bst 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 dna polymerase
    Bst 2 0 DNA Polymerase
    Bst 2 0 DNA Polymerase 8 000 units
    https://www.bioz.com/result/bst dna polymerase/product/New England Biolabs
    Average 99 stars, based on 93 article reviews
    Price from $9.99 to $1999.99
    bst dna polymerase - by Bioz Stars, 2020-08
    99/100 stars

    Images

    1) 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

    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 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

    3) Product Images from "Rapid Identification of Emerging Human-Pathogenic Sporothrix Species with Rolling Circle Amplification"

    Article Title: Rapid Identification of Emerging Human-Pathogenic Sporothrix Species with Rolling Circle Amplification

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2015.01385

    Schematic of RCA of circularized padlock probes . (A) S. brasiliensis (Sbra-RCA) padlock probe design. (B) CAL is amplified by PCR with primers CAL -Fw and CAL -Rv; PCR products are submitted to ligation. Circularization of padlock probes occurs only if both probe arms hybridize correctly to the target sequence. (C) Upon specific hybridization, the phosphorylated 5′ end and the free hydroxyl at the 3′ end of the probe are joined by Pfu DNA ligase. After ligation, non-circularized probes and single-stranded primers are removed with Exo I and Exo III (optional step). (D) Circularized padlock probes serve as DNA template and signal amplification occurs via RCA, using Bst DNA polymerase and the primers RCA1 and RCA2. DNA synthesis occurs continuously for 1 h under isothermal amplification (65°C). DNA products are detected by gel electrophoresis or directly with SYBR Green I.
    Figure Legend Snippet: Schematic of RCA of circularized padlock probes . (A) S. brasiliensis (Sbra-RCA) padlock probe design. (B) CAL is amplified by PCR with primers CAL -Fw and CAL -Rv; PCR products are submitted to ligation. Circularization of padlock probes occurs only if both probe arms hybridize correctly to the target sequence. (C) Upon specific hybridization, the phosphorylated 5′ end and the free hydroxyl at the 3′ end of the probe are joined by Pfu DNA ligase. After ligation, non-circularized probes and single-stranded primers are removed with Exo I and Exo III (optional step). (D) Circularized padlock probes serve as DNA template and signal amplification occurs via RCA, using Bst DNA polymerase and the primers RCA1 and RCA2. DNA synthesis occurs continuously for 1 h under isothermal amplification (65°C). DNA products are detected by gel electrophoresis or directly with SYBR Green I.

    Techniques Used: Amplification, Polymerase Chain Reaction, Ligation, Sequencing, Hybridization, DNA Synthesis, Nucleic Acid Electrophoresis, SYBR Green Assay

    4) Product Images from "Simple and rapid detection of human enterovirus 71 by reverse-transcription and loop-mediated isothermal amplification: cryopreservation affected the detection ability"

    Article Title: Simple and rapid detection of human enterovirus 71 by reverse-transcription and loop-mediated isothermal amplification: cryopreservation affected the detection ability

    Journal: Diagnostic Microbiology and Infectious Disease

    doi: 10.1016/j.diagmicrobio.2011.07.014

    Sensitivity and specificity of RT-LAMP amplification of EV71. (A) RT-LAMP amplification of EV71 RNA. M: DNA marker; −Pol: without Bst DNA polymerase; −RNA: without RNA; −FIP/BIP: without inner pair primers of FIP and BIP; RT-LAMP: complete RT-LAMP. (B) The sensitivity of RT-LAMP amplification of EV71 RNA. The numerical value (5–50,000) over the electrophoresis land indicates the start copies of EV71 RNA. (C) The specificity of RT-LAMP amplification of EV71. Lanes marked with 1, 2, and 3 indicate the EV71 samples, Cox virus, and Coxsackie virus, respectively.
    Figure Legend Snippet: Sensitivity and specificity of RT-LAMP amplification of EV71. (A) RT-LAMP amplification of EV71 RNA. M: DNA marker; −Pol: without Bst DNA polymerase; −RNA: without RNA; −FIP/BIP: without inner pair primers of FIP and BIP; RT-LAMP: complete RT-LAMP. (B) The sensitivity of RT-LAMP amplification of EV71 RNA. The numerical value (5–50,000) over the electrophoresis land indicates the start copies of EV71 RNA. (C) The specificity of RT-LAMP amplification of EV71. Lanes marked with 1, 2, and 3 indicate the EV71 samples, Cox virus, and Coxsackie virus, respectively.

    Techniques Used: Amplification, Marker, Electrophoresis

    5) Product Images from "Simple and rapid detection of human enterovirus 71 by reverse-transcription and loop-mediated isothermal amplification: cryopreservation affected the detection ability "

    Article Title: Simple and rapid detection of human enterovirus 71 by reverse-transcription and loop-mediated isothermal amplification: cryopreservation affected the detection ability

    Journal: Diagnostic Microbiology and Infectious Disease

    doi: 10.1016/j.diagmicrobio.2011.07.014

    Sensitivity and specificity of RT-LAMP amplification of EV71. (A) RT-LAMP amplification of EV71 RNA. M: DNA marker; −Pol: without Bst DNA polymerase; −RNA: without RNA; −FIP/BIP: without inner pair primers of FIP and BIP; RT-LAMP: complete RT-LAMP. (B) The sensitivity of RT-LAMP amplification of EV71 RNA. The numerical value (5–50,000) over the electrophoresis land indicates the start copies of EV71 RNA. (C) The specificity of RT-LAMP amplification of EV71. Lanes marked with 1, 2, and 3 indicate the EV71 samples, Cox virus, and Coxsackie virus, respectively.
    Figure Legend Snippet: Sensitivity and specificity of RT-LAMP amplification of EV71. (A) RT-LAMP amplification of EV71 RNA. M: DNA marker; −Pol: without Bst DNA polymerase; −RNA: without RNA; −FIP/BIP: without inner pair primers of FIP and BIP; RT-LAMP: complete RT-LAMP. (B) The sensitivity of RT-LAMP amplification of EV71 RNA. The numerical value (5–50,000) over the electrophoresis land indicates the start copies of EV71 RNA. (C) The specificity of RT-LAMP amplification of EV71. Lanes marked with 1, 2, and 3 indicate the EV71 samples, Cox virus, and Coxsackie virus, respectively.

    Techniques Used: Amplification, Marker, Electrophoresis

    6) Product Images from "Hinge-initiated Primer-dependent Amplification of Nucleic Acids (HIP) – A New Versatile Isothermal Amplification Method"

    Article Title: Hinge-initiated Primer-dependent Amplification of Nucleic Acids (HIP) – A New Versatile Isothermal Amplification Method

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-08067-x

    Hinge-initiated primer-based replication with one hinge-primer and PCR-like primer. The hinge-primer anneals to the target region and will be extended to double strand DNA by Bst DNA polymerase (1). The refolding to the thermodynamically more stable hairpin structure liberates the initial priming site (2–3). These steps are repeated and the sense DNA strand will be released. The PCR-like primer can anneal to the priming site and will be extended (4).
    Figure Legend Snippet: Hinge-initiated primer-based replication with one hinge-primer and PCR-like primer. The hinge-primer anneals to the target region and will be extended to double strand DNA by Bst DNA polymerase (1). The refolding to the thermodynamically more stable hairpin structure liberates the initial priming site (2–3). These steps are repeated and the sense DNA strand will be released. The PCR-like primer can anneal to the priming site and will be extended (4).

    Techniques Used: Polymerase Chain Reaction

    Mechanism of the symmetric Hinge-initiated primer-dependent replication with two hinge-primer. The first stage (A) starts with initial annealing ( 1 ) of the forward hinge-primer, followed by extension to double strand DNA by Bst DNA polymerase ( 2 ) and refolding to the thermodynamically more stable hairpin structure which liberates ( 3 ) the initial priming site. These steps are repeated following the release of the sense DNA strand ( 4 ). The second stage (B) starts with annealing of the reverse hinge-primer to the newly generated and released single strand ( 5 ) followed by the extension and refolding to a second hairpin structure at both ends ( 6 ). The initial priming sites on the sense and anti-sense strands are thus liberated by refolding ( 7 ). Due to continuous recycling of hinge-primer binding sites, the reaction finally results in DNA product accumulation for specific detection. The “X” in the primer represents a blocking modification for the Bst DNA polymerase. Examples are a dSpacer (abasic furan) or an C12-Spacer (hexaethylenglycol). Optionally, an additional outer primer can be applied to increase the speed of initial single strand template generation.
    Figure Legend Snippet: Mechanism of the symmetric Hinge-initiated primer-dependent replication with two hinge-primer. The first stage (A) starts with initial annealing ( 1 ) of the forward hinge-primer, followed by extension to double strand DNA by Bst DNA polymerase ( 2 ) and refolding to the thermodynamically more stable hairpin structure which liberates ( 3 ) the initial priming site. These steps are repeated following the release of the sense DNA strand ( 4 ). The second stage (B) starts with annealing of the reverse hinge-primer to the newly generated and released single strand ( 5 ) followed by the extension and refolding to a second hairpin structure at both ends ( 6 ). The initial priming sites on the sense and anti-sense strands are thus liberated by refolding ( 7 ). Due to continuous recycling of hinge-primer binding sites, the reaction finally results in DNA product accumulation for specific detection. The “X” in the primer represents a blocking modification for the Bst DNA polymerase. Examples are a dSpacer (abasic furan) or an C12-Spacer (hexaethylenglycol). Optionally, an additional outer primer can be applied to increase the speed of initial single strand template generation.

