −6 ng); Lanes 9, 10: positive controls (plasmid DNA)

    Techniques Used: Nested PCR, Plasmid Preparation, Agarose Gel Electrophoresis, Marker, Amplification, Clone Assay, Negative Control, Positive Control

    8) Product Images from "Unusual isothermal multimerization and amplification by the strand-displacing DNA polymerases with reverse transcription activities"

    Article Title: Unusual isothermal multimerization and amplification by the strand-displacing DNA polymerases with reverse transcription activities

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-13324-0

    Verification of UIMA using different DNA polymerases. All reactions shared the same primer (RL) and template (F*R*) and were incubated for 180 min. The sequences of RL and F*R* were shown in Table S1 . ( A ) Real-time fluorescence change in reactions using a series of Bst DNA polymerases ( Bst LF, Bst 2.0, Bst 2.0 WS, and Bst 3.0) at 63 °C. No-primer controls (NPCs) were shown in Fig. S5 . ( B ) Real-time fluorescence change in reactions using non- Bst polymerases (Bsm, BcaBEST, Vent(exo-), and z-Taq) at 63 °C. No-primer controls (NPCs) were shown in Fig. S5 . ( C ) Temperature gradients assay for the products of reactions using the polymerases with negative results in ( B ). The products were analyzed by 2.5% agarose gel electrophoresis. NTC and NPC for Bsm were performed at 56 °C. NTCs and NPCs for Vent (exo-) and z-Taq were performed at 63 °C. The groping of gels cropped from different gels. Exposure time is 5 s. ( D ) Temperature gradients assay for the products of reactions using the polymerases of Klenow(exo-) and Klenow. The products were analyzed by 2.5% agarose gel electrophoresis. Their NTCs and NPCs were performed at 43 °C. M1 and M2: DNA Marker. NTC: no-target control; NPC: no-primer control. The groping of gels cropped from different gels. Exposure time is 5 s. The full-length gels are presented in Supplementary Figure S7 .
    Figure Legend Snippet: Verification of UIMA using different DNA polymerases. All reactions shared the same primer (RL) and template (F*R*) and were incubated for 180 min. The sequences of RL and F*R* were shown in Table S1 . ( A ) Real-time fluorescence change in reactions using a series of Bst DNA polymerases ( Bst LF, Bst 2.0, Bst 2.0 WS, and Bst 3.0) at 63 °C. No-primer controls (NPCs) were shown in Fig. S5 . ( B ) Real-time fluorescence change in reactions using non- Bst polymerases (Bsm, BcaBEST, Vent(exo-), and z-Taq) at 63 °C. No-primer controls (NPCs) were shown in Fig. S5 . ( C ) Temperature gradients assay for the products of reactions using the polymerases with negative results in ( B ). The products were analyzed by 2.5% agarose gel electrophoresis. NTC and NPC for Bsm were performed at 56 °C. NTCs and NPCs for Vent (exo-) and z-Taq were performed at 63 °C. The groping of gels cropped from different gels. Exposure time is 5 s. ( D ) Temperature gradients assay for the products of reactions using the polymerases of Klenow(exo-) and Klenow. The products were analyzed by 2.5% agarose gel electrophoresis. Their NTCs and NPCs were performed at 43 °C. M1 and M2: DNA Marker. NTC: no-target control; NPC: no-primer control. The groping of gels cropped from different gels. Exposure time is 5 s. The full-length gels are presented in Supplementary Figure S7 .

