phi29 dna polymerase  (New England Biolabs)


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    Name:
    phi29 DNA Polymerase
    Description:
    phi29 DNA Polymerase 1 250 units
    Catalog Number:
    M0269L
    Price:
    228
    Category:
    DNA Polymerases
    Size:
    1 250 units
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    New England Biolabs phi29 dna polymerase
    phi29 DNA Polymerase
    phi29 DNA Polymerase 1 250 units
    https://www.bioz.com/result/phi29 dna polymerase/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    phi29 dna polymerase - by Bioz Stars, 2021-06
    99/100 stars

    Images

    1) Product Images from "Efficient amplification of self-gelling polypod-like structured DNA by rolling circle amplification and enzymatic digestion"

    Article Title: Efficient amplification of self-gelling polypod-like structured DNA by rolling circle amplification and enzymatic digestion

    Journal: Scientific Reports

    doi: 10.1038/srep14979

    Schematic diagram of the mass amplification of tripodna with adhesive 5′-ends. The template oligodeoxynucleotides (template 1) were designed to satisfy the following requirements: ( a ) the polypodna automatically forms by self-assembly; ( b ) each pod of the polypodna contains a 9 base long TspRI restriction digest site; ( c ) Each 5′-terminal end is phosphorylated in order to ligate with 3′-terminal within the polypodna body, and ( d ) connecting chain is added to the 3′-terminal of the polypodna to allow polypodna to be connected to one another. The designed templates were amplified via the following steps. ( 1 ) The template ssODNs were circularized using T4 DNA ligase. ( 2 ) After annealing the primer (primer 1), the DNA template was amplified through rolling circle amplification technique using Phi29 polymerase. ( 3 ) Before enzyme digestion, the RCA product was treated with EDTA and folded. ( 4 ) Long single-stranded DNAs were digested using restriction enzyme. ( 5 ) The target sequences were purified by size chromatography. ( 6 ) The resultant DNAs self-assembled after annealing, and they formed a hydrogel.
    Figure Legend Snippet: Schematic diagram of the mass amplification of tripodna with adhesive 5′-ends. The template oligodeoxynucleotides (template 1) were designed to satisfy the following requirements: ( a ) the polypodna automatically forms by self-assembly; ( b ) each pod of the polypodna contains a 9 base long TspRI restriction digest site; ( c ) Each 5′-terminal end is phosphorylated in order to ligate with 3′-terminal within the polypodna body, and ( d ) connecting chain is added to the 3′-terminal of the polypodna to allow polypodna to be connected to one another. The designed templates were amplified via the following steps. ( 1 ) The template ssODNs were circularized using T4 DNA ligase. ( 2 ) After annealing the primer (primer 1), the DNA template was amplified through rolling circle amplification technique using Phi29 polymerase. ( 3 ) Before enzyme digestion, the RCA product was treated with EDTA and folded. ( 4 ) Long single-stranded DNAs were digested using restriction enzyme. ( 5 ) The target sequences were purified by size chromatography. ( 6 ) The resultant DNAs self-assembled after annealing, and they formed a hydrogel.

    Techniques Used: Amplification, Purification, Chromatography

    2) Product Images from "New approaches to the analysis of palindromic sequences from the human genome: evolution and polymorphism of an intronic site at the NF1 locus"

    Article Title: New approaches to the analysis of palindromic sequences from the human genome: evolution and polymorphism of an intronic site at the NF1 locus

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gni189

    Side-by-side comparison of microbial hosts for their ability to maintain the same plasmid. ( A ) Universal deletion in E.coli Top 10 cells. Plasmids re-isolated from TOP 10 clones transformed with the H4#4 plasmid are deleted. Each was XbaI and PvuII digested. H4#4 DNA (also cut with XbaI and PvuII after a phi-29 amplification) is loaded adjacent to the marker lane. Note, a different 100 bp ladder was used here (New England Biolabs) which has an intense 500 bp rather than 600 bp band as in previous figures. ( B ) Instability of the H4#4 plasmid in E.coli SURE cells. Plasmid DNA from individual SURE H4#4 transformants is a mixture of deleted and apparently non-deleted forms despite the lack of a functional SbcCD nuclease. The gel image was cut to remove one lane. ( C ) Stability of H4#4 plasmid in wild-type yeast. Phi-29 amplified minipreparations of DNA from random wild-type yeast clones transformed with H4#4 DNA were digested with XbaI and PvuII. Full-length inserts are observed. ( D ) Phi-29 amplified minipreparations of DNA from random sae2 yeast clones transformed and analyzed as in (C). In A–D, dots mark samples from colonies that were re-streaked as described in the text.
    Figure Legend Snippet: Side-by-side comparison of microbial hosts for their ability to maintain the same plasmid. ( A ) Universal deletion in E.coli Top 10 cells. Plasmids re-isolated from TOP 10 clones transformed with the H4#4 plasmid are deleted. Each was XbaI and PvuII digested. H4#4 DNA (also cut with XbaI and PvuII after a phi-29 amplification) is loaded adjacent to the marker lane. Note, a different 100 bp ladder was used here (New England Biolabs) which has an intense 500 bp rather than 600 bp band as in previous figures. ( B ) Instability of the H4#4 plasmid in E.coli SURE cells. Plasmid DNA from individual SURE H4#4 transformants is a mixture of deleted and apparently non-deleted forms despite the lack of a functional SbcCD nuclease. The gel image was cut to remove one lane. ( C ) Stability of H4#4 plasmid in wild-type yeast. Phi-29 amplified minipreparations of DNA from random wild-type yeast clones transformed with H4#4 DNA were digested with XbaI and PvuII. Full-length inserts are observed. ( D ) Phi-29 amplified minipreparations of DNA from random sae2 yeast clones transformed and analyzed as in (C). In A–D, dots mark samples from colonies that were re-streaked as described in the text.

    Techniques Used: Plasmid Preparation, Isolation, Clone Assay, Transformation Assay, Amplification, Marker, Functional Assay

    (A) Colony PCR analysis of yeast clones obtained from H1 and H4 genomic DNA by the method shown in Figure 1B . Ligated samples (see legend, Figure 2 ) were used to transform S.cerevisiae . In contrast to results with E.coli ( Figure 2 ), full-length candidate clones were obtained. Lanes with dots are from colonies used in further analyses (see text). The DNA ladder is as in Figures 1 and 2 . ( B ) Verification of clone structure. To confirm the structure of the full-length candidates, yeast minipreps were treated with EcoR1 and subjected to random-primed rolling circle amplification with phi-29 DNA polymerase. As an example, H4#4 is displayed on a 2.5% agarose gel after diagnostic digestion with Xbal and PvuII. Upper and lower arrows indicate the vector backbone and insert bands, respectively. ( C ) Efficacy of the EcoR1 pre-digestion. The EcoR1 pre-treated sample in B (‘+’) is run on a 1% agarose gel alongside an untreated (‘−’) miniprep of H4#4. Treated and untreated samples were phi-29 amplified in parallel and digested with XbaI and PvuII. Bands evident in the untreated lane correspond in size to those predicted for 2 µ circle DNA. Bands observed with EcoR1 pre-treatment (arrows) correspond to the pYes2.1 vector and insert.
    Figure Legend Snippet: (A) Colony PCR analysis of yeast clones obtained from H1 and H4 genomic DNA by the method shown in Figure 1B . Ligated samples (see legend, Figure 2 ) were used to transform S.cerevisiae . In contrast to results with E.coli ( Figure 2 ), full-length candidate clones were obtained. Lanes with dots are from colonies used in further analyses (see text). The DNA ladder is as in Figures 1 and 2 . ( B ) Verification of clone structure. To confirm the structure of the full-length candidates, yeast minipreps were treated with EcoR1 and subjected to random-primed rolling circle amplification with phi-29 DNA polymerase. As an example, H4#4 is displayed on a 2.5% agarose gel after diagnostic digestion with Xbal and PvuII. Upper and lower arrows indicate the vector backbone and insert bands, respectively. ( C ) Efficacy of the EcoR1 pre-digestion. The EcoR1 pre-treated sample in B (‘+’) is run on a 1% agarose gel alongside an untreated (‘−’) miniprep of H4#4. Treated and untreated samples were phi-29 amplified in parallel and digested with XbaI and PvuII. Bands evident in the untreated lane correspond in size to those predicted for 2 µ circle DNA. Bands observed with EcoR1 pre-treatment (arrows) correspond to the pYes2.1 vector and insert.

    Techniques Used: Polymerase Chain Reaction, Clone Assay, Random Primed, Amplification, Agarose Gel Electrophoresis, Diagnostic Assay, Plasmid Preparation

    ( A ) Map (to scale) of the palindromic region within a 3.8 kb EcoR1 fragment of the NF1 gene. Exons are numbered according to L05367 (see text). ‘R1’ denotes EcoR1 sites according to Southern blot data ( 27 ) and the reference human genome sequence (May 2004). ( B ) Cloning strategy. Oligos used for PCR flank the palindromic site. The PCR product is ligated into a commercial vector (see Materials and Methods). ‘X’ and ‘P’ refer to the Xbal and PvuII sites used in diagnostic digests. ( C ) PCR amplification of various DNA templates. Lane ‘M’, markers (Trackit 100 bp ladder, Invitrogen). ‘B’ is a PCR with Bac clone CTD-2370N5; ‘H1’ to ‘H5’ are with human genomic DNA from the indicated individuals. ‘C’ is with chimpanzee DNA. ‘φH4’ and ‘φG’ are from an aliquot of H4 and gorilla genomic DNA that had first been amplified with phi-29 polymerase. The H4 samples demonstrate reproducibility of the PCR as well as the fidelity of phi-29 amplification.
    Figure Legend Snippet: ( A ) Map (to scale) of the palindromic region within a 3.8 kb EcoR1 fragment of the NF1 gene. Exons are numbered according to L05367 (see text). ‘R1’ denotes EcoR1 sites according to Southern blot data ( 27 ) and the reference human genome sequence (May 2004). ( B ) Cloning strategy. Oligos used for PCR flank the palindromic site. The PCR product is ligated into a commercial vector (see Materials and Methods). ‘X’ and ‘P’ refer to the Xbal and PvuII sites used in diagnostic digests. ( C ) PCR amplification of various DNA templates. Lane ‘M’, markers (Trackit 100 bp ladder, Invitrogen). ‘B’ is a PCR with Bac clone CTD-2370N5; ‘H1’ to ‘H5’ are with human genomic DNA from the indicated individuals. ‘C’ is with chimpanzee DNA. ‘φH4’ and ‘φG’ are from an aliquot of H4 and gorilla genomic DNA that had first been amplified with phi-29 polymerase. The H4 samples demonstrate reproducibility of the PCR as well as the fidelity of phi-29 amplification.

    Techniques Used: Southern Blot, Sequencing, Clone Assay, Polymerase Chain Reaction, Plasmid Preparation, Diagnostic Assay, Amplification, BAC Assay

    3) Product Images from "Reconstitution and Coupling of DNA Replication and Segregation in a Biomimetic System"

    Article Title: Reconstitution and Coupling of DNA Replication and Segregation in a Biomimetic System

    Journal: Chembiochem

    doi: 10.1002/cbic.201900299

    Replication, recircularization, and compaction of a plasmid containing the T7 promoter. A) Time course of RCA‐based replication of pRepC plasmid containing the T7 promoter and loxP sites (depicted in Figure S1), measured as fluorescence of the DNA‐binding PicoGreen dye. Reaction mixtures contained, as indicated, Phi29 DNA polymerase, T7 DNA polymerase, and T7 RNA polymerase, as well as specific or random primers. For a control reaction with T7 DNA polymerase and T7 RNA polymerase, a pQE30 plasmid lacking the T7 promoter was used. B) Recircularization of the replicated plasmid, mediated by Cre recombinase. Where indicated, Cre recombinase was added after 16 h of replication, and the reaction mixture was incubated for another 30 min. Reaction mixtures were separated along with a DNA ladder (1 kb) on a Midori‐green stained agarose gel. The lower band migrating below 5 kb corresponds to the circularized DNA, whereas larger products apparently correspond to linear concatamers (Figure S2). C) DNA nanoparticles emerging upon prolonged ( > 12 h) T7 DNA replication reaction. Scale bar: 10 μm.
    Figure Legend Snippet: Replication, recircularization, and compaction of a plasmid containing the T7 promoter. A) Time course of RCA‐based replication of pRepC plasmid containing the T7 promoter and loxP sites (depicted in Figure S1), measured as fluorescence of the DNA‐binding PicoGreen dye. Reaction mixtures contained, as indicated, Phi29 DNA polymerase, T7 DNA polymerase, and T7 RNA polymerase, as well as specific or random primers. For a control reaction with T7 DNA polymerase and T7 RNA polymerase, a pQE30 plasmid lacking the T7 promoter was used. B) Recircularization of the replicated plasmid, mediated by Cre recombinase. Where indicated, Cre recombinase was added after 16 h of replication, and the reaction mixture was incubated for another 30 min. Reaction mixtures were separated along with a DNA ladder (1 kb) on a Midori‐green stained agarose gel. The lower band migrating below 5 kb corresponds to the circularized DNA, whereas larger products apparently correspond to linear concatamers (Figure S2). C) DNA nanoparticles emerging upon prolonged ( > 12 h) T7 DNA replication reaction. Scale bar: 10 μm.

    Techniques Used: Plasmid Preparation, Fluorescence, Binding Assay, Incubation, Staining, Agarose Gel Electrophoresis

    4) Product Images from "Next-Generation DNA Curtains for Single-Molecule Studies of Homologous Recombination"

    Article Title: Next-Generation DNA Curtains for Single-Molecule Studies of Homologous Recombination

    Journal: Methods in enzymology

    doi: 10.1016/bs.mie.2017.03.011

    Nucleoprotein filament dynamics on low sequence complexity ssDNA curtains. (A) Sequences of the two ssDNA oligonucleotides used for rolling circle replication. (B) Schematic of rolling circle replication (RCR) reaction. T4 DNA ligase ligates the template oligo to form a contiguous template strand. Next, phi29 DNA polymerase catalyzes the synthesis of long ssDNA molecules. (C) Agarose gel of several time points along the RCR synthesis reaction. The primer oligonucleotide was 32 P labeled on the 5 ′ -terminus phosphate ( gold star ). (D) Wide-field image of a microfabricated barrier set with double-tethered ssDNA curtains coated with RPA-TagRFP ( magenta ). Arrows and circles denote chromium barriers and pedestals, respectively. (E) Illustration and kymograph showing a single ssDNA molecule coated with ATTO488-RAD51(C319S) ( green ) replaced by RPA-TagRFP ( magenta ). Yellow dashed line denotes the injection of RPA–TagRFP into the flowcell. Buffer controls indicate when the buffer flow was toggled off and on to show that the florescent proteins retract to the Cr barriers simultaneously with the ssDNA molecule. This indicates that RAD51 and RPA are on the ssDNA molecule. Panel A: Adapted from Lee, K. S., Marciel, A. B., Kozlov, A. G., Schroeder, C. M., Lohman, T. M., Ha, T. (2014). Ultrafast redistribution of E. coli SSB along long single-stranded DNA via intersegment transfer. Journal of Molecular Biology, 426 , 2413 – 2421.
    Figure Legend Snippet: Nucleoprotein filament dynamics on low sequence complexity ssDNA curtains. (A) Sequences of the two ssDNA oligonucleotides used for rolling circle replication. (B) Schematic of rolling circle replication (RCR) reaction. T4 DNA ligase ligates the template oligo to form a contiguous template strand. Next, phi29 DNA polymerase catalyzes the synthesis of long ssDNA molecules. (C) Agarose gel of several time points along the RCR synthesis reaction. The primer oligonucleotide was 32 P labeled on the 5 ′ -terminus phosphate ( gold star ). (D) Wide-field image of a microfabricated barrier set with double-tethered ssDNA curtains coated with RPA-TagRFP ( magenta ). Arrows and circles denote chromium barriers and pedestals, respectively. (E) Illustration and kymograph showing a single ssDNA molecule coated with ATTO488-RAD51(C319S) ( green ) replaced by RPA-TagRFP ( magenta ). Yellow dashed line denotes the injection of RPA–TagRFP into the flowcell. Buffer controls indicate when the buffer flow was toggled off and on to show that the florescent proteins retract to the Cr barriers simultaneously with the ssDNA molecule. This indicates that RAD51 and RPA are on the ssDNA molecule. Panel A: Adapted from Lee, K. S., Marciel, A. B., Kozlov, A. G., Schroeder, C. M., Lohman, T. M., Ha, T. (2014). Ultrafast redistribution of E. coli SSB along long single-stranded DNA via intersegment transfer. Journal of Molecular Biology, 426 , 2413 – 2421.