    Techniques Used: Generated, Binding Assay, Blocking Assay, Modification

    7) Product Images from "Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus"

    Article Title: Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007124

    FEN1 protein facilitates cccDNA formation in vitro . (A) Schematic presentation of in vitro cccDNA formation assay. Purified NC-DNA (10 8 copies) was incubated with recombinant FEN1, Bst DNA polymerase, and Taq DNA ligase. Following incubation, the DNA was purified and analyzed (B–F). Regions for qPCR amplification (E and F) were indicated as p. The 5.4-kb PstI fragment in HBV plasmid (Control) has a partial HBV sequence but does not have core and intact P genes. (B) cccDNA-selective qPCR. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; *** P
    Figure Legend Snippet: FEN1 protein facilitates cccDNA formation in vitro . (A) Schematic presentation of in vitro cccDNA formation assay. Purified NC-DNA (10 8 copies) was incubated with recombinant FEN1, Bst DNA polymerase, and Taq DNA ligase. Following incubation, the DNA was purified and analyzed (B–F). Regions for qPCR amplification (E and F) were indicated as p. The 5.4-kb PstI fragment in HBV plasmid (Control) has a partial HBV sequence but does not have core and intact P genes. (B) cccDNA-selective qPCR. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; *** P

    Techniques Used: In Vitro, Tube Formation Assay, Purification, Incubation, Recombinant, Real-time Polymerase Chain Reaction, Amplification, Plasmid Preparation, Sequencing

    8) 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

    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
    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

    10) Product Images from "Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus"

    Article Title: Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007124

    FEN1 protein facilitates cccDNA formation in vitro . (A) Schematic presentation of in vitro cccDNA formation assay. Purified NC-DNA (10 8 copies) was incubated with recombinant FEN1, Bst DNA polymerase, and Taq DNA ligase. Following incubation, the DNA was purified and analyzed (B–F). Regions for qPCR amplification (E and F) were indicated as p. The 5.4-kb PstI fragment in HBV plasmid (Control) has a partial HBV sequence but does not have core and intact P genes. (B) cccDNA-selective qPCR. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; *** P
    Figure Legend Snippet: FEN1 protein facilitates cccDNA formation in vitro . (A) Schematic presentation of in vitro cccDNA formation assay. Purified NC-DNA (10 8 copies) was incubated with recombinant FEN1, Bst DNA polymerase, and Taq DNA ligase. Following incubation, the DNA was purified and analyzed (B–F). Regions for qPCR amplification (E and F) were indicated as p. The 5.4-kb PstI fragment in HBV plasmid (Control) has a partial HBV sequence but does not have core and intact P genes. (B) cccDNA-selective qPCR. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; *** P

    Techniques Used: In Vitro, Tube Formation Assay, Purification, Incubation, Recombinant, Real-time Polymerase Chain Reaction, Amplification, Plasmid Preparation, Sequencing

    11) Product Images from "Evolution of a General RNA-Cleaving FANA Enzyme"

    Article Title: Evolution of a General RNA-Cleaving FANA Enzyme

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07611-1

    FANA transcription and reverse transcription in vitro. a Constitutional structures for 2’-deoxyribonucleic acid (DNA) and 2’-fluoroarabino nucleic acid (FANA). b FANA transcription activity for wild-type archaeal DNA polymerases (exo−) from 9°N, DV, Kod, and Tgo (left panel). Samples were analyzed after 15 and 30 min at 55 °C. FANA reverse transcriptase activity of Bst DNA polymerase LF, 2.0, 3.0, and LF* (right panel). LF* denotes wild-type Bst DNA polymerase, large fragment, expressed and purified from E. coli . Samples were analyzed after 30 min at 50 °C. All samples were resolved on denaturing PAGE and visualized using a LI-COR Odyssey CLx. c Fidelity profile observed for FANA replication using Tgo and Bst LF* polymerases. The mutation profile reveals a mutation rate of 8 × 10 -4 and an overall fidelity of ~99.9%. d Catalytic rates observed for FANA synthesis with Tgo (left panel) and reverse transcription with Bst LF* (right panel)
    Figure Legend Snippet: FANA transcription and reverse transcription in vitro. a Constitutional structures for 2’-deoxyribonucleic acid (DNA) and 2’-fluoroarabino nucleic acid (FANA). b FANA transcription activity for wild-type archaeal DNA polymerases (exo−) from 9°N, DV, Kod, and Tgo (left panel). Samples were analyzed after 15 and 30 min at 55 °C. FANA reverse transcriptase activity of Bst DNA polymerase LF, 2.0, 3.0, and LF* (right panel). LF* denotes wild-type Bst DNA polymerase, large fragment, expressed and purified from E. coli . Samples were analyzed after 30 min at 50 °C. All samples were resolved on denaturing PAGE and visualized using a LI-COR Odyssey CLx. c Fidelity profile observed for FANA replication using Tgo and Bst LF* polymerases. The mutation profile reveals a mutation rate of 8 × 10 -4 and an overall fidelity of ~99.9%. d Catalytic rates observed for FANA synthesis with Tgo (left panel) and reverse transcription with Bst LF* (right panel)

    Techniques Used: In Vitro, Activity Assay, Purification, Polyacrylamide Gel Electrophoresis, Mutagenesis

    12) 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

    13) 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

    14) Product Images from "In vitro selection of an XNA aptamer capable of small-molecule recognition"

    Article Title: In vitro selection of an XNA aptamer capable of small-molecule recognition

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky667

    TNA SELEX to generate OTA-binding aptamers. The initial ssDNA library is amplified using a forward primer modified with a PEG spacer and polyT tail to enable separation and recovery by denaturing PAGE. The PEGylated DNA template is then annealed to the FAM-labelled TNA primer and extended using KOD RI polymerase to generate the TNA library for each selection round. The TNA library is incubated with OTA-functionalized magnetic beads, and bound sequences recovered by either heat (rounds 1–4) or ligand elution (rounds 5–9). These sequences are then treated with DNase I to digest any remaining DNA template. The TNA is then reverse transcribed back into DNA using Bst DNA polymerase and PCR amplified for the next round of selection.
    Figure Legend Snippet: TNA SELEX to generate OTA-binding aptamers. The initial ssDNA library is amplified using a forward primer modified with a PEG spacer and polyT tail to enable separation and recovery by denaturing PAGE. The PEGylated DNA template is then annealed to the FAM-labelled TNA primer and extended using KOD RI polymerase to generate the TNA library for each selection round. The TNA library is incubated with OTA-functionalized magnetic beads, and bound sequences recovered by either heat (rounds 1–4) or ligand elution (rounds 5–9). These sequences are then treated with DNase I to digest any remaining DNA template. The TNA is then reverse transcribed back into DNA using Bst DNA polymerase and PCR amplified for the next round of selection.

    Techniques Used: Binding Assay, Amplification, Modification, Polyacrylamide Gel Electrophoresis, Selection, Incubation, Magnetic Beads, Polymerase Chain Reaction

    15) Product Images from "In vitro selection of an XNA aptamer capable of small-molecule recognition"

    Article Title: In vitro selection of an XNA aptamer capable of small-molecule recognition

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky667

    TNA SELEX to generate OTA-binding aptamers. The initial ssDNA library is amplified using a forward primer modified with a PEG spacer and polyT tail to enable separation and recovery by denaturing PAGE. The PEGylated DNA template is then annealed to the FAM-labelled TNA primer and extended using KOD RI polymerase to generate the TNA library for each selection round. The TNA library is incubated with OTA-functionalized magnetic beads, and bound sequences recovered by either heat (rounds 1–4) or ligand elution (rounds 5–9). These sequences are then treated with DNase I to digest any remaining DNA template. The TNA is then reverse transcribed back into DNA using Bst DNA polymerase and PCR amplified for the next round of selection.
    Figure Legend Snippet: TNA SELEX to generate OTA-binding aptamers. The initial ssDNA library is amplified using a forward primer modified with a PEG spacer and polyT tail to enable separation and recovery by denaturing PAGE. The PEGylated DNA template is then annealed to the FAM-labelled TNA primer and extended using KOD RI polymerase to generate the TNA library for each selection round. The TNA library is incubated with OTA-functionalized magnetic beads, and bound sequences recovered by either heat (rounds 1–4) or ligand elution (rounds 5–9). These sequences are then treated with DNase I to digest any remaining DNA template. The TNA is then reverse transcribed back into DNA using Bst DNA polymerase and PCR amplified for the next round of selection.

    Techniques Used: Binding Assay, Amplification, Modification, Polyacrylamide Gel Electrophoresis, Selection, Incubation, Magnetic Beads, Polymerase Chain Reaction

    16) Product Images from "A library-based method to rapidly analyse chromatin accessibility at multiple genomic regions"

    Article Title: A library-based method to rapidly analyse chromatin accessibility at multiple genomic regions

