bst  (New England Biolabs)


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    Name:
    Bst DNA Polymerase Lg Frag
    Description:
    Bst DNA Polymerase Lg Frag 8 000 units
    Catalog Number:
    M0275L
    Price:
    283
    Category:
    Thermostable DNA Polymerases
    Size:
    8 000 units
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    Structured Review

    New England Biolabs bst
    Bst DNA Polymerase Lg Frag
    Bst DNA Polymerase Lg Frag 8 000 units
    https://www.bioz.com/result/bst/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    bst - by Bioz Stars, 2021-05
    99/100 stars

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    Incubation:

    Article Title: In vitro selection of an XNA aptamer capable of small-molecule recognition
    Article Snippet: The recovered TNA was annealed with 5 pmol of unmodified forward primer in 1× Thermopol buffer as previously described. .. 1.6 U of Bst DNA polymerase I large fragment (New England Biolabs) was added to the primer-template complex and the reaction incubated at 55°C for 3.5 h. The RT reactions were divided into 5 μl aliquots and PCR amplified as previously described for the DNA library using unmodified versions of the forward and reverse PCR primers and an annealing temperature of 50°C. .. This material was purified using MinElute PCR clean up columns and re-amplified using the PEG-forward and FAM-reverse PCR primers to enable strand separation.

    Article Title: A library-based method to rapidly analyse chromatin accessibility at multiple genomic regions
    Article Snippet: Nick repair Nicks at the 3′-junctions between DNA fragments and adaptors were repaired by the strand-displacement activity of Bst DNA polymerase, Large Fragment (Fermentas). .. Fifteen microlitres of the adaptor ligated DNA were incubated for 30 min at 65°C in 1× ThermoPol Reaction Buffer (New England Biolabs), 8 µg Bovine Serum Albumin (BSA) (New England Biolabs), 20 nmol dNTPs and 16 U Bst DNA polymerase, Large Fragment (New England Biolabs) in a total volume of 20 µl. .. Isolation of the single-stranded A-B adaptor fragment library Twenty microlitres of stock M-270 Streptavidin beads (Dynal, Oslo, Norway) were washed twice in a 20 µl volume of 2× Beads buffer and washing buffer (B & W) (10 mM Tris–HCl, pH 7.5, 1 mM EDTA, 2 M NaCl), by precipitating, between the washes, with the magnetic particle concentrator (MPC, Dynal).

    Polymerase Chain Reaction:

    Article Title: In vitro selection of an XNA aptamer capable of small-molecule recognition
    Article Snippet: The recovered TNA was annealed with 5 pmol of unmodified forward primer in 1× Thermopol buffer as previously described. .. 1.6 U of Bst DNA polymerase I large fragment (New England Biolabs) was added to the primer-template complex and the reaction incubated at 55°C for 3.5 h. The RT reactions were divided into 5 μl aliquots and PCR amplified as previously described for the DNA library using unmodified versions of the forward and reverse PCR primers and an annealing temperature of 50°C. .. This material was purified using MinElute PCR clean up columns and re-amplified using the PEG-forward and FAM-reverse PCR primers to enable strand separation.

    Article Title: Novel Bioluminescent Quantitative Detection of Nucleic Acid Amplification in Real-Time
    Article Snippet: .. Materials and reagents Unless otherwise noted, chemicals were purchased from Sigma with the exception of luciferin potassium salt (LH2 ; Europa Biotech, Ely, UK), UltraGlow firefly luciferase (UGrLuc; Promega, WI, USA), adenosine-5′-O-phosphosulphate (APS; Biolog Life Science Institute, Bremen, Germany), Bst DNA polymerase large fragment (Bst) and ThermoPol buffer (New England Biolabs, MA, USA), QuantiTech SYBR Green PCR kit (Qiagen, Hilden, Germany), cloned AMV reverse transcriptase and PicoGreen dsDNA Quantitation kit (Invitrogen, CA, USA). ..

    Amplification:

    Article Title: In vitro selection of an XNA aptamer capable of small-molecule recognition
    Article Snippet: The recovered TNA was annealed with 5 pmol of unmodified forward primer in 1× Thermopol buffer as previously described. .. 1.6 U of Bst DNA polymerase I large fragment (New England Biolabs) was added to the primer-template complex and the reaction incubated at 55°C for 3.5 h. The RT reactions were divided into 5 μl aliquots and PCR amplified as previously described for the DNA library using unmodified versions of the forward and reverse PCR primers and an annealing temperature of 50°C. .. This material was purified using MinElute PCR clean up columns and re-amplified using the PEG-forward and FAM-reverse PCR primers to enable strand separation.

