phi29 dna polymerase  (Thermo Fisher)


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    Name:
    phi29 DNA Polymerase 10 U µL
    Description:
    Thermo Scientific phi29 DNA Polymerase is a highly processive polymerase up to more than 70 kb featuring strong strand displacement activity which allows for highly efficient isothermal DNA amplification phi29 DNA Polymerase also possesses a 3 →5 exonuclease proofreading activity acting preferentially on single stranded DNA or RNA Therefore 3 modified primers are highly recommended Highlights• Highest processivity and strand displacement activity among known DNA polymerases more than 70 kb long DNA stretches can be synthesized• Highly accurate DNA synthesis• Extremely high yields of amplified DNA even from minute amounts of template• Amplification products can be directly used in downstream applications PCR restriction digestion SNP genotyping etc Applications• Rolling circle amplification RCA generation of periodic DNA nanotemplates• Multiple displacement amplification MDA • Unbiased amplification of whole genome WGA see Figure 1 in Supporting Data • amplification of DNA for SNP and STR detection• cell free amplification of DNA from single cells• pathogenic organisms or metagenomes• amplification of DNA from filter paper blood spot samples• DNA template preparation for sequencing• Protein primed DNA amplification• In situ genotyping with padlock probesRecombination based cloning• Cell free cloning of lethal DNA• RNA primed DNA amplificationNoteAddition of Pyrophosphatase to the reaction mixture with phi29 DNA Polymerase may enhance DNA synthesis Use of this enzyme in certain applications may be covered by patents and may require a license
    Catalog Number:
    ep0091
    Price:
    None
    Applications:
    Cloning|DNA & RNA Purification & Analysis
    Category:
    Proteins Enzymes Peptides
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    Structured Review

    Thermo Fisher phi29 dna polymerase
    Size distribution of <t>DNA-magnesium-pyrophosphate</t> particles as a function of multiple displacement amplification reaction times, with and without the heat-inactivation step. At different time points (17, 38 and 66 h) of the MDA reaction in 3-pL droplets at 30 °C, the resulting DNA-Mg-PP i particles were released from droplets and imaged under TEM to measure their size. The first set of measurements (green) was made without heat-inactivation step. The second set of measurements (grey) was made with heat-inactivation step at 65 °C for 15 min that is typically used to inactivate <t>phi29</t> DNA polymerase and terminate the MDA reaction.
    Thermo Scientific phi29 DNA Polymerase is a highly processive polymerase up to more than 70 kb featuring strong strand displacement activity which allows for highly efficient isothermal DNA amplification phi29 DNA Polymerase also possesses a 3 →5 exonuclease proofreading activity acting preferentially on single stranded DNA or RNA Therefore 3 modified primers are highly recommended Highlights• Highest processivity and strand displacement activity among known DNA polymerases more than 70 kb long DNA stretches can be synthesized• Highly accurate DNA synthesis• Extremely high yields of amplified DNA even from minute amounts of template• Amplification products can be directly used in downstream applications PCR restriction digestion SNP genotyping etc Applications• Rolling circle amplification RCA generation of periodic DNA nanotemplates• Multiple displacement amplification MDA • Unbiased amplification of whole genome WGA see Figure 1 in Supporting Data • amplification of DNA for SNP and STR detection• cell free amplification of DNA from single cells• pathogenic organisms or metagenomes• amplification of DNA from filter paper blood spot samples• DNA template preparation for sequencing• Protein primed DNA amplification• In situ genotyping with padlock probesRecombination based cloning• Cell free cloning of lethal DNA• RNA primed DNA amplificationNoteAddition of Pyrophosphatase to the reaction mixture with phi29 DNA Polymerase may enhance DNA synthesis Use of this enzyme in certain applications may be covered by patents and may require a license
    https://www.bioz.com/result/phi29 dna polymerase/product/Thermo Fisher
    Average 96 stars, based on 56 article reviews
    Price from $9.99 to $1999.99
    phi29 dna polymerase - by Bioz Stars, 2020-08
    96/100 stars

    Images

    1) Product Images from "Droplet Microfluidics Approach for Single-DNA Molecule Amplification and Condensation into DNA-Magnesium-Pyrophosphate Particles"

    Article Title: Droplet Microfluidics Approach for Single-DNA Molecule Amplification and Condensation into DNA-Magnesium-Pyrophosphate Particles

    Journal: Micromachines

    doi: 10.3390/mi8020062

    Size distribution of DNA-magnesium-pyrophosphate particles as a function of multiple displacement amplification reaction times, with and without the heat-inactivation step. At different time points (17, 38 and 66 h) of the MDA reaction in 3-pL droplets at 30 °C, the resulting DNA-Mg-PP i particles were released from droplets and imaged under TEM to measure their size. The first set of measurements (green) was made without heat-inactivation step. The second set of measurements (grey) was made with heat-inactivation step at 65 °C for 15 min that is typically used to inactivate phi29 DNA polymerase and terminate the MDA reaction.
    Figure Legend Snippet: Size distribution of DNA-magnesium-pyrophosphate particles as a function of multiple displacement amplification reaction times, with and without the heat-inactivation step. At different time points (17, 38 and 66 h) of the MDA reaction in 3-pL droplets at 30 °C, the resulting DNA-Mg-PP i particles were released from droplets and imaged under TEM to measure their size. The first set of measurements (green) was made without heat-inactivation step. The second set of measurements (grey) was made with heat-inactivation step at 65 °C for 15 min that is typically used to inactivate phi29 DNA polymerase and terminate the MDA reaction.

    Techniques Used: Multiple Displacement Amplification, Transmission Electron Microscopy

    2) Product Images from "The level of activity of the alternative lengthening of telomeres correlates with patient age in IDH-mutant ATRX-loss-of-expression anaplastic astrocytomas"

    Article Title: The level of activity of the alternative lengthening of telomeres correlates with patient age in IDH-mutant ATRX-loss-of-expression anaplastic astrocytomas

    Journal: Acta Neuropathologica Communications

    doi: 10.1186/s40478-019-0833-0

    Measurement of the level of ALT activity levels in human diffuse gliomas. Top panels illustrate the principle of the ALT C-circle assay [ 17 ] and describe its general steps. ALT cells have very long telomeres that have been amplified mainly by homologous recombination that generates partially single-stranded extra-chromosomal circles. Genomic DNA prepared from tumor samples is then incubated with the Phi29 DNA polymerase that specifically amplifies this telomeric DNA. Middle panel illustrates ALT-specific signals measured in tumor DNA samples using this assay, which were detected here on dot blots hybridized with a telomeric 32 P-labeled probe. Genomic DNAs from HeLa (telomerase positive) and U2OS (ALT positive) cells were also probed, representing negative and posititve controls for the C-circle assay, respectively. These assays were systematically performed in duplicates and here dot blot 2, on the right, was loaded with the same tumor samples as dot blot 1, on the left, to insure for reproducibility. Bottom table illustrates examples of duplicate numbers obtained for each of the indicated tumors, real signals of which are represented in the middle panel above. The C-circle score was determined after calculating the intensity of the signal relative to that of the ALT positive U2OS cell line, designated to be 100 arbitrary units (AU). Note that the C-circle assays were performed on representative samples, including those from the two patient groups analyzed in the present study
    Figure Legend Snippet: Measurement of the level of ALT activity levels in human diffuse gliomas. Top panels illustrate the principle of the ALT C-circle assay [ 17 ] and describe its general steps. ALT cells have very long telomeres that have been amplified mainly by homologous recombination that generates partially single-stranded extra-chromosomal circles. Genomic DNA prepared from tumor samples is then incubated with the Phi29 DNA polymerase that specifically amplifies this telomeric DNA. Middle panel illustrates ALT-specific signals measured in tumor DNA samples using this assay, which were detected here on dot blots hybridized with a telomeric 32 P-labeled probe. Genomic DNAs from HeLa (telomerase positive) and U2OS (ALT positive) cells were also probed, representing negative and posititve controls for the C-circle assay, respectively. These assays were systematically performed in duplicates and here dot blot 2, on the right, was loaded with the same tumor samples as dot blot 1, on the left, to insure for reproducibility. Bottom table illustrates examples of duplicate numbers obtained for each of the indicated tumors, real signals of which are represented in the middle panel above. The C-circle score was determined after calculating the intensity of the signal relative to that of the ALT positive U2OS cell line, designated to be 100 arbitrary units (AU). Note that the C-circle assays were performed on representative samples, including those from the two patient groups analyzed in the present study