    Techniques Used: Incubation, Fluorescence, Agarose Gel Electrophoresis, Marker

    Real-time fluorescence and electrophoresis analysis of UIMA. All reactions shared the same primer (RL) or template (F*R*), and were incubated for 180 min. The sequences of RL and F*R* were shown in Table S1 . ( A ) The results of real-time fluorescence obtained from the reactions that contained 10 nM template, 1.6 μM primer, and 3.2 U Bst WS DNA polymerase at 63 °C for 180 min. Each test was in triplicate. ( B ) Time course of the UIMA assay. 2.5% agarose gel electrophoresis shows the products of UIMA. The assay time was varied from 30–120 minutes as indicated above each lane. M1 and M2: DNA marker; NEC, NTC and NPC were all incubated for 120 min. Exposure time is 5 s. ( C ) Extension status of template and primer. Template and primer were labeled with the FAM fluorophore. 1: reaction with FAM-labeled primer and template but no Bst ; 2: with FAM-labeled primer, non-labeled template, and Bst ; 3: with FAM-labeled primer and template and Bst ; 4: with FAM-labeled primer and Bst but no template; 5: with non-labeled primer and template, and Bst ; 6: with non-labeled primer, FAM-labeled template, and Bst ; 7: with non-labeled primer and Bst but no template; 8: with non-labeled template and Bst but no primer; 9: with FAM-labeled template and Bst but no primer. All the reactions were incubated at 63 °C for 180 min. Their products were analyzed by 17% denatured polyacrylamide gel electrophoresis (DPAGE). NEC (no-enzyme control): the reaction without Bst 2.0 WS DNA polymerase. NTC (no-template control): control reaction just lacked the template; NPC (no-primer control): control reaction lacked primer RL. Horizontal arrows denoted the 5′-3′ direction of sequences. Exposure time is 5 s. The full-length gels are presented in Supplementary Figure S1 .
    Figure Legend Snippet: Real-time fluorescence and electrophoresis analysis of UIMA. All reactions shared the same primer (RL) or template (F*R*), and were incubated for 180 min. The sequences of RL and F*R* were shown in Table S1 . ( A ) The results of real-time fluorescence obtained from the reactions that contained 10 nM template, 1.6 μM primer, and 3.2 U Bst WS DNA polymerase at 63 °C for 180 min. Each test was in triplicate. ( B ) Time course of the UIMA assay. 2.5% agarose gel electrophoresis shows the products of UIMA. The assay time was varied from 30–120 minutes as indicated above each lane. M1 and M2: DNA marker; NEC, NTC and NPC were all incubated for 120 min. Exposure time is 5 s. ( C ) Extension status of template and primer. Template and primer were labeled with the FAM fluorophore. 1: reaction with FAM-labeled primer and template but no Bst ; 2: with FAM-labeled primer, non-labeled template, and Bst ; 3: with FAM-labeled primer and template and Bst ; 4: with FAM-labeled primer and Bst but no template; 5: with non-labeled primer and template, and Bst ; 6: with non-labeled primer, FAM-labeled template, and Bst ; 7: with non-labeled primer and Bst but no template; 8: with non-labeled template and Bst but no primer; 9: with FAM-labeled template and Bst but no primer. All the reactions were incubated at 63 °C for 180 min. Their products were analyzed by 17% denatured polyacrylamide gel electrophoresis (DPAGE). NEC (no-enzyme control): the reaction without Bst 2.0 WS DNA polymerase. NTC (no-template control): control reaction just lacked the template; NPC (no-primer control): control reaction lacked primer RL. Horizontal arrows denoted the 5′-3′ direction of sequences. Exposure time is 5 s. The full-length gels are presented in Supplementary Figure S1 .

    Techniques Used: Fluorescence, Electrophoresis, Incubation, Agarose Gel Electrophoresis, Marker, Labeling, Polyacrylamide Gel Electrophoresis

    9) Product Images from "Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Ustilago maydis"

    Article Title: Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Ustilago maydis

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-13881-4

    Single factor experiment of RealAmp. The influence of each variable on the LAMP reaction was analyzed by the amplification curve ( a ) and the melt peak ( b ). For the Bst DNA polymerase optimization, Bst polymerase quantities were adjusted to 2 U, 4 U, 6 U and 8 U, respectively. Ratios of inner to outer primers were set at 2:1, 4:1, 6:1 and 8:1 with the outer primer concentration fixed to 0.2 μM to optimize the primer ratios. Mg 2+ concentrations in the LAMP reactions were varied from 5 mM, to 8 mM for the optimization of Mg 2+ .
    Figure Legend Snippet: Single factor experiment of RealAmp. The influence of each variable on the LAMP reaction was analyzed by the amplification curve ( a ) and the melt peak ( b ). For the Bst DNA polymerase optimization, Bst polymerase quantities were adjusted to 2 U, 4 U, 6 U and 8 U, respectively. Ratios of inner to outer primers were set at 2:1, 4:1, 6:1 and 8:1 with the outer primer concentration fixed to 0.2 μM to optimize the primer ratios. Mg 2+ concentrations in the LAMP reactions were varied from 5 mM, to 8 mM for the optimization of Mg 2+ .