    Article Title: Validation of PfSNP-LAMP-Lateral Flow Dipstick for Detection of Single Nucleotide Polymorphism Associated with Pyrimethamine Resistance in Plasmodium falciparum
    Article Snippet: .. PfSNP-LAMP-LFD Conditions The 25 µL-volume of Pf SNP-LAMP reaction mixture contained the following components: 2 µM each of Pf -snp-FIP and Pf -snp-BIP primers, 0.2 µM each of Pf -snp-F3 and Pf -snp-B3 primers, 1X isothermal amplification buffer (20 mM Tris-HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl2 , 2 mM MgSO4 , 0.1% Triton X-100, pH 8.8), 0.4 M betaine (USB Corporation, Cleveland, OH, USA), 8 mM MgSO4 (Sigma-Aldrich, St. Louis, MO, USA), 1.4 mM dNTP mix (Promega, Madison, WI, USA), 8 units of Bst DNA polymerase, a large fragment or Bst 2.0 DNA polymerase or Bst 2.0 WarmStart DNA polymerase (New England Biolab, Ipswich, MA, USA), and 2 µL of DNA sample. ..

    Concentration Assay:

    Article Title: Large fragment Bst DNA polymerase for whole genome amplification of DNA from formalin-fixed paraffin-embedded tissues
    Article Snippet: .. The reaction mixture was then brought up to 50 μL with 400 μM dNTPs in 1× ThermoPol buffer, 0.35 units/μL Bst DNA polymerase, large fragment (New England Biolabs, Ipswich, MA) and 4% final concentration of DMSO. .. T4 gene 32 protein (Amersham Biosciences, Piscataway, NJ) was added to the reaction in final concentration of 30 ng/μL.

    Luciferase:

    Article Title: Novel Bioluminescent Quantitative Detection of Nucleic Acid Amplification in Real-Time
    Article Snippet: .. Materials and reagents Unless otherwise noted, chemicals were purchased from Sigma with the exception of luciferin potassium salt (LH2 ; Europa Biotech, Ely, UK), UltraGlow firefly luciferase (UGrLuc; Promega, WI, USA), adenosine-5′-O-phosphosulphate (APS; Biolog Life Science Institute, Bremen, Germany), Bst DNA polymerase large fragment (Bst) and ThermoPol buffer (New England Biolabs, MA, USA), QuantiTech SYBR Green PCR kit (Qiagen, Hilden, Germany), cloned AMV reverse transcriptase and PicoGreen dsDNA Quantitation kit (Invitrogen, CA, USA). ..

    SYBR Green Assay:

    Article Title: Novel Bioluminescent Quantitative Detection of Nucleic Acid Amplification in Real-Time
    Article Snippet: .. Materials and reagents Unless otherwise noted, chemicals were purchased from Sigma with the exception of luciferin potassium salt (LH2 ; Europa Biotech, Ely, UK), UltraGlow firefly luciferase (UGrLuc; Promega, WI, USA), adenosine-5′-O-phosphosulphate (APS; Biolog Life Science Institute, Bremen, Germany), Bst DNA polymerase large fragment (Bst) and ThermoPol buffer (New England Biolabs, MA, USA), QuantiTech SYBR Green PCR kit (Qiagen, Hilden, Germany), cloned AMV reverse transcriptase and PicoGreen dsDNA Quantitation kit (Invitrogen, CA, USA). ..

    Clone Assay:

    Article Title: Novel Bioluminescent Quantitative Detection of Nucleic Acid Amplification in Real-Time
    Article Snippet: .. Materials and reagents Unless otherwise noted, chemicals were purchased from Sigma with the exception of luciferin potassium salt (LH2 ; Europa Biotech, Ely, UK), UltraGlow firefly luciferase (UGrLuc; Promega, WI, USA), adenosine-5′-O-phosphosulphate (APS; Biolog Life Science Institute, Bremen, Germany), Bst DNA polymerase large fragment (Bst) and ThermoPol buffer (New England Biolabs, MA, USA), QuantiTech SYBR Green PCR kit (Qiagen, Hilden, Germany), cloned AMV reverse transcriptase and PicoGreen dsDNA Quantitation kit (Invitrogen, CA, USA). ..

    Quantitation Assay:

    Article Title: Novel Bioluminescent Quantitative Detection of Nucleic Acid Amplification in Real-Time
    Article Snippet: .. Materials and reagents Unless otherwise noted, chemicals were purchased from Sigma with the exception of luciferin potassium salt (LH2 ; Europa Biotech, Ely, UK), UltraGlow firefly luciferase (UGrLuc; Promega, WI, USA), adenosine-5′-O-phosphosulphate (APS; Biolog Life Science Institute, Bremen, Germany), Bst DNA polymerase large fragment (Bst) and ThermoPol buffer (New England Biolabs, MA, USA), QuantiTech SYBR Green PCR kit (Qiagen, Hilden, Germany), cloned AMV reverse transcriptase and PicoGreen dsDNA Quantitation kit (Invitrogen, CA, USA). ..

    Activity Assay:

    Article Title: COVID-19 Infection Diagnosis: Potential Impact of Isothermal Amplification Technology to Reduce Community Transmission of SARS-CoV-2
    Article Snippet: .. Essentially, LAMP uses a Bst DNA polymerase with strand-displacement activity, coupled with two inner primers (FIP, BIP) and outer primers (F3, B3) that recognizes six separate regions on a DNA template. .. Additional loop primers (LF, LB) may be added to speed up the reaction by binding to and amplifying newly formed loop amplicons in the reaction.

