cas9 nuclease  (New England Biolabs)


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  • 99
    Name:
    Cas9 Nuclease S pyogenes
    Description:
    Cas9 Nuclease S pyogenes 2 000 pmol
    Catalog Number:
    m0386m
    Price:
    540
    Size:
    2 000 pmol
    Category:
    Other Endonucleases
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    Structured Review

    New England Biolabs cas9 nuclease
    Cas9 Nuclease S pyogenes
    Cas9 Nuclease S pyogenes 2 000 pmol
    https://www.bioz.com/result/cas9 nuclease/product/New England Biolabs
    Average 99 stars, based on 396 article reviews
    Price from $9.99 to $1999.99
    cas9 nuclease - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "The Manipulation of RNA‐Guided Nucleic Acid Cleavage with Ninhydrin Chemistry, The Manipulation of RNA‐Guided Nucleic Acid Cleavage with Ninhydrin Chemistry"

    Article Title: The Manipulation of RNA‐Guided Nucleic Acid Cleavage with Ninhydrin Chemistry, The Manipulation of RNA‐Guided Nucleic Acid Cleavage with Ninhydrin Chemistry

    Journal: Advanced Science

    doi: 10.1002/advs.201903770

    Ninhydrin chemistry to controlling CRISPR/Cas9 system. Reactions have been performed as described in the Experimental Section. All samples have been tested in three biological replicates. Image of representative data is shown here. Uncleaved t‐GFP1 DNA (702 bp) cut to shorter cleavage fragments (469 and 233 bp) are demonstrated. A) The ninhydrin masking of gRNA (gGFP1). The gGFP1 has been incubated with 5 g L −1 ninhydrin for different periods. B) The influence of ninhydrin masking on DNA cleavage. Lane 1: DNA markers; lane 2: no Cas9 control; lane 3: original gGFP1; lanes 4–6: gGFP1 with different masking levels. C) GTP unmasking of ninhydrin‐masked gGFP1. D) The influence of GTP unmasking on DNA cleavage. For (C) and (D), the masked gGFP1 (5 g L −1 ninhydrin, 5 h) has been incubated with different concentrations of GTP for 5 min.
    Figure Legend Snippet: Ninhydrin chemistry to controlling CRISPR/Cas9 system. Reactions have been performed as described in the Experimental Section. All samples have been tested in three biological replicates. Image of representative data is shown here. Uncleaved t‐GFP1 DNA (702 bp) cut to shorter cleavage fragments (469 and 233 bp) are demonstrated. A) The ninhydrin masking of gRNA (gGFP1). The gGFP1 has been incubated with 5 g L −1 ninhydrin for different periods. B) The influence of ninhydrin masking on DNA cleavage. Lane 1: DNA markers; lane 2: no Cas9 control; lane 3: original gGFP1; lanes 4–6: gGFP1 with different masking levels. C) GTP unmasking of ninhydrin‐masked gGFP1. D) The influence of GTP unmasking on DNA cleavage. For (C) and (D), the masked gGFP1 (5 g L −1 ninhydrin, 5 h) has been incubated with different concentrations of GTP for 5 min.

    Techniques Used: CRISPR, Incubation

    2) Product Images from "Novel Bioluminescent Quantitative Detection of Nucleic Acid Amplification in Real-Time"

    Article Title: Novel Bioluminescent Quantitative Detection of Nucleic Acid Amplification in Real-Time