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp037

    Overview of the experimental steps required to create and analyse a chromatin accessibility library. ( A ) Step 1: fungal mycelia pre-grown under specific conditions or isolated DNA ( in vitro controls) are processed as described in Materials and methods section and digested with MNase or restriction enzymes of choice. Step 2: digested DNA is blunt-ended and phosphorylated by subsequent treatment of the chromatin with Klenow fragment polymerase, T4 polynucleotide kinase. This step produces blunt-ended DNA fragments for ligation with adaptors. Step 3: DNA fragments are ligated with double-stranded adaptors A and B, originating from oligonucleotides Adaptor-A short and Adaptor-A long or Adaptor-B short and Adaptor-B long , where adaptor oligonucleotide B long is biotinylated for later retention on the streptavidin beads. In this step, fragments containing all adaptor combinations (A-A, A-B and B-B) are generated. Step 4: the ligation step leaves nicks at the 3′-terminus that are repaired by Bst polymerase treatment. Step 5: all fragments containing biotinylated adaptor B are captured on streptavidin-coated magnetic beads. At this step, adaptor A-A fragments are lost. Step 6: after a washing step, the retained fragments (adaptors A-B and B-B fragments) are denatured at 95°C. The denaturation step results in the release of single strands which exclusively carry A-B adaptor fragments. Step 7: the single-stranded A-B adaptor fragment library is amplified by a nested PCR approach to give the final A-B fragment library. The input and output fragment libraries are quality controlled by amplification with single A and B, as well as mixed A-B primers. Only the A-B primer mix should result in the amplification of fragments in the range of 200–1000 bp (see Panel B). Step 8: the resulting A-B adaptor fragment library is diluted and aliquots are used for analytical PCR amplifications for fragment size analysis of specific loci of interest. In the final analytical PCR step, either gene-specific or adaptor-specific primers can be labelled for subsequent capillary sequencer analysis. The chromatograms are finally analysed by image analysis software. ( B ) Example of quality control of A-B adaptor fragment libraries. Two input chromatin fragment libraries without adaptor ligation (lanes 1 and 2) are compared to two output libraries with adaptor ligation as described in Materials and methods section (lanes 3 and 4). Libraries originating from nitrate-grown cells (lanes 1 and 3) as well as from ammonium-grown cells (lanes 2 and 4) are shown as an example. M, DNA size marker.
    Figure Legend Snippet: Overview of the experimental steps required to create and analyse a chromatin accessibility library. ( A ) Step 1: fungal mycelia pre-grown under specific conditions or isolated DNA ( in vitro controls) are processed as described in Materials and methods section and digested with MNase or restriction enzymes of choice. Step 2: digested DNA is blunt-ended and phosphorylated by subsequent treatment of the chromatin with Klenow fragment polymerase, T4 polynucleotide kinase. This step produces blunt-ended DNA fragments for ligation with adaptors. Step 3: DNA fragments are ligated with double-stranded adaptors A and B, originating from oligonucleotides Adaptor-A short and Adaptor-A long or Adaptor-B short and Adaptor-B long , where adaptor oligonucleotide B long is biotinylated for later retention on the streptavidin beads. In this step, fragments containing all adaptor combinations (A-A, A-B and B-B) are generated. Step 4: the ligation step leaves nicks at the 3′-terminus that are repaired by Bst polymerase treatment. Step 5: all fragments containing biotinylated adaptor B are captured on streptavidin-coated magnetic beads. At this step, adaptor A-A fragments are lost. Step 6: after a washing step, the retained fragments (adaptors A-B and B-B fragments) are denatured at 95°C. The denaturation step results in the release of single strands which exclusively carry A-B adaptor fragments. Step 7: the single-stranded A-B adaptor fragment library is amplified by a nested PCR approach to give the final A-B fragment library. The input and output fragment libraries are quality controlled by amplification with single A and B, as well as mixed A-B primers. Only the A-B primer mix should result in the amplification of fragments in the range of 200–1000 bp (see Panel B). Step 8: the resulting A-B adaptor fragment library is diluted and aliquots are used for analytical PCR amplifications for fragment size analysis of specific loci of interest. In the final analytical PCR step, either gene-specific or adaptor-specific primers can be labelled for subsequent capillary sequencer analysis. The chromatograms are finally analysed by image analysis software. ( B ) Example of quality control of A-B adaptor fragment libraries. Two input chromatin fragment libraries without adaptor ligation (lanes 1 and 2) are compared to two output libraries with adaptor ligation as described in Materials and methods section (lanes 3 and 4). Libraries originating from nitrate-grown cells (lanes 1 and 3) as well as from ammonium-grown cells (lanes 2 and 4) are shown as an example. M, DNA size marker.

    Techniques Used: Isolation, In Vitro, Ligation, Generated, Magnetic Beads, Amplification, Nested PCR, Polymerase Chain Reaction, Software, Marker

    17) 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

    18) 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

    19) 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

    20) Product Images from "Rapid Identification of Emerging Human-Pathogenic Sporothrix Species with Rolling Circle Amplification"

    Article Title: Rapid Identification of Emerging Human-Pathogenic Sporothrix Species with Rolling Circle Amplification

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2015.01385

    Schematic of RCA of circularized padlock probes . (A) S. brasiliensis (Sbra-RCA) padlock probe design. (B) CAL is amplified by PCR with primers CAL -Fw and CAL -Rv; PCR products are submitted to ligation. Circularization of padlock probes occurs only if both probe arms hybridize correctly to the target sequence. (C) Upon specific hybridization, the phosphorylated 5′ end and the free hydroxyl at the 3′ end of the probe are joined by Pfu DNA ligase. After ligation, non-circularized probes and single-stranded primers are removed with Exo I and Exo III (optional step). (D) Circularized padlock probes serve as DNA template and signal amplification occurs via RCA, using Bst DNA polymerase and the primers RCA1 and RCA2. DNA synthesis occurs continuously for 1 h under isothermal amplification (65°C). DNA products are detected by gel electrophoresis or directly with SYBR Green I.
    Figure Legend Snippet: Schematic of RCA of circularized padlock probes . (A) S. brasiliensis (Sbra-RCA) padlock probe design. (B) CAL is amplified by PCR with primers CAL -Fw and CAL -Rv; PCR products are submitted to ligation. Circularization of padlock probes occurs only if both probe arms hybridize correctly to the target sequence. (C) Upon specific hybridization, the phosphorylated 5′ end and the free hydroxyl at the 3′ end of the probe are joined by Pfu DNA ligase. After ligation, non-circularized probes and single-stranded primers are removed with Exo I and Exo III (optional step). (D) Circularized padlock probes serve as DNA template and signal amplification occurs via RCA, using Bst DNA polymerase and the primers RCA1 and RCA2. DNA synthesis occurs continuously for 1 h under isothermal amplification (65°C). DNA products are detected by gel electrophoresis or directly with SYBR Green I.

    Techniques Used: Amplification, Polymerase Chain Reaction, Ligation, Sequencing, Hybridization, DNA Synthesis, Nucleic Acid Electrophoresis, SYBR Green Assay

    21) 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

    Identification of recombinant plasmids by colony PCR. (a) M: 2 kb ladder marker; 1: WT of Bst DNA pol LF gene; (b) M: 10 kb ladder marker; 1: WT of Bst DNA pol LF gene; 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.
    Figure Legend Snippet: Identification of recombinant plasmids by colony PCR. (a) M: 2 kb ladder marker; 1: WT of Bst DNA pol LF gene; (b) M: 10 kb ladder marker; 1: WT of Bst DNA pol LF gene; 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.

    Techniques Used: Recombinant, Polymerase Chain Reaction, Marker, Mutagenesis

    SDS-PAGE analysis of the WT of Bst DNA pol LF. M: protein ladder marker shown in kDa on the left sides of panels; 1: uninduced whole cell sample; 2: supernatant fraction of uninduced sample; 3: pellet fraction of uninduced sample; 4: whole cell sample after induction for 6 h; 5: supernatant fraction after induction for 6 h; 6: pellet fraction after induction for 6 h. Corresponding position of Bst DNA pol LF was marked by black arrow.
    Figure Legend Snippet: SDS-PAGE analysis of the WT of Bst DNA pol LF. M: protein ladder marker shown in kDa on the left sides of panels; 1: uninduced whole cell sample; 2: supernatant fraction of uninduced sample; 3: pellet fraction of uninduced sample; 4: whole cell sample after induction for 6 h; 5: supernatant fraction after induction for 6 h; 6: pellet fraction after induction for 6 h. Corresponding position of Bst DNA pol LF was marked by black arrow.

    Techniques Used: SDS Page, Marker

    22) 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

    23) 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

    24) 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

    25) Product Images from "Development and application of a rapid and visual loop-mediated isothermal amplification for the detection of Sporisorium scitamineum in sugarcane"

    Article Title: Development and application of a rapid and visual loop-mediated isothermal amplification for the detection of Sporisorium scitamineum in sugarcane

    Journal: Scientific Reports

    doi: 10.1038/srep23994

    Optimization of concentration of Bst DNA polymerase in the LAMP reaction. ( A ) LAMP products detected by 1 000×SYBR Green I (Biotek Co., Ltd., Beijing, China). ( B ) Detection of LAMP products by agarose gel electrophoresis stained by EB (ethidium bromide). Tubes and lanes 3, 7, 11 and 15: negative control. Tubes and lanes 4, 8, 12 and 16: blank control. Tubes and lanes 1 and 2, 5 and 6, 9 and 10, 13 and 14: the positive plasmid pMD19-T-Pep1, repeated twice. Tubes and lanes 1–4, 5–8, 9–12 and 13–16: Bst DNA polymerase concentrations were 2.0 U, 4.0 U, 6.0 U and 8.0 U, respectively. Lane M: DL 15 000 + 2 000 DNA marker.
    Figure Legend Snippet: Optimization of concentration of Bst DNA polymerase in the LAMP reaction. ( A ) LAMP products detected by 1 000×SYBR Green I (Biotek Co., Ltd., Beijing, China). ( B ) Detection of LAMP products by agarose gel electrophoresis stained by EB (ethidium bromide). Tubes and lanes 3, 7, 11 and 15: negative control. Tubes and lanes 4, 8, 12 and 16: blank control. Tubes and lanes 1 and 2, 5 and 6, 9 and 10, 13 and 14: the positive plasmid pMD19-T-Pep1, repeated twice. Tubes and lanes 1–4, 5–8, 9–12 and 13–16: Bst DNA polymerase concentrations were 2.0 U, 4.0 U, 6.0 U and 8.0 U, respectively. Lane M: DL 15 000 + 2 000 DNA marker.