    Techniques Used: Sequencing, Agarose Gel Electrophoresis, Labeling, Recombinase Polymerase Amplification, Injection, Flow Cytometry

    5) Product Images from "Quantification and epigenetic evaluation of the residual pool of hepatitis B covalently closed circular DNA in long-term nucleoside analogue-treated patients"

    Article Title: Quantification and epigenetic evaluation of the residual pool of hepatitis B covalently closed circular DNA in long-term nucleoside analogue-treated patients

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-78001-1

    Rolling Circle Amplification (RCA) analysis on patients’ liver biopsies. (a) Workflow of RCA set-up in liver biopsies: DNA extracted from frozen-liver biopsies was first amplified with Phi29 polymerase for 21 h at 30 °C. Amplification products were then either digested with SpeI enzyme and analyzed according to Southern Blot technique using HBV-specific cold probes or assessed following a full-length HBV genomic PCR (P1-P2) followed by gel electrophoresis. (b) Examples of Southern Blot following RCA and SpeI digestion on liver biopsies from patients with different cccDNA concentration measured by qPCR and negative (0; H20) and positive controls (PC; plasmid containing a full-length HBV genome). (c) Examples of gel electrophoresis following full-length HBV genomic PCR (P1-P2) performed on RCA products from patients’ liver biopsies with different cccDNA concentration measured by qPCR and negative (0; H20) and positive control (PC; plasmid containing a full-length HBV genome). MW molecular weight; 0: negative control (H20); PC positive control (plasmid containing a full-length HBV genome).
    Figure Legend Snippet: Rolling Circle Amplification (RCA) analysis on patients’ liver biopsies. (a) Workflow of RCA set-up in liver biopsies: DNA extracted from frozen-liver biopsies was first amplified with Phi29 polymerase for 21 h at 30 °C. Amplification products were then either digested with SpeI enzyme and analyzed according to Southern Blot technique using HBV-specific cold probes or assessed following a full-length HBV genomic PCR (P1-P2) followed by gel electrophoresis. (b) Examples of Southern Blot following RCA and SpeI digestion on liver biopsies from patients with different cccDNA concentration measured by qPCR and negative (0; H20) and positive controls (PC; plasmid containing a full-length HBV genome). (c) Examples of gel electrophoresis following full-length HBV genomic PCR (P1-P2) performed on RCA products from patients’ liver biopsies with different cccDNA concentration measured by qPCR and negative (0; H20) and positive control (PC; plasmid containing a full-length HBV genome). MW molecular weight; 0: negative control (H20); PC positive control (plasmid containing a full-length HBV genome).

    Techniques Used: Amplification, Southern Blot, Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Concentration Assay, Real-time Polymerase Chain Reaction, Plasmid Preparation, Positive Control, Molecular Weight, Negative Control

    6) Product Images from "Limited reverse transcriptase activity of phi29 DNA polymerase"

    Article Title: Limited reverse transcriptase activity of phi29 DNA polymerase

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky190

    Effect of RNA substitutions in circular templates on rolling circle amplification with phi29 DNA polymerase. ( A ) Total amount of RCA products (y-axis) generated for padlock probes with/without a terminal 3′ RNA and in the absence of synthetic RNA ligation template (template -). ( B ) Circles with 0–7 RNA substitutions in the backbone were amplified and digitally counted. The y-axis shows the number of rolling circle products (RCPs); error bars ± S.D.; n = 2. The same RCA reactions with chimeric circles were also monitored in real-time by measuring SYBR Gold incorporation on qPCR instrument ( C and E ). (C) RCA reaction curves of circles with 0, 1 and 2 RNA substitutions. ( D ) RCPs from C were imaged on microscope slides and size and intensity of individual RCPs were quantified. Black line, median; upper whisker, highest value that is within 1.5 the interquartile range of the hinge; lower whisker, lowest value within 1.5 the interquartile range of the hinge. (E) Real-time data of the same RCA reactions as in B with 0–7 RNA substitutes are displayed. Representative samples are presented from a duplicated experiment. To highlight the initial stages of RCA and to show the difference between the samples with low RCA efficiency, fluorescence intensity readout between 3000 and 6000 is presented.
    Figure Legend Snippet: Effect of RNA substitutions in circular templates on rolling circle amplification with phi29 DNA polymerase. ( A ) Total amount of RCA products (y-axis) generated for padlock probes with/without a terminal 3′ RNA and in the absence of synthetic RNA ligation template (template -). ( B ) Circles with 0–7 RNA substitutions in the backbone were amplified and digitally counted. The y-axis shows the number of rolling circle products (RCPs); error bars ± S.D.; n = 2. The same RCA reactions with chimeric circles were also monitored in real-time by measuring SYBR Gold incorporation on qPCR instrument ( C and E ). (C) RCA reaction curves of circles with 0, 1 and 2 RNA substitutions. ( D ) RCPs from C were imaged on microscope slides and size and intensity of individual RCPs were quantified. Black line, median; upper whisker, highest value that is within 1.5 the interquartile range of the hinge; lower whisker, lowest value within 1.5 the interquartile range of the hinge. (E) Real-time data of the same RCA reactions as in B with 0–7 RNA substitutes are displayed. Representative samples are presented from a duplicated experiment. To highlight the initial stages of RCA and to show the difference between the samples with low RCA efficiency, fluorescence intensity readout between 3000 and 6000 is presented.

    Techniques Used: Amplification, Generated, Ligation, Real-time Polymerase Chain Reaction, Microscopy, Whisker Assay, Fluorescence

    Phi29 DNA polymerase exhibits higher RCA rate with circles containing pyrimidine RNA substitutions. ( A ) Real-time RCA curves of circles containing 1, 2, 3 or 4 consecutive RNA substations of rG, rU, rA, rC RNA bases are displayed (number of consecutive substitutions is indicated above plots). Rate of RCA was monitored by measuring fluorescence build-up (y-axis) resulted from SYBR Gold incorporation into RCPs. Averaged fluorescence intensity for each RCA time point was calculated from a duplicated experiment. RCA was conducted in the presence of Mg 2+ and Mn 2+ (solid and dashed lines respectively). ( B ) Linear, early stage RCA velocity (y-axis) is presented for PLPs from (A) in the presence of Mg 2+ (solid lines) and Mn 2+ (dashed lines). ( C ) RCA for the control PLP (non-chimeric DNA circle, with Mg 2+ (solid) and Mn 2+ (dashed line) are displayed.
    Figure Legend Snippet: Phi29 DNA polymerase exhibits higher RCA rate with circles containing pyrimidine RNA substitutions. ( A ) Real-time RCA curves of circles containing 1, 2, 3 or 4 consecutive RNA substations of rG, rU, rA, rC RNA bases are displayed (number of consecutive substitutions is indicated above plots). Rate of RCA was monitored by measuring fluorescence build-up (y-axis) resulted from SYBR Gold incorporation into RCPs. Averaged fluorescence intensity for each RCA time point was calculated from a duplicated experiment. RCA was conducted in the presence of Mg 2+ and Mn 2+ (solid and dashed lines respectively). ( B ) Linear, early stage RCA velocity (y-axis) is presented for PLPs from (A) in the presence of Mg 2+ (solid lines) and Mn 2+ (dashed lines). ( C ) RCA for the control PLP (non-chimeric DNA circle, with Mg 2+ (solid) and Mn 2+ (dashed line) are displayed.

    Techniques Used: Fluorescence, Plasmid Purification

    DNA sequencing-based analysis of rolling circle products reveals reverse transcription activity of phi29 DNA polymerase. ( A ) After RCA, short DNA oligonucleotides were hybridized to an AluI restriction site in the RCA products and RCPs were digested with AluI restriction enzyme, resulting in RCA monomers. Following digestion, monomers were PCR-amplified using primers containing Ilumina adapter sequences. PCR products were extended using IIlumina indexed primers. Finally, sequencing library was prepared using indexed primers-specific P5/7 PCR primers. The region of interest containing RNA substitutions in the original padlock probe sequence is indicated with green boxes. ( B ) Logos showing sequencing frequencies for each position within RCA monomers generated from the control DNA circle (P1 = dG), and circles containing single rG, rU, rA and rC substitutions at the RNA position (P1). Positions P1 and P2 are indicated and position P1 was additionally highlighted with the red box. ( C ) Incorporation of incorrect nucleotides for every position in the sequenced monomers from (B). Error rates, calculated as Incorporation error [%] = 1 – number of reads with expected nucleotide/total number of reads, is presented for padlock probes with single- (upper plot) and double-RNA substitutions (lower plots). P1 position for the first RNA substitution is indicated with the box.
    Figure Legend Snippet: DNA sequencing-based analysis of rolling circle products reveals reverse transcription activity of phi29 DNA polymerase. ( A ) After RCA, short DNA oligonucleotides were hybridized to an AluI restriction site in the RCA products and RCPs were digested with AluI restriction enzyme, resulting in RCA monomers. Following digestion, monomers were PCR-amplified using primers containing Ilumina adapter sequences. PCR products were extended using IIlumina indexed primers. Finally, sequencing library was prepared using indexed primers-specific P5/7 PCR primers. The region of interest containing RNA substitutions in the original padlock probe sequence is indicated with green boxes. ( B ) Logos showing sequencing frequencies for each position within RCA monomers generated from the control DNA circle (P1 = dG), and circles containing single rG, rU, rA and rC substitutions at the RNA position (P1). Positions P1 and P2 are indicated and position P1 was additionally highlighted with the red box. ( C ) Incorporation of incorrect nucleotides for every position in the sequenced monomers from (B). Error rates, calculated as Incorporation error [%] = 1 – number of reads with expected nucleotide/total number of reads, is presented for padlock probes with single- (upper plot) and double-RNA substitutions (lower plots). P1 position for the first RNA substitution is indicated with the box.

    Techniques Used: DNA Sequencing, Activity Assay, Polymerase Chain Reaction, Amplification, Sequencing, Generated

    7) Product Images from "Improvements of rolling circle amplification (RCA) efficiency and accuracy using Thermus thermophilus SSB mutant protein"

    Article Title: Improvements of rolling circle amplification (RCA) efficiency and accuracy using Thermus thermophilus SSB mutant protein

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl350

    Effect of Tth SSB-255 protein on the efficiency and specificity of RCA. ( a ) Top: RCAs were performed in the absence of the SSB proteins using pUC19 DNA as template and phi29 DNA polymerase for the indicated reaction times. Bottom: Signals of spot hybridization of the same samples. ( b ) Top and Bottom: same as (a), except for the absence of template DNA. ( c ) Top and Bottom: same as (a), except for the presence of the Tth SSB protein (3.0 µg/20 µl reaction volume). ( d ) Top and Bottom: same as (c), except for the absence of template DNA. ( e ) Top and Bottom: same as (a), except for the presence of the Tth SSB-255 protein (3.0 µg/20 µl reaction volume). ( f ) Top and Bottom: same as (e), except for the absence of template DNA.
    Figure Legend Snippet: Effect of Tth SSB-255 protein on the efficiency and specificity of RCA. ( a ) Top: RCAs were performed in the absence of the SSB proteins using pUC19 DNA as template and phi29 DNA polymerase for the indicated reaction times. Bottom: Signals of spot hybridization of the same samples. ( b ) Top and Bottom: same as (a), except for the absence of template DNA. ( c ) Top and Bottom: same as (a), except for the presence of the Tth SSB protein (3.0 µg/20 µl reaction volume). ( d ) Top and Bottom: same as (c), except for the absence of template DNA. ( e ) Top and Bottom: same as (a), except for the presence of the Tth SSB-255 protein (3.0 µg/20 µl reaction volume). ( f ) Top and Bottom: same as (e), except for the absence of template DNA.

    Techniques Used: Hybridization

    Effect of Tth SSB-255 protein on RCA assays. ( a ) RCAs were carried out in the absence or presence of the indicated SSB proteins using pUC19 DNA as a template and phi29 DNA polymerase. ( b ) Same as (a), except for using linearized ( Eco RI) pUC19 DNA as the template. ( c ) Same as (a), except that the amplifications were carried out in the absence of template DNA. Lane M: molecular weight markers (100 and 12 kb).
    Figure Legend Snippet: Effect of Tth SSB-255 protein on RCA assays. ( a ) RCAs were carried out in the absence or presence of the indicated SSB proteins using pUC19 DNA as a template and phi29 DNA polymerase. ( b ) Same as (a), except for using linearized ( Eco RI) pUC19 DNA as the template. ( c ) Same as (a), except that the amplifications were carried out in the absence of template DNA. Lane M: molecular weight markers (100 and 12 kb).

    Techniques Used: Molecular Weight

    8) Product Images from "Selective Whole-Genome Amplification Is a Robust Method That Enables Scalable Whole-Genome Sequencing of Plasmodium vivax from Unprocessed Clinical Samples"

    Article Title: Selective Whole-Genome Amplification Is a Robust Method That Enables Scalable Whole-Genome Sequencing of Plasmodium vivax from Unprocessed Clinical Samples

    Journal: mBio

    doi: 10.1128/mBio.02257-16

    Selective whole-genome amplification (SWGA) of Plasmodium vivax genomic DNA (gDNA) from human blood samples. (A) SWGA primers bind frequently to Plasmodium vivax gDNA and infrequently to human gDNA. (B) When phi29 encounters double-stranded gDNA, it displaces the newly synthesized strand, opening new primer binding sites on the synthesized gDNA, leading to selective amplification of templates with frequent primer binding sites. (C) Post-SWGA, the percentage of P. vivax DNA has increased relative to the percentage of host DNA.
    Figure Legend Snippet: Selective whole-genome amplification (SWGA) of Plasmodium vivax genomic DNA (gDNA) from human blood samples. (A) SWGA primers bind frequently to Plasmodium vivax gDNA and infrequently to human gDNA. (B) When phi29 encounters double-stranded gDNA, it displaces the newly synthesized strand, opening new primer binding sites on the synthesized gDNA, leading to selective amplification of templates with frequent primer binding sites. (C) Post-SWGA, the percentage of P. vivax DNA has increased relative to the percentage of host DNA.

    Techniques Used: Whole Genome Amplification, Synthesized, Binding Assay, Amplification

    9) Product Images from "ATRX loss induces multiple hallmarks of the alternative lengthening of telomeres (ALT) phenotype in human glioma cell lines in a cell line-specific manner"

    Article Title: ATRX loss induces multiple hallmarks of the alternative lengthening of telomeres (ALT) phenotype in human glioma cell lines in a cell line-specific manner

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0204159

    Glioma cell lines chosen for ATRX modulation. (A) Immunoblotting for known ALT suppressors ATRX and DAXX in seven glioma cell lines, as well as U2-OS, a known ALT-positive cell line with deletion of ATRX [ 24 ]. Arrowhead indicates band representing wild-type ATRX. (B) TRAP assay shows telomerase activity in all seven glioma cell lines, but not U2-OS. NTC indicates no template control. All seven glioma cell lines lack characteristics of ALT, including (C) c-circles, as measured by phi29-mediated rolling circle amplification and dot blot, and (D) ALT-associated telomere DNA foci (arrow), as assessed by telomere-specific FISH.
    Figure Legend Snippet: Glioma cell lines chosen for ATRX modulation. (A) Immunoblotting for known ALT suppressors ATRX and DAXX in seven glioma cell lines, as well as U2-OS, a known ALT-positive cell line with deletion of ATRX [ 24 ]. Arrowhead indicates band representing wild-type ATRX. (B) TRAP assay shows telomerase activity in all seven glioma cell lines, but not U2-OS. NTC indicates no template control. All seven glioma cell lines lack characteristics of ALT, including (C) c-circles, as measured by phi29-mediated rolling circle amplification and dot blot, and (D) ALT-associated telomere DNA foci (arrow), as assessed by telomere-specific FISH.

    Techniques Used: TRAP Assay, Activity Assay, Amplification, Dot Blot, Fluorescence In Situ Hybridization

    10) Product Images from "Tapping diversity lost in transformations--in vitro amplification of ligation reactions"

    Article Title: Tapping diversity lost in transformations--in vitro amplification of ligation reactions

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl605

    Antibody repertoire by combinatorial ligation. Regions corresponding to CDRs 1/2 and CDR 3 were PCR-amplified and recombined by ligation into a PCR-amplified phagemid vector backbone. The ligation reactions were either directly electroporated or amplified using Phi29 polymerase followed by electroporation into E.coli bacteria.
    Figure Legend Snippet: Antibody repertoire by combinatorial ligation. Regions corresponding to CDRs 1/2 and CDR 3 were PCR-amplified and recombined by ligation into a PCR-amplified phagemid vector backbone. The ligation reactions were either directly electroporated or amplified using Phi29 polymerase followed by electroporation into E.coli bacteria.

