Padlock oligonucleotides, including a 15 nt random sequence (100 nM) were ligated into circular DNA strands in the presence of template (100 nM) in 1x phi29 buffer ((Thermo Scientific), 10 mM Mg-acetate, 66 mM K-acetate, 0.1% (v/v) Tween 20, 1 mM DTT) containing 0.01 U/μl T4 ligase (Thermo Scientific), and 1 mM ATP (Thermo Scientific). The ligation was performed at 37 °C for 30 min. To remove unligated padlock oligonucleotides, exonuclease I and exonuclease III (Thermo Scientific) were added to the ligation mix to a concentration of 0.2 U/μl and 2 U/μl, respectively. The reactions were incubated at 37 °C for 30 min and terminated by incubation at 85 °C for 20 min. Then the RCA primer (the same oligonucleotide used as ligation template) was adjusted to a concentration of 100 nM and incubated at 37 °C for 20 min to reanneal to the circular DNA strands. The RCA reactions were initiated by adding d(A, U, G, C)TP at concentrations of 1 mM and phi29 polymerase (Thermo Scientific) at 0.1 U/μl. RCA was performed at 37 °C for 10 min, and terminated by heating at 65 °C for 10 min. The RCA products were kept at −20 °C until use. The RCA products were characterized with Nanoparticle Tracking Analysis NTA (Malvern Nanosight NS300) according to the manufacturer’s instructions.
Phi29 polymerase
Manufactured by Thermo Fisher Scientific
Sourced in United States
Phi29 polymerase is a DNA-dependent DNA polymerase enzyme derived from the Bacillus subtilis bacteriophage Phi29. The enzyme is capable of performing highly processive and strand-displacement DNA synthesis, making it a useful tool for applications such as whole genome amplification and DNA sequencing.
Automatically generated - may contain errors
Lab products found in correlation
Dntps, by Thermo Fisher Scientific (8 mentions)
Phi29 buffer, by Thermo Fisher Scientific (4 mentions)
Bovine serum albumin (bsa), by New England Biolabs (3 mentions)
T4 ligase, by Thermo Fisher Scientific (2 mentions)
Pyrophosphatase, by Thermo Fisher Scientific (2 mentions)
Exo resistant random primer, by Thermo Fisher Scientific (2 mentions)
Bglii, by New England Biolabs (2 mentions)
Restriction enzyme buffer, by New England Biolabs (2 mentions)
Milliq, by New England Biolabs (2 mentions)
Adenosine triphosphate (atp), by Thermo Fisher Scientific (1 mentions)
Exonuclease 3, by Thermo Fisher Scientific (1 mentions)
Exonuclease 1, by Thermo Fisher Scientific (1 mentions)
Amplitaq gold dna polymerase, by Thermo Fisher Scientific (1 mentions)
T4 pnk, by Thermo Fisher Scientific (1 mentions)
Recombinant tnf α, by R&D Systems (1 mentions)
Cyclosporin a, by Merck Group (1 mentions)
Rpmi 1650, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Jurkat cell, by American Type Culture Collection (1 mentions)
Sybr safe, by Thermo Fisher Scientific (1 mentions)
17 protocols using phi29 polymerase
1
Padlock Ligation and Rolling Circle Amplification
2
Family-based DNA Sequencing and SNP Genotyping
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Blood genomic DNA from seven available individuals of the family (Fig. 1 ) was used for Sanger resequencing. PCR primer sequences are shown in Supplementary Table 2 . PCR assays were performed according to a standard protocol using AmpliTaq Gold DNA polymerase (Life Technologies) as follows: 10 min at 94 °C; followed by 32 cycles of 15 s at 94 °C, 15 s at 58 °C, and 1 min at 72 °C; followed by a final extension of 5 min at 72 °C. The PCR amplicons were sequenced using an ABI3730 DNA sequencer (Genomics Shared Resource, OSU).
SNaPshot assays (Life Technologies) were performed as described18 (link). To amplify genomic regions around the 2 candidate variants (CHD9, SRRM2), PCR was performed followed by a single nucleotide extension reaction.
Screening for the SNP rs149019598 in Ohio PTC patients and controls was carried out by deCODE Genetics in Reykjavik, Iceland, applying the Centaurus (Nanogen) single-track assay54 (link). DNA that had been whole-genome amplified using standard protocols based on the Phi29 polymerase (Thermo Fisher Scientific Inc.) was used. To confirm the T allele at rs149019598 as identified from the single-track assay, we used Sanger sequencing to assay unamplified DNA extracted from whole blood.
SNaPshot assays (Life Technologies) were performed as described18 (link). To amplify genomic regions around the 2 candidate variants (CHD9, SRRM2), PCR was performed followed by a single nucleotide extension reaction.