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

    26) 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

    27) 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

    28) Product Images from "Evolution of a General RNA-Cleaving FANA Enzyme"

    Article Title: Evolution of a General RNA-Cleaving FANA Enzyme

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07611-1

    FANA transcription and reverse transcription in vitro. a Constitutional structures for 2’-deoxyribonucleic acid (DNA) and 2’-fluoroarabino nucleic acid (FANA). b FANA transcription activity for wild-type archaeal DNA polymerases (exo−) from 9°N, DV, Kod, and Tgo (left panel). Samples were analyzed after 15 and 30 min at 55 °C. FANA reverse transcriptase activity of Bst DNA polymerase LF, 2.0, 3.0, and LF* (right panel). LF* denotes wild-type Bst DNA polymerase, large fragment, expressed and purified from E. coli . Samples were analyzed after 30 min at 50 °C. All samples were resolved on denaturing PAGE and visualized using a LI-COR Odyssey CLx. c Fidelity profile observed for FANA replication using Tgo and Bst LF* polymerases. The mutation profile reveals a mutation rate of 8 × 10 -4 and an overall fidelity of ~99.9%. d Catalytic rates observed for FANA synthesis with Tgo (left panel) and reverse transcription with Bst LF* (right panel)
    Figure Legend Snippet: FANA transcription and reverse transcription in vitro. a Constitutional structures for 2’-deoxyribonucleic acid (DNA) and 2’-fluoroarabino nucleic acid (FANA). b FANA transcription activity for wild-type archaeal DNA polymerases (exo−) from 9°N, DV, Kod, and Tgo (left panel). Samples were analyzed after 15 and 30 min at 55 °C. FANA reverse transcriptase activity of Bst DNA polymerase LF, 2.0, 3.0, and LF* (right panel). LF* denotes wild-type Bst DNA polymerase, large fragment, expressed and purified from E. coli . Samples were analyzed after 30 min at 50 °C. All samples were resolved on denaturing PAGE and visualized using a LI-COR Odyssey CLx. c Fidelity profile observed for FANA replication using Tgo and Bst LF* polymerases. The mutation profile reveals a mutation rate of 8 × 10 -4 and an overall fidelity of ~99.9%. d Catalytic rates observed for FANA synthesis with Tgo (left panel) and reverse transcription with Bst LF* (right panel)

    Techniques Used: In Vitro, Activity Assay, Purification, Polyacrylamide Gel Electrophoresis, Mutagenesis

    29) Product Images from "High-efficiency and integrable DNA arithmetic and logic system based on strand displacement synthesis"

    Article Title: High-efficiency and integrable DNA arithmetic and logic system based on strand displacement synthesis

    Journal: Nature Communications

    doi: 10.1038/s41467-019-13310-2

    Construction of six basic dual-rail systems and their performance. a Construction of the AND, OR, NAND, NOR, XOR, and XNOR dual-rail gates. The upper regular octagon shows a single-rail AND gate, and the lower regular hexagon shows a single-rail OR gate. The green and magenta colors mean defined TRUE and FALSE signals, respectively. Superscripts 1 and 0 also indicate the TRUE and FALSE inputs and outputs, respectively. When computations were performed, a set of input strands must be adder, e.g., inputs (1, 0) means adding inputs I A 1 (TURE) and I B 0 (FALSE). Detailed structures can be seen in Fig. S12. Reaction details of AND and XOR gates can be seen in Supplementary Figs. 7 and 8 . b , c Reaction kinetics of the dual-rail AND and XOR gates with all possible combinations of inputs. The reaction was performed with 3.2 U Bst polymerase (large fragment), TRI and FRI at 35 °C. The curve was plotted by transferring the cycle value into the reaction time. The outputs were normalized to the RFU values in the FAM and ROX channels with the highest signals. The FAM and ROX signals correspond to the TRUE and FALSE returns, respectively. Sequences of the DNA strands are listed in the Supplementary Information . d Summary of all the outputs computed by the six basic logic gates constructed from DNA.
    Figure Legend Snippet: Construction of six basic dual-rail systems and their performance. a Construction of the AND, OR, NAND, NOR, XOR, and XNOR dual-rail gates. The upper regular octagon shows a single-rail AND gate, and the lower regular hexagon shows a single-rail OR gate. The green and magenta colors mean defined TRUE and FALSE signals, respectively. Superscripts 1 and 0 also indicate the TRUE and FALSE inputs and outputs, respectively. When computations were performed, a set of input strands must be adder, e.g., inputs (1, 0) means adding inputs I A 1 (TURE) and I B 0 (FALSE). Detailed structures can be seen in Fig. S12. Reaction details of AND and XOR gates can be seen in Supplementary Figs. 7 and 8 . b , c Reaction kinetics of the dual-rail AND and XOR gates with all possible combinations of inputs. The reaction was performed with 3.2 U Bst polymerase (large fragment), TRI and FRI at 35 °C. The curve was plotted by transferring the cycle value into the reaction time. The outputs were normalized to the RFU values in the FAM and ROX channels with the highest signals. The FAM and ROX signals correspond to the TRUE and FALSE returns, respectively. Sequences of the DNA strands are listed in the Supplementary Information . d Summary of all the outputs computed by the six basic logic gates constructed from DNA.

    Techniques Used: Transferring, Construct

    Construction of DNA ALU with our DNA logic gates. a Abstract diagram of a typical ALU. b A typical construction of a 1-bit ALU with a digital logic circuit. A and B: inputs; C in : carry in; S 0 and S 1 : select signals; Y: output; C out : carry out. c Assembling the 4:1 multiplexer with DNA components. The sequences of the strands are listed in the Supplementary Table 2 . The details of the integrated logic gates are shown in Fig. S20. d Function table of the DNA ALU. e Summary of the outputs of the ALU. f , g Reaction kinetics of the ALU with all possible combinations of inputs. The reaction was performed with 12 U Bst polymerase (large fragment), TRIII, FRIII, TRII, and FRII. The curve was plotted by transferring the cycle values into the reaction time. The outputs were normalized to the RFU values in the FAM, ROX, HEX, and Cy5 channels with the highest signals. The TRIII (FAM) and FRIII (ROX) signals correspond to the TRUE and FALSE returns of Y, respectively. The TRII (HEX) and FRII (Cy5) signals correspond to the TRUE and FALSE returns of C out, respectively. The reaction kinetics of the individual gates can be seen in Supplementary Fig. 22 . The sequences of the DNA strands are listed in the Supplementary Table 2 .
    Figure Legend Snippet: Construction of DNA ALU with our DNA logic gates. a Abstract diagram of a typical ALU. b A typical construction of a 1-bit ALU with a digital logic circuit. A and B: inputs; C in : carry in; S 0 and S 1 : select signals; Y: output; C out : carry out. c Assembling the 4:1 multiplexer with DNA components. The sequences of the strands are listed in the Supplementary Table 2 . The details of the integrated logic gates are shown in Fig. S20. d Function table of the DNA ALU. e Summary of the outputs of the ALU. f , g Reaction kinetics of the ALU with all possible combinations of inputs. The reaction was performed with 12 U Bst polymerase (large fragment), TRIII, FRIII, TRII, and FRII. The curve was plotted by transferring the cycle values into the reaction time. The outputs were normalized to the RFU values in the FAM, ROX, HEX, and Cy5 channels with the highest signals. The TRIII (FAM) and FRIII (ROX) signals correspond to the TRUE and FALSE returns of Y, respectively. The TRII (HEX) and FRII (Cy5) signals correspond to the TRUE and FALSE returns of C out, respectively. The reaction kinetics of the individual gates can be seen in Supplementary Fig. 22 . The sequences of the DNA strands are listed in the Supplementary Table 2 .

    Techniques Used: Transferring

    Construction of the full adder and 4:1 multiplexer from our DNA logic gates. a , e A typical construction of the full adder and the 4:1 multiplexer with the digital logic circuit. D 0 –D 3 : inputs; S 0 and S 1 : select signals; Z: output. b , f Constructing a full adder and the 4:1 multiplexer with DNA logic gates. The sequences of the DNA strands are listed in the Supplementary table 2 . c , g Summary of all the outputs computed by the DNA full adder and 4:1 multiplexer. d , h Reaction kinetics of the full adder and the 4:1 multiplexer with all possible combinations of inputs. For the full adder, the reaction was performed with 12 U Bst polymerase (large fragment), TRI, FRI, TRII, and FRII. The outputs were normalized to the RFU values in the FAM, ROX, HEX, and Cy5 channels with the highest signals. For the full adder: The TRI (FAM) and FRI (ROX) signals correspond to TRUE and FALSE returns of S, respectively. The TRII (HEX) and FRII (Cy5) signals correspond to the TRUE and FALSE returns of C out, respectively. For the 4:1 multiplexer, the reaction was performed with 6.4 U Bst polymerase (large fragment), TRIII and FRII. The outputs were normalized to the RFU values in the FAM and ROX channels with the highest signals. The TRIII (FAM) and FRIII (ROX) signals correspond to TRUE and FALSE returns, respectively. The reaction kinetics of the individual gates can be seen in Supplementary Figs. 14 and 19 .
    Figure Legend Snippet: Construction of the full adder and 4:1 multiplexer from our DNA logic gates. a , e A typical construction of the full adder and the 4:1 multiplexer with the digital logic circuit. D 0 –D 3 : inputs; S 0 and S 1 : select signals; Z: output. b , f Constructing a full adder and the 4:1 multiplexer with DNA logic gates. The sequences of the DNA strands are listed in the Supplementary table 2 . c , g Summary of all the outputs computed by the DNA full adder and 4:1 multiplexer. d , h Reaction kinetics of the full adder and the 4:1 multiplexer with all possible combinations of inputs. For the full adder, the reaction was performed with 12 U Bst polymerase (large fragment), TRI, FRI, TRII, and FRII. The outputs were normalized to the RFU values in the FAM, ROX, HEX, and Cy5 channels with the highest signals. For the full adder: The TRI (FAM) and FRI (ROX) signals correspond to TRUE and FALSE returns of S, respectively. The TRII (HEX) and FRII (Cy5) signals correspond to the TRUE and FALSE returns of C out, respectively. For the 4:1 multiplexer, the reaction was performed with 6.4 U Bst polymerase (large fragment), TRIII and FRII. The outputs were normalized to the RFU values in the FAM and ROX channels with the highest signals. The TRIII (FAM) and FRIII (ROX) signals correspond to TRUE and FALSE returns, respectively. The reaction kinetics of the individual gates can be seen in Supplementary Figs. 14 and 19 .