    Techniques Used: Activity Assay, Amplification, Homologous Recombination, Incubation, Labeling, Dot Blot

    3) Product Images from "Droplet Microfluidics Approach for Single-DNA Molecule Amplification and Condensation into DNA-Magnesium-Pyrophosphate Particles"

    Article Title: Droplet Microfluidics Approach for Single-DNA Molecule Amplification and Condensation into DNA-Magnesium-Pyrophosphate Particles

    Journal: Micromachines

    doi: 10.3390/mi8020062

    Size distribution of DNA-magnesium-pyrophosphate particles as a function of multiple displacement amplification reaction times, with and without the heat-inactivation step. At different time points (17, 38 and 66 h) of the MDA reaction in 3-pL droplets at 30 °C, the resulting DNA-Mg-PP i particles were released from droplets and imaged under TEM to measure their size. The first set of measurements (green) was made without heat-inactivation step. The second set of measurements (grey) was made with heat-inactivation step at 65 °C for 15 min that is typically used to inactivate phi29 DNA polymerase and terminate the MDA reaction.
    Figure Legend Snippet: Size distribution of DNA-magnesium-pyrophosphate particles as a function of multiple displacement amplification reaction times, with and without the heat-inactivation step. At different time points (17, 38 and 66 h) of the MDA reaction in 3-pL droplets at 30 °C, the resulting DNA-Mg-PP i particles were released from droplets and imaged under TEM to measure their size. The first set of measurements (green) was made without heat-inactivation step. The second set of measurements (grey) was made with heat-inactivation step at 65 °C for 15 min that is typically used to inactivate phi29 DNA polymerase and terminate the MDA reaction.

    Techniques Used: Multiple Displacement Amplification, Transmission Electron Microscopy

    4) Product Images from "Droplet Microfluidics Approach for Single-DNA Molecule Amplification and Condensation into DNA-Magnesium-Pyrophosphate Particles"

    Article Title: Droplet Microfluidics Approach for Single-DNA Molecule Amplification and Condensation into DNA-Magnesium-Pyrophosphate Particles

    Journal: Micromachines

    doi: 10.3390/mi8020062

    Size distribution of DNA-magnesium-pyrophosphate particles as a function of multiple displacement amplification reaction times, with and without the heat-inactivation step. At different time points (17, 38 and 66 h) of the MDA reaction in 3-pL droplets at 30 °C, the resulting DNA-Mg-PP i particles were released from droplets and imaged under TEM to measure their size. The first set of measurements (green) was made without heat-inactivation step. The second set of measurements (grey) was made with heat-inactivation step at 65 °C for 15 min that is typically used to inactivate phi29 DNA polymerase and terminate the MDA reaction.
    Figure Legend Snippet: Size distribution of DNA-magnesium-pyrophosphate particles as a function of multiple displacement amplification reaction times, with and without the heat-inactivation step. At different time points (17, 38 and 66 h) of the MDA reaction in 3-pL droplets at 30 °C, the resulting DNA-Mg-PP i particles were released from droplets and imaged under TEM to measure their size. The first set of measurements (green) was made without heat-inactivation step. The second set of measurements (grey) was made with heat-inactivation step at 65 °C for 15 min that is typically used to inactivate phi29 DNA polymerase and terminate the MDA reaction.

    Techniques Used: Multiple Displacement Amplification, Transmission Electron Microscopy

    5) Product Images from "Rolling circle replication requires single-stranded DNA binding protein to avoid termination and production of double-stranded DNA"

    Article Title: Rolling circle replication requires single-stranded DNA binding protein to avoid termination and production of double-stranded DNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku737

    RCA assay in a MOSIC method system. ( A ) Schematic representation of the single-stranded oligonucleotide production by MOSIC method. p378 double-stranded circular nicked DNA (i) is amplified by RCA in two possible ways: single-stranded 378 nt ODN sequence repeated in tandem with hairpin structures in between (ii) and then digested as single-stranded product (iii) or double-stranded DNA repeated in tandem which is digested in double-stranded 378 bp DNA fragments. ( B ) Agarose gel of BseGI digestion products from p378 RCA. RCAs of nicked p378 were stopped at different reaction times from 0.5 to 24 h (lanes 1–14) and the amplifications were performed with (+) and without (−) T4 gene 32 protein. L = 100 bp DNA ladder. The digestion products of RCA performed in absence of T4 gene 32 (odd lanes) correspond to the predicted 378 bp dsDNA which means that phi29 DNA polymerase amplifies p378 mostly in double-stranded form. On the other hand the digestion product of RCA performed with the addition of T4 gene 32 (even lanes) corresponds to the predicted 378 nt ODN as also confirmed from the denaturing PAGE in Supplementary Figure S3.
    Figure Legend Snippet: RCA assay in a MOSIC method system. ( A ) Schematic representation of the single-stranded oligonucleotide production by MOSIC method. p378 double-stranded circular nicked DNA (i) is amplified by RCA in two possible ways: single-stranded 378 nt ODN sequence repeated in tandem with hairpin structures in between (ii) and then digested as single-stranded product (iii) or double-stranded DNA repeated in tandem which is digested in double-stranded 378 bp DNA fragments. ( B ) Agarose gel of BseGI digestion products from p378 RCA. RCAs of nicked p378 were stopped at different reaction times from 0.5 to 24 h (lanes 1–14) and the amplifications were performed with (+) and without (−) T4 gene 32 protein. L = 100 bp DNA ladder. The digestion products of RCA performed in absence of T4 gene 32 (odd lanes) correspond to the predicted 378 bp dsDNA which means that phi29 DNA polymerase amplifies p378 mostly in double-stranded form. On the other hand the digestion product of RCA performed with the addition of T4 gene 32 (even lanes) corresponds to the predicted 378 nt ODN as also confirmed from the denaturing PAGE in Supplementary Figure S3.

    Techniques Used: Amplification, Sequencing, Agarose Gel Electrophoresis, Polyacrylamide Gel Electrophoresis

    RCA assay of pUC19 DNA plasmid. ( A ) Agarose gel of pUC19 RCA products. Lanes 1–7 RCA performed with increasing concentrations of T4 gene 32 protein (0,10, 20, 30, 50, 75, 100 ng/μl respectively); lane 8 negative control with no phi29 DNA polymerase in the reaction mixture; 1 kb plus DNA ladders (L). ( B ) Agarose gel of MlyI digestion test. RCA products in (A) were digested by MlyI restriction enzyme and the corresponding digestion products (9-16) were run on agarose gel. 1 kb plus DNA ladders (L). ( C ) Picogreen assay of pUC19 RCA. The amplification is expressed in percentage of relative fluorescence units (RFU) and the signal of the amplification product without T4 gene 32 is taken as 100%. Both MlyI digestion and picogreen assay confirm that rolling circle amplification makes mostly double-stranded DNA but they also suggest that T4 gene 32 SSB protein drastically reduces dsDNA production.
    Figure Legend Snippet: RCA assay of pUC19 DNA plasmid. ( A ) Agarose gel of pUC19 RCA products. Lanes 1–7 RCA performed with increasing concentrations of T4 gene 32 protein (0,10, 20, 30, 50, 75, 100 ng/μl respectively); lane 8 negative control with no phi29 DNA polymerase in the reaction mixture; 1 kb plus DNA ladders (L). ( B ) Agarose gel of MlyI digestion test. RCA products in (A) were digested by MlyI restriction enzyme and the corresponding digestion products (9-16) were run on agarose gel. 1 kb plus DNA ladders (L). ( C ) Picogreen assay of pUC19 RCA. The amplification is expressed in percentage of relative fluorescence units (RFU) and the signal of the amplification product without T4 gene 32 is taken as 100%. Both MlyI digestion and picogreen assay confirm that rolling circle amplification makes mostly double-stranded DNA but they also suggest that T4 gene 32 SSB protein drastically reduces dsDNA production.

    Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis, Negative Control, Picogreen Assay, Amplification, Fluorescence

    Model of template switching and its implications on ds/ssDNA production. ( A ), ( B ) The template switching event in detail. (A) According to the current view on RCA mechanisms, a polymerase, in grey, produces new DNA (in green) from the circular template (in blue) and continually displaces the 5′-end DNA from the template (in red). We propose that strand switching events, that are randomly distributed with an exponential distribution, converts some of the normal RCA templates into templates for double-stranded DNA production in a mechanism like the one depicted in (B)—switching from the circular template, up to the displaced strand. ( C ) This mechanism can be shut down by single-stranded DNA binding proteins (SSBs) that prevent access to the displaced strand for the polymerase. ( D ) Phases of ssDNA versus dsDNA production of a single template. Initially, all templates are assumed to produce ssDNA in our model. Assuming that a template switching event occurs at time t 0 /2, then the polymerase attached to this template will consume the ssDNA that has been produced up to that time while converting it to dsDNA. Assuming that the rate of the polymerase is equal while processing along the single strand, at time t 0 it will stop nucleotide incorporation and fall of the template that is now fully double-stranded. ( E ) Assuming an exponential decay of the templates into dsDNA production with a rate constant of λ , the probability density function will determine the instantaneous flipping probability. To get a figure of the current production rate of dsDNA at time t , one needs to consider that only the templates that have switched between t /2 and t will be currently making dsDNA. The rate of ds-production at t is thus proportional to an integral over the probability density function between t /2 and t times the concentration, n , and the phi29 rate, ø . Once the rate of dsDNA production is known, the rate of ssDNA production can be calculated as the rate of production of all non-switched templates, minus the above ds-DNA rate (because that process consumes already produced ssDNA). Integrating (Supplementary Note S1) the rates, yields expressions for the total mass of produced dsDNA, Nds ( t ), and total mass of produced ssDNA, Nss ( t ). The expressions are plotted in ( F ) using arbitrary units. ( G ) Quantitative fit of the data in Figure 3C to the theoretical curves ( Nds ( t ) and Nss ( t ) not fitted independently with respect to each other) reveals a close resemblance to the measured data and our model. From this we estimate the rate constant λ to be 1.95 ± 0.25 × 10 −5 events per second per template for the switching process.
    Figure Legend Snippet: Model of template switching and its implications on ds/ssDNA production. ( A ), ( B ) The template switching event in detail. (A) According to the current view on RCA mechanisms, a polymerase, in grey, produces new DNA (in green) from the circular template (in blue) and continually displaces the 5′-end DNA from the template (in red). We propose that strand switching events, that are randomly distributed with an exponential distribution, converts some of the normal RCA templates into templates for double-stranded DNA production in a mechanism like the one depicted in (B)—switching from the circular template, up to the displaced strand. ( C ) This mechanism can be shut down by single-stranded DNA binding proteins (SSBs) that prevent access to the displaced strand for the polymerase. ( D ) Phases of ssDNA versus dsDNA production of a single template. Initially, all templates are assumed to produce ssDNA in our model. Assuming that a template switching event occurs at time t 0 /2, then the polymerase attached to this template will consume the ssDNA that has been produced up to that time while converting it to dsDNA. Assuming that the rate of the polymerase is equal while processing along the single strand, at time t 0 it will stop nucleotide incorporation and fall of the template that is now fully double-stranded. ( E ) Assuming an exponential decay of the templates into dsDNA production with a rate constant of λ , the probability density function will determine the instantaneous flipping probability. To get a figure of the current production rate of dsDNA at time t , one needs to consider that only the templates that have switched between t /2 and t will be currently making dsDNA. The rate of ds-production at t is thus proportional to an integral over the probability density function between t /2 and t times the concentration, n , and the phi29 rate, ø . Once the rate of dsDNA production is known, the rate of ssDNA production can be calculated as the rate of production of all non-switched templates, minus the above ds-DNA rate (because that process consumes already produced ssDNA). Integrating (Supplementary Note S1) the rates, yields expressions for the total mass of produced dsDNA, Nds ( t ), and total mass of produced ssDNA, Nss ( t ). The expressions are plotted in ( F ) using arbitrary units. ( G ) Quantitative fit of the data in Figure 3C to the theoretical curves ( Nds ( t ) and Nss ( t ) not fitted independently with respect to each other) reveals a close resemblance to the measured data and our model. From this we estimate the rate constant λ to be 1.95 ± 0.25 × 10 −5 events per second per template for the switching process.

    Techniques Used: DNA Binding Assay, Produced, Concentration Assay

    6) Product Images from "SLX4IP Antagonizes Promiscuous BLM Activity during ALT Maintenance"

    Article Title: SLX4IP Antagonizes Promiscuous BLM Activity during ALT Maintenance

    Journal: Molecular Cell

    doi: 10.1016/j.molcel.2019.07.010

    Loss of SLX4IP in ALT-Positive Cells Increases ALT-Related Phenotypes (A) Genomic DNA was isolated from U2OS cells and processed to detect Phi29-dependent telomere circles. The Phi29 amplification products were detected by Southern blotting using a γ[ 32 P]-labeled telomeric (TTAGGG) probe. (B) Quantification of (A). The extent of [ 32 P] incorporation was quantified from the autoradiograph and normalized to SLX4IP +/+ , which was arbitrarily assigned a value of 1. Data are represented as mean ± SD; n = 3; ∗ p
    Figure Legend Snippet: Loss of SLX4IP in ALT-Positive Cells Increases ALT-Related Phenotypes (A) Genomic DNA was isolated from U2OS cells and processed to detect Phi29-dependent telomere circles. The Phi29 amplification products were detected by Southern blotting using a γ[ 32 P]-labeled telomeric (TTAGGG) probe. (B) Quantification of (A). The extent of [ 32 P] incorporation was quantified from the autoradiograph and normalized to SLX4IP +/+ , which was arbitrarily assigned a value of 1. Data are represented as mean ± SD; n = 3; ∗ p

    Techniques Used: Isolation, Amplification, Southern Blot, Labeling, Autoradiography

    SLX4 Depletion Further Augments the Increase in ALT-Related Phenotypes in SLX4IP −/− Cells (A) U2OS cells were transfected with the indicated siRNAs. Their genomic DNA was then processed to detect Phi29-dependent telomere circles. The Phi29 amplification products were detected by Southern blotting using a γ[ 32 P]-labeled telomeric (TTAGGG) probe. (B) Quantification of (A). The extent of [ 32 P] incorporation was quantified from the autoradiograph and normalized to SLX4IP +/+ siCTRL, which was arbitrarily assigned a value of 1. Data are represented as mean ± SD; n = 3; ∗ p
    Figure Legend Snippet: SLX4 Depletion Further Augments the Increase in ALT-Related Phenotypes in SLX4IP −/− Cells (A) U2OS cells were transfected with the indicated siRNAs. Their genomic DNA was then processed to detect Phi29-dependent telomere circles. The Phi29 amplification products were detected by Southern blotting using a γ[ 32 P]-labeled telomeric (TTAGGG) probe. (B) Quantification of (A). The extent of [ 32 P] incorporation was quantified from the autoradiograph and normalized to SLX4IP +/+ siCTRL, which was arbitrarily assigned a value of 1. Data are represented as mean ± SD; n = 3; ∗ p

    Techniques Used: Transfection, Amplification, Southern Blot, Labeling, Autoradiography

    7) Product Images from "Circular RNA Profiling by Illumina Sequencing via Template-Dependent Multiple Displacement Amplification"

    Article Title: Circular RNA Profiling by Illumina Sequencing via Template-Dependent Multiple Displacement Amplification

    Journal: BioMed Research International

    doi: 10.1155/2019/2756516

    Amplification of cDNA by Phi29 DNA polymerase . Total RNA from N. benthamiana (a) and O. sativa (b) was treated with DNase and RNase R to enrich circRNAs. The enriched circRNAs were converted into cDNA using random hexamer and subjected to amplification by Phi29 DNA polymerase.
    Figure Legend Snippet: Amplification of cDNA by Phi29 DNA polymerase . Total RNA from N. benthamiana (a) and O. sativa (b) was treated with DNase and RNase R to enrich circRNAs. The enriched circRNAs were converted into cDNA using random hexamer and subjected to amplification by Phi29 DNA polymerase.

