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    Nb BsmI
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    Nb BsmI 1 000 units
    Catalog Number:
    R0706L
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    Restriction Enzymes
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    New England Biolabs nb bsmi
    Nb BsmI
    Nb BsmI 1 000 units
    https://www.bioz.com/result/nb bsmi/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    nb bsmi - by Bioz Stars, 2021-05
    95/100 stars

    Images

    1) Product Images from "Sensitive RNA detection by combining three-way junction formation and primer generation-rolling circle amplification"

    Article Title: Sensitive RNA detection by combining three-way junction formation and primer generation-rolling circle amplification

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr909

    Confirmation of 3WJ primer extension and signal primer generation products. 3WJ primer P1, 3WJ template T1b and RNA50 were mixed in different combinations [( A) : P1/T1b/RNA50, ( B ): P1/T1b, ( C ): P1/RNA50 and ( D ): T1b/RNA50] and incubated to form a 3WJ structure. Signal generation reaction was conducted at 60°C for 0, 15 and 30 min by adding an enzyme mix [0.2 U Vent(exo-) DNA polymerase and 1 U Nb.BsmI] to the samples. The reaction products were analyzed on 15% denaturing polyacrylamide gel. P1* indicates a reaction product through primer extension of 3WJ primer and SP indicates signal primer generated from 3WJ structure. The numbers in parentheses indicate the length or expected length of each probe or reaction product. Lane M is 20–100 nt oligonucleotide marker.
    Figure Legend Snippet: Confirmation of 3WJ primer extension and signal primer generation products. 3WJ primer P1, 3WJ template T1b and RNA50 were mixed in different combinations [( A) : P1/T1b/RNA50, ( B ): P1/T1b, ( C ): P1/RNA50 and ( D ): T1b/RNA50] and incubated to form a 3WJ structure. Signal generation reaction was conducted at 60°C for 0, 15 and 30 min by adding an enzyme mix [0.2 U Vent(exo-) DNA polymerase and 1 U Nb.BsmI] to the samples. The reaction products were analyzed on 15% denaturing polyacrylamide gel. P1* indicates a reaction product through primer extension of 3WJ primer and SP indicates signal primer generated from 3WJ structure. The numbers in parentheses indicate the length or expected length of each probe or reaction product. Lane M is 20–100 nt oligonucleotide marker.

    Techniques Used: Incubation, Generated, Marker

    2) Product Images from "Sensitive isothermal detection of nucleic-acid sequence by primer generation-rolling circle amplification"

    Article Title: Sensitive isothermal detection of nucleic-acid sequence by primer generation-rolling circle amplification

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkn1014

    Optimization of reaction conditions for PG–RCA. Threshold time in the presence (open circle) or absence (cross) of 500 zmol sample DNA was compared at various concentrations of each reaction component ( n =2). ( A ) At constant concentrations of Vent (exo-) DNA polymerase (0.4 U), Nb.BsmI (1 U) and circular probe II (7.5 nM), dNTP concentrations were compared among 400 (original), 40, 4 and 0.4 µM. ( B ) At constant concentrations of dNTP (400 µM), Nb.BsmI (1 U) and circular probe II (7.5 nM), Vent (exo-) polymerase concentrations were compared among 0.4 (original), 0.08, 0.04 and 0.02 U. ( C ) At constant concentrations of dNTP (400 µM), Vent (exo-) DNA polymerase (0.4 U) and circular probe II (7.5 nM), Nb.BsmI concentrations were compared among 1 (original), 0.5, 0.25 and 0.125 U. ( D ) At constant concentrations of dNTP (0.4 µM, 1000-fold dilution of the original concentration), Nb.BsmI (1 U) and circular probe II (7.5 nM), Vent (exo-) polymerase concentrations were compared among 0.4, 0.2, 0.1 and 0.05 U.
    Figure Legend Snippet: Optimization of reaction conditions for PG–RCA. Threshold time in the presence (open circle) or absence (cross) of 500 zmol sample DNA was compared at various concentrations of each reaction component ( n =2). ( A ) At constant concentrations of Vent (exo-) DNA polymerase (0.4 U), Nb.BsmI (1 U) and circular probe II (7.5 nM), dNTP concentrations were compared among 400 (original), 40, 4 and 0.4 µM. ( B ) At constant concentrations of dNTP (400 µM), Nb.BsmI (1 U) and circular probe II (7.5 nM), Vent (exo-) polymerase concentrations were compared among 0.4 (original), 0.08, 0.04 and 0.02 U. ( C ) At constant concentrations of dNTP (400 µM), Vent (exo-) DNA polymerase (0.4 U) and circular probe II (7.5 nM), Nb.BsmI concentrations were compared among 1 (original), 0.5, 0.25 and 0.125 U. ( D ) At constant concentrations of dNTP (0.4 µM, 1000-fold dilution of the original concentration), Nb.BsmI (1 U) and circular probe II (7.5 nM), Vent (exo-) polymerase concentrations were compared among 0.4, 0.2, 0.1 and 0.05 U.