    Techniques Used:

    Construction of AND and OR gates with DNA polymerase and their performance. a , c Right: structure of the AND and OR gates. The capital names are the sequence names; the lowercase names refer to the elementary sequences and asterisk indicates a complementary sequence. Each elementary sequence contains 18 bases. The gray parts are 4-mer spacer sequences used to reduce steric hinderance. The sequences of inputs A and B are the same as a and b. Left: abstract diagram of the AND and OR gates. The regular octagon shows the main structure of the DNA components; the two bold lines on the left indicate the binding sites of the two inputs in the DNA component; the vector line on the right indicates the potential output. b , d Mechanism of the AND and OR gates. e The fluorescent reporter used to visualize the devices. The letter Q denotes the quencher, and F denotes the fluorophore. f , g Reaction kinetics of the AND and OR gates with all possible combinations of inputs. The reaction was performed with 3.2 U Bst polymerase (large fragment) and TRI at 35 °C. The curve was plotted by transferring the cycle value into the reaction time. The outputs were normalized to the relative fluorescence unit (RFU) values in the FAM channel with the highest signals. The original signals are plotted in Supplementary Fig. 3 . The sequences of the DNA strands are listed in the Supplementary Table 2 .
    Figure Legend Snippet: Construction of AND and OR gates with DNA polymerase and their performance. a , c Right: structure of the AND and OR gates. The capital names are the sequence names; the lowercase names refer to the elementary sequences and asterisk indicates a complementary sequence. Each elementary sequence contains 18 bases. The gray parts are 4-mer spacer sequences used to reduce steric hinderance. The sequences of inputs A and B are the same as a and b. Left: abstract diagram of the AND and OR gates. The regular octagon shows the main structure of the DNA components; the two bold lines on the left indicate the binding sites of the two inputs in the DNA component; the vector line on the right indicates the potential output. b , d Mechanism of the AND and OR gates. e The fluorescent reporter used to visualize the devices. The letter Q denotes the quencher, and F denotes the fluorophore. f , g Reaction kinetics of the AND and OR gates with all possible combinations of inputs. The reaction was performed with 3.2 U Bst polymerase (large fragment) and TRI at 35 °C. The curve was plotted by transferring the cycle value into the reaction time. The outputs were normalized to the relative fluorescence unit (RFU) values in the FAM channel with the highest signals. The original signals are plotted in Supplementary Fig. 3 . The sequences of the DNA strands are listed in the Supplementary Table 2 .

    Techniques Used: Sequencing, Binding Assay, Plasmid Preparation, Transferring, Fluorescence

    30) Product Images from "Development and application of a rapid and visual loop-mediated isothermal amplification for the detection of Sporisorium scitamineum in sugarcane"

    Article Title: Development and application of a rapid and visual loop-mediated isothermal amplification for the detection of Sporisorium scitamineum in sugarcane

    Journal: Scientific Reports

    doi: 10.1038/srep23994

    Optimization of concentration of Bst DNA polymerase in the LAMP reaction. ( A ) LAMP products detected by 1 000×SYBR Green I (Biotek Co., Ltd., Beijing, China). ( B ) Detection of LAMP products by agarose gel electrophoresis stained by EB (ethidium bromide). Tubes and lanes 3, 7, 11 and 15: negative control. Tubes and lanes 4, 8, 12 and 16: blank control. Tubes and lanes 1 and 2, 5 and 6, 9 and 10, 13 and 14: the positive plasmid pMD19-T-Pep1, repeated twice. Tubes and lanes 1–4, 5–8, 9–12 and 13–16: Bst DNA polymerase concentrations were 2.0 U, 4.0 U, 6.0 U and 8.0 U, respectively. Lane M: DL 15 000 + 2 000 DNA marker.
    Figure Legend Snippet: Optimization of concentration of Bst DNA polymerase in the LAMP reaction. ( A ) LAMP products detected by 1 000×SYBR Green I (Biotek Co., Ltd., Beijing, China). ( B ) Detection of LAMP products by agarose gel electrophoresis stained by EB (ethidium bromide). Tubes and lanes 3, 7, 11 and 15: negative control. Tubes and lanes 4, 8, 12 and 16: blank control. Tubes and lanes 1 and 2, 5 and 6, 9 and 10, 13 and 14: the positive plasmid pMD19-T-Pep1, repeated twice. Tubes and lanes 1–4, 5–8, 9–12 and 13–16: Bst DNA polymerase concentrations were 2.0 U, 4.0 U, 6.0 U and 8.0 U, respectively. Lane M: DL 15 000 + 2 000 DNA marker.

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

    31) Product Images from "Rapid Identification of Emerging Human-Pathogenic Sporothrix Species with Rolling Circle Amplification"

    Article Title: Rapid Identification of Emerging Human-Pathogenic Sporothrix Species with Rolling Circle Amplification

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2015.01385

    Schematic of RCA of circularized padlock probes . (A) S. brasiliensis (Sbra-RCA) padlock probe design. (B) CAL is amplified by PCR with primers CAL -Fw and CAL -Rv; PCR products are submitted to ligation. Circularization of padlock probes occurs only if both probe arms hybridize correctly to the target sequence. (C) Upon specific hybridization, the phosphorylated 5′ end and the free hydroxyl at the 3′ end of the probe are joined by Pfu DNA ligase. After ligation, non-circularized probes and single-stranded primers are removed with Exo I and Exo III (optional step). (D) Circularized padlock probes serve as DNA template and signal amplification occurs via RCA, using Bst DNA polymerase and the primers RCA1 and RCA2. DNA synthesis occurs continuously for 1 h under isothermal amplification (65°C). DNA products are detected by gel electrophoresis or directly with SYBR Green I.
    Figure Legend Snippet: Schematic of RCA of circularized padlock probes . (A) S. brasiliensis (Sbra-RCA) padlock probe design. (B) CAL is amplified by PCR with primers CAL -Fw and CAL -Rv; PCR products are submitted to ligation. Circularization of padlock probes occurs only if both probe arms hybridize correctly to the target sequence. (C) Upon specific hybridization, the phosphorylated 5′ end and the free hydroxyl at the 3′ end of the probe are joined by Pfu DNA ligase. After ligation, non-circularized probes and single-stranded primers are removed with Exo I and Exo III (optional step). (D) Circularized padlock probes serve as DNA template and signal amplification occurs via RCA, using Bst DNA polymerase and the primers RCA1 and RCA2. DNA synthesis occurs continuously for 1 h under isothermal amplification (65°C). DNA products are detected by gel electrophoresis or directly with SYBR Green I.

    Techniques Used: Amplification, Polymerase Chain Reaction, Ligation, Sequencing, Hybridization, DNA Synthesis, Nucleic Acid Electrophoresis, SYBR Green Assay

    32) Product Images from "COVID-19 Infection Diagnosis: Potential Impact of Isothermal Amplification Technology to Reduce Community Transmission of SARS-CoV-2"

    Article Title: COVID-19 Infection Diagnosis: Potential Impact of Isothermal Amplification Technology to Reduce Community Transmission of SARS-CoV-2

    Journal: Diagnostics

    doi: 10.3390/diagnostics10060399

    Schematic representation of loop-mediated amplification reaction and its principle. Unlike PCR primer design, LAMP is characterized with four different primers, specifically designed to recognize six distinct regions of the target DNA. Forward inner primer (FIP) consists of a F2 region at the 3’-end and an F1c region at the 5’-end. While the F3 primer (forward outer primer) consists of a F3 region which is complementary to the F3c region of the template sequence. The Backward Inner primer (BIP) is made up of a B2 region at the 3’-end and a B1c region at the 5’-end. B3 primer (backward outer primer) consists of a B3 region which is complementary to the B3c region of the template sequence. In regards to LAMP reaction, amplification begins when F2 region of FIP anneals to F2c region of the target DNA and initiates complementary strand synthesis, and F3 primer anneals to the F3c region of the target and extends, displacing the FIP linked complementary strand. This displaced strand forms a loop at the 5’-end, which provides the template for BIP, and B2 anneals to B2c region of the template. DNA synthesis is initiated, which results in the formation of a complementary strand and opening of the 5’-end loop. Subsequently, B3 anneals to B3c region of the target DNA and extends, displacing the BIP linked complementary strand, which forms a dumbbell-shaped DNA. The nucleotides are added to the 3’-end of F1 by Bst DNA polymerase, which extends and opens up the loop at the 5’-end. The dumbbell-shaped DNA is converted to a stem–loop structure (a and b), which initiates LAMP cycling (second stage of LAMP reaction). The amplicons formed are a mixture of stem–loop and cauliflower-like structures with multiple loops [ 49 ].
    Figure Legend Snippet: Schematic representation of loop-mediated amplification reaction and its principle. Unlike PCR primer design, LAMP is characterized with four different primers, specifically designed to recognize six distinct regions of the target DNA. Forward inner primer (FIP) consists of a F2 region at the 3’-end and an F1c region at the 5’-end. While the F3 primer (forward outer primer) consists of a F3 region which is complementary to the F3c region of the template sequence. The Backward Inner primer (BIP) is made up of a B2 region at the 3’-end and a B1c region at the 5’-end. B3 primer (backward outer primer) consists of a B3 region which is complementary to the B3c region of the template sequence. In regards to LAMP reaction, amplification begins when F2 region of FIP anneals to F2c region of the target DNA and initiates complementary strand synthesis, and F3 primer anneals to the F3c region of the target and extends, displacing the FIP linked complementary strand. This displaced strand forms a loop at the 5’-end, which provides the template for BIP, and B2 anneals to B2c region of the template. DNA synthesis is initiated, which results in the formation of a complementary strand and opening of the 5’-end loop. Subsequently, B3 anneals to B3c region of the target DNA and extends, displacing the BIP linked complementary strand, which forms a dumbbell-shaped DNA. The nucleotides are added to the 3’-end of F1 by Bst DNA polymerase, which extends and opens up the loop at the 5’-end. The dumbbell-shaped DNA is converted to a stem–loop structure (a and b), which initiates LAMP cycling (second stage of LAMP reaction). The amplicons formed are a mixture of stem–loop and cauliflower-like structures with multiple loops [ 49 ].