    Techniques Used: Amplification, Concentration Assay

    10) Product Images from "Enhancement of Polymerase Activity of the Large Fragment in DNA Polymerase I from Geobacillus stearothermophilus by Site-Directed Mutagenesis at the Active Site"

    Article Title: Enhancement of Polymerase Activity of the Large Fragment in DNA Polymerase I from Geobacillus stearothermophilus by Site-Directed Mutagenesis at the Active Site

    Journal: BioMed Research International

    doi: 10.1155/2016/2906484

    SDS-PAGE analysis of recombinant Bst DNA pol LF and mutant enzymes purified by one-step affinity chromatography. M: protein ladder marker shown in kDa on the left sides of panels; 1: WT Bst DNA pol LF; 2: LF mutant D540A; 3: LF mutant D540E; 4: LF mutant G310A; 5: LF mutant G310L; 6: LF mutant R412A; 7: LF mutant R412E; 8: LF mutant K416A; 9: LF mutant K416D; 10: LF mutant G310A-D540E; 11: LF mutant G310L-D540E; 12: commercial Bst 2.0 DNA polymerase.
    Figure Legend Snippet: SDS-PAGE analysis of recombinant Bst DNA pol LF and mutant enzymes purified by one-step affinity chromatography. M: protein ladder marker shown in kDa on the left sides of panels; 1: WT Bst DNA pol LF; 2: LF mutant D540A; 3: LF mutant D540E; 4: LF mutant G310A; 5: LF mutant G310L; 6: LF mutant R412A; 7: LF mutant R412E; 8: LF mutant K416A; 9: LF mutant K416D; 10: LF mutant G310A-D540E; 11: LF mutant G310L-D540E; 12: commercial Bst 2.0 DNA polymerase.

    Techniques Used: SDS Page, Recombinant, Mutagenesis, Purification, Affinity Chromatography, Marker

    Visual IMSA assay and sensitivity evaluation of IMSA assay to test EV71. (a) Visual detection was performed with IMSA assay by adding HNB dye prior to amplification procedure. The color of sky blue demonstrates positive reactions while the color of violet demonstrates negative reactions. The number of the tube indicates IMSA reaction, respectively, as follows: 1: commercial Bst 2.0 DNA polymerase; 2: WT of Bst DNA pol LF; 3: LF mutant D540E; 4: LF mutant G310A; 5: LF mutant G310L; 6: LF mutant D540A; 7: LF mutant R412A; 8: LF mutant R412E; 9: LF mutant K416A; 10: LF mutant K416D; 11: LF mutant G310A-D540E; 12: LF mutant G310L-D540E; 13: negative control. (b) Fluorescence signals on real-time PCR instrument. Fluorescence values and curves were evaluated with Deaou-308C constant temperature fluorescence detection equipment. The reaction order in (b) table was arranged the same as tubes number in (a). The sign of “+” indicates positive reactions while “−” indicates negative reactions. Reactions 1–5 were able to amplify VP1 gene to detect EV71. The curves in different colors represent distinct proteins in IMSA reaction. Curve in black and “reaction 1” represent commercial Bst 2.0 DNA polymerase. Curve in green and “reaction 2” represent WT of Bst DNA pol LF. Curve in orange and “reaction 3” represent LF mutant D540E. Curve in pink and “reaction 4” represent LF mutant G310A. Curve in red and “reaction 5” represent LF mutant G310L.
    Figure Legend Snippet: Visual IMSA assay and sensitivity evaluation of IMSA assay to test EV71. (a) Visual detection was performed with IMSA assay by adding HNB dye prior to amplification procedure. The color of sky blue demonstrates positive reactions while the color of violet demonstrates negative reactions. The number of the tube indicates IMSA reaction, respectively, as follows: 1: commercial Bst 2.0 DNA polymerase; 2: WT of Bst DNA pol LF; 3: LF mutant D540E; 4: LF mutant G310A; 5: LF mutant G310L; 6: LF mutant D540A; 7: LF mutant R412A; 8: LF mutant R412E; 9: LF mutant K416A; 10: LF mutant K416D; 11: LF mutant G310A-D540E; 12: LF mutant G310L-D540E; 13: negative control. (b) Fluorescence signals on real-time PCR instrument. Fluorescence values and curves were evaluated with Deaou-308C constant temperature fluorescence detection equipment. The reaction order in (b) table was arranged the same as tubes number in (a). The sign of “+” indicates positive reactions while “−” indicates negative reactions. Reactions 1–5 were able to amplify VP1 gene to detect EV71. The curves in different colors represent distinct proteins in IMSA reaction. Curve in black and “reaction 1” represent commercial Bst 2.0 DNA polymerase. Curve in green and “reaction 2” represent WT of Bst DNA pol LF. Curve in orange and “reaction 3” represent LF mutant D540E. Curve in pink and “reaction 4” represent LF mutant G310A. Curve in red and “reaction 5” represent LF mutant G310L.