    Techniques Used: Concentration Assay

    PG–RCA under optimized reaction condition. ( A ) Fluorescent intensity of PG–RCA reaction at the optimized reaction condition (0.4 µM dNTP, 0.05 U Vent (exo-) DNA polymerase, 1 U Nb.BsmI and 7.5 nM circular probe II) was monitored in real time. Sample DNA concentration in each reaction was prepared by 10-fold serial dilution from 5 fmol to 0.5 ymol and their signal amplification curves were indicated by colored lines (dark blue, blue, light blue, purple, dark green, green, brown, orange, yellow, red and gray, respectively) ( n =2). Negative controls are indicated by black lines ( n =2). ( B ) Threshold time ( T T ) was plotted against the sample DNA concentration (S) of the reaction. Solid line indicates linear least squares fitting between 5 fmol and 0.5 zmol sample DNA and its formulation is T T =−19.2 log 10 (S)+75.6 ( R 2 =0.998). Perforated line indicates average T T value of the negative controls. Limit of detection is 84.5 ymol or 50.7 molecules of sample DNA by calculation from the intersection of both lines.
    Figure Legend Snippet: PG–RCA under optimized reaction condition. ( A ) Fluorescent intensity of PG–RCA reaction at the optimized reaction condition (0.4 µM dNTP, 0.05 U Vent (exo-) DNA polymerase, 1 U Nb.BsmI and 7.5 nM circular probe II) was monitored in real time. Sample DNA concentration in each reaction was prepared by 10-fold serial dilution from 5 fmol to 0.5 ymol and their signal amplification curves were indicated by colored lines (dark blue, blue, light blue, purple, dark green, green, brown, orange, yellow, red and gray, respectively) ( n =2). Negative controls are indicated by black lines ( n =2). ( B ) Threshold time ( T T ) was plotted against the sample DNA concentration (S) of the reaction. Solid line indicates linear least squares fitting between 5 fmol and 0.5 zmol sample DNA and its formulation is T T =−19.2 log 10 (S)+75.6 ( R 2 =0.998). Perforated line indicates average T T value of the negative controls. Limit of detection is 84.5 ymol or 50.7 molecules of sample DNA by calculation from the intersection of both lines.

    Techniques Used: Concentration Assay, Serial Dilution, Amplification

    Real-time product analysis of PG–RCA. ( A ) Fluorescent intensity of PG–RCA reaction was monitored in real time. PG–RCA was conducted at 60°C with 400 µM each dNTP, 0.4 U Vent (exo-) DNA polymerase, 1 U Nb.BsmI and 7.5 nM circular probe II as described in Materials and methods section. Sample DNA concentration in each reaction was prepared by 10-fold serial dilution from 500 amol to 0.5 zmol, and their signal amplification curves were indicated by colored lines (blue, light blue, purple, dark green, green, brown and orange, respectively) ( n =3). Negative controls are indicated by black lines ( n =3). ( B ) Threshold time T T (the reaction time when fluorescent intensity of each reaction exceeds a threshold, indicated by a perforated line in Figure 3A) was plotted against the sample DNA concentration (S) of the reaction. Solid line indicates linear least squares fitting between 500 and 0.5 amol sample DNA and its formulation is T T =−8.65 log 10 (S)+28.5 (R 2 =0.997). Perforated line indicates average T T value of the negative controls. Limit of detection is 58.4 zmol or 3.50 × 10 4 molecules of sample DNA by calculation from the intersection of both lines.
    Figure Legend Snippet: Real-time product analysis of PG–RCA. ( A ) Fluorescent intensity of PG–RCA reaction was monitored in real time. PG–RCA was conducted at 60°C with 400 µM each dNTP, 0.4 U Vent (exo-) DNA polymerase, 1 U Nb.BsmI and 7.5 nM circular probe II as described in Materials and methods section. Sample DNA concentration in each reaction was prepared by 10-fold serial dilution from 500 amol to 0.5 zmol, and their signal amplification curves were indicated by colored lines (blue, light blue, purple, dark green, green, brown and orange, respectively) ( n =3). Negative controls are indicated by black lines ( n =3). ( B ) Threshold time T T (the reaction time when fluorescent intensity of each reaction exceeds a threshold, indicated by a perforated line in Figure 3A) was plotted against the sample DNA concentration (S) of the reaction. Solid line indicates linear least squares fitting between 500 and 0.5 amol sample DNA and its formulation is T T =−8.65 log 10 (S)+28.5 (R 2 =0.997). Perforated line indicates average T T value of the negative controls. Limit of detection is 58.4 zmol or 3.50 × 10 4 molecules of sample DNA by calculation from the intersection of both lines.