    Techniques Used: Amplification, Polymerase Chain Reaction, Sequencing, DNA Synthesis

    33) Product Images from "Diagnosis of Brugian Filariasis by Loop-Mediated Isothermal Amplification"

    Article Title: Diagnosis of Brugian Filariasis by Loop-Mediated Isothermal Amplification

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0001948

    Species-specificity of Hha I LAMP assay. (A) Each curve represents the calculated average of triplicate turbidity curves generated with various genomic DNAs (0. 1 ng) using Bst 2.0 DNA polymerase without loop primers. Turbidity was observed using B. malayi or B. timori DNA. (B) As a positive control, an actin gene fragment was PCR amplified from B. malayi (Bma), D. immitis (Dim), O. volvulus (Ovo), the mosquito Aedes albopictus (Aal), W. bancrofti (Wba), human (Hsa) and B. timori (Bti) DNAs using degenerate primers. Agarose gel showing amplification of a 244 bp fragment of the actin gene. The 100 bp DNA Ladder (New England Biolabs) was used as the molecular weight marker (MWM). Water was used in the non-template controls (NTC) in (A) and (B).
    Figure Legend Snippet: Species-specificity of Hha I LAMP assay. (A) Each curve represents the calculated average of triplicate turbidity curves generated with various genomic DNAs (0. 1 ng) using Bst 2.0 DNA polymerase without loop primers. Turbidity was observed using B. malayi or B. timori DNA. (B) As a positive control, an actin gene fragment was PCR amplified from B. malayi (Bma), D. immitis (Dim), O. volvulus (Ovo), the mosquito Aedes albopictus (Aal), W. bancrofti (Wba), human (Hsa) and B. timori (Bti) DNAs using degenerate primers. Agarose gel showing amplification of a 244 bp fragment of the actin gene. The 100 bp DNA Ladder (New England Biolabs) was used as the molecular weight marker (MWM). Water was used in the non-template controls (NTC) in (A) and (B).

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

    Sensitivity of Hha I LAMP assay. Ten-fold serial dilutions of B. malayi genomic DNA amplified with the Hha I primer set alone (A) or in the presence of loop primers (B) with Bst DNA polymerase, large fragment (wt Bst LF), Bst 2.0 DNA polymerase ( Bst 2.0) and Bst 2.0 WarmStart DNA polymerase ( Bst 2.0 WS). Data points represent the average of three samples and the error bars represent the standard deviation at each point. For each enzyme, 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 amount of starting material. (C) UV detection (365 nm) of products generated within 60 minutes using Bst 2.0 in the presence of loop primers and Fluorescent Detection Reagent. The amount of starting material in ng is shown below the photograph. Positive samples fluoresce green while negative samples remain dark.
    Figure Legend Snippet: Sensitivity of Hha I LAMP assay. Ten-fold serial dilutions of B. malayi genomic DNA amplified with the Hha I primer set alone (A) or in the presence of loop primers (B) with Bst DNA polymerase, large fragment (wt Bst LF), Bst 2.0 DNA polymerase ( Bst 2.0) and Bst 2.0 WarmStart DNA polymerase ( Bst 2.0 WS). Data points represent the average of three samples and the error bars represent the standard deviation at each point. For each enzyme, 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 amount of starting material. (C) UV detection (365 nm) of products generated within 60 minutes using Bst 2.0 in the presence of loop primers and Fluorescent Detection Reagent. The amount of starting material in ng is shown below the photograph. Positive samples fluoresce green while negative samples remain dark.

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

    Hha I LAMP assay for the detection of B. malayi infected blood samples. A set of serial dilutions (two-fold) of microfilariae in blood was prepared and DNA was isolated from each dilution. Three experiments were performed using a different but overlapping range of DNA dilutions. One µl of DNA from each dilution was used in LAMP reactions with Bst 2.0 DNA polymerase. Samples from each experimental set-up were performed in triplicate (experiments 1 and 2) or duplicate (experiment 3). Average threshold times and standard deviations were plotted against the approximate number of mf/µl DNA solution.
    Figure Legend Snippet: Hha I LAMP assay for the detection of B. malayi infected blood samples. A set of serial dilutions (two-fold) of microfilariae in blood was prepared and DNA was isolated from each dilution. Three experiments were performed using a different but overlapping range of DNA dilutions. One µl of DNA from each dilution was used in LAMP reactions with Bst 2.0 DNA polymerase. Samples from each experimental set-up were performed in triplicate (experiments 1 and 2) or duplicate (experiment 3). Average threshold times and standard deviations were plotted against the approximate number of mf/µl DNA solution.

    Techniques Used: Lamp Assay, Infection, Isolation

    34) Product Images from "Large fragment Bst DNA polymerase for whole genome amplification of DNA from formalin-fixed paraffin-embedded tissues"

    Article Title: Large fragment Bst DNA polymerase for whole genome amplification of DNA from formalin-fixed paraffin-embedded tissues

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-7-312

    Detection of Skp2 amplification in NSCLC samples following whole genome amplification by Bst DNA polymerase . The ratios of Skp2 to PIK3R1 gene were maintained in Bst amplified vs. non-amplified NSCLC samples. Error bars represent SD.
    Figure Legend Snippet: Detection of Skp2 amplification in NSCLC samples following whole genome amplification by Bst DNA polymerase . The ratios of Skp2 to PIK3R1 gene were maintained in Bst amplified vs. non-amplified NSCLC samples. Error bars represent SD.

    Techniques Used: Amplification, Whole Genome Amplification

    Gel electrophoresis of Bst DNA polymerase amplification products . From left to right: (1) Lambda DNA-Hind III digested ladder; FFPE samples: (2, 3) normal lung 3 4; (4, 5) neuroblastoma xenografts LAN-5 SK-N-BE (2) and (6, 7) NSCLC 3 4; (8) Commercial DNA; (9) Negative control. Samples were analyzed in 0.5% agarose gel, stained with SYBR-green II. 10% by volume of the amplification product was used for the gel electrophoresis.
    Figure Legend Snippet: Gel electrophoresis of Bst DNA polymerase amplification products . From left to right: (1) Lambda DNA-Hind III digested ladder; FFPE samples: (2, 3) normal lung 3 4; (4, 5) neuroblastoma xenografts LAN-5 SK-N-BE (2) and (6, 7) NSCLC 3 4; (8) Commercial DNA; (9) Negative control. Samples were analyzed in 0.5% agarose gel, stained with SYBR-green II. 10% by volume of the amplification product was used for the gel electrophoresis.

    Techniques Used: Nucleic Acid Electrophoresis, Amplification, Lambda DNA Preparation, Formalin-fixed Paraffin-Embedded, Negative Control, Agarose Gel Electrophoresis, Staining, SYBR Green Assay

    N- myc gene content in Bst amplified vs. non-amplified neuroblastoma xenografts . For neuroblastoma xenografts, where N- myc gene is highly amplified, relative gene content in Bst amplified samples was comparable to the respective values in non-amplified samples and the representational distortion was negligible. Note: NBL-S is a neuroblastoma cell line that lacks N- myc amplification and appropriately the calculated copy numbers were 1.12 ± 0.03 for non-amplified DNA and 1.14 ± 0.35 for Bst amplified DNA. Error bars represent SD.
    Figure Legend Snippet: N- myc gene content in Bst amplified vs. non-amplified neuroblastoma xenografts . For neuroblastoma xenografts, where N- myc gene is highly amplified, relative gene content in Bst amplified samples was comparable to the respective values in non-amplified samples and the representational distortion was negligible. Note: NBL-S is a neuroblastoma cell line that lacks N- myc amplification and appropriately the calculated copy numbers were 1.12 ± 0.03 for non-amplified DNA and 1.14 ± 0.35 for Bst amplified DNA. Error bars represent SD.

    Techniques Used: Amplification

    Mean amplification of DNA by Bst polymerase . All reactions started with 10 ng of target DNA. FFPE samples: Lung 1–5, neuroblastoma xenografts (LAN-5, NUB-7, SK-N-BE(2), NBL-S) and NSCLC 1–7. Intact DNA samples: FL (Frozen Lung) 1–4 and Positive C. (Control). Negative C. (Control) contained water in lieu of target DNA. For each sample the mean and SD of 2–6 independent experiments is shown.
    Figure Legend Snippet: Mean amplification of DNA by Bst polymerase . All reactions started with 10 ng of target DNA. FFPE samples: Lung 1–5, neuroblastoma xenografts (LAN-5, NUB-7, SK-N-BE(2), NBL-S) and NSCLC 1–7. Intact DNA samples: FL (Frozen Lung) 1–4 and Positive C. (Control). Negative C. (Control) contained water in lieu of target DNA. For each sample the mean and SD of 2–6 independent experiments is shown.

    Techniques Used: Amplification, Formalin-fixed Paraffin-Embedded

    35) Product Images from "Evaluation of the stability of lyophilized loop-mediated isothermal amplification reagents for the detection of Coxiella burnetii"

    Article Title: Evaluation of the stability of lyophilized loop-mediated isothermal amplification reagents for the detection of Coxiella burnetii

    Journal: Heliyon

    doi: 10.1016/j.heliyon.2017.e00415

    Lyophilized LAMP reagents for the detection of Coxiella burnetii . The lyophilized reagents were inside a 0.2 ml vial containing Bst DNA polymerase, SYBR green, primers, and dNTPs. Vials were packaged in a zipped aluminum foil bag.
    Figure Legend Snippet: Lyophilized LAMP reagents for the detection of Coxiella burnetii . The lyophilized reagents were inside a 0.2 ml vial containing Bst DNA polymerase, SYBR green, primers, and dNTPs. Vials were packaged in a zipped aluminum foil bag.