    Techniques Used: Amplification, Mutagenesis, Negative Control, Fluorescence, Real-time Polymerase Chain Reaction

    HPLC analysis of polymerization efficiency of Bst DNA polymerases in IMSA assay. (a) Negative control: retention time of dCTP is 16.583 min and the peak area is 2459.42; (b) the LF mutant G310L: retention time of dCTP is 17.447 min and the peak area is 1781.62; (c) Bst DNA pol WT: retention time of dCTP is 17.059 min and the peak area is 1840.69; (d) commercialized Bst 2.0 DNA polymerase: retention time of dCTP is 17.454 min and the peak area is 1941.52.
    Figure Legend Snippet: HPLC analysis of polymerization efficiency of Bst DNA polymerases in IMSA assay. (a) Negative control: retention time of dCTP is 16.583 min and the peak area is 2459.42; (b) the LF mutant G310L: retention time of dCTP is 17.447 min and the peak area is 1781.62; (c) Bst DNA pol WT: retention time of dCTP is 17.059 min and the peak area is 1840.69; (d) commercialized Bst 2.0 DNA polymerase: retention time of dCTP is 17.454 min and the peak area is 1941.52.

    Techniques Used: High Performance Liquid Chromatography, Negative Control, Mutagenesis

    Related Articles

    Polymerase Chain Reaction:

    Article Title: A nuclease-polymerase chain reaction enables amplification of probes used for capture-based DNA target enrichment
    Article Snippet: .. BST–DSN reaction using PCR products as input BST 2.0 DNA polymerase (BST) and DSN were purchased from NEB and Sapphire North America, respectively. ..

    Amplification:

    Article Title: A novel loop-mediated isothermal amplification-based test for detecting Neospora caninum DNA
    Article Snippet: .. LAMP reaction Bst 2.0 DNA Polymerase (New England Biolabs, Herts, UK), having strand displacement activity, was used for LAMP assay amplification at 25 μl final reaction volume. .. Optimization of LAMP assay conditions was carried out through evaluation of reagents concentrations following ranges reported in the literature for pathogen detection [ , ] and the manufacturer’s instructions.

    Activity Assay:

    Article Title: A novel loop-mediated isothermal amplification-based test for detecting Neospora caninum DNA
    Article Snippet: .. LAMP reaction Bst 2.0 DNA Polymerase (New England Biolabs, Herts, UK), having strand displacement activity, was used for LAMP assay amplification at 25 μl final reaction volume. .. Optimization of LAMP assay conditions was carried out through evaluation of reagents concentrations following ranges reported in the literature for pathogen detection [ , ] and the manufacturer’s instructions.