Screening for the SNP rs149019598 in Ohio PTC patients and controls was carried out by deCODE Genetics in Reykjavik, Iceland, applying the Centaurus (Nanogen) single-track assay54 (link). DNA that had been whole-genome amplified using standard protocols based on the Phi29 polymerase (Thermo Fisher Scientific Inc.) was used. To confirm the T allele at rs149019598 as identified from the single-track assay, we used Sanger sequencing to assay unamplified DNA extracted from whole blood.
3
RCA Reaction and Oligonucleotide Hybridization
4
Multi-Aptamer Enrichment and Detection
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All DNA sequences used in this study were purchased from Integrated DNA Technologies, Inc. (Coralville, IA). Materials (FITC-dUTPs, dNTPs, T4 PNK, phi29 polymerase, T4 ligase, and buffers) used for rolling circle amplification were purchased from Thermo Scientific (Waltham, MA). 100K centrifugal devices to purify Multi-Aptamer products were purchased from Pall Life Sciences (Port Washington, NY). Jurkat cells were obtained from ATCC and cultured following manufacturer’s protocol; human brain endothelial cells were from Cell Systems and cultured following manufacturer’s protocol. RPMI-1650 was obtained from Gibco and fetal bovine serum (FBS) from Atlantic Biologicals. Cyclosporin A and phorbol myristate acetate (PMA) were purchased from Sigma Aldrich (St. Louis, MO); recombinant TNFα was from R&D Systems (Minneapolis, MN). The non-blocking anti-L-selectin antibody (clone 4G8), and the enzyme-linked immunosorbent assay (ELISA) for detection of soluble L-selectin were purchased from R&D Systems. The FITC-labeled anti-L-selectin blocking antibody (DREG56) and the antibodies (N-18 anti-L-selectin and HRP-conjugated secondary antibodies) used for Western blots were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX). Penicillin-streptomycin, SYBR Safe, and FITC annexin V cell apoptosis kit were purchased from Life Technologies (Carlsbad, CA).
5
Circular DNA Amplification and Analysis
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Circular DNA obtained by the circularization reaction was combined with 12 μl 5× Annealing buffer (50 mM Tris @ pH 7.5–8.0, 250 mM NaCl, 5 mM EDTA) and 1 μl Exo-resistant random primers (Thermofisher), heated for 5 min at 98 °C and then cooled down at room temperature. Subsequently, the RCA mix (previous reaction mixture, 10 μl 10× Phi29 Buffer (Thermofisher), 2 μl BSA (New England Biolabs), 10 μl dNTPs (Thermofisher), 4 μl pyrophosphatase (Thermofisher), 2 μl Phi29 Polymerase (Thermofisher), and MQ (to a volume of 100 μl)) was prepared. RCA was performed overnight at 30 °C. The RCA-reaction was inactivated by 10 min incubation at 70 °C.
To test whether CyclomicsSeq worked, 4 μl of RCA mixture was incubated with a restriction enzyme that specifically cuts backbone-backbone interactions, but not backbone-insert interactions. Briefly, 4 μl of RCA mixture was combined with 4 μl Restriction enzyme buffer (New England Biolabs), 13 μl MilliQ, and 1 μl BglII (New England Biolabs). The reaction mixture was incubated for 1 h at 37 °C and then ran on a 1.5% Agarose gel.
To test whether CyclomicsSeq worked, 4 μl of RCA mixture was incubated with a restriction enzyme that specifically cuts backbone-backbone interactions, but not backbone-insert interactions. Briefly, 4 μl of RCA mixture was combined with 4 μl Restriction enzyme buffer (New England Biolabs), 13 μl MilliQ, and 1 μl BglII (New England Biolabs). The reaction mixture was incubated for 1 h at 37 °C and then ran on a 1.5% Agarose gel.
6
Circular DNA Enrichment from Urine
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To remove the linear portions of urinary cfDNA, 40 μl of urinary cfDNA with added spike‐in plasmid control (∼ 100 copies of P895 and ∼ 100 copies of P1035) was treated with 25 units of Plasmid‐Safe Dnase (Epicenter) at 37°C for 24 hours. The Plasmid‐Safe Dnase was then inactivated by incubating samples at 70°C for 30 minutes. The digestion products were cleaned up from the reaction mixes using 2.0 × WAHTS® DNA clean beads (Vazyme) and eluted in 30 μl RNase‐free water. Approximately 50% (14 μl) of the total volume of resultant circular DNA was subjected to a RCA reaction with incubation at 30°C for 16 hours. The RCA reaction system involved 1 μl Phi29 polymerase (Thermo), 4 μl 10 × Phi29 buffer (Thermo), 2 μl exonuclease‐resistant random primer (Thermo), 4 μl 2.5 μM dNTP mixture (Takara), .8 μl 100 mM DTT and 14.2 μl RNase‐free water. The phi29‐amplified products were recovered by 2.0 × WAHTS® DNA clean beads, eluted in 80 μl RNase‐free water and quantified by Qubit 3.0.