    Techniques Used: Amplification, Random Hexamer Labeling

    8) Product Images from "Improved efficiency of in situ protein analysis by proximity ligation using UnFold probes"

    Article Title: Improved efficiency of in situ protein analysis by proximity ligation using UnFold probes

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-23582-1

    Schematic illustration of in situ PLA using conventional and UnFold probes. ( a ) Conventional in situ PLA. ( b ) In situ PLA using UnFold probes. (i) After pairs of primary antibodies have bound a pair of interacting proteins (red and green) followed by washes, secondary conventional or UnFold in situ PLA probes are added, followed after an incubation by renewed washes. (ii) In the conventional design under ( a ) two more oligonucleotides are then added that can form a DNA circle. Using the UnFold design in ( b ) the probe carrying a hairpin-loop oligonucleotide is cleaved at the U residues, liberating a free 5′ end capable of being ligated to the 3′ end of the same DNA strand. Meanwhile, the U residues in the hairpin DNA strand of the other UnFold probe are cleaved presenting a single-stranded template for the enzymatic joining of the ends of the strand on the first UnFold probe. (iii) A DNA ligase is added to form DNA circles in the two variants of in situ PLA. (iv) Finally, phi29 DNA polymerase is added to initiate RCA primed by oligonucleotides on one of the antibodies, and fluorescent oligonucleotides are used to visualize the RCA products.
    Figure Legend Snippet: Schematic illustration of in situ PLA using conventional and UnFold probes. ( a ) Conventional in situ PLA. ( b ) In situ PLA using UnFold probes. (i) After pairs of primary antibodies have bound a pair of interacting proteins (red and green) followed by washes, secondary conventional or UnFold in situ PLA probes are added, followed after an incubation by renewed washes. (ii) In the conventional design under ( a ) two more oligonucleotides are then added that can form a DNA circle. Using the UnFold design in ( b ) the probe carrying a hairpin-loop oligonucleotide is cleaved at the U residues, liberating a free 5′ end capable of being ligated to the 3′ end of the same DNA strand. Meanwhile, the U residues in the hairpin DNA strand of the other UnFold probe are cleaved presenting a single-stranded template for the enzymatic joining of the ends of the strand on the first UnFold probe. (iii) A DNA ligase is added to form DNA circles in the two variants of in situ PLA. (iv) Finally, phi29 DNA polymerase is added to initiate RCA primed by oligonucleotides on one of the antibodies, and fluorescent oligonucleotides are used to visualize the RCA products.

    Techniques Used: In Situ, Proximity Ligation Assay, Incubation

    9) Product Images from "Duality of polynucleotide substrates for Phi29 DNA polymerase: 3′→5′ RNase activity of the enzyme"

    Article Title: Duality of polynucleotide substrates for Phi29 DNA polymerase: 3′→5′ RNase activity of the enzyme

    Journal: RNA

    doi: 10.1261/rna.622108

    The polarity of Phi29 DNA polymerase exoribonuclease activity. RNA hydrolysis studies were carried out under the conditions described in “Materials and Methods,” using 5′-end labeled ( A ) or 3′-end labeled ( B ) RNA1 oligonucleotides
    Figure Legend Snippet: The polarity of Phi29 DNA polymerase exoribonuclease activity. RNA hydrolysis studies were carried out under the conditions described in “Materials and Methods,” using 5′-end labeled ( A ) or 3′-end labeled ( B ) RNA1 oligonucleotides

    Techniques Used: Activity Assay, Labeling

    3′→5′ Exonucleolytic activity of Phi29 DNA polymerase on RNA and DNA substrates. The experiments were performed under the conditions described in “Materials and Methods,” using 5′-end-labeled 16-mer DNA
    Figure Legend Snippet: 3′→5′ Exonucleolytic activity of Phi29 DNA polymerase on RNA and DNA substrates. The experiments were performed under the conditions described in “Materials and Methods,” using 5′-end-labeled 16-mer DNA

    Techniques Used: Activity Assay, Labeling

    Phi29 DNA polymerase 3′→5′ exoribonuclease activity on the RNA–DNA hybrids. The RNA–DNA hybrid hydrolysis studies were carried out under the conditions described in “Materials and Methods,” using
    Figure Legend Snippet: Phi29 DNA polymerase 3′→5′ exoribonuclease activity on the RNA–DNA hybrids. The RNA–DNA hybrid hydrolysis studies were carried out under the conditions described in “Materials and Methods,” using

    Techniques Used: Activity Assay

    Sequence comparison and active sites superposition of Phi29 DNA polymerase and RNaseT orthologs. ( A ), Escherichia
    Figure Legend Snippet: Sequence comparison and active sites superposition of Phi29 DNA polymerase and RNaseT orthologs. ( A ), Escherichia

    Techniques Used: Sequencing

    The target RNA conversion into a primer for RCA. The experiments ( A ) were performed under the conditions described in “Materials and Methods.” The 5′-end-labeled RNA1–DNA hybrids were incubated with Phi29 DNA polymerase
    Figure Legend Snippet: The target RNA conversion into a primer for RCA. The experiments ( A ) were performed under the conditions described in “Materials and Methods.” The 5′-end-labeled RNA1–DNA hybrids were incubated with Phi29 DNA polymerase

    Techniques Used: Labeling, Incubation

    ( A ) Sequencing of Phi29 DNA polymerase 3′→5′ exoribonuclecleolytic degradation products. The experiments were performed under the conditions described in “Materials and Methods.” The RNA degradation products of
    Figure Legend Snippet: ( A ) Sequencing of Phi29 DNA polymerase 3′→5′ exoribonuclecleolytic degradation products. The experiments were performed under the conditions described in “Materials and Methods.” The RNA degradation products of

    Techniques Used: Sequencing

    10) Product Images from "Droplet Microfluidics Approach for Single-DNA Molecule Amplification and Condensation into DNA-Magnesium-Pyrophosphate Particles"

    Article Title: Droplet Microfluidics Approach for Single-DNA Molecule Amplification and Condensation into DNA-Magnesium-Pyrophosphate Particles

    Journal: Micromachines

    doi: 10.3390/mi8020062

    Size distribution of DNA-magnesium-pyrophosphate particles as a function of multiple displacement amplification reaction times, with and without the heat-inactivation step. At different time points (17, 38 and 66 h) of the MDA reaction in 3-pL droplets at 30 °C, the resulting DNA-Mg-PP i particles were released from droplets and imaged under TEM to measure their size. The first set of measurements (green) was made without heat-inactivation step. The second set of measurements (grey) was made with heat-inactivation step at 65 °C for 15 min that is typically used to inactivate phi29 DNA polymerase and terminate the MDA reaction.
    Figure Legend Snippet: Size distribution of DNA-magnesium-pyrophosphate particles as a function of multiple displacement amplification reaction times, with and without the heat-inactivation step. At different time points (17, 38 and 66 h) of the MDA reaction in 3-pL droplets at 30 °C, the resulting DNA-Mg-PP i particles were released from droplets and imaged under TEM to measure their size. The first set of measurements (green) was made without heat-inactivation step. The second set of measurements (grey) was made with heat-inactivation step at 65 °C for 15 min that is typically used to inactivate phi29 DNA polymerase and terminate the MDA reaction.

    Techniques Used: Multiple Displacement Amplification, Transmission Electron Microscopy

    11) Product Images from "Improved efficiency of in situ protein analysis by proximity ligation using UnFold probes"

    Article Title: Improved efficiency of in situ protein analysis by proximity ligation using UnFold probes

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-23582-1

    Schematic illustration of in situ PLA using conventional and UnFold probes. ( a ) Conventional in situ PLA. ( b ) In situ PLA using UnFold probes. (i) After pairs of primary antibodies have bound a pair of interacting proteins (red and green) followed by washes, secondary conventional or UnFold in situ PLA probes are added, followed after an incubation by renewed washes. (ii) In the conventional design under ( a ) two more oligonucleotides are then added that can form a DNA circle. Using the UnFold design in ( b ) the probe carrying a hairpin-loop oligonucleotide is cleaved at the U residues, liberating a free 5′ end capable of being ligated to the 3′ end of the same DNA strand. Meanwhile, the U residues in the hairpin DNA strand of the other UnFold probe are cleaved presenting a single-stranded template for the enzymatic joining of the ends of the strand on the first UnFold probe. (iii) A DNA ligase is added to form DNA circles in the two variants of in situ PLA. (iv) Finally, phi29 DNA polymerase is added to initiate RCA primed by oligonucleotides on one of the antibodies, and fluorescent oligonucleotides are used to visualize the RCA products.
    Figure Legend Snippet: Schematic illustration of in situ PLA using conventional and UnFold probes. ( a ) Conventional in situ PLA. ( b ) In situ PLA using UnFold probes. (i) After pairs of primary antibodies have bound a pair of interacting proteins (red and green) followed by washes, secondary conventional or UnFold in situ PLA probes are added, followed after an incubation by renewed washes. (ii) In the conventional design under ( a ) two more oligonucleotides are then added that can form a DNA circle. Using the UnFold design in ( b ) the probe carrying a hairpin-loop oligonucleotide is cleaved at the U residues, liberating a free 5′ end capable of being ligated to the 3′ end of the same DNA strand. Meanwhile, the U residues in the hairpin DNA strand of the other UnFold probe are cleaved presenting a single-stranded template for the enzymatic joining of the ends of the strand on the first UnFold probe. (iii) A DNA ligase is added to form DNA circles in the two variants of in situ PLA. (iv) Finally, phi29 DNA polymerase is added to initiate RCA primed by oligonucleotides on one of the antibodies, and fluorescent oligonucleotides are used to visualize the RCA products.