    Lamp Assay:

    Article Title: A novel loop-mediated isothermal amplification-based test for detecting Neospora caninum DNA
    Article Snippet: .. LAMP reaction Bst 2.0 DNA Polymerase (New England Biolabs, Herts, UK), having strand displacement activity, was used for LAMP assay amplification at 25 μl final reaction volume. .. Optimization of LAMP assay conditions was carried out through evaluation of reagents concentrations following ranges reported in the literature for pathogen detection [ , ] and the manufacturer’s instructions.

    Article Title: A Simple Isothermal DNA Amplification Method to Screen Black Flies for Onchocerca volvulus Infection
    Article Snippet: .. LAMP assay LAMP reactions were performed in a final volume of 25 µL reaction buffer [10 mM Tris–HCl (pH 8.8), 50 mM KCl, 10 mM (NH4 )2 SO4 , 8 mM MgSO4 , and 0.1% Tween 20], 8 U Bst 2.0 DNA polymerase (New England Biolabs, Ipswich, MA, USA), (1.4 mM) of each deoxynucleoside triphosphate (dNTP), 1.6 mM of each FIP and BIP primer, 0.2 mM of each F3 and B3 primer, 0.4 mM of FLP and BLP, and 2 µL of target DNA. .. Reactions were carried out using either a Loop Amp Realtime Turbidimeter (LA-320c, Eiken Chemical Co, Japan) or a 2720 Thermocycler (Applied Biosystems, USA) set at a constant temperature for colorimetric detection.

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    New England Biolabs bst 2 0 dna polymerase
    Sensitivity of the L . loa RF4-based LAMP assay. Dilutions of genomic L . loa <t>DNA</t> were amplified with the RF4 primer set and Bst 2.0 DNA polymerase in the absence (blue) or presence (red) of the V/DEF additive. Two ul of each dilution was added to LAMP reactions. The average threshold time, defined as the time at which the change in turbidity over time (dT/dt) reaches a value of 0.1, is plotted against the concentration of L . loa DNA (ng/ml). All reactions were performed in triplicate. Error bars represent the standard deviation at each point.
    Bst 2 0 Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 135 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sensitivity of the L . loa RF4-based LAMP assay. Dilutions of genomic L . loa DNA were amplified with the RF4 primer set and Bst 2.0 DNA polymerase in the absence (blue) or presence (red) of the V/DEF additive. Two ul of each dilution was added to LAMP reactions. The average threshold time, defined as the time at which the change in turbidity over time (dT/dt) reaches a value of 0.1, is plotted against the concentration of L . loa DNA (ng/ml). All reactions were performed in triplicate. Error bars represent the standard deviation at each point.

    Journal: PLoS ONE

    Article Title: Genome Filtering for New DNA Biomarkers of Loa loa Infection Suitable for Loop-Mediated Isothermal Amplification

    doi: 10.1371/journal.pone.0139286

    Figure Lengend Snippet: Sensitivity of the L . loa RF4-based LAMP assay. Dilutions of genomic L . loa DNA were amplified with the RF4 primer set and Bst 2.0 DNA polymerase in the absence (blue) or presence (red) of the V/DEF additive. Two ul of each dilution was added to LAMP reactions. The average threshold time, defined as the time at which the change in turbidity over time (dT/dt) reaches a value of 0.1, is plotted against the concentration of L . loa DNA (ng/ml). All reactions were performed in triplicate. Error bars represent the standard deviation at each point.

    Article Snippet: LAMP Assays LAMP reactions contained 1.6 μM each of primers FIP and BIP, 0.2 μM each of F3 and B3, 0.4 μM each of LF and FB, 1.4 mM of each dNTP, 20 mM Tris-HCl (pH 8.8), 50 mM KCl, 10 mM (NH4 )2 SO4 , 8 mM MgSO4 , 0.1% Tween-20 and 8 U of Bst 2.0 DNA Polymerase (New England Biolabs) mixed with one of several template DNAs, or 10 mM Tris-HCl (pH 8), 0.1 mM EDTA for non-template controls (NTC), in a total volume of 25 μl.