    Techniques Used: Ligation, Polymerase Chain Reaction, Amplification, Plasmid Preparation, Electroporation

    In vitro amplification of ligation reactions. Linear fragments were joined into recombinant, circular units by treatment with DNA ligase. Hexamer primers were annealed and Phi29 polymerase added. This causes extensive amplification of circular species through rolling-circle replication and the formation of extended linear concatemers. The concatemers were cleaved by restriction digestion and re-circularized using DNA ligase.
    Figure Legend Snippet: In vitro amplification of ligation reactions. Linear fragments were joined into recombinant, circular units by treatment with DNA ligase. Hexamer primers were annealed and Phi29 polymerase added. This causes extensive amplification of circular species through rolling-circle replication and the formation of extended linear concatemers. The concatemers were cleaved by restriction digestion and re-circularized using DNA ligase.

    Techniques Used: In Vitro, Amplification, Ligation, Recombinant

    11) Product Images from "Dynamics of Staphylococcus aureus Cas9 in DNA target Association and Dissociation"

    Article Title: Dynamics of Staphylococcus aureus Cas9 in DNA target Association and Dissociation

    Journal: EMBO Reports

    doi: 10.15252/embr.202050184

    DNA ‐tracking motors were stalled by DNA ‐bound dSaC as9 DNA unwinding by DnaB was initiated from the upstream side of the PAM in the presence of dSaCas9 ( n = 10). Representative traces show the number of unwound base pairs versus the time under an assisting force of 12 pN in the absence (black) or presence (red) of the prebound dSaCas9. For clarity, the traces have been shifted along the time axis. The dashed lines indicate the expected dSaCas9‐binding positions. DNA unwinding by DnaB was initiated from the downstream side of the PAM in the presence of dSaCas9 ( n = 11). Representative traces show the number of unwound base pairs versus the time under an assisting force of 12 pN in the absence (black) or presence (red) of the prebound dSaCas9. DNA unwinding by BLM was initiated from the upstream side of the PAM in the presence of dSaCas9 ( n = 12). Representative traces show the number of unwound base pairs versus the time under an assisting force of 12 pN in the absence (black) or presence (red) of the prebound dSaCas9. DNA unwinding by BLM was initiated from the downstream side of the PAM in the presence of dSaCas9 ( n = 14). Representative traces show the number of unwound base pairs versus the time under an assisting force of 12 pN in the absence (black) or presence (red) of the prebound dSaCas9. Phi29 DNAP strand‐displacement synthesis was initiated from the upstream side of the PAM ( n = 15). Representative traces show the number of unwound/synthesized base pairs versus the time under an assisting force of 12 pN in the absence (black) or presence (red) of the prebound dSaCas9. Phi29 DNAP strand‐displacement synthesis was initiated from the downstream side of the PAM ( n = 19). Representative traces show the number of unwound/synthesized base pairs versus the time under an assisting force of 12 pN in the absence (black) or presence (red) of the prebound dSaCas9.
    Figure Legend Snippet: DNA ‐tracking motors were stalled by DNA ‐bound dSaC as9 DNA unwinding by DnaB was initiated from the upstream side of the PAM in the presence of dSaCas9 ( n = 10). Representative traces show the number of unwound base pairs versus the time under an assisting force of 12 pN in the absence (black) or presence (red) of the prebound dSaCas9. For clarity, the traces have been shifted along the time axis. The dashed lines indicate the expected dSaCas9‐binding positions. DNA unwinding by DnaB was initiated from the downstream side of the PAM in the presence of dSaCas9 ( n = 11). Representative traces show the number of unwound base pairs versus the time under an assisting force of 12 pN in the absence (black) or presence (red) of the prebound dSaCas9. DNA unwinding by BLM was initiated from the upstream side of the PAM in the presence of dSaCas9 ( n = 12). Representative traces show the number of unwound base pairs versus the time under an assisting force of 12 pN in the absence (black) or presence (red) of the prebound dSaCas9. DNA unwinding by BLM was initiated from the downstream side of the PAM in the presence of dSaCas9 ( n = 14). Representative traces show the number of unwound base pairs versus the time under an assisting force of 12 pN in the absence (black) or presence (red) of the prebound dSaCas9. Phi29 DNAP strand‐displacement synthesis was initiated from the upstream side of the PAM ( n = 15). Representative traces show the number of unwound/synthesized base pairs versus the time under an assisting force of 12 pN in the absence (black) or presence (red) of the prebound dSaCas9. Phi29 DNAP strand‐displacement synthesis was initiated from the downstream side of the PAM ( n = 19). Representative traces show the number of unwound/synthesized base pairs versus the time under an assisting force of 12 pN in the absence (black) or presence (red) of the prebound dSaCas9.

    Techniques Used: Binding Assay, Synthesized

    The dissociation of SaCas9 after DNA cleavage Representative traces of the reverse DNA unzipping in the absence (gray) and presence (blue) of wild‐type SaCas9 showing the force versus number of base pairs unzipped. In total, 23 traces were collected in this assay. The dashed line shows the expected interaction sites between dSaCas9 and DNA. The blue arrow indicates the force peak of the post‐PAM interaction. Representative traces of the forward DNA unzipping in the absence (gray) and presence (black) of wild‐type SaCas9 showing the force versus number of base pairs unzipped. In total, 49 traces were collected in this assay. The dashed line shows the expected interaction sites between dSaCas9 and DNA. Confocal images of fluorescently labeled λ DNA with dSaCas9 ( n = 11) and wild‐type SaCas9 ( n = 16) bound to its DNA target. DNA‐bound SaCas9 was visualized by labeling the 5’ end of the crRNA with Cy3. The λ DNA molecule was either suspended between two microspheres held by two optical traps under 5 pN or stretched by laminar flow. The fluorescence intensity of Cy3 alongside the image is shown to indicate the on‐target binding of SaCas9. DNA unwinding by DnaB was initiated from the downstream side of the PAM after the association of the SaCas9 protein ( n = 11). Representative traces show the number of unwound base pairs versus time under an assisting force of 12 pN. For clarity, the traces have been shifted along the time axis. The dotted lines indicate the expected SaCas9‐binding positions. Phi29 DNAP‐mediated strand‐displacement synthesis was initiated from the downstream side of the PAM after the association of the SaCas9 protein ( n = 13). Representative traces show the number of unwound/synthesized base pairs versus time under an assisting force of 12 pN. DNA unwinding by BLM was initiated from the downstream side of the PAM after the association of the SaCas9 protein ( n = 23). Representative traces show the number of unwound base pairs versus time under an assisting force of 12 pN.
    Figure Legend Snippet: The dissociation of SaCas9 after DNA cleavage Representative traces of the reverse DNA unzipping in the absence (gray) and presence (blue) of wild‐type SaCas9 showing the force versus number of base pairs unzipped. In total, 23 traces were collected in this assay. The dashed line shows the expected interaction sites between dSaCas9 and DNA. The blue arrow indicates the force peak of the post‐PAM interaction. Representative traces of the forward DNA unzipping in the absence (gray) and presence (black) of wild‐type SaCas9 showing the force versus number of base pairs unzipped. In total, 49 traces were collected in this assay. The dashed line shows the expected interaction sites between dSaCas9 and DNA. Confocal images of fluorescently labeled λ DNA with dSaCas9 ( n = 11) and wild‐type SaCas9 ( n = 16) bound to its DNA target. DNA‐bound SaCas9 was visualized by labeling the 5’ end of the crRNA with Cy3. The λ DNA molecule was either suspended between two microspheres held by two optical traps under 5 pN or stretched by laminar flow. The fluorescence intensity of Cy3 alongside the image is shown to indicate the on‐target binding of SaCas9. DNA unwinding by DnaB was initiated from the downstream side of the PAM after the association of the SaCas9 protein ( n = 11). Representative traces show the number of unwound base pairs versus time under an assisting force of 12 pN. For clarity, the traces have been shifted along the time axis. The dotted lines indicate the expected SaCas9‐binding positions. Phi29 DNAP‐mediated strand‐displacement synthesis was initiated from the downstream side of the PAM after the association of the SaCas9 protein ( n = 13). Representative traces show the number of unwound/synthesized base pairs versus time under an assisting force of 12 pN. DNA unwinding by BLM was initiated from the downstream side of the PAM after the association of the SaCas9 protein ( n = 23). Representative traces show the number of unwound base pairs versus time under an assisting force of 12 pN.

    Techniques Used: Labeling, Fluorescence, Binding Assay, Synthesized

    12) Product Images from "Selective Whole-Genome Amplification Is a Robust Method That Enables Scalable Whole-Genome Sequencing of Plasmodium vivax from Unprocessed Clinical Samples"

    Article Title: Selective Whole-Genome Amplification Is a Robust Method That Enables Scalable Whole-Genome Sequencing of Plasmodium vivax from Unprocessed Clinical Samples

    Journal: mBio

    doi: 10.1128/mBio.02257-16

    Selective whole-genome amplification (SWGA) of Plasmodium vivax genomic DNA (gDNA) from human blood samples. (A) SWGA primers bind frequently to Plasmodium vivax gDNA and infrequently to human gDNA. (B) When phi29 encounters double-stranded gDNA, it displaces the newly synthesized strand, opening new primer binding sites on the synthesized gDNA, leading to selective amplification of templates with frequent primer binding sites. (C) Post-SWGA, the percentage of P. vivax DNA has increased relative to the percentage of host DNA.
    Figure Legend Snippet: Selective whole-genome amplification (SWGA) of Plasmodium vivax genomic DNA (gDNA) from human blood samples. (A) SWGA primers bind frequently to Plasmodium vivax gDNA and infrequently to human gDNA. (B) When phi29 encounters double-stranded gDNA, it displaces the newly synthesized strand, opening new primer binding sites on the synthesized gDNA, leading to selective amplification of templates with frequent primer binding sites. (C) Post-SWGA, the percentage of P. vivax DNA has increased relative to the percentage of host DNA.

    Techniques Used: Whole Genome Amplification, Synthesized, Binding Assay, Amplification

    13) Product Images from "Tapping diversity lost in transformations--in vitro amplification of ligation reactions"

    Article Title: Tapping diversity lost in transformations--in vitro amplification of ligation reactions

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl605

    Antibody repertoire by combinatorial ligation. Regions corresponding to CDRs 1/2 and CDR 3 were PCR-amplified and recombined by ligation into a PCR-amplified phagemid vector backbone. The ligation reactions were either directly electroporated or amplified using Phi29 polymerase followed by electroporation into E.coli bacteria.
    Figure Legend Snippet: Antibody repertoire by combinatorial ligation. Regions corresponding to CDRs 1/2 and CDR 3 were PCR-amplified and recombined by ligation into a PCR-amplified phagemid vector backbone. The ligation reactions were either directly electroporated or amplified using Phi29 polymerase followed by electroporation into E.coli bacteria.

    Techniques Used: Ligation, Polymerase Chain Reaction, Amplification, Plasmid Preparation, Electroporation

    In vitro amplification of ligation reactions. Linear fragments were joined into recombinant, circular units by treatment with DNA ligase. Hexamer primers were annealed and Phi29 polymerase added. This causes extensive amplification of circular species through rolling-circle replication and the formation of extended linear concatemers. The concatemers were cleaved by restriction digestion and re-circularized using DNA ligase.
    Figure Legend Snippet: In vitro amplification of ligation reactions. Linear fragments were joined into recombinant, circular units by treatment with DNA ligase. Hexamer primers were annealed and Phi29 polymerase added. This causes extensive amplification of circular species through rolling-circle replication and the formation of extended linear concatemers. The concatemers were cleaved by restriction digestion and re-circularized using DNA ligase.

    Techniques Used: In Vitro, Amplification, Ligation, Recombinant

    14) Product Images from "Uracil DNA Glycosylase Counteracts APOBEC3G-Induced Hypermutation of Hepatitis B Viral Genomes: Excision Repair of Covalently Closed Circular DNA"

    Article Title: Uracil DNA Glycosylase Counteracts APOBEC3G-Induced Hypermutation of Hepatitis B Viral Genomes: Excision Repair of Covalently Closed Circular DNA

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003361

    UNG inhibition decreases the replication activity of DHBV cccDNA in the presence of A3G expression. (A) RCA products from the cccDNAs. Expression vectors of A3G, UGI, and GFP were used for transfection of LMH cells together with the pCSD3.5ΔS replicon plasmid. After 7 days of cultivation, cccDNAs were purified from the nuclear fraction by Hirt extraction and treated with DpnI to digest transfected plasmids. The cccDNAs were amplified with phi29 DNA polymerase. The DHBV replicon plasmid (pCSD3.5ΔS) was also reacted as a control. RCA concatemeric products (indicated by an arrow) were digested with EcoRI and electrophoresed on agarose gel to verify successful amplification of the 3.0-kb full-length DHBV genomic DNA (left side). The 4.7-kb fragment represents the pCSD3.5 backbone (see Figure S3A for the plasmid construct). (B) qPCR analysis to assess replication activity of reconstructed replicon plasmids. The amplified full-length genomes from cccDNA were cloned into a pCSD3.5 backbone. Resulting reconstructed clones were used to transfect LMH cells without any other vectors (see Figure S5 for the experimental design). DHBV NC-DNA was purified and quantified 3 days later. The graph shows the relative DHBV DNA level; the level of GFP transfectants was set as 1. ***P
    Figure Legend Snippet: UNG inhibition decreases the replication activity of DHBV cccDNA in the presence of A3G expression. (A) RCA products from the cccDNAs. Expression vectors of A3G, UGI, and GFP were used for transfection of LMH cells together with the pCSD3.5ΔS replicon plasmid. After 7 days of cultivation, cccDNAs were purified from the nuclear fraction by Hirt extraction and treated with DpnI to digest transfected plasmids. The cccDNAs were amplified with phi29 DNA polymerase. The DHBV replicon plasmid (pCSD3.5ΔS) was also reacted as a control. RCA concatemeric products (indicated by an arrow) were digested with EcoRI and electrophoresed on agarose gel to verify successful amplification of the 3.0-kb full-length DHBV genomic DNA (left side). The 4.7-kb fragment represents the pCSD3.5 backbone (see Figure S3A for the plasmid construct). (B) qPCR analysis to assess replication activity of reconstructed replicon plasmids. The amplified full-length genomes from cccDNA were cloned into a pCSD3.5 backbone. Resulting reconstructed clones were used to transfect LMH cells without any other vectors (see Figure S5 for the experimental design). DHBV NC-DNA was purified and quantified 3 days later. The graph shows the relative DHBV DNA level; the level of GFP transfectants was set as 1. ***P

    Techniques Used: Inhibition, Activity Assay, Expressing, Transfection, Plasmid Preparation, Purification, Amplification, Agarose Gel Electrophoresis, Construct, Real-time Polymerase Chain Reaction, Clone Assay

    15) Product Images from "A transcription and translation-coupled DNA replication system using rolling-circle replication"

    Article Title: A transcription and translation-coupled DNA replication system using rolling-circle replication

    Journal: Scientific Reports

    doi: 10.1038/srep10404

    Transcription- and translation-coupled DNA (TTcDR) replication. To perform the TTcDR reaction, circular plasmid DNA encoding phi29 DNA polymerase was incubated with the translation system optimized in a previous study 11 , including dNTPs, yeast ppiase, T7 RNA polymerase, and [ 32 P]-dCTP, for 12 h at 30 °C. An aliquot of the mixture after incubation was used in 1% agarose gel electrophoresis and autoradiography. The arrowhead indicates the product of the TTcDR reaction. Lane 1: lambda-BstPI marker. Lane 2: TTcDR reaction without plasmid DNA. Lane 3: TTcDR reaction with plasmid DNA. Lane 4: DNA polymerization with a purified phi29 in phi29 standard buffer.
    Figure Legend Snippet: Transcription- and translation-coupled DNA (TTcDR) replication. To perform the TTcDR reaction, circular plasmid DNA encoding phi29 DNA polymerase was incubated with the translation system optimized in a previous study 11 , including dNTPs, yeast ppiase, T7 RNA polymerase, and [ 32 P]-dCTP, for 12 h at 30 °C. An aliquot of the mixture after incubation was used in 1% agarose gel electrophoresis and autoradiography. The arrowhead indicates the product of the TTcDR reaction. Lane 1: lambda-BstPI marker. Lane 2: TTcDR reaction without plasmid DNA. Lane 3: TTcDR reaction with plasmid DNA. Lane 4: DNA polymerization with a purified phi29 in phi29 standard buffer.

    Techniques Used: Plasmid Preparation, Incubation, Agarose Gel Electrophoresis, Autoradiography, Marker, Purification

    Translation of phi29 DNA polymerase from newly synthesized DNA in the TTcDR reaction. A ) Experimental procedure. First, we performed the optimized TTcDR reaction without [ 35 S]-methionine in the presence or absence of dNTPs, and one-tenth of the mixture was transferred to the second reaction mixture, which contained [ 35 S]-methionine, to detect translation from the replicated DNA product in the first reaction. After incubation at 30 °C for 12 h, an aliquot was used for 10% SDS-PAGE and autoradiography. B ) Translation results. Increased translation of the DNA polymerase was detected when the first reaction contained dNTPs, indicating that the translation occurred from the DNA produced in the first reaction.
    Figure Legend Snippet: Translation of phi29 DNA polymerase from newly synthesized DNA in the TTcDR reaction. A ) Experimental procedure. First, we performed the optimized TTcDR reaction without [ 35 S]-methionine in the presence or absence of dNTPs, and one-tenth of the mixture was transferred to the second reaction mixture, which contained [ 35 S]-methionine, to detect translation from the replicated DNA product in the first reaction. After incubation at 30 °C for 12 h, an aliquot was used for 10% SDS-PAGE and autoradiography. B ) Translation results. Increased translation of the DNA polymerase was detected when the first reaction contained dNTPs, indicating that the translation occurred from the DNA produced in the first reaction.