    Techniques Used: Concentration Assay, Serial Dilution, Amplification

    Endpoint product analysis of PG–RCA. ( A ) Endpoint products in the presence or absence of 500 amol sample DNA were analyzed on 1.0% agarose gel electrophoresis. The reaction was conducted at 60°C for 30, 45, 60, 75, 90, 105 and 120 min and each reaction was analyzed separately. Lane M was loaded with a 100 bp DNA marker (100, 200, 300, 400, 500/517, 600, 700, 800, 900, 1000, 1200 and 1517 bp from the bottom). ( B ) Endpoint products of 90-min reactions with all or partial reaction components were analyzed on 1.0% agarose gel electrophoresis. DNA, POL and NICK indicate sample DNA, Vent (exo-) DNA polymerase and Nb.BsmI, respectively. Lane M is a 100 bp DNA marker. ( C ) Endpoint product of a 120-min PG–RCA reaction was digested with either BsmI or BbvCI and analyzed on 1.5% agarose gel electrophoresis. Lane ‘-’ was loaded with the 120 min reaction product before restriction enzyme digestion. Lane M is a 100 bp DNA marker.
    Figure Legend Snippet: Endpoint product analysis of PG–RCA. ( A ) Endpoint products in the presence or absence of 500 amol sample DNA were analyzed on 1.0% agarose gel electrophoresis. The reaction was conducted at 60°C for 30, 45, 60, 75, 90, 105 and 120 min and each reaction was analyzed separately. Lane M was loaded with a 100 bp DNA marker (100, 200, 300, 400, 500/517, 600, 700, 800, 900, 1000, 1200 and 1517 bp from the bottom). ( B ) Endpoint products of 90-min reactions with all or partial reaction components were analyzed on 1.0% agarose gel electrophoresis. DNA, POL and NICK indicate sample DNA, Vent (exo-) DNA polymerase and Nb.BsmI, respectively. Lane M is a 100 bp DNA marker. ( C ) Endpoint product of a 120-min PG–RCA reaction was digested with either BsmI or BbvCI and analyzed on 1.5% agarose gel electrophoresis. Lane ‘-’ was loaded with the 120 min reaction product before restriction enzyme digestion. Lane M is a 100 bp DNA marker.

    Techniques Used: Agarose Gel Electrophoresis, Marker

    Real-time quantification of hly gene in L. monocytogenes genomic DNA by PG–RCA. ( A ) Circular probe LM for detection of pathogenic L. monocytogenes genomic DNA. The probe targets the complementary strand of virulence gene, hly (GeneBank GeneID 2797098), encoding a cholesterol-dependent cytolysin, listeriolysin O (LLO). Circular probe LM contains three repeats of a 26-base sequence complementary to the gene including a nicking site for Nb.BsmI. Since these repeat sequences have 5-base overlaps each other, the circular probe comprises three repeats of a 21-base sequence (red, blue and green). ( B ) Genomic DNA from L. monocytogenes (0.1–100 pg) was analyzed by real-time PG–RCA with circular probe LM. Threshold time ( T T ) was plotted against the L. monocytogenes genomic DNA concentration (S) of the reaction. Solid line indicates linear least squares fitting between 0.1 and 100 pg L. monocytogenes genomic DNA and its formulation is T T = −19.1 log 10 (S) + 233 ( R 2 =0.964). Perforated line indicates average T T value of the negative controls ( n =2). Limit of detection is 0.163 pg (∼60 molecules) of L. monocytogenes genomic DNA by calculation from the intersection of both lines. ( C ) Genomic DNA (100 pg) from L. monocytogenes , L. innocua , E. coli and S. enterica were analyzed by real-time PG–RCA with circular probe LM and their threshold times were compared with the values for L. monocytogenes (100 pg). ‘No DNA’ indicates the negative controls.
    Figure Legend Snippet: Real-time quantification of hly gene in L. monocytogenes genomic DNA by PG–RCA. ( A ) Circular probe LM for detection of pathogenic L. monocytogenes genomic DNA. The probe targets the complementary strand of virulence gene, hly (GeneBank GeneID 2797098), encoding a cholesterol-dependent cytolysin, listeriolysin O (LLO). Circular probe LM contains three repeats of a 26-base sequence complementary to the gene including a nicking site for Nb.BsmI. Since these repeat sequences have 5-base overlaps each other, the circular probe comprises three repeats of a 21-base sequence (red, blue and green). ( B ) Genomic DNA from L. monocytogenes (0.1–100 pg) was analyzed by real-time PG–RCA with circular probe LM. Threshold time ( T T ) was plotted against the L. monocytogenes genomic DNA concentration (S) of the reaction. Solid line indicates linear least squares fitting between 0.1 and 100 pg L. monocytogenes genomic DNA and its formulation is T T = −19.1 log 10 (S) + 233 ( R 2 =0.964). Perforated line indicates average T T value of the negative controls ( n =2). Limit of detection is 0.163 pg (∼60 molecules) of L. monocytogenes genomic DNA by calculation from the intersection of both lines. ( C ) Genomic DNA (100 pg) from L. monocytogenes , L. innocua , E. coli and S. enterica were analyzed by real-time PG–RCA with circular probe LM and their threshold times were compared with the values for L. monocytogenes (100 pg). ‘No DNA’ indicates the negative controls.