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0014155

    Effect of dNTPs, ATP, PPi and APS on light output in BART. Simulation of effects of different ingredients on the light output in LAMP-BART in a “deficient mix” lacking primers and Bst polymerase but containing all other components as described in each case below. (A) Light output detected using varying concentrations of an equimolar mixture of four dNTPs. Light output peaks at 500 µM total dNTP concentration. (B) Light output detected using varying concentrations of ATP in the presence of 250 µM equimolar dNTPs. Light output is higher than in panel (A) and reaches saturation at 100 µM ATP, showing greater sensitivity to ATP. (C) Inhibitory effect of different concentrations of PPi on the light emission in the presence of 250 µM dNTPs and 100 µM ATP. (D) Stimulatory effect of increasing concentrations of APS on the light emission in the presence of 250 µM dNTPs and 100 µM PPi. (E) Effect of different concentrations of APS on BART curves in complete LAMP-BART formulation with 10 7 ChAT target DNA (red – 100 µM, navy – 200 µM, brown – 500 µM, green – 750 µM, blue - 1000 µM). As APS concentration is increased, there is little effect on peaking time but more PP i is converted to ATP resulting in a lower rate of inhibition of luciferase and a slower “switch off” of light output.
    Figure Legend Snippet: Effect of dNTPs, ATP, PPi and APS on light output in BART. Simulation of effects of different ingredients on the light output in LAMP-BART in a “deficient mix” lacking primers and Bst polymerase but containing all other components as described in each case below. (A) Light output detected using varying concentrations of an equimolar mixture of four dNTPs. Light output peaks at 500 µM total dNTP concentration. (B) Light output detected using varying concentrations of ATP in the presence of 250 µM equimolar dNTPs. Light output is higher than in panel (A) and reaches saturation at 100 µM ATP, showing greater sensitivity to ATP. (C) Inhibitory effect of different concentrations of PPi on the light emission in the presence of 250 µM dNTPs and 100 µM ATP. (D) Stimulatory effect of increasing concentrations of APS on the light emission in the presence of 250 µM dNTPs and 100 µM PPi. (E) Effect of different concentrations of APS on BART curves in complete LAMP-BART formulation with 10 7 ChAT target DNA (red – 100 µM, navy – 200 µM, brown – 500 µM, green – 750 µM, blue - 1000 µM). As APS concentration is increased, there is little effect on peaking time but more PP i is converted to ATP resulting in a lower rate of inhibition of luciferase and a slower “switch off” of light output.

    Techniques Used: Concentration Assay, Inhibition, Luciferase

    3) 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

    Related Articles

    Multiplex Assay:

    Article Title: Defining CRISPR-Cas9 genome-wide nuclease activities with CIRCLE-seq
    Article Snippet: .. E3110K) Plasmid-Safe 10× Reaction Buffer (Epicentre), supplied with Plasmid-Safe ATP-dependent DNase 25mM ATP solution (Epicentre), supplied with Plasmid-Safe ATP-dependent DNase Cas9 nuclease S. pyogenes (New England BioLabs, cat.no M0386M) 10× Cas9 buffer (New England BioLabs), supplied with Cas9 nuclease S. pyogenes NEBNext® Multiplex Oligos for Illumina® (Dual Index Primers Set 1) (New England BioLabs, cat.no. .. E7600S) NEBNext adapter for Illumina (New England BioLabs), supplied with NEBNext® Multiplex Oligos for Illumina® Qubit dsDNA BR Assay Kit (Thermo Fisher Scientific, cat.no. )

    Agarose Gel Electrophoresis:

    Article Title: SLALOM: A Simple and Rapid Method for Enzymatic Synthesis of CRISPR-Cas9 sgRNA Libraries
    Article Snippet: .. In vitro Digestion with Cas9 In vitro digestion of DNA fragments was accomplished by adding 200 ng of DNA, 70 pmol Cas9 Nuclease, S. pyogenes (1,000 nM) (New England Biolabs, Ipswich, USA), and 100 ng of the sgRNA in NEBuffer 3.1 (New England Biolabs, Ipswich, USA) for 20 minutes at 37°C and 10 minutes at 65°C, followed by adding 1 μL of proteinase K (800 units/ml) (NEB) and incubating at room temperature for 10 minutes before being run on a 1.7% agarose gel with a 100 bp ladder for 35 minutes. .. DNA adapters were prepared by resuspending complimentary oligos at a final concentration of 10 μM of each in CutSmart® buffer (New England Biolabs, Ipswich, USA).