    Techniques Used: Synthesized, Incubation, SDS Page, Autoradiography, Produced

    DNA replication with or without random hexamers in the absence of TTcDR components. DNA replication was performed by purified phi29 DNA polymerase with or without random hexamers in the TTcDR mixtures in which some of the components (NTP, tRNA, T7 polymerase, ribosome, and translation proteins) were omitted, and the amount of replicated DNA was measured as described in the Methods section. The translation proteins contained all protein factors in the translation system (e.g., IFs, EFs, RFs, and aminoacyl-tRNA synthetases). In the experiments with random hexamers, the template plasmid was heated with the hexamers at 95 °C for 3 min and then cooled immediately.
    Figure Legend Snippet: DNA replication with or without random hexamers in the absence of TTcDR components. DNA replication was performed by purified phi29 DNA polymerase with or without random hexamers in the TTcDR mixtures in which some of the components (NTP, tRNA, T7 polymerase, ribosome, and translation proteins) were omitted, and the amount of replicated DNA was measured as described in the Methods section. The translation proteins contained all protein factors in the translation system (e.g., IFs, EFs, RFs, and aminoacyl-tRNA synthetases). In the experiments with random hexamers, the template plasmid was heated with the hexamers at 95 °C for 3 min and then cooled immediately.

    Techniques Used: Purification, Plasmid Preparation

    Schematic representation of the transcription- and translation-coupled DNA replication system. Circular DNA encoding phi29 DNA polymerase under control of the T7 promoter is incubated with the reconstituted translation system including T7 RNA polymerase. mRNA is transcribed from the DNA, and phi29 DNA polymerase is translated. The polymerase attaches to the circular DNA and initiates the polymerization of a long single-stranded RNA in a rolling-circle manner. The polymerase further synthesizes the complementary strand to produce double-stranded DNA, which is a long repeat of the circular DNA sequence. The next round of transcription and translation occurs from the double-stranded DNA.
    Figure Legend Snippet: Schematic representation of the transcription- and translation-coupled DNA replication system. Circular DNA encoding phi29 DNA polymerase under control of the T7 promoter is incubated with the reconstituted translation system including T7 RNA polymerase. mRNA is transcribed from the DNA, and phi29 DNA polymerase is translated. The polymerase attaches to the circular DNA and initiates the polymerization of a long single-stranded RNA in a rolling-circle manner. The polymerase further synthesizes the complementary strand to produce double-stranded DNA, which is a long repeat of the circular DNA sequence. The next round of transcription and translation occurs from the double-stranded DNA.

    Techniques Used: Incubation, Sequencing

    16) Product Images from "Advanced microRNA-based cancer diagnostics using amplified time-gated FRET microRNA-based cancer diagnostics using amplified time-gated FRET †Electronic supplementary information (ESI) available. See DOI: 10.1039/c8sc03121e"

    Article Title: Advanced microRNA-based cancer diagnostics using amplified time-gated FRET microRNA-based cancer diagnostics using amplified time-gated FRET †Electronic supplementary information (ESI) available. See DOI: 10.1039/c8sc03121e

    Journal: Chemical Science

    doi: 10.1039/c8sc03121e

    Principle of miRNA detection by amplified TG-FRET. (A) After specific recognition of miRNA by a linear padlock DNA (1), the DNA padlock nick is ligated over the miRNA target splint using SplintR ligase (2) and the miRNA becomes a primer for a phi29 polymerase to synthesize and displace (by RCA) complimentary DNA around the circularized padlock DNA (3). After stopping RCA, the rolling circle product (RCP) is incubated with Tb (Lumi4-Tb) donor and Cy5.5 acceptor labeled ssDNA, which hybridize to specific sequences that exist more than 1000-fold on the amplified RCP concatemer. The close distance of Lumi4-Tb and Cy5.5 in the RCP allows for Tb-to-Cy5.5 FRET, which is not possible if both are free in solution (not hybridized to the RCP). Thus, the TG-FRET signal can be used for quantifying miRNA without any washing or separation steps. (B) Ratiometric TG-FRET, which measures the ratio of FRET-sensitized Cy5.5 photoluminescence (PL) and FRET-quenched Tb PL within a specific time-window after pulsed excitation (to suppress autofluorescence), is used to quantify the miRNA target in a 140 μl microwell within 5 seconds.
    Figure Legend Snippet: Principle of miRNA detection by amplified TG-FRET. (A) After specific recognition of miRNA by a linear padlock DNA (1), the DNA padlock nick is ligated over the miRNA target splint using SplintR ligase (2) and the miRNA becomes a primer for a phi29 polymerase to synthesize and displace (by RCA) complimentary DNA around the circularized padlock DNA (3). After stopping RCA, the rolling circle product (RCP) is incubated with Tb (Lumi4-Tb) donor and Cy5.5 acceptor labeled ssDNA, which hybridize to specific sequences that exist more than 1000-fold on the amplified RCP concatemer. The close distance of Lumi4-Tb and Cy5.5 in the RCP allows for Tb-to-Cy5.5 FRET, which is not possible if both are free in solution (not hybridized to the RCP). Thus, the TG-FRET signal can be used for quantifying miRNA without any washing or separation steps. (B) Ratiometric TG-FRET, which measures the ratio of FRET-sensitized Cy5.5 photoluminescence (PL) and FRET-quenched Tb PL within a specific time-window after pulsed excitation (to suppress autofluorescence), is used to quantify the miRNA target in a 140 μl microwell within 5 seconds.

    Techniques Used: Amplification, Incubation, Labeling

    17) Product Images from "Electrochemical Biosensors Combined with Isothermal Amplification for Quantitative Detection of Nucleic Acids"

    Article Title: Electrochemical Biosensors Combined with Isothermal Amplification for Quantitative Detection of Nucleic Acids

    Journal: Biosensors and Biodetection

    doi: 10.1007/978-1-4939-6911-1_10

    RCA-CC biosensor for microRNA detection. ( a ) Integrated gold electrodes were fabricated on polystyrene substrate by chemical plating. ( b ) Schematic illustration of three-gold electrode substrate. ( c ) After immobilization of DNA probe 1 on the working electrode, microRNA and DNA probe 2, which are partly complementary, hybridize to it, and then solid-phase RCA is initiated by phi29 DNA polymerase at 30 °C. If no hybridization takes place, the RCA reaction does not proceed
    Figure Legend Snippet: RCA-CC biosensor for microRNA detection. ( a ) Integrated gold electrodes were fabricated on polystyrene substrate by chemical plating. ( b ) Schematic illustration of three-gold electrode substrate. ( c ) After immobilization of DNA probe 1 on the working electrode, microRNA and DNA probe 2, which are partly complementary, hybridize to it, and then solid-phase RCA is initiated by phi29 DNA polymerase at 30 °C. If no hybridization takes place, the RCA reaction does not proceed

    Techniques Used: Hybridization

    18) Product Images from "Effects of acetaldehyde-induced DNA lesions on DNA metabolism"

    Article Title: Effects of acetaldehyde-induced DNA lesions on DNA metabolism

    Journal: Genes and Environment

    doi: 10.1186/s41021-019-0142-7

    DNA replication reaction in acetaldehyde-treated plasmids. a In the absence of DNA damage, phi29 DNA polymerase and random primers generate new DNA synthesis products from the template. If acetaldehyde damages DNA, the resulting lesions inhibit DNA synthesis, as phi29 DNA polymerase cannot synthesize new DNA products from damaged templates, and products will not be detected. b Agarose gel (1%) demonstrating the presence of an acetaldehyde-induced lesion. The phi29 DNA polymerase and non-acetaldehyde treated DNA template/random primer complexes (lane 1) or acetaldehyde treated DNA template/random primer complexes (lane 5) were incubated for the indicated times (0,1, 2, and 4 h: lanes 1–4 or lanes 5–8). Rp is random primers and triangles are incubation time. c Quantification of DNA synthesis products via 1% agarose gel analysis (b)
    Figure Legend Snippet: DNA replication reaction in acetaldehyde-treated plasmids. a In the absence of DNA damage, phi29 DNA polymerase and random primers generate new DNA synthesis products from the template. If acetaldehyde damages DNA, the resulting lesions inhibit DNA synthesis, as phi29 DNA polymerase cannot synthesize new DNA products from damaged templates, and products will not be detected. b Agarose gel (1%) demonstrating the presence of an acetaldehyde-induced lesion. The phi29 DNA polymerase and non-acetaldehyde treated DNA template/random primer complexes (lane 1) or acetaldehyde treated DNA template/random primer complexes (lane 5) were incubated for the indicated times (0,1, 2, and 4 h: lanes 1–4 or lanes 5–8). Rp is random primers and triangles are incubation time. c Quantification of DNA synthesis products via 1% agarose gel analysis (b)

    Techniques Used: DNA Synthesis, Agarose Gel Electrophoresis, Incubation

    19) Product Images from "Limited reverse transcriptase activity of phi29 DNA polymerase"

    Article Title: Limited reverse transcriptase activity of phi29 DNA polymerase

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky190

    Effect of RNA substitutions in circular templates on rolling circle amplification with phi29 DNA polymerase. ( A ) Total amount of RCA products (y-axis) generated for padlock probes with/without a terminal 3′ RNA and in the absence of synthetic RNA ligation template (template -). ( B ) Circles with 0–7 RNA substitutions in the backbone were amplified and digitally counted. The y-axis shows the number of rolling circle products (RCPs); error bars ± S.D.; n = 2. The same RCA reactions with chimeric circles were also monitored in real-time by measuring SYBR Gold incorporation on qPCR instrument ( C and E ). (C) RCA reaction curves of circles with 0, 1 and 2 RNA substitutions. ( D ) RCPs from C were imaged on microscope slides and size and intensity of individual RCPs were quantified. Black line, median; upper whisker, highest value that is within 1.5 the interquartile range of the hinge; lower whisker, lowest value within 1.5 the interquartile range of the hinge. (E) Real-time data of the same RCA reactions as in B with 0–7 RNA substitutes are displayed. Representative samples are presented from a duplicated experiment. To highlight the initial stages of RCA and to show the difference between the samples with low RCA efficiency, fluorescence intensity readout between 3000 and 6000 is presented.
    Figure Legend Snippet: Effect of RNA substitutions in circular templates on rolling circle amplification with phi29 DNA polymerase. ( A ) Total amount of RCA products (y-axis) generated for padlock probes with/without a terminal 3′ RNA and in the absence of synthetic RNA ligation template (template -). ( B ) Circles with 0–7 RNA substitutions in the backbone were amplified and digitally counted. The y-axis shows the number of rolling circle products (RCPs); error bars ± S.D.; n = 2. The same RCA reactions with chimeric circles were also monitored in real-time by measuring SYBR Gold incorporation on qPCR instrument ( C and E ). (C) RCA reaction curves of circles with 0, 1 and 2 RNA substitutions. ( D ) RCPs from C were imaged on microscope slides and size and intensity of individual RCPs were quantified. Black line, median; upper whisker, highest value that is within 1.5 the interquartile range of the hinge; lower whisker, lowest value within 1.5 the interquartile range of the hinge. (E) Real-time data of the same RCA reactions as in B with 0–7 RNA substitutes are displayed. Representative samples are presented from a duplicated experiment. To highlight the initial stages of RCA and to show the difference between the samples with low RCA efficiency, fluorescence intensity readout between 3000 and 6000 is presented.

    Techniques Used: Amplification, Generated, Ligation, Real-time Polymerase Chain Reaction, Microscopy, Whisker Assay, Fluorescence

    Phi29 DNA polymerase exhibits higher RCA rate with circles containing pyrimidine RNA substitutions. ( A ) Real-time RCA curves of circles containing 1, 2, 3 or 4 consecutive RNA substations of rG, rU, rA, rC RNA bases are displayed (number of consecutive substitutions is indicated above plots). Rate of RCA was monitored by measuring fluorescence build-up (y-axis) resulted from SYBR Gold incorporation into RCPs. Averaged fluorescence intensity for each RCA time point was calculated from a duplicated experiment. RCA was conducted in the presence of Mg 2+ and Mn 2+ (solid and dashed lines respectively). ( B ) Linear, early stage RCA velocity (y-axis) is presented for PLPs from (A) in the presence of Mg 2+ (solid lines) and Mn 2+ (dashed lines). ( C ) RCA for the control PLP (non-chimeric DNA circle, with Mg 2+ (solid) and Mn 2+ (dashed line) are displayed.
    Figure Legend Snippet: Phi29 DNA polymerase exhibits higher RCA rate with circles containing pyrimidine RNA substitutions. ( A ) Real-time RCA curves of circles containing 1, 2, 3 or 4 consecutive RNA substations of rG, rU, rA, rC RNA bases are displayed (number of consecutive substitutions is indicated above plots). Rate of RCA was monitored by measuring fluorescence build-up (y-axis) resulted from SYBR Gold incorporation into RCPs. Averaged fluorescence intensity for each RCA time point was calculated from a duplicated experiment. RCA was conducted in the presence of Mg 2+ and Mn 2+ (solid and dashed lines respectively). ( B ) Linear, early stage RCA velocity (y-axis) is presented for PLPs from (A) in the presence of Mg 2+ (solid lines) and Mn 2+ (dashed lines). ( C ) RCA for the control PLP (non-chimeric DNA circle, with Mg 2+ (solid) and Mn 2+ (dashed line) are displayed.

    Techniques Used: Fluorescence, Plasmid Purification

    DNA sequencing-based analysis of rolling circle products reveals reverse transcription activity of phi29 DNA polymerase. ( A ) After RCA, short DNA oligonucleotides were hybridized to an AluI restriction site in the RCA products and RCPs were digested with AluI restriction enzyme, resulting in RCA monomers. Following digestion, monomers were PCR-amplified using primers containing Ilumina adapter sequences. PCR products were extended using IIlumina indexed primers. Finally, sequencing library was prepared using indexed primers-specific P5/7 PCR primers. The region of interest containing RNA substitutions in the original padlock probe sequence is indicated with green boxes. ( B ) Logos showing sequencing frequencies for each position within RCA monomers generated from the control DNA circle (P1 = dG), and circles containing single rG, rU, rA and rC substitutions at the RNA position (P1). Positions P1 and P2 are indicated and position P1 was additionally highlighted with the red box. ( C ) Incorporation of incorrect nucleotides for every position in the sequenced monomers from (B). Error rates, calculated as Incorporation error [%] = 1 – number of reads with expected nucleotide/total number of reads, is presented for padlock probes with single- (upper plot) and double-RNA substitutions (lower plots). P1 position for the first RNA substitution is indicated with the box.
    Figure Legend Snippet: DNA sequencing-based analysis of rolling circle products reveals reverse transcription activity of phi29 DNA polymerase. ( A ) After RCA, short DNA oligonucleotides were hybridized to an AluI restriction site in the RCA products and RCPs were digested with AluI restriction enzyme, resulting in RCA monomers. Following digestion, monomers were PCR-amplified using primers containing Ilumina adapter sequences. PCR products were extended using IIlumina indexed primers. Finally, sequencing library was prepared using indexed primers-specific P5/7 PCR primers. The region of interest containing RNA substitutions in the original padlock probe sequence is indicated with green boxes. ( B ) Logos showing sequencing frequencies for each position within RCA monomers generated from the control DNA circle (P1 = dG), and circles containing single rG, rU, rA and rC substitutions at the RNA position (P1). Positions P1 and P2 are indicated and position P1 was additionally highlighted with the red box. ( C ) Incorporation of incorrect nucleotides for every position in the sequenced monomers from (B). Error rates, calculated as Incorporation error [%] = 1 – number of reads with expected nucleotide/total number of reads, is presented for padlock probes with single- (upper plot) and double-RNA substitutions (lower plots). P1 position for the first RNA substitution is indicated with the box.