    Techniques: Lamp Assay, Amplification, Concentration Assay, Standard Deviation

    Detection of L . loa DNA in spiked blood samples. A two-fold dilution series of genomic L . loa DNA was prepared using uninfected human whole blood. NTCs only contained uninfected human whole blood. After DNA isolation, two μl of each dilution (or NTC) was used in LAMP reactions containing the V/DEF additive with Bst 2.0 DNA polymerase. For each experiment, all samples were assayed in triplicate. Average threshold times and standard deviations are plotted against ng DNA/ml of elution buffer.

    Journal: PLoS ONE

    Article Title: Genome Filtering for New DNA Biomarkers of Loa loa Infection Suitable for Loop-Mediated Isothermal Amplification

    doi: 10.1371/journal.pone.0139286

    Figure Lengend Snippet: Detection of L . loa DNA in spiked blood samples. A two-fold dilution series of genomic L . loa DNA was prepared using uninfected human whole blood. NTCs only contained uninfected human whole blood. After DNA isolation, two μl of each dilution (or NTC) was used in LAMP reactions containing the V/DEF additive with Bst 2.0 DNA polymerase. For each experiment, all samples were assayed in triplicate. Average threshold times and standard deviations are plotted against ng DNA/ml of elution buffer.

    Article Snippet: LAMP Assays LAMP reactions contained 1.6 μM each of primers FIP and BIP, 0.2 μM each of F3 and B3, 0.4 μM each of LF and FB, 1.4 mM of each dNTP, 20 mM Tris-HCl (pH 8.8), 50 mM KCl, 10 mM (NH4 )2 SO4 , 8 mM MgSO4 , 0.1% Tween-20 and 8 U of Bst 2.0 DNA Polymerase (New England Biolabs) mixed with one of several template DNAs, or 10 mM Tris-HCl (pH 8), 0.1 mM EDTA for non-template controls (NTC), in a total volume of 25 μl.

    Techniques: DNA Extraction

    Single factor experiment of RealAmp. The influence of each variable on the LAMP reaction was analyzed by the amplification curve ( a ) and the melt peak ( b ). For the Bst DNA polymerase optimization, Bst polymerase quantities were adjusted to 2 U, 4 U, 6

    Journal: Scientific Reports

    Article Title: Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Ustilago maydis

    doi: 10.1038/s41598-017-13881-4

    Figure Lengend Snippet: Single factor experiment of RealAmp. The influence of each variable on the LAMP reaction was analyzed by the amplification curve ( a ) and the melt peak ( b ). For the Bst DNA polymerase optimization, Bst polymerase quantities were adjusted to 2 U, 4 U, 6

    Article Snippet: This optimized reaction mixture (total, 25 μl) contained 12.5 μl 2 × Bst DNA polymerase (NEB, Ipswich, MA, USA) reaction buffer [1 × containing 1.6 mM dNTPs, 1 M betaine, 4 mM MgSO4 , 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2 SO4 , and 0.1% Triton X-20 (Sigma-Aldrich Inc., Saint Louis, USA), Double Helix Tech.

    Techniques: Amplification

    Specific amplification in digital single-molecule experiments using Bst 2.0. ( A ) Fluorescence micrographs of individual partitions are traced over time. For simplicity, we illustrate a subset of 250 of 20,000 possible partitions at three time points (0, 20 and 45 min). Of the 250 partitions in this micrograph, 30 partitions amplified. Partitions A and B are visible at 20 min; partition C becomes visible at 45 min. ( B ) Fluorescence micrographs of individual partitions are traced across temperatures during an HRM experiment. As the double-stranded DNA in each partition de-hybridizes, the intercalating dye is released and fluorescence decreases. ( C ) Plotting the fluorescence intensity as a function of time generates the standard amplification traces of individual partitions generated during a 90-min LAMP experiment. Orange curves correspond to partitions A–C from panel A. ( D ) Traces of fluorescence intensity as a function of temperature for individual partitions during melting experiments. By quantifying real-time intensity of individual partitions as temperature increases, melting traces are obtained. Temperature resolution is 1°C from 55–90°C, and 0.5°C from 90–95°C. ( E ) The derivative plot of panel D generates the standard melting curve. The temperature at which the derivative maximum occurs corresponds to the ‘melting point’ of the LAMP products in the individual partition. ( F ) The time each partition reached a fluorescence intensity of 250 RFU (TTP) as a function of temperature. ( G ) Maximum rate as a function of T m for each partition. ( H ) TTP as a function of maximum rate for each partition.