    Techniques Used: Sequencing, Concentration Assay

    3) Product Images from "Multiplex Digital MicroRNA Detection Using Cross-Inhibitory DNA Circuits"

    Article Title: Multiplex Digital MicroRNA Detection Using Cross-Inhibitory DNA Circuits

    Journal: ACS Sensors

    doi: 10.1021/acssensors.0c00593

    Tetrastable system built from two cross-inhibitory bistable switches. (a) Schematic of the tetrastable DNA circuit. Two microRNA-sensing circuits (cT, aT, pT, and rT) are interconnected by kT αkβ and βkα, which repress unwanted cross-activation. (b) Detailed mechanism of the five kinds of templates (pol. = Vent(exo-), nick. 1 = Nt.BstNBI, nick. 2 = Nb.BsmI, RE = BsmI, and exo. = ttRecJ). cTs convert the complementary microRNA target to a signal strand (α or β). Autocataytic templates (aTs) exponentially amplify the signal strands. pTs, by deactivating a fraction of signal strands, suppress background amplification stemming from biochemical noise. Reporting templates (rTs) transduce the molecular signal (α or β) to a detectable fluorescence signal (green = Oregon green fluorophore, red = Atto633 fluorophore). From the α or β strands, killer templates (kTs) produce pTs of the opposite switch, mitigating unspecific cross-talks. All produced strands are continuously degraded by the exonuclease to maintain the system dynamics and avoid system poisoning by the accumulation of DNA strands. Only one half of the tetrastable circuit is represented here, the second half being obtained by substituting α by β and conversely.
    Figure Legend Snippet: Tetrastable system built from two cross-inhibitory bistable switches. (a) Schematic of the tetrastable DNA circuit. Two microRNA-sensing circuits (cT, aT, pT, and rT) are interconnected by kT αkβ and βkα, which repress unwanted cross-activation. (b) Detailed mechanism of the five kinds of templates (pol. = Vent(exo-), nick. 1 = Nt.BstNBI, nick. 2 = Nb.BsmI, RE = BsmI, and exo. = ttRecJ). cTs convert the complementary microRNA target to a signal strand (α or β). Autocataytic templates (aTs) exponentially amplify the signal strands. pTs, by deactivating a fraction of signal strands, suppress background amplification stemming from biochemical noise. Reporting templates (rTs) transduce the molecular signal (α or β) to a detectable fluorescence signal (green = Oregon green fluorophore, red = Atto633 fluorophore). From the α or β strands, killer templates (kTs) produce pTs of the opposite switch, mitigating unspecific cross-talks. All produced strands are continuously degraded by the exonuclease to maintain the system dynamics and avoid system poisoning by the accumulation of DNA strands. Only one half of the tetrastable circuit is represented here, the second half being obtained by substituting α by β and conversely.

    Techniques Used: Activation Assay, Amplification, Fluorescence, Produced

    Related Articles

    other:

    Article Title: Sensitive isothermal detection of nucleic-acid sequence by primer generation-rolling circle amplification
    Article Snippet: Vent (exo-) DNA polymerase, Nb.BsmI, BsmI, BbvCI and exonuclease III were purchased from New England Biolabs.

    Plasmid Preparation:

    Article Title: Separable functions of Tof1/Timeless in intra-S-checkpoint signalling, replisome stability and DNA topological stress
    Article Snippet: .. For plasmid DNA catenation analysis of pRS316, purified DNA was nicked with Nb.Bsm1 (New England Biolabs, R0706S) according to manufacturer's instructions. .. For fork pausing analysis of pRS426-RFB, purified DNA was digested with BamHI-HF (New England Biolabs, R3136S) and SnaBI (New England Biolabs, R0130L) or SnaBI alone according to manufacturer's instructions.

    Article Title: RNA polymerase I passage through nucleosomes depends on its lobe binding subunits
    Article Snippet: The transcription were performed as described above on PCR-derived templates containing a 3’overhang. .. Biotinylated oligos downstream of the tail in reverse direction (oligos 4018 and 4220 for 1 kb and 2 kb transcripts, respectively) and an oligo containing a Nb.BsmI (NEB) nicking site (oligo 4220a) were used to generate templates of 1 and 2 kb using plasmid 2316 as template. .. PCR products were cut with Nb.BsmI and heat inactivated at 80°C for 20 min. After 10 min, a competitor oligo 4220a with the same sequence as the 24 nt 3’overhang was added in excess to anneal with released cleaved 5’Nb.BsmI fragment.

    Article Title: A teaching protocol demonstrating the use of EasyClone and CRISPR/Cas9 for metabolic engineering of Saccharomyces cerevisiae and Yarrowia lipolytica.
    Article Snippet: Measure the concentration using NanoDrop (or similar). c. Digest the whole purified digested plasmid with Nb.BsmI at 65◦C for 1 h as follows: i. .. 1 U Nb.BsmI per μg of digested vector (typically 1 μL/μg) ii. .. Appropriate volume of NEB buffer 3.1 d. Gel purify as per previous step.

    Purification:

    Article Title: Separable functions of Tof1/Timeless in intra-S-checkpoint signalling, replisome stability and DNA topological stress
    Article Snippet: .. For plasmid DNA catenation analysis of pRS316, purified DNA was nicked with Nb.Bsm1 (New England Biolabs, R0706S) according to manufacturer's instructions. .. For fork pausing analysis of pRS426-RFB, purified DNA was digested with BamHI-HF (New England Biolabs, R3136S) and SnaBI (New England Biolabs, R0130L) or SnaBI alone according to manufacturer's instructions.

