Journal: Micromachines

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

doi: 10.3390/mi8020062

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

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

Techniques: Multiple Displacement Amplification, Transmission Electron Microscopy

Journal: RNA

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

doi: 10.1261/rna.622108

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

Article Snippet: BSA, glycogen, DNaseI, and DNaseI buffer with MgCl2 (10×), DEPC-treated water, Phi29 DNA polymerase, Phi29 DNA polymerase exo− mutant (D12A + D66A), Ribolock ribonuclease inhibitor, RNaseT1, T4 polynucleotide kinase (T4 PNK), T4 DNA ligase, T4 RNA ligase, Tango buffer (10×), TBE buffer (10×), and RNA Loading Dye Solution (2×) were products of Fermentas UAB.

Techniques: Activity Assay, Labeling

Journal: RNA

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

doi: 10.1261/rna.622108

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

Article Snippet: BSA, glycogen, DNaseI, and DNaseI buffer with MgCl2 (10×), DEPC-treated water, Phi29 DNA polymerase, Phi29 DNA polymerase exo− mutant (D12A + D66A), Ribolock ribonuclease inhibitor, RNaseT1, T4 polynucleotide kinase (T4 PNK), T4 DNA ligase, T4 RNA ligase, Tango buffer (10×), TBE buffer (10×), and RNA Loading Dye Solution (2×) were products of Fermentas UAB.

Techniques: Activity Assay, Labeling

Journal: RNA

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

doi: 10.1261/rna.622108

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

Article Snippet: BSA, glycogen, DNaseI, and DNaseI buffer with MgCl2 (10×), DEPC-treated water, Phi29 DNA polymerase, Phi29 DNA polymerase exo− mutant (D12A + D66A), Ribolock ribonuclease inhibitor, RNaseT1, T4 polynucleotide kinase (T4 PNK), T4 DNA ligase, T4 RNA ligase, Tango buffer (10×), TBE buffer (10×), and RNA Loading Dye Solution (2×) were products of Fermentas UAB.

Techniques: Activity Assay

Journal: RNA

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

doi: 10.1261/rna.622108

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

Article Snippet: BSA, glycogen, DNaseI, and DNaseI buffer with MgCl2 (10×), DEPC-treated water, Phi29 DNA polymerase, Phi29 DNA polymerase exo− mutant (D12A + D66A), Ribolock ribonuclease inhibitor, RNaseT1, T4 polynucleotide kinase (T4 PNK), T4 DNA ligase, T4 RNA ligase, Tango buffer (10×), TBE buffer (10×), and RNA Loading Dye Solution (2×) were products of Fermentas UAB.

Techniques: Sequencing

Journal: RNA

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

doi: 10.1261/rna.622108

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

Article Snippet: BSA, glycogen, DNaseI, and DNaseI buffer with MgCl2 (10×), DEPC-treated water, Phi29 DNA polymerase, Phi29 DNA polymerase exo− mutant (D12A + D66A), Ribolock ribonuclease inhibitor, RNaseT1, T4 polynucleotide kinase (T4 PNK), T4 DNA ligase, T4 RNA ligase, Tango buffer (10×), TBE buffer (10×), and RNA Loading Dye Solution (2×) were products of Fermentas UAB.

Techniques: Labeling, Incubation

Journal: RNA

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

doi: 10.1261/rna.622108

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

Article Snippet: BSA, glycogen, DNaseI, and DNaseI buffer with MgCl2 (10×), DEPC-treated water, Phi29 DNA polymerase, Phi29 DNA polymerase exo− mutant (D12A + D66A), Ribolock ribonuclease inhibitor, RNaseT1, T4 polynucleotide kinase (T4 PNK), T4 DNA ligase, T4 RNA ligase, Tango buffer (10×), TBE buffer (10×), and RNA Loading Dye Solution (2×) were products of Fermentas UAB.

Techniques: Sequencing

Journal: BioTechniques

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

doi: 10.2144/000114566

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

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

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

Journal: RNA

Article Title: Direct detection of RNA in vitro and in situ by target-primed RCA: The impact of E. coli RNase III on the detection efficiency of RNA sequences distanced far from the 3?-end

doi: 10.1261/rna.2068510

Figure Lengend Snippet: Target RNA conversion into a primer for RCA, the impact of E. coli RNase III. The reactions were carried out under the conditions described in Materials and Methods. The 5′-end-labeled ( A ) RNA2-PP1 (panel 1 ), ( B ) RNA2-PP2 (panel 1 ), and RNA1-PP1 (panel 2 ) hybrids were incubated with Phi29 DNA polymerase in the absence or presence of dNTP. ( A , panel 2 ) The experiments with E. coli RNase III were performed using RNA2-PP1 duplex. The RNA degradation and RCA products were analyzed by electrophoresis through denaturing 8% polyacrylamide gels. Different RNA-PP duplexes are shown above the gels. The radioactive label is indicated by the star. The contents of samples are shown above the gel lines. Reaction products are labeled as “ a ” for the 5′-end-labeled RNA1 hydrolysis product serving as an RNA primer for RCA and “ b ” for the 5′-end-labeled RCA product. ( C ) The reaction scheme represents the conversions of target RNA.