    In Vitro:

    Article Title: Whole exome sequencing of ENU-induced thrombosis modifier mutations in the mouse
    Article Snippet: .. In vitro digestion of target DNA was carried out by complexes of synthetic sgRNA and S . pyogenes Cas9 Nuclease (New England BioLabs) according to manufacturer's recommendations. .. Agarose gel electrophoresis of the reaction products was used to identify sgRNA molecules that mediated template cleavage by Cas9 protein ( ).

    Article Title: SLALOM: A Simple and Rapid Method for Enzymatic Synthesis of CRISPR-Cas9 sgRNA Libraries
    Article Snippet: .. In vitro Digestion with Cas9 In vitro digestion of DNA fragments was accomplished by adding 200 ng of DNA, 70 pmol Cas9 Nuclease, S. pyogenes (1,000 nM) (New England Biolabs, Ipswich, USA), and 100 ng of the sgRNA in NEBuffer 3.1 (New England Biolabs, Ipswich, USA) for 20 minutes at 37°C and 10 minutes at 65°C, followed by adding 1 μL of proteinase K (800 units/ml) (NEB) and incubating at room temperature for 10 minutes before being run on a 1.7% agarose gel with a 100 bp ladder for 35 minutes. .. DNA adapters were prepared by resuspending complimentary oligos at a final concentration of 10 μM of each in CutSmart® buffer (New England Biolabs, Ipswich, USA).

    Synthesized:

    Article Title: USH2A Gene Editing Using the CRISPR System
    Article Snippet: .. For the RNP complexes, 15 μg purified Cas9 endonuclease (no. M0646T; New England Biolabs) was blended with 20 μg IVT synthesized sgRNA (PNA Bio, CA, USA) by 10-min incubation at room temperature with 1× Cas9 Nuclease Reaction Buffer (New England Biolabs). .. For the RNP complexes, 15 μg purified Cas9 endonuclease (no. M0646T; New England Biolabs) was blended with 20 μg IVT synthesized sgRNA (PNA Bio, CA, USA) by 10-min incubation at room temperature with 1× Cas9 Nuclease Reaction Buffer (New England Biolabs).

    Purification:

    Article Title: USH2A Gene Editing Using the CRISPR System
    Article Snippet: .. For the RNP complexes, 15 μg purified Cas9 endonuclease (no. M0646T; New England Biolabs) was blended with 20 μg IVT synthesized sgRNA (PNA Bio, CA, USA) by 10-min incubation at room temperature with 1× Cas9 Nuclease Reaction Buffer (New England Biolabs). .. For the RNP complexes, 15 μg purified Cas9 endonuclease (no. M0646T; New England Biolabs) was blended with 20 μg IVT synthesized sgRNA (PNA Bio, CA, USA) by 10-min incubation at room temperature with 1× Cas9 Nuclease Reaction Buffer (New England Biolabs).

    Article Title: Staphylococcus aureus Cas9 is a multiple-turnover enzyme
    Article Snippet: .. Both S. pyogenes and S. aureus Cas9 were purified at New England Biolabs using standard liquid chromatography protein purification techniques. .. Protein stock concentration for both Spy- and SauCas9 was measured by absorbance of 280 nm light on a NanoDrop instrument (A280 ) as well as Bio-Rad Bradford assays per manufacturer protocol.

    Protein Purification:

    Article Title: Staphylococcus aureus Cas9 is a multiple-turnover enzyme
    Article Snippet: .. Both S. pyogenes and S. aureus Cas9 were purified at New England Biolabs using standard liquid chromatography protein purification techniques. .. Protein stock concentration for both Spy- and SauCas9 was measured by absorbance of 280 nm light on a NanoDrop instrument (A280 ) as well as Bio-Rad Bradford assays per manufacturer protocol.