    Techniques Used: DNA Sequencing, Activity Assay, Polymerase Chain Reaction, Amplification, Sequencing, Generated

    20) Product Images from "Droplet Tn-Seq combines microfluidics with Tn-Seq for identifying complex single-cell phenotypes"

    Article Title: Droplet Tn-Seq combines microfluidics with Tn-Seq for identifying complex single-cell phenotypes

    Journal: Nature Communications

    doi: 10.1038/s41467-019-13719-9

    Schematic overview of droplet Tn-Seq. a A microfluidic device encapsulates single bacterial cells into droplets containing growth medium. Bacteria are allowed to grow within droplets, genomic DNA (gDNA) is isolated at the start of the experiment (t1) and after growth (t2). Importantly, while growth for each transposon mutant takes place in isolation, gDNA is isolated from the pooled population, enabling screening of all mutants simultaneously. b gDNA is then amplified with DNA polymerase phi29, digested with MmeI, an adapter is ligated, a ~180 bp fragment is produced which contains ~16 nucleotides of bacterial gDNA, defining the transposon-insertion location, followed by Illumina sequencing. Reads are demultiplexed based on the barcode in the adapter and a potential second barcode in primer 1, mapped to the genome, and fitness is calculated for each defined region.
    Figure Legend Snippet: Schematic overview of droplet Tn-Seq. a A microfluidic device encapsulates single bacterial cells into droplets containing growth medium. Bacteria are allowed to grow within droplets, genomic DNA (gDNA) is isolated at the start of the experiment (t1) and after growth (t2). Importantly, while growth for each transposon mutant takes place in isolation, gDNA is isolated from the pooled population, enabling screening of all mutants simultaneously. b gDNA is then amplified with DNA polymerase phi29, digested with MmeI, an adapter is ligated, a ~180 bp fragment is produced which contains ~16 nucleotides of bacterial gDNA, defining the transposon-insertion location, followed by Illumina sequencing. Reads are demultiplexed based on the barcode in the adapter and a potential second barcode in primer 1, mapped to the genome, and fitness is calculated for each defined region.

    Techniques Used: Isolation, Mutagenesis, Amplification, Produced, Sequencing

    Unbiased whole-genome amplification of low-quantity genomic DNA. a , b gDNA was prepared by two different methods for transposon sequencing. For the WGA sample, 10 ng of gDNA was amplified first with DNA polymerase phi29 before MmeI digestion and adapter ligation. For the standard sample, 1 μg of gDNA was digested with MmeI, followed by adapter ligation. There is a strong correlation between fitness values obtained from WGA preparation compared with standard Tn-Seq library preparation a , and WGA preparation is highly reproducible b .
    Figure Legend Snippet: Unbiased whole-genome amplification of low-quantity genomic DNA. a , b gDNA was prepared by two different methods for transposon sequencing. For the WGA sample, 10 ng of gDNA was amplified first with DNA polymerase phi29 before MmeI digestion and adapter ligation. For the standard sample, 1 μg of gDNA was digested with MmeI, followed by adapter ligation. There is a strong correlation between fitness values obtained from WGA preparation compared with standard Tn-Seq library preparation a , and WGA preparation is highly reproducible b .

    Techniques Used: Whole Genome Amplification, Sequencing, Amplification, Ligation

    21) Product Images from "Reconstitution and Coupling of DNA Replication and Segregation in a Biomimetic System"

    Article Title: Reconstitution and Coupling of DNA Replication and Segregation in a Biomimetic System

    Journal: Chembiochem

    doi: 10.1002/cbic.201900299

    Replication, recircularization, and compaction of a plasmid containing the T7 promoter. A) Time course of RCA‐based replication of pRepC plasmid containing the T7 promoter and loxP sites (depicted in Figure S1), measured as fluorescence of the DNA‐binding PicoGreen dye. Reaction mixtures contained, as indicated, Phi29 DNA polymerase, T7 DNA polymerase, and T7 RNA polymerase, as well as specific or random primers. For a control reaction with T7 DNA polymerase and T7 RNA polymerase, a pQE30 plasmid lacking the T7 promoter was used. B) Recircularization of the replicated plasmid, mediated by Cre recombinase. Where indicated, Cre recombinase was added after 16 h of replication, and the reaction mixture was incubated for another 30 min. Reaction mixtures were separated along with a DNA ladder (1 kb) on a Midori‐green stained agarose gel. The lower band migrating below 5 kb corresponds to the circularized DNA, whereas larger products apparently correspond to linear concatamers (Figure S2). C) DNA nanoparticles emerging upon prolonged ( > 12 h) T7 DNA replication reaction. Scale bar: 10 μm.
    Figure Legend Snippet: Replication, recircularization, and compaction of a plasmid containing the T7 promoter. A) Time course of RCA‐based replication of pRepC plasmid containing the T7 promoter and loxP sites (depicted in Figure S1), measured as fluorescence of the DNA‐binding PicoGreen dye. Reaction mixtures contained, as indicated, Phi29 DNA polymerase, T7 DNA polymerase, and T7 RNA polymerase, as well as specific or random primers. For a control reaction with T7 DNA polymerase and T7 RNA polymerase, a pQE30 plasmid lacking the T7 promoter was used. B) Recircularization of the replicated plasmid, mediated by Cre recombinase. Where indicated, Cre recombinase was added after 16 h of replication, and the reaction mixture was incubated for another 30 min. Reaction mixtures were separated along with a DNA ladder (1 kb) on a Midori‐green stained agarose gel. The lower band migrating below 5 kb corresponds to the circularized DNA, whereas larger products apparently correspond to linear concatamers (Figure S2). C) DNA nanoparticles emerging upon prolonged ( > 12 h) T7 DNA replication reaction. Scale bar: 10 μm.

    Techniques Used: Plasmid Preparation, Fluorescence, Binding Assay, Incubation, Staining, Agarose Gel Electrophoresis

    22) Product Images from "A Comparison of Methods for the Extraction of Plasmids Capable of Conferring Antibiotic Resistance in a Human Pathogen From Complex Broiler Cecal Samples"

    Article Title: A Comparison of Methods for the Extraction of Plasmids Capable of Conferring Antibiotic Resistance in a Human Pathogen From Complex Broiler Cecal Samples

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.01731

    Plasmid DNA from the cecal sample after amplification with phi29 polymerase. 1 , 1 kb ladder and 2 , Plasmid DNA amplified with Phi29 DNA polymerase.
    Figure Legend Snippet: Plasmid DNA from the cecal sample after amplification with phi29 polymerase. 1 , 1 kb ladder and 2 , Plasmid DNA amplified with Phi29 DNA polymerase.

    Techniques Used: Plasmid Preparation, Amplification

    Digested plasmid DNA extracted from E. coli transformants after electroporation with the phi29 polymerase amplified DNA. 1 , 1 kb ladder; Plasmid DNA extracted from transformants selected on agar plates containing; 2 , ampicillin 32 mg/L (M_Amp_BC); 3 , ampicillin 32 mg/L (M_Amp_SC); 4 , tetracycline 16 mg/L (M_Tet_BC); 5 , tetracycline 16 mg/L (M_Tet_SC); 6 , kanamycin 25 mg/L (M_Kan); 7 , ciprofloxacin 16 mg/L (M_Cip). BC and SC refer to the two different colony morphology types, big or small colonies, on the same antibiotic plate.
    Figure Legend Snippet: Digested plasmid DNA extracted from E. coli transformants after electroporation with the phi29 polymerase amplified DNA. 1 , 1 kb ladder; Plasmid DNA extracted from transformants selected on agar plates containing; 2 , ampicillin 32 mg/L (M_Amp_BC); 3 , ampicillin 32 mg/L (M_Amp_SC); 4 , tetracycline 16 mg/L (M_Tet_BC); 5 , tetracycline 16 mg/L (M_Tet_SC); 6 , kanamycin 25 mg/L (M_Kan); 7 , ciprofloxacin 16 mg/L (M_Cip). BC and SC refer to the two different colony morphology types, big or small colonies, on the same antibiotic plate.

    Techniques Used: Plasmid Preparation, Electroporation, Amplification

    23) Product Images from "Target-fueled DNA walker for highly selective miRNA detection DNA walker for highly selective miRNA detection †Electronic supplementary information (ESI) available: DNA strand structure and sequences, assembly of DNA strands as noted in the text. See DOI: 10.1039/c5sc02784eClick here for additional data file."

    Article Title: Target-fueled DNA walker for highly selective miRNA detection DNA walker for highly selective miRNA detection †Electronic supplementary information (ESI) available: DNA strand structure and sequences, assembly of DNA strands as noted in the text. See DOI: 10.1039/c5sc02784eClick here for additional data file.

    Journal: Chemical Science

    doi: 10.1039/c5sc02784e

    (A) Optimization of T4 DNA ligase concentration and time. (B) Optimization of phi29 DNA polymerase concentration and time. (C) Optimization of Nb.Mva1269I concentration and time. The assays were carried out in the reaction buffer, containing 10 nM let-7a, and 200 nM MB.
    Figure Legend Snippet: (A) Optimization of T4 DNA ligase concentration and time. (B) Optimization of phi29 DNA polymerase concentration and time. (C) Optimization of Nb.Mva1269I concentration and time. The assays were carried out in the reaction buffer, containing 10 nM let-7a, and 200 nM MB.

    Techniques Used: Concentration Assay

    24) Product Images from "Droplet Tn-Seq combines microfluidics with Tn-Seq for identifying complex single-cell phenotypes"

    Article Title: Droplet Tn-Seq combines microfluidics with Tn-Seq for identifying complex single-cell phenotypes

    Journal: Nature Communications

    doi: 10.1038/s41467-019-13719-9

    Schematic overview of droplet Tn-Seq. a A microfluidic device encapsulates single bacterial cells into droplets containing growth medium. Bacteria are allowed to grow within droplets, genomic DNA (gDNA) is isolated at the start of the experiment (t1) and after growth (t2). Importantly, while growth for each transposon mutant takes place in isolation, gDNA is isolated from the pooled population, enabling screening of all mutants simultaneously. b gDNA is then amplified with DNA polymerase phi29, digested with MmeI, an adapter is ligated, a ~180 bp fragment is produced which contains ~16 nucleotides of bacterial gDNA, defining the transposon-insertion location, followed by Illumina sequencing. Reads are demultiplexed based on the barcode in the adapter and a potential second barcode in primer 1, mapped to the genome, and fitness is calculated for each defined region.
    Figure Legend Snippet: Schematic overview of droplet Tn-Seq. a A microfluidic device encapsulates single bacterial cells into droplets containing growth medium. Bacteria are allowed to grow within droplets, genomic DNA (gDNA) is isolated at the start of the experiment (t1) and after growth (t2). Importantly, while growth for each transposon mutant takes place in isolation, gDNA is isolated from the pooled population, enabling screening of all mutants simultaneously. b gDNA is then amplified with DNA polymerase phi29, digested with MmeI, an adapter is ligated, a ~180 bp fragment is produced which contains ~16 nucleotides of bacterial gDNA, defining the transposon-insertion location, followed by Illumina sequencing. Reads are demultiplexed based on the barcode in the adapter and a potential second barcode in primer 1, mapped to the genome, and fitness is calculated for each defined region.

    Techniques Used: Isolation, Mutagenesis, Amplification, Produced, Sequencing

    Unbiased whole-genome amplification of low-quantity genomic DNA. a , b gDNA was prepared by two different methods for transposon sequencing. For the WGA sample, 10 ng of gDNA was amplified first with DNA polymerase phi29 before MmeI digestion and adapter ligation. For the standard sample, 1 μg of gDNA was digested with MmeI, followed by adapter ligation. There is a strong correlation between fitness values obtained from WGA preparation compared with standard Tn-Seq library preparation a , and WGA preparation is highly reproducible b .
    Figure Legend Snippet: Unbiased whole-genome amplification of low-quantity genomic DNA. a , b gDNA was prepared by two different methods for transposon sequencing. For the WGA sample, 10 ng of gDNA was amplified first with DNA polymerase phi29 before MmeI digestion and adapter ligation. For the standard sample, 1 μg of gDNA was digested with MmeI, followed by adapter ligation. There is a strong correlation between fitness values obtained from WGA preparation compared with standard Tn-Seq library preparation a , and WGA preparation is highly reproducible b .

    Techniques Used: Whole Genome Amplification, Sequencing, Amplification, Ligation

    25) Product Images from "Dual-Signal Amplification Strategy for Sensitive MicroRNA Detection Based on Rolling Circle Amplification and Enzymatic Repairing Amplification"

    Article Title: Dual-Signal Amplification Strategy for Sensitive MicroRNA Detection Based on Rolling Circle Amplification and Enzymatic Repairing Amplification

    Journal: ACS Omega

    doi: 10.1021/acsomega.0c05141

    (A) Fluorescence emission spectra of the miRNA analysis under different conditions. (a) Padlock probe + T4 RNA ligase 2 + miR-21 (500 pM) + phi29 DNA polymerase + dNTPs + TaqMan probe + Endo IV, (b) control with no miR-21, (c) control with no padlock probe, and (d) control with no Endo IV. (B) Gel electrophoresis image for RCA products. Lane M: DNA marker (10–300 bp); Lane 1: 2 μM miR-21; Lane 2: 1 μM padlock probe; Lane 3: 1 μM padlock probe + 1 μM miR-21 + 2 U T4 RNA ligase 2; Lane 4: 100 nM padlock probe + 2 U T4 RNA ligase 2 + 10 U phi29 DNA polymerase + 0.5 mM dNTPs. Lane 5: 100 nM padlock probe + 10 nM miR-21 + 10 U phi29 DNA polymerase + 0.5 mM dNTPs; Lane 6: 100 nM padlock probe + 10 nM miR-21 + 2 U T4 RNA ligase 2 + 10 U phi29 DNA polymerase + 0.5 mM dNTPs.
    Figure Legend Snippet: (A) Fluorescence emission spectra of the miRNA analysis under different conditions. (a) Padlock probe + T4 RNA ligase 2 + miR-21 (500 pM) + phi29 DNA polymerase + dNTPs + TaqMan probe + Endo IV, (b) control with no miR-21, (c) control with no padlock probe, and (d) control with no Endo IV. (B) Gel electrophoresis image for RCA products. Lane M: DNA marker (10–300 bp); Lane 1: 2 μM miR-21; Lane 2: 1 μM padlock probe; Lane 3: 1 μM padlock probe + 1 μM miR-21 + 2 U T4 RNA ligase 2; Lane 4: 100 nM padlock probe + 2 U T4 RNA ligase 2 + 10 U phi29 DNA polymerase + 0.5 mM dNTPs. Lane 5: 100 nM padlock probe + 10 nM miR-21 + 10 U phi29 DNA polymerase + 0.5 mM dNTPs; Lane 6: 100 nM padlock probe + 10 nM miR-21 + 2 U T4 RNA ligase 2 + 10 U phi29 DNA polymerase + 0.5 mM dNTPs.

    Techniques Used: Fluorescence, Nucleic Acid Electrophoresis, Marker

    26) Product Images from "Dual-Signal Amplification Strategy for Sensitive MicroRNA Detection Based on Rolling Circle Amplification and Enzymatic Repairing Amplification"

    Article Title: Dual-Signal Amplification Strategy for Sensitive MicroRNA Detection Based on Rolling Circle Amplification and Enzymatic Repairing Amplification

    Journal: ACS Omega

    doi: 10.1021/acsomega.0c05141

    (A) Fluorescence emission spectra of the miRNA analysis under different conditions. (a) Padlock probe + T4 RNA ligase 2 + miR-21 (500 pM) + phi29 DNA polymerase + dNTPs + TaqMan probe + Endo IV, (b) control with no miR-21, (c) control with no padlock probe, and (d) control with no Endo IV. (B) Gel electrophoresis image for RCA products. Lane M: DNA marker (10–300 bp); Lane 1: 2 μM miR-21; Lane 2: 1 μM padlock probe; Lane 3: 1 μM padlock probe + 1 μM miR-21 + 2 U T4 RNA ligase 2; Lane 4: 100 nM padlock probe + 2 U T4 RNA ligase 2 + 10 U phi29 DNA polymerase + 0.5 mM dNTPs. Lane 5: 100 nM padlock probe + 10 nM miR-21 + 10 U phi29 DNA polymerase + 0.5 mM dNTPs; Lane 6: 100 nM padlock probe + 10 nM miR-21 + 2 U T4 RNA ligase 2 + 10 U phi29 DNA polymerase + 0.5 mM dNTPs.
    Figure Legend Snippet: (A) Fluorescence emission spectra of the miRNA analysis under different conditions. (a) Padlock probe + T4 RNA ligase 2 + miR-21 (500 pM) + phi29 DNA polymerase + dNTPs + TaqMan probe + Endo IV, (b) control with no miR-21, (c) control with no padlock probe, and (d) control with no Endo IV. (B) Gel electrophoresis image for RCA products. Lane M: DNA marker (10–300 bp); Lane 1: 2 μM miR-21; Lane 2: 1 μM padlock probe; Lane 3: 1 μM padlock probe + 1 μM miR-21 + 2 U T4 RNA ligase 2; Lane 4: 100 nM padlock probe + 2 U T4 RNA ligase 2 + 10 U phi29 DNA polymerase + 0.5 mM dNTPs. Lane 5: 100 nM padlock probe + 10 nM miR-21 + 10 U phi29 DNA polymerase + 0.5 mM dNTPs; Lane 6: 100 nM padlock probe + 10 nM miR-21 + 2 U T4 RNA ligase 2 + 10 U phi29 DNA polymerase + 0.5 mM dNTPs.