    Techniques Used: SYBR Green Assay

    36) 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

    37) 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

    38) Product Images from "A novel multiplex isothermal amplification method for rapid detection and identification of viruses"

    Article Title: A novel multiplex isothermal amplification method for rapid detection and identification of viruses

    Journal: Scientific Reports

    doi: 10.1038/srep17925

    Position of oligonucleotides, oligofluorophores hybridization, and mechanism of detection illustrated on the HCV 5′-NCR. (A) Illustration of the positions of oligonucleotides and oligofluorophores used for sequence-specific detection on the 5′-NCR of the HCV genome. (B) Biomolecular mechanism of the bi-labeled Loop Reverse oligonucleotide (LRp) covalently linked with 6-FAM as reporter on the 5′ end and BHQ1 as quencher on the 3′end. In step 1, in the absence of hybridization to amplified product (target sequence), the reporter remains quenched, with no fluorescence emission due to the close proximity of the quencher to the fluorophore. In the presence of amplified product in steps 2 and 3, the LRp hybridizes to the specific target between the R1c and R2 segments of the gene sequence, thus fluorophore begins to fluoresce as it separates from the quencher. From step 3 to step 4 as Bst DNA Polymerase catalyzes the reaction, quenching is disrupted and fluorescence is increased due to the increased distance (separation) between the reporter and quencher when the fluorooligonucleotide hybridizes and also due to fluorescence resonance energy transfer (FRET). The emitted fluorescence which corresponds to the amount of amplified nucleic acid is measured by spectrofluorometer in relative fluorescence units (RFU) and visualized by the naked-eye under UV-illumination.
    Figure Legend Snippet: Position of oligonucleotides, oligofluorophores hybridization, and mechanism of detection illustrated on the HCV 5′-NCR. (A) Illustration of the positions of oligonucleotides and oligofluorophores used for sequence-specific detection on the 5′-NCR of the HCV genome. (B) Biomolecular mechanism of the bi-labeled Loop Reverse oligonucleotide (LRp) covalently linked with 6-FAM as reporter on the 5′ end and BHQ1 as quencher on the 3′end. In step 1, in the absence of hybridization to amplified product (target sequence), the reporter remains quenched, with no fluorescence emission due to the close proximity of the quencher to the fluorophore. In the presence of amplified product in steps 2 and 3, the LRp hybridizes to the specific target between the R1c and R2 segments of the gene sequence, thus fluorophore begins to fluoresce as it separates from the quencher. From step 3 to step 4 as Bst DNA Polymerase catalyzes the reaction, quenching is disrupted and fluorescence is increased due to the increased distance (separation) between the reporter and quencher when the fluorooligonucleotide hybridizes and also due to fluorescence resonance energy transfer (FRET). The emitted fluorescence which corresponds to the amount of amplified nucleic acid is measured by spectrofluorometer in relative fluorescence units (RFU) and visualized by the naked-eye under UV-illumination.

    Techniques Used: Hybridization, Sequencing, Labeling, Amplification, Fluorescence, Förster Resonance Energy Transfer

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    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
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  • 99
    New England Biolabs bst e ii digested lambda dna
    Taq polymerase alone yields a S/N of 10 but can be improved Bridging PCR with Taq polymerase and the improvement of the S/N by addition of gp32 and (NH 4 ) 2 SO 4 . The cut or intact templates was prepared by PCR with Taq polymerase and primers b1 and Jp on double-stranded plasmid pBS(-). BPCR was carried out with Taq polymerase and primers b1 and M13 pI. (See Figure 2 for location of primers and Table 1 for their sequence.) The other template was Phagescript phage particles. The 1 × PCR buffer for lanes 1–10 was 50 mM KCl, 10 mM Tris-HCl (pH 8.8 at 25°C), 1.5 mM MgCl 2 . The 1 × PCR buffer with (NH 4 ) 2 SO 4 , (lanes 11 to 20) was 10 mM KCl, 20 mM Tris-HCl (pH 8.8 at 25°C), 2.0 mM MgSO 4 , 10 mM (NH 4 ) 2 SO 4 , 0.1% Triton X-100 and 0.1 mg/ml bovine serum albumin. The ethidium bromide fluorescence image of the 1.0% agarose electrophoresis gel was photographed. The photograph was digitized and processed with Scion Image Adobe Photoshop software on a Macintosh computer. The image intensity was inverted. Each lane has two rows which are labeled as cut, i.e. Pvu II digested (upper row), or intact (lower row). The BPCR product bands are about 1261 bp, as expected from the template sequences (see figure 2 ). Lanes 1 to 5 show BPCR with Taq polymerase and no other additions. Lanes 6 to 10 show the enhancement of the S/N of BPCR by the addition of 0.01% gp32. Lanes 11 to 15, show the enhancement of BPCR by the addition of 10 mM (NH 4 ) 2 SO 4 . Lanes 16 to 20, show BPCR with gp32 and (NH 4 ) 2 SO 4 . The addition of both gp32 and (NH 4 ) 2 SO 4 did not improve the S/N. Each lane has two samples separately loaded into the two separate loading wells, one on each row. In the figure, the upper row is of source template cut with Pvu II, and the lower <t>DNA</t> sample is of the same source template intact (without Pvu II cut). The source template concentrations in the two samples were equal. Lanes 2, 3, 4 and 5 are serial template dilutions of lane 1. Lanes 7, 8, 9 and 10 are serial template dilutions of lane 6. Lanes 12, 13, 14 and 15 are serial template dilutions of lane 11. Lanes 17, 18, 19 and 20 are serial template dilutions of lane 16. Lane 21 contains DNA size markers made from <t>Lambda</t> DNA, <t>Bst</t> E II digested, 125 ng. The source template concentrations of lanes 1, 6, 11 and 17 are adjusted to equal to 1 × 10 -17 mol/μl PCR solution.
    Bst E Ii Digested Lambda Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    New England Biolabs general information bst dna polymerase large fragment
    Verification of UIMA using different <t>DNA</t> 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 <t>Bst</t> 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 .
    General Information Bst Dna Polymerase Large Fragment, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    New England Biolabs g stearothermophilus bst dna polymerase
    FANA transcription and reverse transcription in vitro. a Constitutional structures for 2’-deoxyribonucleic acid <t>(DNA)</t> and 2’-fluoroarabino nucleic acid (FANA). b FANA transcription activity for wild-type archaeal DNA polymerases (exo−) from 9°N, DV, Kod, and Tgo (left panel). Samples were analyzed after 15 and 30 min at 55 °C. FANA reverse transcriptase activity of <t>Bst</t> DNA polymerase LF, 2.0, 3.0, and LF* (right panel). LF* denotes wild-type Bst DNA polymerase, large fragment, expressed and purified from E. coli . Samples were analyzed after 30 min at 50 °C. All samples were resolved on denaturing PAGE and visualized using a LI-COR Odyssey CLx. c Fidelity profile observed for FANA replication using Tgo and Bst LF* polymerases. The mutation profile reveals a mutation rate of 8 × 10 -4 and an overall fidelity of ~99.9%. d Catalytic rates observed for FANA synthesis with Tgo (left panel) and reverse transcription with Bst LF* (right panel)
    G Stearothermophilus Bst Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Taq polymerase alone yields a S/N of 10 but can be improved Bridging PCR with Taq polymerase and the improvement of the S/N by addition of gp32 and (NH 4 ) 2 SO 4 . The cut or intact templates was prepared by PCR with Taq polymerase and primers b1 and Jp on double-stranded plasmid pBS(-). BPCR was carried out with Taq polymerase and primers b1 and M13 pI. (See Figure 2 for location of primers and Table 1 for their sequence.) The other template was Phagescript phage particles. The 1 × PCR buffer for lanes 1–10 was 50 mM KCl, 10 mM Tris-HCl (pH 8.8 at 25°C), 1.5 mM MgCl 2 . The 1 × PCR buffer with (NH 4 ) 2 SO 4 , (lanes 11 to 20) was 10 mM KCl, 20 mM Tris-HCl (pH 8.8 at 25°C), 2.0 mM MgSO 4 , 10 mM (NH 4 ) 2 SO 4 , 0.1% Triton X-100 and 0.1 mg/ml bovine serum albumin. The ethidium bromide fluorescence image of the 1.0% agarose electrophoresis gel was photographed. The photograph was digitized and processed with Scion Image Adobe Photoshop software on a Macintosh computer. The image intensity was inverted. Each lane has two rows which are labeled as cut, i.e. Pvu II digested (upper row), or intact (lower row). The BPCR product bands are about 1261 bp, as expected from the template sequences (see figure 2 ). Lanes 1 to 5 show BPCR with Taq polymerase and no other additions. Lanes 6 to 10 show the enhancement of the S/N of BPCR by the addition of 0.01% gp32. Lanes 11 to 15, show the enhancement of BPCR by the addition of 10 mM (NH 4 ) 2 SO 4 . Lanes 16 to 20, show BPCR with gp32 and (NH 4 ) 2 SO 4 . The addition of both gp32 and (NH 4 ) 2 SO 4 did not improve the S/N. Each lane has two samples separately loaded into the two separate loading wells, one on each row. In the figure, the upper row is of source template cut with Pvu II, and the lower DNA sample is of the same source template intact (without Pvu II cut). The source template concentrations in the two samples were equal. Lanes 2, 3, 4 and 5 are serial template dilutions of lane 1. Lanes 7, 8, 9 and 10 are serial template dilutions of lane 6. Lanes 12, 13, 14 and 15 are serial template dilutions of lane 11. Lanes 17, 18, 19 and 20 are serial template dilutions of lane 16. Lane 21 contains DNA size markers made from Lambda DNA, Bst E II digested, 125 ng. The source template concentrations of lanes 1, 6, 11 and 17 are adjusted to equal to 1 × 10 -17 mol/μl PCR solution.

    Journal: BMC Biotechnology

    Article Title: Signal and noise in bridging PCR

    doi: 10.1186/1472-6750-2-13

    Figure Lengend Snippet: Taq polymerase alone yields a S/N of 10 but can be improved Bridging PCR with Taq polymerase and the improvement of the S/N by addition of gp32 and (NH 4 ) 2 SO 4 . The cut or intact templates was prepared by PCR with Taq polymerase and primers b1 and Jp on double-stranded plasmid pBS(-). BPCR was carried out with Taq polymerase and primers b1 and M13 pI. (See Figure 2 for location of primers and Table 1 for their sequence.) The other template was Phagescript phage particles. The 1 × PCR buffer for lanes 1–10 was 50 mM KCl, 10 mM Tris-HCl (pH 8.8 at 25°C), 1.5 mM MgCl 2 . The 1 × PCR buffer with (NH 4 ) 2 SO 4 , (lanes 11 to 20) was 10 mM KCl, 20 mM Tris-HCl (pH 8.8 at 25°C), 2.0 mM MgSO 4 , 10 mM (NH 4 ) 2 SO 4 , 0.1% Triton X-100 and 0.1 mg/ml bovine serum albumin. The ethidium bromide fluorescence image of the 1.0% agarose electrophoresis gel was photographed. The photograph was digitized and processed with Scion Image Adobe Photoshop software on a Macintosh computer. The image intensity was inverted. Each lane has two rows which are labeled as cut, i.e. Pvu II digested (upper row), or intact (lower row). The BPCR product bands are about 1261 bp, as expected from the template sequences (see figure 2 ). Lanes 1 to 5 show BPCR with Taq polymerase and no other additions. Lanes 6 to 10 show the enhancement of the S/N of BPCR by the addition of 0.01% gp32. Lanes 11 to 15, show the enhancement of BPCR by the addition of 10 mM (NH 4 ) 2 SO 4 . Lanes 16 to 20, show BPCR with gp32 and (NH 4 ) 2 SO 4 . The addition of both gp32 and (NH 4 ) 2 SO 4 did not improve the S/N. Each lane has two samples separately loaded into the two separate loading wells, one on each row. In the figure, the upper row is of source template cut with Pvu II, and the lower DNA sample is of the same source template intact (without Pvu II cut). The source template concentrations in the two samples were equal. Lanes 2, 3, 4 and 5 are serial template dilutions of lane 1. Lanes 7, 8, 9 and 10 are serial template dilutions of lane 6. Lanes 12, 13, 14 and 15 are serial template dilutions of lane 11. Lanes 17, 18, 19 and 20 are serial template dilutions of lane 16. Lane 21 contains DNA size markers made from Lambda DNA, Bst E II digested, 125 ng. The source template concentrations of lanes 1, 6, 11 and 17 are adjusted to equal to 1 × 10 -17 mol/μl PCR solution.