    Incubation:

    Article Title: USH2A Gene Editing Using the CRISPR System
    Article Snippet: .. For the RNP complexes, 15 μg purified Cas9 endonuclease (no. M0646T; New England Biolabs) was blended with 20 μg IVT synthesized sgRNA (PNA Bio, CA, USA) by 10-min incubation at room temperature with 1× Cas9 Nuclease Reaction Buffer (New England Biolabs). .. For the RNP complexes, 15 μg purified Cas9 endonuclease (no. M0646T; New England Biolabs) was blended with 20 μg IVT synthesized sgRNA (PNA Bio, CA, USA) by 10-min incubation at room temperature with 1× Cas9 Nuclease Reaction Buffer (New England Biolabs).

    other:

    Article Title: CRISPR-typing PCR (ctPCR), a new Cas9-based DNA detection method
    Article Snippet: The results indicated that the HPV16 and HPV18 L1 genes could be specifically targeted by their corresponding sgRNA and cut by the guided Cas9 nuclease (Fig. ).

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Defining CRISPR-Cas9 genome-wide nuclease activities with CIRCLE-seq
    Article Snippet: .. E3110K) Plasmid-Safe 10× Reaction Buffer (Epicentre), supplied with Plasmid-Safe ATP-dependent DNase 25mM ATP solution (Epicentre), supplied with Plasmid-Safe ATP-dependent DNase Cas9 nuclease S. pyogenes (New England BioLabs, cat.no M0386M) 10× Cas9 buffer (New England BioLabs), supplied with Cas9 nuclease S. pyogenes NEBNext® Multiplex Oligos for Illumina® (Dual Index Primers Set 1) (New England BioLabs, cat.no. .. E7600S) NEBNext adapter for Illumina (New England BioLabs), supplied with NEBNext® Multiplex Oligos for Illumina® Qubit dsDNA BR Assay Kit (Thermo Fisher Scientific, cat.no. )

    Liquid Chromatography:

    Article Title: Staphylococcus aureus Cas9 is a multiple-turnover enzyme
    Article Snippet: .. Both S. pyogenes and S. aureus Cas9 were purified at New England Biolabs using standard liquid chromatography protein purification techniques. .. Protein stock concentration for both Spy- and SauCas9 was measured by absorbance of 280 nm light on a NanoDrop instrument (A280 ) as well as Bio-Rad Bradford assays per manufacturer protocol.

    Plasmid Preparation:

    Article Title: Defining CRISPR-Cas9 genome-wide nuclease activities with CIRCLE-seq
    Article Snippet: .. E3110K) Plasmid-Safe 10× Reaction Buffer (Epicentre), supplied with Plasmid-Safe ATP-dependent DNase 25mM ATP solution (Epicentre), supplied with Plasmid-Safe ATP-dependent DNase Cas9 nuclease S. pyogenes (New England BioLabs, cat.no M0386M) 10× Cas9 buffer (New England BioLabs), supplied with Cas9 nuclease S. pyogenes NEBNext® Multiplex Oligos for Illumina® (Dual Index Primers Set 1) (New England BioLabs, cat.no. .. E7600S) NEBNext adapter for Illumina (New England BioLabs), supplied with NEBNext® Multiplex Oligos for Illumina® Qubit dsDNA BR Assay Kit (Thermo Fisher Scientific, cat.no. )