    Techniques Used: In Situ, Proximity Ligation Assay, Incubation

    12) Product Images from "SLX4IP Antagonizes Promiscuous BLM Activity during ALT Maintenance"

    Article Title: SLX4IP Antagonizes Promiscuous BLM Activity during ALT Maintenance

    Journal: Molecular Cell

    doi: 10.1016/j.molcel.2019.07.010

    Loss of SLX4IP in ALT-Positive Cells Increases ALT-Related Phenotypes (A) Genomic DNA was isolated from U2OS cells and processed to detect Phi29-dependent telomere circles. The Phi29 amplification products were detected by Southern blotting using a γ[ 32 P]-labeled telomeric (TTAGGG) probe. (B) Quantification of (A). The extent of [ 32 P] incorporation was quantified from the autoradiograph and normalized to SLX4IP +/+ , which was arbitrarily assigned a value of 1. Data are represented as mean ± SD; n = 3; ∗ p
    Figure Legend Snippet: Loss of SLX4IP in ALT-Positive Cells Increases ALT-Related Phenotypes (A) Genomic DNA was isolated from U2OS cells and processed to detect Phi29-dependent telomere circles. The Phi29 amplification products were detected by Southern blotting using a γ[ 32 P]-labeled telomeric (TTAGGG) probe. (B) Quantification of (A). The extent of [ 32 P] incorporation was quantified from the autoradiograph and normalized to SLX4IP +/+ , which was arbitrarily assigned a value of 1. Data are represented as mean ± SD; n = 3; ∗ p

    Techniques Used: Isolation, Amplification, Southern Blot, Labeling, Autoradiography

    SLX4 Depletion Further Augments the Increase in ALT-Related Phenotypes in SLX4IP −/− Cells (A) U2OS cells were transfected with the indicated siRNAs. Their genomic DNA was then processed to detect Phi29-dependent telomere circles. The Phi29 amplification products were detected by Southern blotting using a γ[ 32 P]-labeled telomeric (TTAGGG) probe. (B) Quantification of (A). The extent of [ 32 P] incorporation was quantified from the autoradiograph and normalized to SLX4IP +/+ siCTRL, which was arbitrarily assigned a value of 1. Data are represented as mean ± SD; n = 3; ∗ p
    Figure Legend Snippet: SLX4 Depletion Further Augments the Increase in ALT-Related Phenotypes in SLX4IP −/− Cells (A) U2OS cells were transfected with the indicated siRNAs. Their genomic DNA was then processed to detect Phi29-dependent telomere circles. The Phi29 amplification products were detected by Southern blotting using a γ[ 32 P]-labeled telomeric (TTAGGG) probe. (B) Quantification of (A). The extent of [ 32 P] incorporation was quantified from the autoradiograph and normalized to SLX4IP +/+ siCTRL, which was arbitrarily assigned a value of 1. Data are represented as mean ± SD; n = 3; ∗ p

    Techniques Used: Transfection, Amplification, Southern Blot, Labeling, Autoradiography

    13) Product Images from "Droplet Microfluidics Approach for Single-DNA Molecule Amplification and Condensation into DNA-Magnesium-Pyrophosphate Particles"

    Article Title: Droplet Microfluidics Approach for Single-DNA Molecule Amplification and Condensation into DNA-Magnesium-Pyrophosphate Particles

    Journal: Micromachines

    doi: 10.3390/mi8020062

    Size distribution of DNA-magnesium-pyrophosphate particles as a function of multiple displacement amplification reaction times, with and without the heat-inactivation step. At different time points (17, 38 and 66 h) of the MDA reaction in 3-pL droplets at 30 °C, the resulting DNA-Mg-PP i particles were released from droplets and imaged under TEM to measure their size. The first set of measurements (green) was made without heat-inactivation step. The second set of measurements (grey) was made with heat-inactivation step at 65 °C for 15 min that is typically used to inactivate phi29 DNA polymerase and terminate the MDA reaction.
    Figure Legend Snippet: Size distribution of DNA-magnesium-pyrophosphate particles as a function of multiple displacement amplification reaction times, with and without the heat-inactivation step. At different time points (17, 38 and 66 h) of the MDA reaction in 3-pL droplets at 30 °C, the resulting DNA-Mg-PP i particles were released from droplets and imaged under TEM to measure their size. The first set of measurements (green) was made without heat-inactivation step. The second set of measurements (grey) was made with heat-inactivation step at 65 °C for 15 min that is typically used to inactivate phi29 DNA polymerase and terminate the MDA reaction.

    Techniques Used: Multiple Displacement Amplification, Transmission Electron Microscopy

    14) Product Images from "The level of activity of the alternative lengthening of telomeres correlates with patient age in IDH-mutant ATRX-loss-of-expression anaplastic astrocytomas"

    Article Title: The level of activity of the alternative lengthening of telomeres correlates with patient age in IDH-mutant ATRX-loss-of-expression anaplastic astrocytomas

    Journal: Acta Neuropathologica Communications

    doi: 10.1186/s40478-019-0833-0

    Measurement of the level of ALT activity levels in human diffuse gliomas. Top panels illustrate the principle of the ALT C-circle assay [ 17 ] and describe its general steps. ALT cells have very long telomeres that have been amplified mainly by homologous recombination that generates partially single-stranded extra-chromosomal circles. Genomic DNA prepared from tumor samples is then incubated with the Phi29 DNA polymerase that specifically amplifies this telomeric DNA. Middle panel illustrates ALT-specific signals measured in tumor DNA samples using this assay, which were detected here on dot blots hybridized with a telomeric 32 P-labeled probe. Genomic DNAs from HeLa (telomerase positive) and U2OS (ALT positive) cells were also probed, representing negative and posititve controls for the C-circle assay, respectively. These assays were systematically performed in duplicates and here dot blot 2, on the right, was loaded with the same tumor samples as dot blot 1, on the left, to insure for reproducibility. Bottom table illustrates examples of duplicate numbers obtained for each of the indicated tumors, real signals of which are represented in the middle panel above. The C-circle score was determined after calculating the intensity of the signal relative to that of the ALT positive U2OS cell line, designated to be 100 arbitrary units (AU). Note that the C-circle assays were performed on representative samples, including those from the two patient groups analyzed in the present study
    Figure Legend Snippet: Measurement of the level of ALT activity levels in human diffuse gliomas. Top panels illustrate the principle of the ALT C-circle assay [ 17 ] and describe its general steps. ALT cells have very long telomeres that have been amplified mainly by homologous recombination that generates partially single-stranded extra-chromosomal circles. Genomic DNA prepared from tumor samples is then incubated with the Phi29 DNA polymerase that specifically amplifies this telomeric DNA. Middle panel illustrates ALT-specific signals measured in tumor DNA samples using this assay, which were detected here on dot blots hybridized with a telomeric 32 P-labeled probe. Genomic DNAs from HeLa (telomerase positive) and U2OS (ALT positive) cells were also probed, representing negative and posititve controls for the C-circle assay, respectively. These assays were systematically performed in duplicates and here dot blot 2, on the right, was loaded with the same tumor samples as dot blot 1, on the left, to insure for reproducibility. Bottom table illustrates examples of duplicate numbers obtained for each of the indicated tumors, real signals of which are represented in the middle panel above. The C-circle score was determined after calculating the intensity of the signal relative to that of the ALT positive U2OS cell line, designated to be 100 arbitrary units (AU). Note that the C-circle assays were performed on representative samples, including those from the two patient groups analyzed in the present study

    Techniques Used: Activity Assay, Amplification, Homologous Recombination, Incubation, Labeling, Dot Blot

    15) Product Images from "A dumbbell probe-mediated rolling circle amplification strategy for highly sensitive microRNA detection"

    Article Title: A dumbbell probe-mediated rolling circle amplification strategy for highly sensitive microRNA detection

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq556

    The D-RCA strategy for miRNA detection. A dumbbell probe contains three domains, an MBD, an SGBD and a loop domain. The binding of miRNA to MBD initiates RCA in the presence of T4 DNA ligase and phi29 polymerase, which generates a long DNA sequence that contains many SGBD for an amplified fluorescent readout.
    Figure Legend Snippet: The D-RCA strategy for miRNA detection. A dumbbell probe contains three domains, an MBD, an SGBD and a loop domain. The binding of miRNA to MBD initiates RCA in the presence of T4 DNA ligase and phi29 polymerase, which generates a long DNA sequence that contains many SGBD for an amplified fluorescent readout.