    Article Snippet: Materials and Methods The sources of materials used in this study: Phagescript and pBS(-) plasmid were from Stratagene; Taq polymerase and the Stoffel fragment of Taq were from Perkin Elmer Cetus; gp32 was from Pharmacia; Bst E II-digested Lambda DNA was from New England Biolabs.

    Techniques: Polymerase Chain Reaction, Plasmid Preparation, Sequencing, Fluorescence, Electrophoresis, Software, Labeling, Lambda DNA Preparation

    Stoffel fragment of Taq yields a S/N of 100 The Stoffel fragment of Taq polymerase was used in BPCR. The gel was 1% agarose containing ethidium bromide. The fluorescent image of the gel was acquired and processed with a Macintosh computer, a video camera and the software Scion Image 1.59 (from NIH). The gel image intensity was inverted, and the background was subtracted (horizontal 1D). The band intensity of the 328 bp PCR product was recorded. The intensity profile curve of the 328 bp band is aligned and plotted below the gel lanes. The relative area under each peak is given below the peak. Area values are equalized to Lane 10, whose area value is set as 1.0. Lane 1, DNA size markers: a mixture of three separate PCR products: 235 bp (with primers b1 and M13 revI and template pBS(-)), 495 bp (primers M13 pIII and M13 revI and template Phagescript) and 736 bp (primers b1 and b2 and template pBS (-)). Lanes 2, size markers of Lambda DNA, Bst E II-digested, 75 ng/lane. Lanes 3, 4, 5 and 6, PCR with intact source template. Each lane had 5 μl PCR product. Lanes 7, 8, 9, 10 and 11, PCR with source template cut with Pvu II. Each lane had 5 μl PCR product. The concentrations of source template of lanes 3 and 7 are adjusted to be equal, 1 × 10 -16 mol/μl PCR solution. Lanes 4, 5 and 6 are serial template dilutions of lane 3. Lanes 8, 9, 10 and 11 are serial template dilutions of lane 7. BPCR was carried out with the Stoffel fragment of Taq polymerase and primers b1 and M13 pV. (See Figure 2 for location of primers and Table 1 for their sequence.) The cut or intact template was made by PCR with the Stoffel fragment, primers b1 and Jp and a plasmid preparation of pBS(-). The other template (reference template) was ssDNA from Phagescript particles. The 1 × PCR buffer contains 10 mM KCl, 10 mM Tris-HCl (pH 8.3 at 25°C) and 2.5 mM MgCl 2 .

    Journal: BMC Biotechnology

    Article Title: Signal and noise in bridging PCR

    doi: 10.1186/1472-6750-2-13

    Figure Lengend Snippet: Stoffel fragment of Taq yields a S/N of 100 The Stoffel fragment of Taq polymerase was used in BPCR. The gel was 1% agarose containing ethidium bromide. The fluorescent image of the gel was acquired and processed with a Macintosh computer, a video camera and the software Scion Image 1.59 (from NIH). The gel image intensity was inverted, and the background was subtracted (horizontal 1D). The band intensity of the 328 bp PCR product was recorded. The intensity profile curve of the 328 bp band is aligned and plotted below the gel lanes. The relative area under each peak is given below the peak. Area values are equalized to Lane 10, whose area value is set as 1.0. Lane 1, DNA size markers: a mixture of three separate PCR products: 235 bp (with primers b1 and M13 revI and template pBS(-)), 495 bp (primers M13 pIII and M13 revI and template Phagescript) and 736 bp (primers b1 and b2 and template pBS (-)). Lanes 2, size markers of Lambda DNA, Bst E II-digested, 75 ng/lane. Lanes 3, 4, 5 and 6, PCR with intact source template. Each lane had 5 μl PCR product. Lanes 7, 8, 9, 10 and 11, PCR with source template cut with Pvu II. Each lane had 5 μl PCR product. The concentrations of source template of lanes 3 and 7 are adjusted to be equal, 1 × 10 -16 mol/μl PCR solution. Lanes 4, 5 and 6 are serial template dilutions of lane 3. Lanes 8, 9, 10 and 11 are serial template dilutions of lane 7. BPCR was carried out with the Stoffel fragment of Taq polymerase and primers b1 and M13 pV. (See Figure 2 for location of primers and Table 1 for their sequence.) The cut or intact template was made by PCR with the Stoffel fragment, primers b1 and Jp and a plasmid preparation of pBS(-). The other template (reference template) was ssDNA from Phagescript particles. The 1 × PCR buffer contains 10 mM KCl, 10 mM Tris-HCl (pH 8.3 at 25°C) and 2.5 mM MgCl 2 .

    Article Snippet: Materials and Methods The sources of materials used in this study: Phagescript and pBS(-) plasmid were from Stratagene; Taq polymerase and the Stoffel fragment of Taq were from Perkin Elmer Cetus; gp32 was from Pharmacia; Bst E II-digested Lambda DNA was from New England Biolabs.

    Techniques: Software, Polymerase Chain Reaction, Lambda DNA Preparation, Sequencing, Plasmid Preparation

    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 .

    Journal: Scientific Reports

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

    doi: 10.1038/s41598-017-13324-0

    Figure Lengend 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 .

    Article Snippet: General information Bst DNA polymerase Large fragment (Bst LF), Bst 2.0 DNA polymerase (Bst 2.0), Bst 2.0 WarmStart DNA polymerase (Bst 2.0 WS), Bst 3.0 DNA polymerase (Bst 3.0), Klenow fragment polymerase (Klenow), Klenow fragment exo- polymerase (Klenow (exo-)), Vent exo- DNA polymerase (Vent (exo-)), and dNTP Mix were purchased from New England Biolabs.

    Techniques: 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 .

    Journal: Scientific Reports

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

    doi: 10.1038/s41598-017-13324-0

    Figure Lengend 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 .

    Article Snippet: General information Bst DNA polymerase Large fragment (Bst LF), Bst 2.0 DNA polymerase (Bst 2.0), Bst 2.0 WarmStart DNA polymerase (Bst 2.0 WS), Bst 3.0 DNA polymerase (Bst 3.0), Klenow fragment polymerase (Klenow), Klenow fragment exo- polymerase (Klenow (exo-)), Vent exo- DNA polymerase (Vent (exo-)), and dNTP Mix were purchased from New England Biolabs.

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

    FANA transcription and reverse transcription in vitro. a Constitutional structures for 2’-deoxyribonucleic acid (DNA) and 2’-fluoroarabino nucleic acid (FANA). b FANA transcription activity for wild-type archaeal DNA polymerases (exo−) from 9°N, DV, Kod, and Tgo (left panel). Samples were analyzed after 15 and 30 min at 55 °C. FANA reverse transcriptase activity of Bst DNA polymerase LF, 2.0, 3.0, and LF* (right panel). LF* denotes wild-type Bst DNA polymerase, large fragment, expressed and purified from E. coli . Samples were analyzed after 30 min at 50 °C. All samples were resolved on denaturing PAGE and visualized using a LI-COR Odyssey CLx. c Fidelity profile observed for FANA replication using Tgo and Bst LF* polymerases. The mutation profile reveals a mutation rate of 8 × 10 -4 and an overall fidelity of ~99.9%. d Catalytic rates observed for FANA synthesis with Tgo (left panel) and reverse transcription with Bst LF* (right panel)

    Journal: Nature Communications

    Article Title: Evolution of a General RNA-Cleaving FANA Enzyme

    doi: 10.1038/s41467-018-07611-1

    Figure Lengend Snippet: FANA transcription and reverse transcription in vitro. a Constitutional structures for 2’-deoxyribonucleic acid (DNA) and 2’-fluoroarabino nucleic acid (FANA). b FANA transcription activity for wild-type archaeal DNA polymerases (exo−) from 9°N, DV, Kod, and Tgo (left panel). Samples were analyzed after 15 and 30 min at 55 °C. FANA reverse transcriptase activity of Bst DNA polymerase LF, 2.0, 3.0, and LF* (right panel). LF* denotes wild-type Bst DNA polymerase, large fragment, expressed and purified from E. coli . Samples were analyzed after 30 min at 50 °C. All samples were resolved on denaturing PAGE and visualized using a LI-COR Odyssey CLx. c Fidelity profile observed for FANA replication using Tgo and Bst LF* polymerases. The mutation profile reveals a mutation rate of 8 × 10 -4 and an overall fidelity of ~99.9%. d Catalytic rates observed for FANA synthesis with Tgo (left panel) and reverse transcription with Bst LF* (right panel)

    Article Snippet: ThermoPol buffer, Taq DNA polymerase, G. stearothermophilus Bst DNA polymerase, LF, and its variants Bst 2.0 DNA polymerase (2.0), and Bst 3.0 DNA polymerase (3.0), DH5α competent cells, and Monarch DNA gel extraction kits were purchased from New England Biolabs (Ipswich, MA).

    Techniques: In Vitro, Activity Assay, Purification, Polyacrylamide Gel Electrophoresis, Mutagenesis