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    New England Biolabs bst dna polymerase large fragment bst
    Effect of dNTPs, ATP, PPi and APS on light output in BART. Simulation of effects of different ingredients on the light output in LAMP-BART in a “deficient mix” lacking primers and <t>Bst</t> polymerase but containing all other components as described in each case below. (A) Light output detected using varying concentrations of an equimolar mixture of four dNTPs. Light output peaks at 500 µM total dNTP concentration. (B) Light output detected using varying concentrations of ATP in the presence of 250 µM equimolar dNTPs. Light output is higher than in panel (A) and reaches saturation at 100 µM ATP, showing greater sensitivity to ATP. (C) Inhibitory effect of different concentrations of PPi on the light emission in the presence of 250 µM dNTPs and 100 µM ATP. (D) Stimulatory effect of increasing concentrations of APS on the light emission in the presence of 250 µM dNTPs and 100 µM PPi. (E) Effect of different concentrations of APS on BART curves in complete LAMP-BART formulation with 10 7 ChAT target <t>DNA</t> (red – 100 µM, navy – 200 µM, brown – 500 µM, green – 750 µM, blue - 1000 µM). As APS concentration is increased, there is little effect on peaking time but more PP i is converted to ATP resulting in a lower rate of inhibition of luciferase and a slower “switch off” of light output.
    Bst Dna Polymerase Large Fragment Bst, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 97 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bst dna polymerase large fragment bst/product/New England Biolabs
    Average 97 stars, based on 97 article reviews
    Price from $9.99 to $1999.99
    bst dna polymerase large fragment bst - by Bioz Stars, 2020-09
    97/100 stars
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    Effect of dNTPs, ATP, PPi and APS on light output in BART. Simulation of effects of different ingredients on the light output in LAMP-BART in a “deficient mix” lacking primers and Bst polymerase but containing all other components as described in each case below. (A) Light output detected using varying concentrations of an equimolar mixture of four dNTPs. Light output peaks at 500 µM total dNTP concentration. (B) Light output detected using varying concentrations of ATP in the presence of 250 µM equimolar dNTPs. Light output is higher than in panel (A) and reaches saturation at 100 µM ATP, showing greater sensitivity to ATP. (C) Inhibitory effect of different concentrations of PPi on the light emission in the presence of 250 µM dNTPs and 100 µM ATP. (D) Stimulatory effect of increasing concentrations of APS on the light emission in the presence of 250 µM dNTPs and 100 µM PPi. (E) Effect of different concentrations of APS on BART curves in complete LAMP-BART formulation with 10 7 ChAT target DNA (red – 100 µM, navy – 200 µM, brown – 500 µM, green – 750 µM, blue - 1000 µM). As APS concentration is increased, there is little effect on peaking time but more PP i is converted to ATP resulting in a lower rate of inhibition of luciferase and a slower “switch off” of light output.

    Journal: PLoS ONE

    Article Title: Novel Bioluminescent Quantitative Detection of Nucleic Acid Amplification in Real-Time

    doi: 10.1371/journal.pone.0014155

    Figure Lengend Snippet: Effect of dNTPs, ATP, PPi and APS on light output in BART. Simulation of effects of different ingredients on the light output in LAMP-BART in a “deficient mix” lacking primers and Bst polymerase but containing all other components as described in each case below. (A) Light output detected using varying concentrations of an equimolar mixture of four dNTPs. Light output peaks at 500 µM total dNTP concentration. (B) Light output detected using varying concentrations of ATP in the presence of 250 µM equimolar dNTPs. Light output is higher than in panel (A) and reaches saturation at 100 µM ATP, showing greater sensitivity to ATP. (C) Inhibitory effect of different concentrations of PPi on the light emission in the presence of 250 µM dNTPs and 100 µM ATP. (D) Stimulatory effect of increasing concentrations of APS on the light emission in the presence of 250 µM dNTPs and 100 µM PPi. (E) Effect of different concentrations of APS on BART curves in complete LAMP-BART formulation with 10 7 ChAT target DNA (red – 100 µM, navy – 200 µM, brown – 500 µM, green – 750 µM, blue - 1000 µM). As APS concentration is increased, there is little effect on peaking time but more PP i is converted to ATP resulting in a lower rate of inhibition of luciferase and a slower “switch off” of light output.

    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).

    Techniques: Concentration Assay, Inhibition, Luciferase

    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