    Produced:

    Article Title: RNA polymerase I (Pol I) passage through nucleosomes depends on Pol I subunits binding its lobe structure
    Article Snippet: The reference template was generated by PCR using plasmid 1573 and oligonucleotides 2115 and 4234. .. 3′-overhangs were produced as described above using cleavage by Nb.BsmI. ..

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    New England Biolabs nb bsmi
    Confirmation of 3WJ primer extension and signal primer generation products. 3WJ primer P1, 3WJ template T1b and RNA50 were mixed in different combinations [( A) : P1/T1b/RNA50, ( B ): P1/T1b, ( C ): P1/RNA50 and ( D ): T1b/RNA50] and incubated to form a 3WJ structure. Signal generation reaction was conducted at 60°C for 0, 15 and 30 min by adding an enzyme mix [0.2 U Vent(exo-) <t>DNA</t> polymerase and 1 U <t>Nb.BsmI]</t> to the samples. The reaction products were analyzed on 15% denaturing polyacrylamide gel. P1* indicates a reaction product through primer extension of 3WJ primer and SP indicates signal primer generated from 3WJ structure. The numbers in parentheses indicate the length or expected length of each probe or reaction product. Lane M is 20–100 nt oligonucleotide marker.
    Nb Bsmi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nb bsmi/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
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    Confirmation of 3WJ primer extension and signal primer generation products. 3WJ primer P1, 3WJ template T1b and RNA50 were mixed in different combinations [( A) : P1/T1b/RNA50, ( B ): P1/T1b, ( C ): P1/RNA50 and ( D ): T1b/RNA50] and incubated to form a 3WJ structure. Signal generation reaction was conducted at 60°C for 0, 15 and 30 min by adding an enzyme mix [0.2 U Vent(exo-) DNA polymerase and 1 U Nb.BsmI] to the samples. The reaction products were analyzed on 15% denaturing polyacrylamide gel. P1* indicates a reaction product through primer extension of 3WJ primer and SP indicates signal primer generated from 3WJ structure. The numbers in parentheses indicate the length or expected length of each probe or reaction product. Lane M is 20–100 nt oligonucleotide marker.

    Journal: Nucleic Acids Research

    Article Title: Sensitive RNA detection by combining three-way junction formation and primer generation-rolling circle amplification

    doi: 10.1093/nar/gkr909

    Figure Lengend Snippet: Confirmation of 3WJ primer extension and signal primer generation products. 3WJ primer P1, 3WJ template T1b and RNA50 were mixed in different combinations [( A) : P1/T1b/RNA50, ( B ): P1/T1b, ( C ): P1/RNA50 and ( D ): T1b/RNA50] and incubated to form a 3WJ structure. Signal generation reaction was conducted at 60°C for 0, 15 and 30 min by adding an enzyme mix [0.2 U Vent(exo-) DNA polymerase and 1 U Nb.BsmI] to the samples. The reaction products were analyzed on 15% denaturing polyacrylamide gel. P1* indicates a reaction product through primer extension of 3WJ primer and SP indicates signal primer generated from 3WJ structure. The numbers in parentheses indicate the length or expected length of each probe or reaction product. Lane M is 20–100 nt oligonucleotide marker.

    Article Snippet: Vent (exo-) DNA polymerase, Nb.BsmI and exonuclease III were purchased from New England Biolabs.

    Techniques: Incubation, Generated, Marker

    Optimization of reaction conditions for PG–RCA. Threshold time in the presence (open circle) or absence (cross) of 500 zmol sample DNA was compared at various concentrations of each reaction component ( n =2). ( A ) At constant concentrations of Vent (exo-) DNA polymerase (0.4 U), Nb.BsmI (1 U) and circular probe II (7.5 nM), dNTP concentrations were compared among 400 (original), 40, 4 and 0.4 µM. ( B ) At constant concentrations of dNTP (400 µM), Nb.BsmI (1 U) and circular probe II (7.5 nM), Vent (exo-) polymerase concentrations were compared among 0.4 (original), 0.08, 0.04 and 0.02 U. ( C ) At constant concentrations of dNTP (400 µM), Vent (exo-) DNA polymerase (0.4 U) and circular probe II (7.5 nM), Nb.BsmI concentrations were compared among 1 (original), 0.5, 0.25 and 0.125 U. ( D ) At constant concentrations of dNTP (0.4 µM, 1000-fold dilution of the original concentration), Nb.BsmI (1 U) and circular probe II (7.5 nM), Vent (exo-) polymerase concentrations were compared among 0.4, 0.2, 0.1 and 0.05 U.