    Techniques Used: Fluorescence, Nucleic Acid Electrophoresis, Marker

    27) Product Images from "SNES: single nucleus exome sequencing"

    Article Title: SNES: single nucleus exome sequencing

    Journal: Genome Biology

    doi: 10.1186/s13059-015-0616-2

    SNES method and WGA quality control. (a) Nuclear suspensions were prepared from tissues, stained with DAPI and flow-sorted. Single nuclei were isolated by gating the G1/0 or G2/M ploidy distributions and deposited nuclei singly into a 96-well plate. Multiple-displacement-amplification is performed using Φ29 to perform WGA. (b) Time-course of WGA showing total DNA yield from single nuclei. (c) Quality control assay using a panel of 22 chromosome-specific qPCR primers to determine the WGA amplification efficiency of each single nucleus.
    Figure Legend Snippet: SNES method and WGA quality control. (a) Nuclear suspensions were prepared from tissues, stained with DAPI and flow-sorted. Single nuclei were isolated by gating the G1/0 or G2/M ploidy distributions and deposited nuclei singly into a 96-well plate. Multiple-displacement-amplification is performed using Φ29 to perform WGA. (b) Time-course of WGA showing total DNA yield from single nuclei. (c) Quality control assay using a panel of 22 chromosome-specific qPCR primers to determine the WGA amplification efficiency of each single nucleus.

    Techniques Used: Whole Genome Amplification, Staining, Flow Cytometry, Isolation, Multiple Displacement Amplification, Control Assay, Real-time Polymerase Chain Reaction, Amplification

    28) Product Images from "Improvements of rolling circle amplification (RCA) efficiency and accuracy using Thermus thermophilus SSB mutant protein"

    Article Title: Improvements of rolling circle amplification (RCA) efficiency and accuracy using Thermus thermophilus SSB mutant protein

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl350

    Effect of Tth SSB-255 protein on the efficiency and specificity of RCA. ( a ) Top: RCAs were performed in the absence of the SSB proteins using pUC19 DNA as template and phi29 DNA polymerase for the indicated reaction times. Bottom: Signals of spot hybridization of the same samples. ( b ) Top and Bottom: same as (a), except for the absence of template DNA. ( c ) Top and Bottom: same as (a), except for the presence of the Tth SSB protein (3.0 µg/20 µl reaction volume). ( d ) Top and Bottom: same as (c), except for the absence of template DNA. ( e ) Top and Bottom: same as (a), except for the presence of the Tth SSB-255 protein (3.0 µg/20 µl reaction volume). ( f ) Top and Bottom: same as (e), except for the absence of template DNA.
    Figure Legend Snippet: Effect of Tth SSB-255 protein on the efficiency and specificity of RCA. ( a ) Top: RCAs were performed in the absence of the SSB proteins using pUC19 DNA as template and phi29 DNA polymerase for the indicated reaction times. Bottom: Signals of spot hybridization of the same samples. ( b ) Top and Bottom: same as (a), except for the absence of template DNA. ( c ) Top and Bottom: same as (a), except for the presence of the Tth SSB protein (3.0 µg/20 µl reaction volume). ( d ) Top and Bottom: same as (c), except for the absence of template DNA. ( e ) Top and Bottom: same as (a), except for the presence of the Tth SSB-255 protein (3.0 µg/20 µl reaction volume). ( f ) Top and Bottom: same as (e), except for the absence of template DNA.

    Techniques Used: Hybridization

    Effect of Tth SSB-255 protein on RCA assays. ( a ) RCAs were carried out in the absence or presence of the indicated SSB proteins using pUC19 DNA as a template and phi29 DNA polymerase. ( b ) Same as (a), except for using linearized ( Eco RI) pUC19 DNA as the template. ( c ) Same as (a), except that the amplifications were carried out in the absence of template DNA. Lane M: molecular weight markers (100 and 12 kb).
    Figure Legend Snippet: Effect of Tth SSB-255 protein on RCA assays. ( a ) RCAs were carried out in the absence or presence of the indicated SSB proteins using pUC19 DNA as a template and phi29 DNA polymerase. ( b ) Same as (a), except for using linearized ( Eco RI) pUC19 DNA as the template. ( c ) Same as (a), except that the amplifications were carried out in the absence of template DNA. Lane M: molecular weight markers (100 and 12 kb).

    Techniques Used: Molecular Weight

    29) Product Images from "TERRA transcription destabilizes telomere integrity to initiate break-induced replication in human ALT cells"

    Article Title: TERRA transcription destabilizes telomere integrity to initiate break-induced replication in human ALT cells

    Journal: bioRxiv

    doi: 10.1101/2021.02.18.431840

    C-circle analysis in T-TALE cells. ( a ) C-circle assay analysis of genomic DNA from the indicated cells treated with dox for up to 72 hours. Reaction products were dot-blotted and hybridized to a radiolabeled telomeric probe. Control reactions were performed in absence of phi29 polymerase (-Φ29). ( b ) Quantifications of C-circle signals from experiments as in a . Bars and error bars are means and SEMs from 3 independent experiments. Circles are single data points.
    Figure Legend Snippet: C-circle analysis in T-TALE cells. ( a ) C-circle assay analysis of genomic DNA from the indicated cells treated with dox for up to 72 hours. Reaction products were dot-blotted and hybridized to a radiolabeled telomeric probe. Control reactions were performed in absence of phi29 polymerase (-Φ29). ( b ) Quantifications of C-circle signals from experiments as in a . Bars and error bars are means and SEMs from 3 independent experiments. Circles are single data points.

    Techniques Used:

    30) Product Images from "A Comparison of Methods for the Extraction of Plasmids Capable of Conferring Antibiotic Resistance in a Human Pathogen From Complex Broiler Cecal Samples"

    Article Title: A Comparison of Methods for the Extraction of Plasmids Capable of Conferring Antibiotic Resistance in a Human Pathogen From Complex Broiler Cecal Samples

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.01731

    Plasmid DNA from the cecal sample after amplification with phi29 polymerase. 1 , 1 kb ladder and 2 , Plasmid DNA amplified with Phi29 DNA polymerase.
    Figure Legend Snippet: Plasmid DNA from the cecal sample after amplification with phi29 polymerase. 1 , 1 kb ladder and 2 , Plasmid DNA amplified with Phi29 DNA polymerase.

    Techniques Used: Plasmid Preparation, Amplification

    Digested plasmid DNA extracted from E. coli transformants after electroporation with the phi29 polymerase amplified DNA. 1 , 1 kb ladder; Plasmid DNA extracted from transformants selected on agar plates containing; 2 , ampicillin 32 mg/L (M_Amp_BC); 3 , ampicillin 32 mg/L (M_Amp_SC); 4 , tetracycline 16 mg/L (M_Tet_BC); 5 , tetracycline 16 mg/L (M_Tet_SC); 6 , kanamycin 25 mg/L (M_Kan); 7 , ciprofloxacin 16 mg/L (M_Cip). BC and SC refer to the two different colony morphology types, big or small colonies, on the same antibiotic plate.
    Figure Legend Snippet: Digested plasmid DNA extracted from E. coli transformants after electroporation with the phi29 polymerase amplified DNA. 1 , 1 kb ladder; Plasmid DNA extracted from transformants selected on agar plates containing; 2 , ampicillin 32 mg/L (M_Amp_BC); 3 , ampicillin 32 mg/L (M_Amp_SC); 4 , tetracycline 16 mg/L (M_Tet_BC); 5 , tetracycline 16 mg/L (M_Tet_SC); 6 , kanamycin 25 mg/L (M_Kan); 7 , ciprofloxacin 16 mg/L (M_Cip). BC and SC refer to the two different colony morphology types, big or small colonies, on the same antibiotic plate.

    Techniques Used: Plasmid Preparation, Electroporation, Amplification

    31) Product Images from "Assessing Protein Dynamics on Low-Complexity Single-Stranded DNA Curtains"

    Article Title: Assessing Protein Dynamics on Low-Complexity Single-Stranded DNA Curtains

    Journal: Langmuir : the ACS journal of surfaces and colloids

    doi: 10.1021/acs.langmuir.8b01812

    Assembly of low-complexity ssDNA curtains. (A) A phosphorylated template (black) and a biotinylated primer (green) are annealed and treated with T4 DNA ligase to make minicircles. Low-complexity ssDNA composed solely of thymidine and cytidine is synthesized via rolling circle replication by phi29 DNAP. (B) Low-complexity ssDNA curtains with fluorescent end labeling. The 3′ end of the ssDNA was labeled with a fluorescent antibody. (C) RPA-GFP (green)-coated ssDNA with fluorescent end labeling (magenta). (D) Kymograph of a representative ssDNA in panel (C) with buffer flow on and off, indicating that the ssDNA is anchored to the surface via the 5′-biotin tether.
    Figure Legend Snippet: Assembly of low-complexity ssDNA curtains. (A) A phosphorylated template (black) and a biotinylated primer (green) are annealed and treated with T4 DNA ligase to make minicircles. Low-complexity ssDNA composed solely of thymidine and cytidine is synthesized via rolling circle replication by phi29 DNAP. (B) Low-complexity ssDNA curtains with fluorescent end labeling. The 3′ end of the ssDNA was labeled with a fluorescent antibody. (C) RPA-GFP (green)-coated ssDNA with fluorescent end labeling (magenta). (D) Kymograph of a representative ssDNA in panel (C) with buffer flow on and off, indicating that the ssDNA is anchored to the surface via the 5′-biotin tether.

    Techniques Used: Synthesized, End Labeling, Labeling, Recombinase Polymerase Amplification, Flow Cytometry

    32) Product Images from "Template-dependent multiple displacement amplification for profiling human circulating RNA"

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

    Journal: BioTechniques

    doi: 10.2144/000114566

    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.
    Figure Legend 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.

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

    33) Product Images from "High-Resolution Phylogenetic and Population Genetic Analysis of Microbial Communities with RoC-ITS"

    Article Title: High-Resolution Phylogenetic and Population Genetic Analysis of Microbial Communities with RoC-ITS

    Journal: bioRxiv

    doi: 10.1101/2020.10.16.342691

    Diagram illustrating the RoC-ITS strategy. The 16S-ITS region is isolated using PCR and Gibson Assembly is used to circularize the products. A long single-stranded DNA product consisting of multiple iterations of the same 16S-ITS region is generated using the phi29 polymerase. Finally, the linear product is used to create an Oxford Nanopore DNA library for sequencing.
    Figure Legend Snippet: Diagram illustrating the RoC-ITS strategy. The 16S-ITS region is isolated using PCR and Gibson Assembly is used to circularize the products. A long single-stranded DNA product consisting of multiple iterations of the same 16S-ITS region is generated using the phi29 polymerase. Finally, the linear product is used to create an Oxford Nanopore DNA library for sequencing.

    Techniques Used: Isolation, Polymerase Chain Reaction, Generated, Sequencing

    34) Product Images from "Advanced microRNA-based cancer diagnostics using amplified time-gated FRET Advanced microRNA-based cancer diagnostics using amplified time-gated FRET †Electronic supplementary information (ESI) available. See DOI: 10.1039/c8sc03121e"

    Article Title: Advanced microRNA-based cancer diagnostics using amplified time-gated FRET Advanced microRNA-based cancer diagnostics using amplified time-gated FRET †Electronic supplementary information (ESI) available. See DOI: 10.1039/c8sc03121e

    Journal: Chemical Science

    doi: 10.1039/c8sc03121e

    Principle of miRNA detection by amplified TG-FRET. (A) After specific recognition of miRNA by a linear padlock DNA (1), the DNA padlock nick is ligated over the miRNA target splint using SplintR ligase (2) and the miRNA becomes a primer for a phi29 polymerase to synthesize and displace (by RCA) complimentary DNA around the circularized padlock DNA (3). After stopping RCA, the rolling circle product (RCP) is incubated with Tb (Lumi4-Tb) donor and Cy5.5 acceptor labeled ssDNA, which hybridize to specific sequences that exist more than 1000-fold on the amplified RCP concatemer. The close distance of Lumi4-Tb and Cy5.5 in the RCP allows for Tb-to-Cy5.5 FRET, which is not possible if both are free in solution (not hybridized to the RCP). Thus, the TG-FRET signal can be used for quantifying miRNA without any washing or separation steps. (B) Ratiometric TG-FRET, which measures the ratio of FRET-sensitized Cy5.5 photoluminescence (PL) and FRET-quenched Tb PL within a specific time-window after pulsed excitation (to suppress autofluorescence), is used to quantify the miRNA target in a 140 μl microwell within 5 seconds.
    Figure Legend Snippet: Principle of miRNA detection by amplified TG-FRET. (A) After specific recognition of miRNA by a linear padlock DNA (1), the DNA padlock nick is ligated over the miRNA target splint using SplintR ligase (2) and the miRNA becomes a primer for a phi29 polymerase to synthesize and displace (by RCA) complimentary DNA around the circularized padlock DNA (3). After stopping RCA, the rolling circle product (RCP) is incubated with Tb (Lumi4-Tb) donor and Cy5.5 acceptor labeled ssDNA, which hybridize to specific sequences that exist more than 1000-fold on the amplified RCP concatemer. The close distance of Lumi4-Tb and Cy5.5 in the RCP allows for Tb-to-Cy5.5 FRET, which is not possible if both are free in solution (not hybridized to the RCP). Thus, the TG-FRET signal can be used for quantifying miRNA without any washing or separation steps. (B) Ratiometric TG-FRET, which measures the ratio of FRET-sensitized Cy5.5 photoluminescence (PL) and FRET-quenched Tb PL within a specific time-window after pulsed excitation (to suppress autofluorescence), is used to quantify the miRNA target in a 140 μl microwell within 5 seconds.

    Techniques Used: Amplification, Incubation, Labeling

    35) Product Images from "A Comparison of Methods for the Extraction of Plasmids Capable of Conferring Antibiotic Resistance in a Human Pathogen From Complex Broiler Cecal Samples"

    Article Title: A Comparison of Methods for the Extraction of Plasmids Capable of Conferring Antibiotic Resistance in a Human Pathogen From Complex Broiler Cecal Samples

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.01731

    Plasmid DNA from the cecal sample after amplification with phi29 polymerase. 1 , 1 kb ladder and 2 , Plasmid DNA amplified with Phi29 DNA polymerase.
    Figure Legend Snippet: Plasmid DNA from the cecal sample after amplification with phi29 polymerase. 1 , 1 kb ladder and 2 , Plasmid DNA amplified with Phi29 DNA polymerase.

    Techniques Used: Plasmid Preparation, Amplification

    Digested plasmid DNA extracted from E. coli transformants after electroporation with the phi29 polymerase amplified DNA. 1 , 1 kb ladder; Plasmid DNA extracted from transformants selected on agar plates containing; 2 , ampicillin 32 mg/L (M_Amp_BC); 3 , ampicillin 32 mg/L (M_Amp_SC); 4 , tetracycline 16 mg/L (M_Tet_BC); 5 , tetracycline 16 mg/L (M_Tet_SC); 6 , kanamycin 25 mg/L (M_Kan); 7 , ciprofloxacin 16 mg/L (M_Cip). BC and SC refer to the two different colony morphology types, big or small colonies, on the same antibiotic plate.
    Figure Legend Snippet: Digested plasmid DNA extracted from E. coli transformants after electroporation with the phi29 polymerase amplified DNA. 1 , 1 kb ladder; Plasmid DNA extracted from transformants selected on agar plates containing; 2 , ampicillin 32 mg/L (M_Amp_BC); 3 , ampicillin 32 mg/L (M_Amp_SC); 4 , tetracycline 16 mg/L (M_Tet_BC); 5 , tetracycline 16 mg/L (M_Tet_SC); 6 , kanamycin 25 mg/L (M_Kan); 7 , ciprofloxacin 16 mg/L (M_Cip). BC and SC refer to the two different colony morphology types, big or small colonies, on the same antibiotic plate.