    Techniques Used: Binding Assay, Sequencing, Amplification

    16) Product Images from "Droplet Microfluidics Approach for Single-DNA Molecule Amplification and Condensation into DNA-Magnesium-Pyrophosphate Particles"

    Article Title: Droplet Microfluidics Approach for Single-DNA Molecule Amplification and Condensation into DNA-Magnesium-Pyrophosphate Particles

    Journal: Micromachines

    doi: 10.3390/mi8020062

    Size distribution of DNA-magnesium-pyrophosphate particles as a function of multiple displacement amplification reaction times, with and without the heat-inactivation step. At different time points (17, 38 and 66 h) of the MDA reaction in 3-pL droplets at 30 °C, the resulting DNA-Mg-PP i particles were released from droplets and imaged under TEM to measure their size. The first set of measurements (green) was made without heat-inactivation step. The second set of measurements (grey) was made with heat-inactivation step at 65 °C for 15 min that is typically used to inactivate phi29 DNA polymerase and terminate the MDA reaction.
    Figure Legend Snippet: Size distribution of DNA-magnesium-pyrophosphate particles as a function of multiple displacement amplification reaction times, with and without the heat-inactivation step. At different time points (17, 38 and 66 h) of the MDA reaction in 3-pL droplets at 30 °C, the resulting DNA-Mg-PP i particles were released from droplets and imaged under TEM to measure their size. The first set of measurements (green) was made without heat-inactivation step. The second set of measurements (grey) was made with heat-inactivation step at 65 °C for 15 min that is typically used to inactivate phi29 DNA polymerase and terminate the MDA reaction.

    Techniques Used: Multiple Displacement Amplification, Transmission Electron Microscopy

    Related Articles

    Amplification:

    Article Title: Droplet Microfluidics Approach for Single-DNA Molecule Amplification and Condensation into DNA-Magnesium-Pyrophosphate Particles
    Article Snippet: .. In this context, DNA amplification driven by phi29 DNA polymerase provides an alternative approach to amplify long DNA molecules and because of isothermal reaction conditions [ , ], the potential problems associated with emulsion stability become irrelevant. .. Moreover, the ability not only to amplify individual DNA molecules, but also to express genes from the amplified template opens many interesting possibilities for high throughput screening applications.

    Article Title: Rolling circle replication requires single-stranded DNA binding protein to avoid termination and production of double-stranded DNA
    Article Snippet: .. RCA of DNA plasmids and digestion of RCA products We incubated pUC19 plasmid DNA (1 ng/μl, Invitrogen) with nicking endonuclease Nt.BspQI (0.5 U/μl, New England Biolabs (NEB)) in 1× NEB3 buffer for 2 h at 50°C, and then we heat inactivated the enzyme incubating the reaction mixture at 80°C for 20 min. We then amplified the resulting nicked DNA (0.25 ng/μl) by using phi29 DNA polymerase (0.5 U/μl, Fermentas) for 24 h at 30°C in a 1× phi29 reaction buffer (33 mM Tris acetate, 10 mM magnesium acetate, 66 mM potassium acetate, 0.1% (v/v) Tween 20 and 1 mM Dithiothreitol (DTT); Fermentas) containing dNTP mix (1 mM each; Fermentas) and increasing concentration of single stranded binding protein T4 gene 32 (0–100 ng/μl, NEB). .. We also performed nicking and amplification reactions with pBluescript II SK (+) (with and without T4 gene 32 protein) in the same conditions.

    Article Title: Droplet Microfluidics Approach for Single-DNA Molecule Amplification and Condensation into DNA-Magnesium-Pyrophosphate Particles
    Article Snippet: .. Single-DNA Molecule Encapsulation and Amplification The MDA reaction mix contained pIVEX2.2-lacZ-his plasmid, 1× phi29 reaction buffer (33 mM Tris-acetate (pH 7.9), 10 mM Mg-acetate, 66 mM K-acetate, 0.1% (v /v ) Tween 20, 1 mM dithiothreitol (DTT)), 50 μM exo-nuclease resistant hexanucleotide primers, 1 mM of each deoxynucleoside triphosphates (dNTP), 0.4% (w /v ) Pluronic F-127 and 0.8 U/μL phi29 DNA polymerase (Thermo Fisher Scientific, Vilnius, Lithuania). .. The reaction components were mixed in DNA LoBind tubes (Eppendorf) by adding DNA template, nuclease-free water, Pluronic F-127 and hexamers and then heated to 90 °C for 20 s to allow primer annealing.

    Article Title: Droplet Microfluidics Approach for Single-DNA Molecule Amplification and Condensation into DNA-Magnesium-Pyrophosphate Particles
    Article Snippet: .. High droplet generation speed (~4000 s−1 ) allowed the collection of over 107 droplets in the form of an emulsion in less than 1 h. Droplets loaded with MDA reaction mix were collected off-chip into a collection tube and incubated for 16 h at 30 °C to initiate the isothermal DNA amplification reaction by phi29 DNA polymerase. .. After, off-chip incubation droplets were heated for 10 min at 65 °C and stained with SYBR Green I dye, which passively migrates between the droplets and becomes fluorescent upon binding dsDNA ( ).

    Article Title: The level of activity of the alternative lengthening of telomeres correlates with patient age in IDH-mutant ATRX-loss-of-expression anaplastic astrocytomas
    Article Snippet: .. ALT cells, but not telomerase positive cells, generate extra-chromosomal telomeric single-stranded DNA called C-circles, which can be amplified using the Phi29 DNA polymerase [ ] (Fig. ). ..

    Concentration Assay:

    Article Title: Rolling circle replication requires single-stranded DNA binding protein to avoid termination and production of double-stranded DNA
    Article Snippet: .. RCA of DNA plasmids and digestion of RCA products We incubated pUC19 plasmid DNA (1 ng/μl, Invitrogen) with nicking endonuclease Nt.BspQI (0.5 U/μl, New England Biolabs (NEB)) in 1× NEB3 buffer for 2 h at 50°C, and then we heat inactivated the enzyme incubating the reaction mixture at 80°C for 20 min. We then amplified the resulting nicked DNA (0.25 ng/μl) by using phi29 DNA polymerase (0.5 U/μl, Fermentas) for 24 h at 30°C in a 1× phi29 reaction buffer (33 mM Tris acetate, 10 mM magnesium acetate, 66 mM potassium acetate, 0.1% (v/v) Tween 20 and 1 mM Dithiothreitol (DTT); Fermentas) containing dNTP mix (1 mM each; Fermentas) and increasing concentration of single stranded binding protein T4 gene 32 (0–100 ng/μl, NEB). .. We also performed nicking and amplification reactions with pBluescript II SK (+) (with and without T4 gene 32 protein) in the same conditions.

    Multiple Displacement Amplification:

    Article Title: Template-dependent multiple displacement amplification for profiling human circulating RNA
    Article Snippet: .. First, using the standard MDA protocol, we evaluated the purity of phi29 DNA polymerases from various vendors, including Lucigen (Middleton, WI), Epicentre (Madison, WI), Thermo Scientific (St. Louis, MO), and New England Biolabs (Ipswich, MA). .. Decontamination through UV irradiation as described by Woyke et al. was also adopted for the reagents, including phi29 DNA polymerase, dNTPs, and primers ( ).