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    New England Biolabs bst dna polymerase
    Detection of Skp2 amplification in NSCLC samples following whole genome amplification by <t>Bst</t> <t>DNA</t> polymerase . The ratios of Skp2 to PIK3R1 gene were maintained in Bst amplified vs. non-amplified NSCLC samples. Error bars represent SD.
    Bst Dna Polymerase, 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
    https://www.bioz.com/result/bst dna polymerase/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    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.

    Journal: BMC Genomics

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

    doi: 10.1186/1471-2164-7-312

    Figure Lengend 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.

    Article Snippet: The reaction mixture was then brought up to 50 μL with 400 μM dNTPs in 1× ThermoPol buffer, 0.35 units/μL Bst DNA polymerase, large fragment (New England Biolabs, Ipswich, MA) and 4% final concentration of DMSO.

    Techniques: 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.

    Journal: BMC Genomics

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

    doi: 10.1186/1471-2164-7-312

    Figure Lengend 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.

    Article Snippet: The reaction mixture was then brought up to 50 μL with 400 μM dNTPs in 1× ThermoPol buffer, 0.35 units/μL Bst DNA polymerase, large fragment (New England Biolabs, Ipswich, MA) and 4% final concentration of DMSO.

    Techniques: 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.

    Journal: BMC Genomics

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

    doi: 10.1186/1471-2164-7-312

    Figure Lengend 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.

    Article Snippet: The reaction mixture was then brought up to 50 μL with 400 μM dNTPs in 1× ThermoPol buffer, 0.35 units/μL Bst DNA polymerase, large fragment (New England Biolabs, Ipswich, MA) and 4% final concentration of DMSO.

    Techniques: 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.

    Journal: BMC Genomics

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

    doi: 10.1186/1471-2164-7-312

    Figure Lengend 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.

    Article Snippet: The reaction mixture was then brought up to 50 μL with 400 μM dNTPs in 1× ThermoPol buffer, 0.35 units/μL Bst DNA polymerase, large fragment (New England Biolabs, Ipswich, MA) and 4% final concentration of DMSO.

    Techniques: Amplification, Formalin-fixed Paraffin-Embedded

    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).

    Journal: PLoS Neglected Tropical Diseases

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

    doi: 10.1371/journal.pntd.0001948

    Figure Lengend 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).

    Article Snippet: LAMP Assays LAMP reactions with Bst DNA polymerase, large fragment (LF, New England Biolabs) contained 1.6 µM each of FIP and BIP, 0.2 µM each of F3 and B3, 1.4 mM of each dNTP, 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4 )2 SO4 , 8 mM MgSO4 , 0.1% Tween-20 and 8 U of enzyme mixed with 1 µl of various genomic DNAs in a total volume of 25 µl.

    Techniques: 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.

    Journal: PLoS Neglected Tropical Diseases

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

    doi: 10.1371/journal.pntd.0001948

    Figure Lengend 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.

    Article Snippet: LAMP Assays LAMP reactions with Bst DNA polymerase, large fragment (LF, New England Biolabs) contained 1.6 µM each of FIP and BIP, 0.2 µM each of F3 and B3, 1.4 mM of each dNTP, 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4 )2 SO4 , 8 mM MgSO4 , 0.1% Tween-20 and 8 U of enzyme mixed with 1 µl of various genomic DNAs in a total volume of 25 µl.

    Techniques: 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.

    Journal: PLoS Neglected Tropical Diseases

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

    doi: 10.1371/journal.pntd.0001948

    Figure Lengend 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.

    Article Snippet: LAMP Assays LAMP reactions with Bst DNA polymerase, large fragment (LF, New England Biolabs) contained 1.6 µM each of FIP and BIP, 0.2 µM each of F3 and B3, 1.4 mM of each dNTP, 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4 )2 SO4 , 8 mM MgSO4 , 0.1% Tween-20 and 8 U of enzyme mixed with 1 µl of various genomic DNAs in a total volume of 25 µl.

    Techniques: Lamp Assay, Infection, Isolation

    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.

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkp037

    Figure Lengend 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.

    Article Snippet: Fifteen microlitres of the adaptor ligated DNA were incubated for 30 min at 65°C in 1× ThermoPol Reaction Buffer (New England Biolabs), 8 µg Bovine Serum Albumin (BSA) (New England Biolabs), 20 nmol dNTPs and 16 U Bst DNA polymerase, Large Fragment (New England Biolabs) in a total volume of 20 µl.

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

    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 ].

    Journal: Diagnostics

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

    doi: 10.3390/diagnostics10060399

    Figure Lengend 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 ].

    Article Snippet: Essentially, LAMP uses a Bst DNA polymerase with strand-displacement activity, coupled with two inner primers (FIP, BIP) and outer primers (F3, B3) that recognizes six separate regions on a DNA template.

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