    Journal: Nucleic Acids Research

    Article Title: Real-time kinetics and high-resolution melt curves in single-molecule digital LAMP to differentiate and study specific and non-specific amplification

    doi: 10.1093/nar/gkaa099

    Figure Lengend Snippet: Specific amplification in digital single-molecule experiments using Bst 2.0. ( A ) Fluorescence micrographs of individual partitions are traced over time. For simplicity, we illustrate a subset of 250 of 20,000 possible partitions at three time points (0, 20 and 45 min). Of the 250 partitions in this micrograph, 30 partitions amplified. Partitions A and B are visible at 20 min; partition C becomes visible at 45 min. ( B ) Fluorescence micrographs of individual partitions are traced across temperatures during an HRM experiment. As the double-stranded DNA in each partition de-hybridizes, the intercalating dye is released and fluorescence decreases. ( C ) Plotting the fluorescence intensity as a function of time generates the standard amplification traces of individual partitions generated during a 90-min LAMP experiment. Orange curves correspond to partitions A–C from panel A. ( D ) Traces of fluorescence intensity as a function of temperature for individual partitions during melting experiments. By quantifying real-time intensity of individual partitions as temperature increases, melting traces are obtained. Temperature resolution is 1°C from 55–90°C, and 0.5°C from 90–95°C. ( E ) The derivative plot of panel D generates the standard melting curve. The temperature at which the derivative maximum occurs corresponds to the ‘melting point’ of the LAMP products in the individual partition. ( F ) The time each partition reached a fluorescence intensity of 250 RFU (TTP) as a function of temperature. ( G ) Maximum rate as a function of T m for each partition. ( H ) TTP as a function of maximum rate for each partition.

    Article Snippet: LAMP reagents IsoAmp I (#B0537S), IsoAmp II (#B0374S), MgSO4 (#B1003S), deoxynucleotide solution (#N0447S), Bovine Serum Albumen (BSA, #B9000S0), Bst 2.0 (8,000 U/ml, #M0537S) and Bst 3.0 (8000 U/ml, #M0374S) were purchased from New England Biolabs (Ipswich, MA, USA).

    Techniques: Amplification, Fluorescence, Generated

    Impacts of host (human) genomic DNA in human haploid genome equivalents (HHGE) on specific and non-specific amplification. Plots of T m as a function TTP using Bst 2.0 at ( A ) 0 HHGE per μl; ( B ) 0.01 HHGE per μL, ( C ) 1 HHGE per μl, ( D ) 100 HHGE per μl and ( E ) 5000 HHGE per μl; and using Bst 3.0 at ( F ) 0 HHGE per μl, ( G ) 0.01 HHGE per μl, ( H ) 1 HHGE per μl, ( I ) 100 HHGE per μl ( J ) 5000 HHGE per μl in the presence of template (blue) and NTC (red). N = 3 for all conditions, except Bst 3.0 at 0 and 100 HHGE per μl in the presence of template, where N = 6.

    Journal: Nucleic Acids Research

    Article Title: Real-time kinetics and high-resolution melt curves in single-molecule digital LAMP to differentiate and study specific and non-specific amplification

    doi: 10.1093/nar/gkaa099

    Figure Lengend Snippet: Impacts of host (human) genomic DNA in human haploid genome equivalents (HHGE) on specific and non-specific amplification. Plots of T m as a function TTP using Bst 2.0 at ( A ) 0 HHGE per μl; ( B ) 0.01 HHGE per μL, ( C ) 1 HHGE per μl, ( D ) 100 HHGE per μl and ( E ) 5000 HHGE per μl; and using Bst 3.0 at ( F ) 0 HHGE per μl, ( G ) 0.01 HHGE per μl, ( H ) 1 HHGE per μl, ( I ) 100 HHGE per μl ( J ) 5000 HHGE per μl in the presence of template (blue) and NTC (red). N = 3 for all conditions, except Bst 3.0 at 0 and 100 HHGE per μl in the presence of template, where N = 6.