    Article Title: Droplet Microfluidics Approach for Single-DNA Molecule Amplification and Condensation into DNA-Magnesium-Pyrophosphate Particles
    Article Snippet: .. Single-DNA Molecule Encapsulation and Amplification The MDA reaction mix contained pIVEX2.2-lacZ-his plasmid, 1× phi29 reaction buffer (33 mM Tris-acetate (pH 7.9), 10 mM Mg-acetate, 66 mM K-acetate, 0.1% (v /v ) Tween 20, 1 mM dithiothreitol (DTT)), 50 μM exo-nuclease resistant hexanucleotide primers, 1 mM of each deoxynucleoside triphosphates (dNTP), 0.4% (w /v ) Pluronic F-127 and 0.8 U/μL phi29 DNA polymerase (Thermo Fisher Scientific, Vilnius, Lithuania). .. The reaction components were mixed in DNA LoBind tubes (Eppendorf) by adding DNA template, nuclease-free water, Pluronic F-127 and hexamers and then heated to 90 °C for 20 s to allow primer annealing.

    Article Title: Droplet Microfluidics Approach for Single-DNA Molecule Amplification and Condensation into DNA-Magnesium-Pyrophosphate Particles
    Article Snippet: .. High droplet generation speed (~4000 s−1 ) allowed the collection of over 107 droplets in the form of an emulsion in less than 1 h. Droplets loaded with MDA reaction mix were collected off-chip into a collection tube and incubated for 16 h at 30 °C to initiate the isothermal DNA amplification reaction by phi29 DNA polymerase. .. After, off-chip incubation droplets were heated for 10 min at 65 °C and stained with SYBR Green I dye, which passively migrates between the droplets and becomes fluorescent upon binding dsDNA ( ).

    Mutagenesis:

    Article Title: Duality of polynucleotide substrates for Phi29 DNA polymerase: 3′→5′ RNase activity of the enzyme
    Article Snippet: .. BSA, glycogen, DNaseI, and DNaseI buffer with MgCl2 (10×), DEPC-treated water, Phi29 DNA polymerase, Phi29 DNA polymerase exo− mutant (D12A + D66A), Ribolock ribonuclease inhibitor, RNaseT1, T4 polynucleotide kinase (T4 PNK), T4 DNA ligase, T4 RNA ligase, Tango buffer (10×), TBE buffer (10×), and RNA Loading Dye Solution (2×) were products of Fermentas UAB. .. RNA labeling KinaseMax kit was purchased from Ambion; yeast tRNA was obtained from Serva.

    Incubation:

    Article Title: Rolling circle replication requires single-stranded DNA binding protein to avoid termination and production of double-stranded DNA
    Article Snippet: .. RCA of DNA plasmids and digestion of RCA products We incubated pUC19 plasmid DNA (1 ng/μl, Invitrogen) with nicking endonuclease Nt.BspQI (0.5 U/μl, New England Biolabs (NEB)) in 1× NEB3 buffer for 2 h at 50°C, and then we heat inactivated the enzyme incubating the reaction mixture at 80°C for 20 min. We then amplified the resulting nicked DNA (0.25 ng/μl) by using phi29 DNA polymerase (0.5 U/μl, Fermentas) for 24 h at 30°C in a 1× phi29 reaction buffer (33 mM Tris acetate, 10 mM magnesium acetate, 66 mM potassium acetate, 0.1% (v/v) Tween 20 and 1 mM Dithiothreitol (DTT); Fermentas) containing dNTP mix (1 mM each; Fermentas) and increasing concentration of single stranded binding protein T4 gene 32 (0–100 ng/μl, NEB). .. We also performed nicking and amplification reactions with pBluescript II SK (+) (with and without T4 gene 32 protein) in the same conditions.

    Article Title: Droplet Microfluidics Approach for Single-DNA Molecule Amplification and Condensation into DNA-Magnesium-Pyrophosphate Particles
    Article Snippet: .. High droplet generation speed (~4000 s−1 ) allowed the collection of over 107 droplets in the form of an emulsion in less than 1 h. Droplets loaded with MDA reaction mix were collected off-chip into a collection tube and incubated for 16 h at 30 °C to initiate the isothermal DNA amplification reaction by phi29 DNA polymerase. .. After, off-chip incubation droplets were heated for 10 min at 65 °C and stained with SYBR Green I dye, which passively migrates between the droplets and becomes fluorescent upon binding dsDNA ( ).

    Binding Assay:

    Article Title: Rolling circle replication requires single-stranded DNA binding protein to avoid termination and production of double-stranded DNA
    Article Snippet: .. RCA of DNA plasmids and digestion of RCA products We incubated pUC19 plasmid DNA (1 ng/μl, Invitrogen) with nicking endonuclease Nt.BspQI (0.5 U/μl, New England Biolabs (NEB)) in 1× NEB3 buffer for 2 h at 50°C, and then we heat inactivated the enzyme incubating the reaction mixture at 80°C for 20 min. We then amplified the resulting nicked DNA (0.25 ng/μl) by using phi29 DNA polymerase (0.5 U/μl, Fermentas) for 24 h at 30°C in a 1× phi29 reaction buffer (33 mM Tris acetate, 10 mM magnesium acetate, 66 mM potassium acetate, 0.1% (v/v) Tween 20 and 1 mM Dithiothreitol (DTT); Fermentas) containing dNTP mix (1 mM each; Fermentas) and increasing concentration of single stranded binding protein T4 gene 32 (0–100 ng/μl, NEB). .. We also performed nicking and amplification reactions with pBluescript II SK (+) (with and without T4 gene 32 protein) in the same conditions.

    Plasmid Preparation:

    Article Title: Rolling circle replication requires single-stranded DNA binding protein to avoid termination and production of double-stranded DNA
    Article Snippet: .. RCA of DNA plasmids and digestion of RCA products We incubated pUC19 plasmid DNA (1 ng/μl, Invitrogen) with nicking endonuclease Nt.BspQI (0.5 U/μl, New England Biolabs (NEB)) in 1× NEB3 buffer for 2 h at 50°C, and then we heat inactivated the enzyme incubating the reaction mixture at 80°C for 20 min. We then amplified the resulting nicked DNA (0.25 ng/μl) by using phi29 DNA polymerase (0.5 U/μl, Fermentas) for 24 h at 30°C in a 1× phi29 reaction buffer (33 mM Tris acetate, 10 mM magnesium acetate, 66 mM potassium acetate, 0.1% (v/v) Tween 20 and 1 mM Dithiothreitol (DTT); Fermentas) containing dNTP mix (1 mM each; Fermentas) and increasing concentration of single stranded binding protein T4 gene 32 (0–100 ng/μl, NEB). .. We also performed nicking and amplification reactions with pBluescript II SK (+) (with and without T4 gene 32 protein) in the same conditions.

    Article Title: Droplet Microfluidics Approach for Single-DNA Molecule Amplification and Condensation into DNA-Magnesium-Pyrophosphate Particles
    Article Snippet: .. Single-DNA Molecule Encapsulation and Amplification The MDA reaction mix contained pIVEX2.2-lacZ-his plasmid, 1× phi29 reaction buffer (33 mM Tris-acetate (pH 7.9), 10 mM Mg-acetate, 66 mM K-acetate, 0.1% (v /v ) Tween 20, 1 mM dithiothreitol (DTT)), 50 μM exo-nuclease resistant hexanucleotide primers, 1 mM of each deoxynucleoside triphosphates (dNTP), 0.4% (w /v ) Pluronic F-127 and 0.8 U/μL phi29 DNA polymerase (Thermo Fisher Scientific, Vilnius, Lithuania). .. The reaction components were mixed in DNA LoBind tubes (Eppendorf) by adding DNA template, nuclease-free water, Pluronic F-127 and hexamers and then heated to 90 °C for 20 s to allow primer annealing.