    Journal: Nucleic Acids Research

    Article Title: Sensitive isothermal detection of nucleic-acid sequence by primer generation-rolling circle amplification

    doi: 10.1093/nar/gkn1014

    Figure Lengend Snippet: Optimization of reaction conditions for PG–RCA. Threshold time in the presence (open circle) or absence (cross) of 500 zmol sample DNA was compared at various concentrations of each reaction component ( n =2). ( A ) At constant concentrations of Vent (exo-) DNA polymerase (0.4 U), Nb.BsmI (1 U) and circular probe II (7.5 nM), dNTP concentrations were compared among 400 (original), 40, 4 and 0.4 µM. ( B ) At constant concentrations of dNTP (400 µM), Nb.BsmI (1 U) and circular probe II (7.5 nM), Vent (exo-) polymerase concentrations were compared among 0.4 (original), 0.08, 0.04 and 0.02 U. ( C ) At constant concentrations of dNTP (400 µM), Vent (exo-) DNA polymerase (0.4 U) and circular probe II (7.5 nM), Nb.BsmI concentrations were compared among 1 (original), 0.5, 0.25 and 0.125 U. ( D ) At constant concentrations of dNTP (0.4 µM, 1000-fold dilution of the original concentration), Nb.BsmI (1 U) and circular probe II (7.5 nM), Vent (exo-) polymerase concentrations were compared among 0.4, 0.2, 0.1 and 0.05 U.

    Article Snippet: Vent (exo-) DNA polymerase, Nb.BsmI, BsmI, BbvCI and exonuclease III were purchased from New England Biolabs.

    Techniques: Concentration Assay

    PG–RCA under optimized reaction condition. ( A ) Fluorescent intensity of PG–RCA reaction at the optimized reaction condition (0.4 µM dNTP, 0.05 U Vent (exo-) DNA polymerase, 1 U Nb.BsmI and 7.5 nM circular probe II) was monitored in real time. Sample DNA concentration in each reaction was prepared by 10-fold serial dilution from 5 fmol to 0.5 ymol and their signal amplification curves were indicated by colored lines (dark blue, blue, light blue, purple, dark green, green, brown, orange, yellow, red and gray, respectively) ( n =2). Negative controls are indicated by black lines ( n =2). ( B ) Threshold time ( T T ) was plotted against the sample DNA concentration (S) of the reaction. Solid line indicates linear least squares fitting between 5 fmol and 0.5 zmol sample DNA and its formulation is T T =−19.2 log 10 (S)+75.6 ( R 2 =0.998). Perforated line indicates average T T value of the negative controls. Limit of detection is 84.5 ymol or 50.7 molecules of sample DNA by calculation from the intersection of both lines.

    Journal: Nucleic Acids Research

    Article Title: Sensitive isothermal detection of nucleic-acid sequence by primer generation-rolling circle amplification

    doi: 10.1093/nar/gkn1014

    Figure Lengend Snippet: PG–RCA under optimized reaction condition. ( A ) Fluorescent intensity of PG–RCA reaction at the optimized reaction condition (0.4 µM dNTP, 0.05 U Vent (exo-) DNA polymerase, 1 U Nb.BsmI and 7.5 nM circular probe II) was monitored in real time. Sample DNA concentration in each reaction was prepared by 10-fold serial dilution from 5 fmol to 0.5 ymol and their signal amplification curves were indicated by colored lines (dark blue, blue, light blue, purple, dark green, green, brown, orange, yellow, red and gray, respectively) ( n =2). Negative controls are indicated by black lines ( n =2). ( B ) Threshold time ( T T ) was plotted against the sample DNA concentration (S) of the reaction. Solid line indicates linear least squares fitting between 5 fmol and 0.5 zmol sample DNA and its formulation is T T =−19.2 log 10 (S)+75.6 ( R 2 =0.998). Perforated line indicates average T T value of the negative controls. Limit of detection is 84.5 ymol or 50.7 molecules of sample DNA by calculation from the intersection of both lines.

    Article Snippet: Vent (exo-) DNA polymerase, Nb.BsmI, BsmI, BbvCI and exonuclease III were purchased from New England Biolabs.

    Techniques: Concentration Assay, Serial Dilution, Amplification

    Real-time product analysis of PG–RCA. ( A ) Fluorescent intensity of PG–RCA reaction was monitored in real time. PG–RCA was conducted at 60°C with 400 µM each dNTP, 0.4 U Vent (exo-) DNA polymerase, 1 U Nb.BsmI and 7.5 nM circular probe II as described in Materials and methods section. Sample DNA concentration in each reaction was prepared by 10-fold serial dilution from 500 amol to 0.5 zmol, and their signal amplification curves were indicated by colored lines (blue, light blue, purple, dark green, green, brown and orange, respectively) ( n =3). Negative controls are indicated by black lines ( n =3). ( B ) Threshold time T T (the reaction time when fluorescent intensity of each reaction exceeds a threshold, indicated by a perforated line in Figure 3A) was plotted against the sample DNA concentration (S) of the reaction. Solid line indicates linear least squares fitting between 500 and 0.5 amol sample DNA and its formulation is T T =−8.65 log 10 (S)+28.5 (R 2 =0.997). Perforated line indicates average T T value of the negative controls. Limit of detection is 58.4 zmol or 3.50 × 10 4 molecules of sample DNA by calculation from the intersection of both lines.