7
DNA Oligomer Preparation and Labeling
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The DNA oligomers were purchased from Integrated DNA Technology (IDT, Coralville, IA) and stored at −20°C. T4 DNA ligase and Exonucleases I and III were obtained from New England Biolab (NEB, Ipswich, MA). Terminal transferase and Phi 29 polymerase were respectively obtained from Thermo Fischer (Waltham, MA) and Mclab (South San Francisco, CA). Digoxigenin dUTP (Dig-dUTP) was purchased from Roche (Swiss). Streptavidin and digoxigenin (Dig) antibody-coated polystyrene beads were purchased from Spherotech (Lake Forest, IL).
8
Sensitive Dengue Virus Detection Protocol
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Phi29 polymerase, phi29 buffer, dNTP mix, bovine serum albumin (BSA), GeneRuler low range DNA ladder, SYBR Gold nucleic acid gel stain, Tris-HCl buffer (1 M, pH 8.0) and Tris-acetate-EDTA buffer (TAE, 50 ×) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). CircLigase II ssDNA ligase, together with other ligation reagents (buffer, MnCl2, betaine), was purchased from Biosearch Technologies (Novato, CA, USA). Nb.BtsI nickase, thermolabile exonuclease I, exonuclease III and loading buffer were purchased from New England BioLabs (Ipswich, MA, USA). Recombinant Acidaminococcus sp. BV3L6 Cas12a nuclease was purchased from Integrated DNA Technologies (Coralville, IA, USA). Fetal bovine serum (FBS), salmon sperm DNA and agarose were purchased from Sigma-Aldrich (St. Louis, MO, USA). Streptavidin-coated cross-linked starch iron oxide composite particles (100 nm size MNP) were purchased from Micromod Partikeltechnologie GmbH (Rostock, Germany). DNA and RNA sequences were synthesized by Integrated DNA Technologies and diluted in 50 mM Tris-HCl (pH 8.0). Sequences of targets (target dengue sequence and target B for amplifying detection loop and reference loop, respectively), linear templates (linear detection template and linear reference template), crRNA and detection probes (DP-DL-I, DP-DL-II, DP-RL-I and DP-RL-II) are listed in Supplementary Table S1 .
9
Quantitative RCA Reaction Monitoring
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7.5 µL of the noted concentrations of c.c. or reacted dumbbell substrates were moved to quantitative PCR (qPCR) tubes and the buffer adjusted to 1 × RCA buffer (33 mM Tris-acetate (pH 7.9), 10 mM Mg-actate, 66 mM K-acetate, 1% (v/v) Tween 20, and 10 mM DTT). Then 15 nM Phi29 Polymerase (ThermoFisher Scientific, Waltham, MA, USA), 1 mM dNTP (Roche , Basel, Switerland), 0.1 µg/µL BSA (Sigma, Saint Louis, MO, USA), and 1 µM of each of the molecular beacons were added to a final volume of 10 µL. The samples were placed in the qPCR machine (Mx3000P, Agilent Technologies, Inc., Santa Clara, CA, USA) and incubated at 30 °C for up to 18 h. Fluorescence emission was measured every 1 min using the FAM (U-FBNA FL filter cube (Excitation: BP470-495, DM505, Emission: BP510-550) and TAMRA (U-FGNA FL filter cube (Excitation: BP540-550, DM570, Emission: BP575-625). The TAMRA filter was used for CAL-Fluor-Red-590. Data from the run was processed in Microsoft Excel as described below.
10
Quantifying Genetic Modifications in A549 Cells
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A549 cells (1 × 106) were transduced with LV(GFP), NILV(GFP), and NILV‐S/MAR(GFP) at 5 IFU/cell. Untransduced cells served as negative control. The cells were puromycin‐selected and cultured for 2–4 weeks before being transferred to staining slides (Superfrost Plus, ThermoFisher Scientific, Waltham, MA). The slides were harvested 1 day after seeding and assayed (Weibrecht et al, 2012 ) with slight modifications. Cells were fixed and permeabilized. They were then treated with nicking enzyme (Nt.BspQI, New England Biolabs, Ipswich, MA) followed by lambda exonuclease (ThermoFisher Scientific) to digest the nicked DNA strand (Appendix Fig S4 ). A specific hybridization padlock probe pPadlock (Invitrogen) was applied followed by ligation using T4 DNA ligase (Invitrogen). Rolling circle amplification (RCA) reaction was performed using phi29 polymerase (ThermoFisher Scientific) and the pRCA primer (Invitrogen) for signal enhancement, and the products were further hybridized with the Cy3‐labeled detection oligo pDetect (Invitrogen). Nuclei were stained with Hoechst 33342 (Invitrogen). Slides were imaged in a fluorescence microscope AxioImager M2 (Zeiss, German).
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