    Article Snippet: LAMP reagents IsoAmp I (#B0537S), IsoAmp II (#B0374S), MgSO4 (#B1003S), deoxynucleotide solution (#N0447S), Bovine Serum Albumen (BSA, #B9000S0), Bst 2.0 (8,000 U/ml, #M0537S) and Bst 3.0 (8000 U/ml, #M0374S) were purchased from New England Biolabs (Ipswich, MA, USA).

    Techniques: Amplification

    Schematic diagram of PRSeq (Promoter RNA Sequencing) method. ( A ) DNA pool preparation. Synthetic single-stranded DNA with a 3′-terminal hairpin structure is subjected to intra-strand fill-in DNA elongation to generate double-stranded recognition sequence for nicking enzyme, Nt.AlwI (green). The randomized promoter region is indicated by red color. After the nicking reaction, the DNA is subjected to DNA elongation employing Bst 2.0 DNA polymerase which has strong strand displacement activity. In this reaction, the 5′-DNA fragment generated by the nicking acts as a primer, and the random sequence in the promoter region is copied to downstream of the promoter. Finally, a primer sequence for reverse transcription is attached by ligation. Note that a part of non-randomized region of the promoter is not copied, and the copied sequence should not act as the promoter. ( B ) In vitro transcription (IVTX) of the DNA pool. The DNA pool prepared by the reactions in ( A ) is applied for IVTX. The template DNA molecules with strong and weak promoters (shown by red and purple colors) generate high and low copies of RNA, respectively, whereas most of the DNA with non-functional promoter variant (shown by blue color) do not. Since the RNA copy number is in proportion to the promoter activity linearly, activity of each promoter sequence can be evaluated by RNA sequencing of the transcript pool.

    Journal: Scientific Reports

    Article Title: Promoter RNA sequencing (PRSeq) for the massive and quantitative promoter analysis in vitro

    doi: 10.1038/s41598-019-39892-x

    Figure Lengend Snippet: Schematic diagram of PRSeq (Promoter RNA Sequencing) method. ( A ) DNA pool preparation. Synthetic single-stranded DNA with a 3′-terminal hairpin structure is subjected to intra-strand fill-in DNA elongation to generate double-stranded recognition sequence for nicking enzyme, Nt.AlwI (green). The randomized promoter region is indicated by red color. After the nicking reaction, the DNA is subjected to DNA elongation employing Bst 2.0 DNA polymerase which has strong strand displacement activity. In this reaction, the 5′-DNA fragment generated by the nicking acts as a primer, and the random sequence in the promoter region is copied to downstream of the promoter. Finally, a primer sequence for reverse transcription is attached by ligation. Note that a part of non-randomized region of the promoter is not copied, and the copied sequence should not act as the promoter. ( B ) In vitro transcription (IVTX) of the DNA pool. The DNA pool prepared by the reactions in ( A ) is applied for IVTX. The template DNA molecules with strong and weak promoters (shown by red and purple colors) generate high and low copies of RNA, respectively, whereas most of the DNA with non-functional promoter variant (shown by blue color) do not. Since the RNA copy number is in proportion to the promoter activity linearly, activity of each promoter sequence can be evaluated by RNA sequencing of the transcript pool.

    Article Snippet: Next, nicking and strand displacement elongation reactions in 200-µL volume were performed with Nt.Alw I (New England Biolabs, Ipswich, MA, USA) and Bst 2.0 DNA polymerase (New England Biolabs), respectively, according to the manufacture’s protocols.

    Techniques: RNA Sequencing Assay, Sequencing, Activity Assay, Generated, Ligation, Activated Clotting Time Assay, In Vitro, Functional Assay, Variant Assay