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    Thermo Fisher phi29 dna polymerases
    Real-time reverse transcription–template dependent multiple displacement amplification (RT-tdMDA) using three hepatitis C virus (HCV) patient serum samples and a negative control (H 2 O) These samples covered the range of the RNA yield extracted from 200 μL serum (7.5–19.2 ng), as quantitated in the final 14 μL of elution by the QIAGEN miRNA kit prior to HL-DNase digestion. An aliquot of 10.6 μL RNA was used for RT in a reaction containing 200 U SuperScript III, 80 μM 5′-end-blocked random pentamer primer (5′-/iSpC3/NNN*N*N-3′; asterisks denote phosphorothioate bonds), and 2 mM dNTPs in a 20-μL volume. An aliquot of 4 μL of the RT reaction was used in a 40-μL tdMDA reaction containing 300 U <t>phi29</t> <t>DNA</t> polymerase (Epicentre), 80 μM primer, and 0.1× SYBR Green I (Thermo Fisher Scientific). The reaction was incubated at 28°C for 24 h on the ABI TaqMan 7500, in which fluorescent intensities were monitored through the SYBR Green channel. Estimated amount of cDNA input in tdMDA = [10.6 / (14 + 0.5 (HL-DNase) + 1.4 (buffer)] × [total RNA amount] × 0.2. Template-independent amplification was completely inhibited, as indicated by the negative control.
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    Real-time reverse transcription–template dependent multiple displacement amplification (RT-tdMDA) using three hepatitis C virus (HCV) patient serum samples and a negative control (H 2 O) These samples covered the range of the RNA yield extracted from 200 μL serum (7.5–19.2 ng), as quantitated in the final 14 μL of elution by the QIAGEN miRNA kit prior to HL-DNase digestion. An aliquot of 10.6 μL RNA was used for RT in a reaction containing 200 U SuperScript III, 80 μM 5′-end-blocked random pentamer primer (5′-/iSpC3/NNN*N*N-3′; asterisks denote phosphorothioate bonds), and 2 mM dNTPs in a 20-μL volume. An aliquot of 4 μL of the RT reaction was used in a 40-μL tdMDA reaction containing 300 U phi29 DNA polymerase (Epicentre), 80 μM primer, and 0.1× SYBR Green I (Thermo Fisher Scientific). The reaction was incubated at 28°C for 24 h on the ABI TaqMan 7500, in which fluorescent intensities were monitored through the SYBR Green channel. Estimated amount of cDNA input in tdMDA = [10.6 / (14 + 0.5 (HL-DNase) + 1.4 (buffer)] × [total RNA amount] × 0.2. Template-independent amplification was completely inhibited, as indicated by the negative control.

    Journal: BioTechniques

    Article Title: Template-dependent multiple displacement amplification for profiling human circulating RNA

    doi: 10.2144/000114566

    Figure Lengend Snippet: Real-time reverse transcription–template dependent multiple displacement amplification (RT-tdMDA) using three hepatitis C virus (HCV) patient serum samples and a negative control (H 2 O) These samples covered the range of the RNA yield extracted from 200 μL serum (7.5–19.2 ng), as quantitated in the final 14 μL of elution by the QIAGEN miRNA kit prior to HL-DNase digestion. An aliquot of 10.6 μL RNA was used for RT in a reaction containing 200 U SuperScript III, 80 μM 5′-end-blocked random pentamer primer (5′-/iSpC3/NNN*N*N-3′; asterisks denote phosphorothioate bonds), and 2 mM dNTPs in a 20-μL volume. An aliquot of 4 μL of the RT reaction was used in a 40-μL tdMDA reaction containing 300 U phi29 DNA polymerase (Epicentre), 80 μM primer, and 0.1× SYBR Green I (Thermo Fisher Scientific). The reaction was incubated at 28°C for 24 h on the ABI TaqMan 7500, in which fluorescent intensities were monitored through the SYBR Green channel. Estimated amount of cDNA input in tdMDA = [10.6 / (14 + 0.5 (HL-DNase) + 1.4 (buffer)] × [total RNA amount] × 0.2. Template-independent amplification was completely inhibited, as indicated by the negative control.

    Article Snippet: First, using the standard MDA protocol, we evaluated the purity of phi29 DNA polymerases from various vendors, including Lucigen (Middleton, WI), Epicentre (Madison, WI), Thermo Scientific (St. Louis, MO), and New England Biolabs (Ipswich, MA).

    Techniques: Multiple Displacement Amplification, Negative Control, SYBR Green Assay, Incubation, Amplification

    Size distribution of DNA-magnesium-pyrophosphate particles as a function of multiple displacement amplification reaction times, with and without the heat-inactivation step. At different time points (17, 38 and 66 h) of the MDA reaction in 3-pL droplets at 30 °C, the resulting DNA-Mg-PP i particles were released from droplets and imaged under TEM to measure their size. The first set of measurements (green) was made without heat-inactivation step. The second set of measurements (grey) was made with heat-inactivation step at 65 °C for 15 min that is typically used to inactivate phi29 DNA polymerase and terminate the MDA reaction.

    Journal: Micromachines

    Article Title: Droplet Microfluidics Approach for Single-DNA Molecule Amplification and Condensation into DNA-Magnesium-Pyrophosphate Particles

    doi: 10.3390/mi8020062

    Figure Lengend Snippet: Size distribution of DNA-magnesium-pyrophosphate particles as a function of multiple displacement amplification reaction times, with and without the heat-inactivation step. At different time points (17, 38 and 66 h) of the MDA reaction in 3-pL droplets at 30 °C, the resulting DNA-Mg-PP i particles were released from droplets and imaged under TEM to measure their size. The first set of measurements (green) was made without heat-inactivation step. The second set of measurements (grey) was made with heat-inactivation step at 65 °C for 15 min that is typically used to inactivate phi29 DNA polymerase and terminate the MDA reaction.

    Article Snippet: In this context, DNA amplification driven by phi29 DNA polymerase provides an alternative approach to amplify long DNA molecules and because of isothermal reaction conditions [ , ], the potential problems associated with emulsion stability become irrelevant.

    Techniques: Multiple Displacement Amplification, Transmission Electron Microscopy

    Measurement of the level of ALT activity levels in human diffuse gliomas. Top panels illustrate the principle of the ALT C-circle assay [ 17 ] and describe its general steps. ALT cells have very long telomeres that have been amplified mainly by homologous recombination that generates partially single-stranded extra-chromosomal circles. Genomic DNA prepared from tumor samples is then incubated with the Phi29 DNA polymerase that specifically amplifies this telomeric DNA. Middle panel illustrates ALT-specific signals measured in tumor DNA samples using this assay, which were detected here on dot blots hybridized with a telomeric 32 P-labeled probe. Genomic DNAs from HeLa (telomerase positive) and U2OS (ALT positive) cells were also probed, representing negative and posititve controls for the C-circle assay, respectively. These assays were systematically performed in duplicates and here dot blot 2, on the right, was loaded with the same tumor samples as dot blot 1, on the left, to insure for reproducibility. Bottom table illustrates examples of duplicate numbers obtained for each of the indicated tumors, real signals of which are represented in the middle panel above. The C-circle score was determined after calculating the intensity of the signal relative to that of the ALT positive U2OS cell line, designated to be 100 arbitrary units (AU). Note that the C-circle assays were performed on representative samples, including those from the two patient groups analyzed in the present study

    Journal: Acta Neuropathologica Communications

    Article Title: The level of activity of the alternative lengthening of telomeres correlates with patient age in IDH-mutant ATRX-loss-of-expression anaplastic astrocytomas

    doi: 10.1186/s40478-019-0833-0

    Figure Lengend Snippet: Measurement of the level of ALT activity levels in human diffuse gliomas. Top panels illustrate the principle of the ALT C-circle assay [ 17 ] and describe its general steps. ALT cells have very long telomeres that have been amplified mainly by homologous recombination that generates partially single-stranded extra-chromosomal circles. Genomic DNA prepared from tumor samples is then incubated with the Phi29 DNA polymerase that specifically amplifies this telomeric DNA. Middle panel illustrates ALT-specific signals measured in tumor DNA samples using this assay, which were detected here on dot blots hybridized with a telomeric 32 P-labeled probe. Genomic DNAs from HeLa (telomerase positive) and U2OS (ALT positive) cells were also probed, representing negative and posititve controls for the C-circle assay, respectively. These assays were systematically performed in duplicates and here dot blot 2, on the right, was loaded with the same tumor samples as dot blot 1, on the left, to insure for reproducibility. Bottom table illustrates examples of duplicate numbers obtained for each of the indicated tumors, real signals of which are represented in the middle panel above. The C-circle score was determined after calculating the intensity of the signal relative to that of the ALT positive U2OS cell line, designated to be 100 arbitrary units (AU). Note that the C-circle assays were performed on representative samples, including those from the two patient groups analyzed in the present study

    Article Snippet: ALT cells, but not telomerase positive cells, generate extra-chromosomal telomeric single-stranded DNA called C-circles, which can be amplified using the Phi29 DNA polymerase [ ] (Fig. ).

    Techniques: Activity Assay, Amplification, Homologous Recombination, Incubation, Labeling, Dot Blot