    Techniques Used: Plasmid Preparation, Electroporation, Amplification

    36) Product Images from "DeNAno: selectable deoxyribonucleic acid nanoparticle libraries"

    Article Title: DeNAno: selectable deoxyribonucleic acid nanoparticle libraries

    Journal: Journal of biotechnology

    doi: 10.1016/j.jbiotec.2009.12.002

    DNA nanoparticle iterative selection scheme. ssDNA libraries are ligated with T4 ligase and polymerized with phi29 DNA polymerase. 3'–5' exonuclease activity of phi29 DNA polymerase ensures nanoparticle purity from extraneous DNA. Immature DCs
    Figure Legend Snippet: DNA nanoparticle iterative selection scheme. ssDNA libraries are ligated with T4 ligase and polymerized with phi29 DNA polymerase. 3'–5' exonuclease activity of phi29 DNA polymerase ensures nanoparticle purity from extraneous DNA. Immature DCs

    Techniques Used: Selection, Activity Assay

    37) Product Images from "Tapping diversity lost in transformations--in vitro amplification of ligation reactions"

    Article Title: Tapping diversity lost in transformations--in vitro amplification of ligation reactions

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl605

    Antibody repertoire by combinatorial ligation. Regions corresponding to CDRs 1/2 and CDR 3 were PCR-amplified and recombined by ligation into a PCR-amplified phagemid vector backbone. The ligation reactions were either directly electroporated or amplified using Phi29 polymerase followed by electroporation into E.coli bacteria.
    Figure Legend Snippet: Antibody repertoire by combinatorial ligation. Regions corresponding to CDRs 1/2 and CDR 3 were PCR-amplified and recombined by ligation into a PCR-amplified phagemid vector backbone. The ligation reactions were either directly electroporated or amplified using Phi29 polymerase followed by electroporation into E.coli bacteria.

    Techniques Used: Ligation, Polymerase Chain Reaction, Amplification, Plasmid Preparation, Electroporation

    In vitro amplification of ligation reactions. Linear fragments were joined into recombinant, circular units by treatment with DNA ligase. Hexamer primers were annealed and Phi29 polymerase added. This causes extensive amplification of circular species through rolling-circle replication and the formation of extended linear concatemers. The concatemers were cleaved by restriction digestion and re-circularized using DNA ligase.
    Figure Legend Snippet: In vitro amplification of ligation reactions. Linear fragments were joined into recombinant, circular units by treatment with DNA ligase. Hexamer primers were annealed and Phi29 polymerase added. This causes extensive amplification of circular species through rolling-circle replication and the formation of extended linear concatemers. The concatemers were cleaved by restriction digestion and re-circularized using DNA ligase.

    Techniques Used: In Vitro, Amplification, Ligation, Recombinant

    38) Product Images from "Tapping diversity lost in transformations--in vitro amplification of ligation reactions"

    Article Title: Tapping diversity lost in transformations--in vitro amplification of ligation reactions

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl605

    Antibody repertoire by combinatorial ligation. Regions corresponding to CDRs 1/2 and CDR 3 were PCR-amplified and recombined by ligation into a PCR-amplified phagemid vector backbone. The ligation reactions were either directly electroporated or amplified using Phi29 polymerase followed by electroporation into E.coli bacteria.
    Figure Legend Snippet: Antibody repertoire by combinatorial ligation. Regions corresponding to CDRs 1/2 and CDR 3 were PCR-amplified and recombined by ligation into a PCR-amplified phagemid vector backbone. The ligation reactions were either directly electroporated or amplified using Phi29 polymerase followed by electroporation into E.coli bacteria.

    Techniques Used: Ligation, Polymerase Chain Reaction, Amplification, Plasmid Preparation, Electroporation

    In vitro amplification of ligation reactions. Linear fragments were joined into recombinant, circular units by treatment with DNA ligase. Hexamer primers were annealed and Phi29 polymerase added. This causes extensive amplification of circular species through rolling-circle replication and the formation of extended linear concatemers. The concatemers were cleaved by restriction digestion and re-circularized using DNA ligase.
    Figure Legend Snippet: In vitro amplification of ligation reactions. Linear fragments were joined into recombinant, circular units by treatment with DNA ligase. Hexamer primers were annealed and Phi29 polymerase added. This causes extensive amplification of circular species through rolling-circle replication and the formation of extended linear concatemers. The concatemers were cleaved by restriction digestion and re-circularized using DNA ligase.

    Techniques Used: In Vitro, Amplification, Ligation, Recombinant

    39) Product Images from "A Comparison of Methods for the Extraction of Plasmids Capable of Conferring Antibiotic Resistance in a Human Pathogen From Complex Broiler Cecal Samples"

    Article Title: A Comparison of Methods for the Extraction of Plasmids Capable of Conferring Antibiotic Resistance in a Human Pathogen From Complex Broiler Cecal Samples

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.01731

    Plasmid DNA from the cecal sample after amplification with phi29 polymerase. 1 , 1 kb ladder and 2 , Plasmid DNA amplified with Phi29 DNA polymerase.
    Figure Legend Snippet: Plasmid DNA from the cecal sample after amplification with phi29 polymerase. 1 , 1 kb ladder and 2 , Plasmid DNA amplified with Phi29 DNA polymerase.

    Techniques Used: Plasmid Preparation, Amplification

    Digested plasmid DNA extracted from E. coli transformants after electroporation with the phi29 polymerase amplified DNA. 1 , 1 kb ladder; Plasmid DNA extracted from transformants selected on agar plates containing; 2 , ampicillin 32 mg/L (M_Amp_BC); 3 , ampicillin 32 mg/L (M_Amp_SC); 4 , tetracycline 16 mg/L (M_Tet_BC); 5 , tetracycline 16 mg/L (M_Tet_SC); 6 , kanamycin 25 mg/L (M_Kan); 7 , ciprofloxacin 16 mg/L (M_Cip). BC and SC refer to the two different colony morphology types, big or small colonies, on the same antibiotic plate.
    Figure Legend Snippet: Digested plasmid DNA extracted from E. coli transformants after electroporation with the phi29 polymerase amplified DNA. 1 , 1 kb ladder; Plasmid DNA extracted from transformants selected on agar plates containing; 2 , ampicillin 32 mg/L (M_Amp_BC); 3 , ampicillin 32 mg/L (M_Amp_SC); 4 , tetracycline 16 mg/L (M_Tet_BC); 5 , tetracycline 16 mg/L (M_Tet_SC); 6 , kanamycin 25 mg/L (M_Kan); 7 , ciprofloxacin 16 mg/L (M_Cip). BC and SC refer to the two different colony morphology types, big or small colonies, on the same antibiotic plate.

    Techniques Used: Plasmid Preparation, Electroporation, Amplification

    Related Articles

    Amplification:

    Article Title: Improvements of rolling circle amplification (RCA) efficiency and accuracy using Thermus thermophilus SSB mutant protein
    Article Snippet: Template DNA was mixed with 0.5 µl hexamers (400 ng/µl, Sigma Genosys) and 0.5 µl binding buffer (400 mM Tris–HCl at pH 8.0 and 160 mM KCl) and denatured at 95°C for 4 min. .. The denatured DNA was amplified using 0.3 µl Phi29 DNA polymerase (10 U/µl, New England Biolabs) complemented with 2 µl of 10× Phi29 DNA polymerase buffer, 0.2 µl of 100× BSA, 3.2 µl of 2.5 mM dNTP and 1 µl of 20% DMSO (Sigma Aldrich) in a volume of 20 µl at 30°C for ∼24 h. The phi29 DNA polymerase was inactivated at 65°C for 10 min and the amplification product was purified using a spin-column (Sephadex G-50, Amersham Biosciences) to eliminate the un-reacted primers. .. DNA spot hybridization To perform DNA spot hybridizations, DNA samples were mixed with 30 µl of DNA denaturation solution.

    Article Title: New approaches to the analysis of palindromic sequences from the human genome: evolution and polymorphism of an intronic site at the NF1 locus
    Article Snippet: .. The amplification mix contained 50 µM exonuclease-resistant random hexamers (‘machine-mixed’ thiophosphate-modified random hexamers, 5′-NpNpNpNps Nps N-3′; Integrated DNA Technologies), 600 µM dNTP (Pharmacia), 100 µg/ml BSA (New England Biolabs) and 3 U of phi-29 polymerase (New England BioLabs) in 1× buffer 4 (as above). ..

    Article Title: Efficient amplification of self-gelling polypod-like structured DNA by rolling circle amplification and enzymatic digestion
    Article Snippet: .. The resultant mixture (10 μM) was amplified by incubating at 30 °C for 16 h in a solution containing 2.5 U/μL phi29 DNA polymerase (New England Biolabs, Ipswich, MA, USA), 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2 , 10 mM (NH4 )2 SO4, 4 mM DTT, 200 μg/ml BSA, and 2.5 mM dNTP (Invitrogen, Carlsbad, CA, USA). .. Polypodna production by restriction digestionThe highly viscous RCA product was incubated in 2 mM EDTA (Sigma-Aldrich, St. Louis, MO, USA) at 80 °C for 15 min to solubilize the product.

    Article Title: Quantification and epigenetic evaluation of the residual pool of hepatitis B covalently closed circular DNA in long-term nucleoside analogue-treated patients
    Article Snippet: We assessed the presence of intrahepatic cccDNA in qPCR-negative samples using this one-step PCR method without additional steps of completion and ligation, as previously described using primers RCA1-8 listed in Supplementary Table and Supplementary Fig. (Fig. a) , . .. The amplification of circular HBV DNA was performed using Phi29 polymerase (New England Biolabs, Ipswich, MA, USA) for 21 h at 30 °C on total DNA extracted from frozen liver samples. ..

    Purification:

    Article Title: Improvements of rolling circle amplification (RCA) efficiency and accuracy using Thermus thermophilus SSB mutant protein
    Article Snippet: Template DNA was mixed with 0.5 µl hexamers (400 ng/µl, Sigma Genosys) and 0.5 µl binding buffer (400 mM Tris–HCl at pH 8.0 and 160 mM KCl) and denatured at 95°C for 4 min. .. The denatured DNA was amplified using 0.3 µl Phi29 DNA polymerase (10 U/µl, New England Biolabs) complemented with 2 µl of 10× Phi29 DNA polymerase buffer, 0.2 µl of 100× BSA, 3.2 µl of 2.5 mM dNTP and 1 µl of 20% DMSO (Sigma Aldrich) in a volume of 20 µl at 30°C for ∼24 h. The phi29 DNA polymerase was inactivated at 65°C for 10 min and the amplification product was purified using a spin-column (Sephadex G-50, Amersham Biosciences) to eliminate the un-reacted primers. .. DNA spot hybridization To perform DNA spot hybridizations, DNA samples were mixed with 30 µl of DNA denaturation solution.

    Article Title: Next-Generation DNA Curtains for Single-Molecule Studies of Homologous Recombination
    Article Snippet: Below, we use a previously characterized fluorescent RAD51(C319S) to demonstrate efficient RAD51 binding independent of other secondary-structure melting factors ( ) ( ). .. TE buffer: 10m M Tris–HCl [pH 8.0]; 0.1m M EDTA RAD51 buffer: 40m M Tris–HCl [pH 8.0]; 1m M MgCl2 ; 5m M CaCl2 ; 100m M KCl; 1m M DTT; 1m M ATP; 0.2 mgmL−1 BSA; 1m M Trolox (Sigma-Aldrich); 1.0% glucose (w/v); 500units catalase (Sigma-Aldrich); 70units glucose oxidase (Sigma-Aldrich) 10× T4 DNA ligase reaction buffer (B0202S; NEB) T4 DNA ligase (M0202; NEB) Primer oligo (/Biosg/TC TCC TCC TTC T—HPLC purified; Integrated DNA Technologies) Template oligo (/5Phos/AG GAG AAA AAG AAA AAA AGA AAA GAA GG—PAGE purified; Integrated DNA Technologies) Nuclease-free water BSA, Molecular Biology Grade (B9000S; NEB) Thermocycler (Mastercycler pro S; Eppendorf ) 10× phi29 DNA polymerase reaction buffer (B0269S; NEB) phi29 DNA polymerase (homemade 5 μ M stock) Deoxynucleotide (dNTP) solution set (N0446S; NEB) .. Prepare a 49 μL ligation reaction containing: (i) 5 μL 10× T4 ligase reaction buffer; (ii) 2 μL template oligo (10 μ M stock in TE buffer); (iii) 1.8 μL primer oligo (10 μ M stock in TE buffer); and (iv) 40.2 μL nuclease-free water.

    Concentration Assay:

    Article Title: Limited reverse transcriptase activity of phi29 DNA polymerase
    Article Snippet: First, RCA products from the real-time RCA measurements, as described above, were diluted in PBS-Tween 0.05% to a concentration of 100 pM. .. Next, RCA products were digested with AluI restriction enzyme in a reaction mixture containing 1 × phi29 DNA polymerase buffer, 0.2 μg/μl BSA, 100 nM restriction oligonucleotide , 120 mU/μl AluI (NEB) and RCA products at a final concentration of 10 pM during 10 min incubation at 37°C. .. Subsequently, the enzyme was heat inactivated at 65°C for 2 min. After complete digestion of the 10 pM RCA products, the RCA monomer concentration is approximately 10 nM (1 h RCA of an 80 base circle yields ∼1000× amplification).

    Incubation:

    Article Title: Limited reverse transcriptase activity of phi29 DNA polymerase
    Article Snippet: First, RCA products from the real-time RCA measurements, as described above, were diluted in PBS-Tween 0.05% to a concentration of 100 pM. .. Next, RCA products were digested with AluI restriction enzyme in a reaction mixture containing 1 × phi29 DNA polymerase buffer, 0.2 μg/μl BSA, 100 nM restriction oligonucleotide , 120 mU/μl AluI (NEB) and RCA products at a final concentration of 10 pM during 10 min incubation at 37°C. .. Subsequently, the enzyme was heat inactivated at 65°C for 2 min. After complete digestion of the 10 pM RCA products, the RCA monomer concentration is approximately 10 nM (1 h RCA of an 80 base circle yields ∼1000× amplification).

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    New England Biolabs phi29 dna polymerase
    Schematic diagram of the mass amplification of tripodna with adhesive 5′-ends. The template oligodeoxynucleotides (template 1) were designed to satisfy the following requirements: ( a ) the polypodna automatically forms by self-assembly; ( b ) each pod of the polypodna contains a 9 base long TspRI restriction digest site; ( c ) Each 5′-terminal end is phosphorylated in order to ligate with 3′-terminal within the polypodna body, and ( d ) connecting chain is added to the 3′-terminal of the polypodna to allow polypodna to be connected to one another. The designed templates were amplified via the following steps. ( 1 ) The template ssODNs were circularized using T4 <t>DNA</t> ligase. ( 2 ) After annealing the primer (primer 1), the DNA template was amplified through rolling circle amplification technique using <t>Phi29</t> polymerase. ( 3 ) Before enzyme digestion, the RCA product was treated with EDTA and folded. ( 4 ) Long single-stranded DNAs were digested using restriction enzyme. ( 5 ) The target sequences were purified by size chromatography. ( 6 ) The resultant DNAs self-assembled after annealing, and they formed a hydrogel.
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    Schematic diagram of the mass amplification of tripodna with adhesive 5′-ends. The template oligodeoxynucleotides (template 1) were designed to satisfy the following requirements: ( a ) the polypodna automatically forms by self-assembly; ( b ) each pod of the polypodna contains a 9 base long TspRI restriction digest site; ( c ) Each 5′-terminal end is phosphorylated in order to ligate with 3′-terminal within the polypodna body, and ( d ) connecting chain is added to the 3′-terminal of the polypodna to allow polypodna to be connected to one another. The designed templates were amplified via the following steps. ( 1 ) The template ssODNs were circularized using T4 DNA ligase. ( 2 ) After annealing the primer (primer 1), the DNA template was amplified through rolling circle amplification technique using Phi29 polymerase. ( 3 ) Before enzyme digestion, the RCA product was treated with EDTA and folded. ( 4 ) Long single-stranded DNAs were digested using restriction enzyme. ( 5 ) The target sequences were purified by size chromatography. ( 6 ) The resultant DNAs self-assembled after annealing, and they formed a hydrogel.

    Journal: Scientific Reports

    Article Title: Efficient amplification of self-gelling polypod-like structured DNA by rolling circle amplification and enzymatic digestion

    doi: 10.1038/srep14979

    Figure Lengend Snippet: Schematic diagram of the mass amplification of tripodna with adhesive 5′-ends. The template oligodeoxynucleotides (template 1) were designed to satisfy the following requirements: ( a ) the polypodna automatically forms by self-assembly; ( b ) each pod of the polypodna contains a 9 base long TspRI restriction digest site; ( c ) Each 5′-terminal end is phosphorylated in order to ligate with 3′-terminal within the polypodna body, and ( d ) connecting chain is added to the 3′-terminal of the polypodna to allow polypodna to be connected to one another. The designed templates were amplified via the following steps. ( 1 ) The template ssODNs were circularized using T4 DNA ligase. ( 2 ) After annealing the primer (primer 1), the DNA template was amplified through rolling circle amplification technique using Phi29 polymerase. ( 3 ) Before enzyme digestion, the RCA product was treated with EDTA and folded. ( 4 ) Long single-stranded DNAs were digested using restriction enzyme. ( 5 ) The target sequences were purified by size chromatography. ( 6 ) The resultant DNAs self-assembled after annealing, and they formed a hydrogel.