Article Snippet: BSA, DEPC-treated water, DNase I, DNA polymerases (Taq, Phi29), glycogen, RevertAid H Minus First Strand cDNA Synthesis Kit, REases (Mva1269I, LguI), Ribolock RNase inhibitor, T4 DNA ligase; PBS buffer (10×), SSC buffer (20×), Tango buffer (10×), TBE buffer (10×), and RNA Loading Dye Solution (2×) were products of Fermentas UAB.

Techniques: Labeling, Incubation, Electrophoresis

Journal: PLoS Genetics

Article Title: A Recessive Founder Mutation in Regulator of Telomere Elongation Helicase 1, RTEL1, Underlies Severe Immunodeficiency and Features of Hoyeraal Hreidarsson Syndrome

doi: 10.1371/journal.pgen.1003695

Figure Lengend Snippet: Inhibiting DNA replication blocks T-circle formation in MSK-41 RTEL1 R1264H cells. (A) Phi29-dependent T-circles in BJ hTERT and MSK-41. (B) Phi29-dependent T-circles in RTEL1 floxed/- MEFs ± Cre, BJ hTERT and MSK-41. (C) Phi29-dependent T-circles in BJ hTERT and MSK-41 ± aphidicolin (APD; 5 µM). (D) Dot blot of the Phi29-dependent T-circles in BJ hTERT and MSK-41 ± aphidicolin (APD; 5 µM). (E) Quantification of the fold increase in intensity of Phi29-dependent T-circles in the different cell lines subjected to the indicated treatments. Intensity mean and standard deviation were calculated over two independent experiments; statistical analysis (one-way ANOVA) was calculated with Prism (GraphPad).

Article Snippet: DNA was double digested by AluI/HinfI restriction enzymes overnight before starting TCA assay and then Southern Blot as described with minor modifications to Phi29 DNA polymerization (MBI Fermentas) with a mammalian telomeric primer and a mammalian telomeric probe for hybridization.

Techniques: Dot Blot, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Limited reverse transcriptase activity of phi29 DNA polymerase

doi: 10.1093/nar/gky190

Figure Lengend 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.

Article Snippet: Following the ligation, 2 μl ligation volume (circles) was added to 18 μl RCA reaction mix containing 0.1 U/μl phi29 DNA polymerase (Monserate Biotechnology Group) 1 × phi29 reaction buffer (Thermo Fisher), 125 μM dNTP (DNA Gdansk), 0.2 μg/μl BSA (NEB) and 1 × SYBR Gold (S11194, Invitrogen) to a final circles’ concentration of 1 nM.

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

Journal: Nucleic Acids Research

Article Title: Limited reverse transcriptase activity of phi29 DNA polymerase

doi: 10.1093/nar/gky190

Figure Lengend 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.

Article Snippet: Following the ligation, 2 μl ligation volume (circles) was added to 18 μl RCA reaction mix containing 0.1 U/μl phi29 DNA polymerase (Monserate Biotechnology Group) 1 × phi29 reaction buffer (Thermo Fisher), 125 μM dNTP (DNA Gdansk), 0.2 μg/μl BSA (NEB) and 1 × SYBR Gold (S11194, Invitrogen) to a final circles’ concentration of 1 nM.

Techniques: Fluorescence, Plasmid Purification

Journal: Nucleic Acids Research

Article Title: Limited reverse transcriptase activity of phi29 DNA polymerase

doi: 10.1093/nar/gky190

Figure Lengend 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.

Article Snippet: Following the ligation, 2 μl ligation volume (circles) was added to 18 μl RCA reaction mix containing 0.1 U/μl phi29 DNA polymerase (Monserate Biotechnology Group) 1 × phi29 reaction buffer (Thermo Fisher), 125 μM dNTP (DNA Gdansk), 0.2 μg/μl BSA (NEB) and 1 × SYBR Gold (S11194, Invitrogen) to a final circles’ concentration of 1 nM.

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