    Journal: Nucleic Acids Research

    Article Title: Sensitive isothermal detection of nucleic-acid sequence by primer generation-rolling circle amplification

    doi: 10.1093/nar/gkn1014

    Figure Lengend Snippet: Real-time product analysis of PG–RCA. ( A ) Fluorescent intensity of PG–RCA reaction was monitored in real time. PG–RCA was conducted at 60°C with 400 µM each dNTP, 0.4 U Vent (exo-) DNA polymerase, 1 U Nb.BsmI and 7.5 nM circular probe II as described in Materials and methods section. Sample DNA concentration in each reaction was prepared by 10-fold serial dilution from 500 amol to 0.5 zmol, and their signal amplification curves were indicated by colored lines (blue, light blue, purple, dark green, green, brown and orange, respectively) ( n =3). Negative controls are indicated by black lines ( n =3). ( B ) Threshold time T T (the reaction time when fluorescent intensity of each reaction exceeds a threshold, indicated by a perforated line in Figure 3A) was plotted against the sample DNA concentration (S) of the reaction. Solid line indicates linear least squares fitting between 500 and 0.5 amol sample DNA and its formulation is T T =−8.65 log 10 (S)+28.5 (R 2 =0.997). Perforated line indicates average T T value of the negative controls. Limit of detection is 58.4 zmol or 3.50 × 10 4 molecules of sample DNA by calculation from the intersection of both lines.

    Article Snippet: Vent (exo-) DNA polymerase, Nb.BsmI, BsmI, BbvCI and exonuclease III were purchased from New England Biolabs.

    Techniques: Concentration Assay, Serial Dilution, Amplification

    Endpoint product analysis of PG–RCA. ( A ) Endpoint products in the presence or absence of 500 amol sample DNA were analyzed on 1.0% agarose gel electrophoresis. The reaction was conducted at 60°C for 30, 45, 60, 75, 90, 105 and 120 min and each reaction was analyzed separately. Lane M was loaded with a 100 bp DNA marker (100, 200, 300, 400, 500/517, 600, 700, 800, 900, 1000, 1200 and 1517 bp from the bottom). ( B ) Endpoint products of 90-min reactions with all or partial reaction components were analyzed on 1.0% agarose gel electrophoresis. DNA, POL and NICK indicate sample DNA, Vent (exo-) DNA polymerase and Nb.BsmI, respectively. Lane M is a 100 bp DNA marker. ( C ) Endpoint product of a 120-min PG–RCA reaction was digested with either BsmI or BbvCI and analyzed on 1.5% agarose gel electrophoresis. Lane ‘-’ was loaded with the 120 min reaction product before restriction enzyme digestion. Lane M is a 100 bp DNA marker.

    Journal: Nucleic Acids Research

    Article Title: Sensitive isothermal detection of nucleic-acid sequence by primer generation-rolling circle amplification

    doi: 10.1093/nar/gkn1014

    Figure Lengend Snippet: Endpoint product analysis of PG–RCA. ( A ) Endpoint products in the presence or absence of 500 amol sample DNA were analyzed on 1.0% agarose gel electrophoresis. The reaction was conducted at 60°C for 30, 45, 60, 75, 90, 105 and 120 min and each reaction was analyzed separately. Lane M was loaded with a 100 bp DNA marker (100, 200, 300, 400, 500/517, 600, 700, 800, 900, 1000, 1200 and 1517 bp from the bottom). ( B ) Endpoint products of 90-min reactions with all or partial reaction components were analyzed on 1.0% agarose gel electrophoresis. DNA, POL and NICK indicate sample DNA, Vent (exo-) DNA polymerase and Nb.BsmI, respectively. Lane M is a 100 bp DNA marker. ( C ) Endpoint product of a 120-min PG–RCA reaction was digested with either BsmI or BbvCI and analyzed on 1.5% agarose gel electrophoresis. Lane ‘-’ was loaded with the 120 min reaction product before restriction enzyme digestion. Lane M is a 100 bp DNA marker.

    Article Snippet: Vent (exo-) DNA polymerase, Nb.BsmI, BsmI, BbvCI and exonuclease III were purchased from New England Biolabs.

    Techniques: Agarose Gel Electrophoresis, Marker

    Real-time quantification of hly gene in L. monocytogenes genomic DNA by PG–RCA. ( A ) Circular probe LM for detection of pathogenic L. monocytogenes genomic DNA. The probe targets the complementary strand of virulence gene, hly (GeneBank GeneID 2797098), encoding a cholesterol-dependent cytolysin, listeriolysin O (LLO). Circular probe LM contains three repeats of a 26-base sequence complementary to the gene including a nicking site for Nb.BsmI. Since these repeat sequences have 5-base overlaps each other, the circular probe comprises three repeats of a 21-base sequence (red, blue and green). ( B ) Genomic DNA from L. monocytogenes (0.1–100 pg) was analyzed by real-time PG–RCA with circular probe LM. Threshold time ( T T ) was plotted against the L. monocytogenes genomic DNA concentration (S) of the reaction. Solid line indicates linear least squares fitting between 0.1 and 100 pg L. monocytogenes genomic DNA and its formulation is T T = −19.1 log 10 (S) + 233 ( R 2 =0.964). Perforated line indicates average T T value of the negative controls ( n =2). Limit of detection is 0.163 pg (∼60 molecules) of L. monocytogenes genomic DNA by calculation from the intersection of both lines. ( C ) Genomic DNA (100 pg) from L. monocytogenes , L. innocua , E. coli and S. enterica were analyzed by real-time PG–RCA with circular probe LM and their threshold times were compared with the values for L. monocytogenes (100 pg). ‘No DNA’ indicates the negative controls.