    Article Snippet: The resultant mixture (10 μM) was amplified by incubating at 30 °C for 16 h in a solution containing 2.5 U/μL phi29 DNA polymerase (New England Biolabs, Ipswich, MA, USA), 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2 , 10 mM (NH4 )2 SO4 , 4 mM DTT, 200 μg/ml BSA, and 2.5 mM dNTP (Invitrogen, Carlsbad, CA, USA).

    Techniques: Amplification, Purification, Chromatography

    Side-by-side comparison of microbial hosts for their ability to maintain the same plasmid. ( A ) Universal deletion in E.coli Top 10 cells. Plasmids re-isolated from TOP 10 clones transformed with the H4#4 plasmid are deleted. Each was XbaI and PvuII digested. H4#4 DNA (also cut with XbaI and PvuII after a phi-29 amplification) is loaded adjacent to the marker lane. Note, a different 100 bp ladder was used here (New England Biolabs) which has an intense 500 bp rather than 600 bp band as in previous figures. ( B ) Instability of the H4#4 plasmid in E.coli SURE cells. Plasmid DNA from individual SURE H4#4 transformants is a mixture of deleted and apparently non-deleted forms despite the lack of a functional SbcCD nuclease. The gel image was cut to remove one lane. ( C ) Stability of H4#4 plasmid in wild-type yeast. Phi-29 amplified minipreparations of DNA from random wild-type yeast clones transformed with H4#4 DNA were digested with XbaI and PvuII. Full-length inserts are observed. ( D ) Phi-29 amplified minipreparations of DNA from random sae2 yeast clones transformed and analyzed as in (C). In A–D, dots mark samples from colonies that were re-streaked as described in the text.

    Journal: Nucleic Acids Research

    Article Title: New approaches to the analysis of palindromic sequences from the human genome: evolution and polymorphism of an intronic site at the NF1 locus

    doi: 10.1093/nar/gni189

    Figure Lengend Snippet: Side-by-side comparison of microbial hosts for their ability to maintain the same plasmid. ( A ) Universal deletion in E.coli Top 10 cells. Plasmids re-isolated from TOP 10 clones transformed with the H4#4 plasmid are deleted. Each was XbaI and PvuII digested. H4#4 DNA (also cut with XbaI and PvuII after a phi-29 amplification) is loaded adjacent to the marker lane. Note, a different 100 bp ladder was used here (New England Biolabs) which has an intense 500 bp rather than 600 bp band as in previous figures. ( B ) Instability of the H4#4 plasmid in E.coli SURE cells. Plasmid DNA from individual SURE H4#4 transformants is a mixture of deleted and apparently non-deleted forms despite the lack of a functional SbcCD nuclease. The gel image was cut to remove one lane. ( C ) Stability of H4#4 plasmid in wild-type yeast. Phi-29 amplified minipreparations of DNA from random wild-type yeast clones transformed with H4#4 DNA were digested with XbaI and PvuII. Full-length inserts are observed. ( D ) Phi-29 amplified minipreparations of DNA from random sae2 yeast clones transformed and analyzed as in (C). In A–D, dots mark samples from colonies that were re-streaked as described in the text.

    Article Snippet: The amplification mix contained 50 µM exonuclease-resistant random hexamers (‘machine-mixed’ thiophosphate-modified random hexamers, 5′-NpNpNpNps Nps N-3′; Integrated DNA Technologies), 600 µM dNTP (Pharmacia), 100 µg/ml BSA (New England Biolabs) and 3 U of phi-29 polymerase (New England BioLabs) in 1× buffer 4 (as above).

    Techniques: Plasmid Preparation, Isolation, Clone Assay, Transformation Assay, Amplification, Marker, Functional Assay

    (A) Colony PCR analysis of yeast clones obtained from H1 and H4 genomic DNA by the method shown in Figure 1B . Ligated samples (see legend, Figure 2 ) were used to transform S.cerevisiae . In contrast to results with E.coli ( Figure 2 ), full-length candidate clones were obtained. Lanes with dots are from colonies used in further analyses (see text). The DNA ladder is as in Figures 1 and 2 . ( B ) Verification of clone structure. To confirm the structure of the full-length candidates, yeast minipreps were treated with EcoR1 and subjected to random-primed rolling circle amplification with phi-29 DNA polymerase. As an example, H4#4 is displayed on a 2.5% agarose gel after diagnostic digestion with Xbal and PvuII. Upper and lower arrows indicate the vector backbone and insert bands, respectively. ( C ) Efficacy of the EcoR1 pre-digestion. The EcoR1 pre-treated sample in B (‘+’) is run on a 1% agarose gel alongside an untreated (‘−’) miniprep of H4#4. Treated and untreated samples were phi-29 amplified in parallel and digested with XbaI and PvuII. Bands evident in the untreated lane correspond in size to those predicted for 2 µ circle DNA. Bands observed with EcoR1 pre-treatment (arrows) correspond to the pYes2.1 vector and insert.

    Journal: Nucleic Acids Research

    Article Title: New approaches to the analysis of palindromic sequences from the human genome: evolution and polymorphism of an intronic site at the NF1 locus

    doi: 10.1093/nar/gni189

    Figure Lengend Snippet: (A) Colony PCR analysis of yeast clones obtained from H1 and H4 genomic DNA by the method shown in Figure 1B . Ligated samples (see legend, Figure 2 ) were used to transform S.cerevisiae . In contrast to results with E.coli ( Figure 2 ), full-length candidate clones were obtained. Lanes with dots are from colonies used in further analyses (see text). The DNA ladder is as in Figures 1 and 2 . ( B ) Verification of clone structure. To confirm the structure of the full-length candidates, yeast minipreps were treated with EcoR1 and subjected to random-primed rolling circle amplification with phi-29 DNA polymerase. As an example, H4#4 is displayed on a 2.5% agarose gel after diagnostic digestion with Xbal and PvuII. Upper and lower arrows indicate the vector backbone and insert bands, respectively. ( C ) Efficacy of the EcoR1 pre-digestion. The EcoR1 pre-treated sample in B (‘+’) is run on a 1% agarose gel alongside an untreated (‘−’) miniprep of H4#4. Treated and untreated samples were phi-29 amplified in parallel and digested with XbaI and PvuII. Bands evident in the untreated lane correspond in size to those predicted for 2 µ circle DNA. Bands observed with EcoR1 pre-treatment (arrows) correspond to the pYes2.1 vector and insert.

    Article Snippet: The amplification mix contained 50 µM exonuclease-resistant random hexamers (‘machine-mixed’ thiophosphate-modified random hexamers, 5′-NpNpNpNps Nps N-3′; Integrated DNA Technologies), 600 µM dNTP (Pharmacia), 100 µg/ml BSA (New England Biolabs) and 3 U of phi-29 polymerase (New England BioLabs) in 1× buffer 4 (as above).

    Techniques: Polymerase Chain Reaction, Clone Assay, Random Primed, Amplification, Agarose Gel Electrophoresis, Diagnostic Assay, Plasmid Preparation

    ( A ) Map (to scale) of the palindromic region within a 3.8 kb EcoR1 fragment of the NF1 gene. Exons are numbered according to L05367 (see text). ‘R1’ denotes EcoR1 sites according to Southern blot data ( 27 ) and the reference human genome sequence (May 2004). ( B ) Cloning strategy. Oligos used for PCR flank the palindromic site. The PCR product is ligated into a commercial vector (see Materials and Methods). ‘X’ and ‘P’ refer to the Xbal and PvuII sites used in diagnostic digests. ( C ) PCR amplification of various DNA templates. Lane ‘M’, markers (Trackit 100 bp ladder, Invitrogen). ‘B’ is a PCR with Bac clone CTD-2370N5; ‘H1’ to ‘H5’ are with human genomic DNA from the indicated individuals. ‘C’ is with chimpanzee DNA. ‘φH4’ and ‘φG’ are from an aliquot of H4 and gorilla genomic DNA that had first been amplified with phi-29 polymerase. The H4 samples demonstrate reproducibility of the PCR as well as the fidelity of phi-29 amplification.

    Journal: Nucleic Acids Research

    Article Title: New approaches to the analysis of palindromic sequences from the human genome: evolution and polymorphism of an intronic site at the NF1 locus

    doi: 10.1093/nar/gni189

    Figure Lengend Snippet: ( A ) Map (to scale) of the palindromic region within a 3.8 kb EcoR1 fragment of the NF1 gene. Exons are numbered according to L05367 (see text). ‘R1’ denotes EcoR1 sites according to Southern blot data ( 27 ) and the reference human genome sequence (May 2004). ( B ) Cloning strategy. Oligos used for PCR flank the palindromic site. The PCR product is ligated into a commercial vector (see Materials and Methods). ‘X’ and ‘P’ refer to the Xbal and PvuII sites used in diagnostic digests. ( C ) PCR amplification of various DNA templates. Lane ‘M’, markers (Trackit 100 bp ladder, Invitrogen). ‘B’ is a PCR with Bac clone CTD-2370N5; ‘H1’ to ‘H5’ are with human genomic DNA from the indicated individuals. ‘C’ is with chimpanzee DNA. ‘φH4’ and ‘φG’ are from an aliquot of H4 and gorilla genomic DNA that had first been amplified with phi-29 polymerase. The H4 samples demonstrate reproducibility of the PCR as well as the fidelity of phi-29 amplification.

    Article Snippet: The amplification mix contained 50 µM exonuclease-resistant random hexamers (‘machine-mixed’ thiophosphate-modified random hexamers, 5′-NpNpNpNps Nps N-3′; Integrated DNA Technologies), 600 µM dNTP (Pharmacia), 100 µg/ml BSA (New England Biolabs) and 3 U of phi-29 polymerase (New England BioLabs) in 1× buffer 4 (as above).

    Techniques: Southern Blot, Sequencing, Clone Assay, Polymerase Chain Reaction, Plasmid Preparation, Diagnostic Assay, Amplification, BAC Assay

    Replication, recircularization, and compaction of a plasmid containing the T7 promoter. A) Time course of RCA‐based replication of pRepC plasmid containing the T7 promoter and loxP sites (depicted in Figure S1), measured as fluorescence of the DNA‐binding PicoGreen dye. Reaction mixtures contained, as indicated, Phi29 DNA polymerase, T7 DNA polymerase, and T7 RNA polymerase, as well as specific or random primers. For a control reaction with T7 DNA polymerase and T7 RNA polymerase, a pQE30 plasmid lacking the T7 promoter was used. B) Recircularization of the replicated plasmid, mediated by Cre recombinase. Where indicated, Cre recombinase was added after 16 h of replication, and the reaction mixture was incubated for another 30 min. Reaction mixtures were separated along with a DNA ladder (1 kb) on a Midori‐green stained agarose gel. The lower band migrating below 5 kb corresponds to the circularized DNA, whereas larger products apparently correspond to linear concatamers (Figure S2). C) DNA nanoparticles emerging upon prolonged ( > 12 h) T7 DNA replication reaction. Scale bar: 10 μm.

    Journal: Chembiochem

    Article Title: Reconstitution and Coupling of DNA Replication and Segregation in a Biomimetic System

    doi: 10.1002/cbic.201900299

    Figure Lengend Snippet: Replication, recircularization, and compaction of a plasmid containing the T7 promoter. A) Time course of RCA‐based replication of pRepC plasmid containing the T7 promoter and loxP sites (depicted in Figure S1), measured as fluorescence of the DNA‐binding PicoGreen dye. Reaction mixtures contained, as indicated, Phi29 DNA polymerase, T7 DNA polymerase, and T7 RNA polymerase, as well as specific or random primers. For a control reaction with T7 DNA polymerase and T7 RNA polymerase, a pQE30 plasmid lacking the T7 promoter was used. B) Recircularization of the replicated plasmid, mediated by Cre recombinase. Where indicated, Cre recombinase was added after 16 h of replication, and the reaction mixture was incubated for another 30 min. Reaction mixtures were separated along with a DNA ladder (1 kb) on a Midori‐green stained agarose gel. The lower band migrating below 5 kb corresponds to the circularized DNA, whereas larger products apparently correspond to linear concatamers (Figure S2). C) DNA nanoparticles emerging upon prolonged ( > 12 h) T7 DNA replication reaction. Scale bar: 10 μm.

    Article Snippet: For Phi29 DNA replication, Phi29 DNA polymerase (NEB, 1 U) was used with random primers (2.5 μm ) and dNTPs (1.25 mm ).

    Techniques: Plasmid Preparation, Fluorescence, Binding Assay, Incubation, Staining, Agarose Gel Electrophoresis

    Nucleoprotein filament dynamics on low sequence complexity ssDNA curtains. (A) Sequences of the two ssDNA oligonucleotides used for rolling circle replication. (B) Schematic of rolling circle replication (RCR) reaction. T4 DNA ligase ligates the template oligo to form a contiguous template strand. Next, phi29 DNA polymerase catalyzes the synthesis of long ssDNA molecules. (C) Agarose gel of several time points along the RCR synthesis reaction. The primer oligonucleotide was 32 P labeled on the 5 ′ -terminus phosphate ( gold star ). (D) Wide-field image of a microfabricated barrier set with double-tethered ssDNA curtains coated with RPA-TagRFP ( magenta ). Arrows and circles denote chromium barriers and pedestals, respectively. (E) Illustration and kymograph showing a single ssDNA molecule coated with ATTO488-RAD51(C319S) ( green ) replaced by RPA-TagRFP ( magenta ). Yellow dashed line denotes the injection of RPA–TagRFP into the flowcell. Buffer controls indicate when the buffer flow was toggled off and on to show that the florescent proteins retract to the Cr barriers simultaneously with the ssDNA molecule. This indicates that RAD51 and RPA are on the ssDNA molecule. Panel A: Adapted from Lee, K. S., Marciel, A. B., Kozlov, A. G., Schroeder, C. M., Lohman, T. M., Ha, T. (2014). Ultrafast redistribution of E. coli SSB along long single-stranded DNA via intersegment transfer. Journal of Molecular Biology, 426 , 2413 – 2421.

    Journal: Methods in enzymology

    Article Title: Next-Generation DNA Curtains for Single-Molecule Studies of Homologous Recombination

    doi: 10.1016/bs.mie.2017.03.011

    Figure Lengend Snippet: Nucleoprotein filament dynamics on low sequence complexity ssDNA curtains. (A) Sequences of the two ssDNA oligonucleotides used for rolling circle replication. (B) Schematic of rolling circle replication (RCR) reaction. T4 DNA ligase ligates the template oligo to form a contiguous template strand. Next, phi29 DNA polymerase catalyzes the synthesis of long ssDNA molecules. (C) Agarose gel of several time points along the RCR synthesis reaction. The primer oligonucleotide was 32 P labeled on the 5 ′ -terminus phosphate ( gold star ). (D) Wide-field image of a microfabricated barrier set with double-tethered ssDNA curtains coated with RPA-TagRFP ( magenta ). Arrows and circles denote chromium barriers and pedestals, respectively. (E) Illustration and kymograph showing a single ssDNA molecule coated with ATTO488-RAD51(C319S) ( green ) replaced by RPA-TagRFP ( magenta ). Yellow dashed line denotes the injection of RPA–TagRFP into the flowcell. Buffer controls indicate when the buffer flow was toggled off and on to show that the florescent proteins retract to the Cr barriers simultaneously with the ssDNA molecule. This indicates that RAD51 and RPA are on the ssDNA molecule. Panel A: Adapted from Lee, K. S., Marciel, A. B., Kozlov, A. G., Schroeder, C. M., Lohman, T. M., Ha, T. (2014). Ultrafast redistribution of E. coli SSB along long single-stranded DNA via intersegment transfer. Journal of Molecular Biology, 426 , 2413 – 2421.

    Article Snippet: TE buffer: 10m M Tris–HCl [pH 8.0]; 0.1m M EDTA RAD51 buffer: 40m M Tris–HCl [pH 8.0]; 1m M MgCl2 ; 5m M CaCl2 ; 100m M KCl; 1m M DTT; 1m M ATP; 0.2 mgmL−1 BSA; 1m M Trolox (Sigma-Aldrich); 1.0% glucose (w/v); 500units catalase (Sigma-Aldrich); 70units glucose oxidase (Sigma-Aldrich) 10× T4 DNA ligase reaction buffer (B0202S; NEB) T4 DNA ligase (M0202; NEB) Primer oligo (/Biosg/TC TCC TCC TTC T—HPLC purified; Integrated DNA Technologies) Template oligo (/5Phos/AG GAG AAA AAG AAA AAA AGA AAA GAA GG—PAGE purified; Integrated DNA Technologies) Nuclease-free water BSA, Molecular Biology Grade (B9000S; NEB) Thermocycler (Mastercycler pro S; Eppendorf ) 10× phi29 DNA polymerase reaction buffer (B0269S; NEB) phi29 DNA polymerase (homemade 5 μ M stock) Deoxynucleotide (dNTP) solution set (N0446S; NEB)

    Techniques: Sequencing, Agarose Gel Electrophoresis, Labeling, Recombinase Polymerase Amplification, Injection, Flow Cytometry