    Journal: Nucleic Acids Research

    Article Title: Sensitive isothermal detection of nucleic-acid sequence by primer generation-rolling circle amplification

    doi: 10.1093/nar/gkn1014

    Figure Lengend Snippet: Real-time quantification of hly gene in L. monocytogenes genomic DNA by PG–RCA. ( A ) Circular probe LM for detection of pathogenic L. monocytogenes genomic DNA. The probe targets the complementary strand of virulence gene, hly (GeneBank GeneID 2797098), encoding a cholesterol-dependent cytolysin, listeriolysin O (LLO). Circular probe LM contains three repeats of a 26-base sequence complementary to the gene including a nicking site for Nb.BsmI. Since these repeat sequences have 5-base overlaps each other, the circular probe comprises three repeats of a 21-base sequence (red, blue and green). ( B ) Genomic DNA from L. monocytogenes (0.1–100 pg) was analyzed by real-time PG–RCA with circular probe LM. Threshold time ( T T ) was plotted against the L. monocytogenes genomic DNA concentration (S) of the reaction. Solid line indicates linear least squares fitting between 0.1 and 100 pg L. monocytogenes genomic DNA and its formulation is T T = −19.1 log 10 (S) + 233 ( R 2 =0.964). Perforated line indicates average T T value of the negative controls ( n =2). Limit of detection is 0.163 pg (∼60 molecules) of L. monocytogenes genomic DNA by calculation from the intersection of both lines. ( C ) Genomic DNA (100 pg) from L. monocytogenes , L. innocua , E. coli and S. enterica were analyzed by real-time PG–RCA with circular probe LM and their threshold times were compared with the values for L. monocytogenes (100 pg). ‘No DNA’ indicates the negative controls.

    Article Snippet: Vent (exo-) DNA polymerase, Nb.BsmI, BsmI, BbvCI and exonuclease III were purchased from New England Biolabs.

    Techniques: Sequencing, Concentration Assay

    Tetrastable system built from two cross-inhibitory bistable switches. (a) Schematic of the tetrastable DNA circuit. Two microRNA-sensing circuits (cT, aT, pT, and rT) are interconnected by kT αkβ and βkα, which repress unwanted cross-activation. (b) Detailed mechanism of the five kinds of templates (pol. = Vent(exo-), nick. 1 = Nt.BstNBI, nick. 2 = Nb.BsmI, RE = BsmI, and exo. = ttRecJ). cTs convert the complementary microRNA target to a signal strand (α or β). Autocataytic templates (aTs) exponentially amplify the signal strands. pTs, by deactivating a fraction of signal strands, suppress background amplification stemming from biochemical noise. Reporting templates (rTs) transduce the molecular signal (α or β) to a detectable fluorescence signal (green = Oregon green fluorophore, red = Atto633 fluorophore). From the α or β strands, killer templates (kTs) produce pTs of the opposite switch, mitigating unspecific cross-talks. All produced strands are continuously degraded by the exonuclease to maintain the system dynamics and avoid system poisoning by the accumulation of DNA strands. Only one half of the tetrastable circuit is represented here, the second half being obtained by substituting α by β and conversely.

    Journal: ACS Sensors

    Article Title: Multiplex Digital MicroRNA Detection Using Cross-Inhibitory DNA Circuits

    doi: 10.1021/acssensors.0c00593

    Figure Lengend Snippet: Tetrastable system built from two cross-inhibitory bistable switches. (a) Schematic of the tetrastable DNA circuit. Two microRNA-sensing circuits (cT, aT, pT, and rT) are interconnected by kT αkβ and βkα, which repress unwanted cross-activation. (b) Detailed mechanism of the five kinds of templates (pol. = Vent(exo-), nick. 1 = Nt.BstNBI, nick. 2 = Nb.BsmI, RE = BsmI, and exo. = ttRecJ). cTs convert the complementary microRNA target to a signal strand (α or β). Autocataytic templates (aTs) exponentially amplify the signal strands. pTs, by deactivating a fraction of signal strands, suppress background amplification stemming from biochemical noise. Reporting templates (rTs) transduce the molecular signal (α or β) to a detectable fluorescence signal (green = Oregon green fluorophore, red = Atto633 fluorophore). From the α or β strands, killer templates (kTs) produce pTs of the opposite switch, mitigating unspecific cross-talks. All produced strands are continuously degraded by the exonuclease to maintain the system dynamics and avoid system poisoning by the accumulation of DNA strands. Only one half of the tetrastable circuit is represented here, the second half being obtained by substituting α by β and conversely.

    Article Snippet: The nicking enzymes Nb.BsmI and Nt.bstNBI, the restriction enzyme BsmI, the DNA polymerase Vent(exo-), BSA, and dNTP were obtained from New England Biolabs (NEB).

    Techniques: Activation Assay, Amplification, Fluorescence, Produced