pbs t rolling circle amplification rca  (Thermo Fisher)

Journal: bioRxiv

Article Title: Multiplexed CRISPR/Cas9 Editing of Tumor Suppressor Genes Recapitulates Molecular and Morphological Features of High-Risk Endometrial Cancer

doi: 10.1101/2025.11.28.691175

Figure Lengend Snippet: (A) Graphic representation of the spatial mRNA-based method used for validation of tumor heterogeneity generated with CRISPR/Cas9. Briefly, a padlock probe is hybridized in the mRNA of the specific gene, designed at the Cas9-cutting site. Then, SplintR ligase is used to ligate de padlock probe, but if there is not 100% homology between the padlock probe and the mRNA (edited sequence), ligation does not occur. Next step is Rolling Circle Amplification (RCA), where EquiPhi29 polymerase amplifies the padlock sequence. Finally, specific regions of different padlock probes are detected with fluorochrome-labelled probes. If the detection is individual for a single gene, generic probes are used. But if the detection is pooled, a fluorochrome code is used. (B) Individual detection of selected TSGs ( Fbxw7, Pten, Trp53, Ppp2r1a, Arhgap35, Arid1a, Pik3r1, Muc16, Kmt2d and Chd4 ) in wildtype epithelial endometrial cells detected with generic probes (green). Hoechst (blue) is used for nuclear staining. Scale bars: 50µm (40X magnification). (C) Pooled detection of mRNA from selected TSGs in wildtype epithelial endometrial cells detected with generic probes (green). Hoechst (blue) is used for nuclear staining. Scale bar: 50µm (40X magnification). (D) Individual detection of Actb mRNA with generic probes. Scale bar: 50µm (40X magnification). (E) Quantification of individual and pooled detections of selected TSG in wildtype cells, shown in (B) and (C) . (F) Representative image of pooled detection of selected TSG in non-electroporated (control) epithelial endometrial cells, detected with fluorochrome code. Images show detection with Atto488 (green), Cy3 (orange), Atto647 (red) and/or AlexaFluor750 (grey). Hoechst (blue) is used for nuclear staining. Scale bars: 50µm (60X magnification). (G) Representative image of pooled detection of selected TSG in electroporated with a pool of RNPs (EP: 10 TSG), detected with fluorochrome code. Images show detection with Atto488 (green), Cy3 (orange), Atto647 (red) and/or AlexaFluor750 (grey). Hoechst (blue) is used for nuclear staining. Scale bars: 50µm (60X magnification). (H) Quantification and co-localization of RNA spots according to the fluorochrome code at a single cell resolution. Binary heatmap represents the presence (green) or absence (yellow) of Fbxw7, Pten, Trp53, Ppp2r1a, Arhgap35, Arid1a, Pik3r1, Muc16, Kmt2d or Chd4 detection in each cell (rows).

Article Snippet: To wash the samples, they were rinsed twice with PBS-T. Rolling Circle Amplification (RCA) was performed for 2 hours at 42°C using 0.5U/μl EquiPhi29 DNA polymerase (A39391, Thermo Fisher Scientific) in a reaction containing 1X EquiPhi29 buffer, 1mM dithiothreitol (DTT) and 1mM dNTPs.

Techniques: Biomarker Discovery, Generated, CRISPR, Sequencing, Ligation, Amplification, Staining, Control

Journal: Applied and Environmental Microbiology

Article Title: High-throughput nanopore DNA sequencing of large insert fosmid clones directly from bacterial colonies

doi: 10.1128/aem.00243-24

Figure Lengend Snippet: Amplification of 96 fosmids using phi29-XT. A total of 96 clones (lanes 1–96) from a T. kodakarensis genomic fosmid library were amplified using phi29-XT. Amplification products were digested with SacI and XhoI, and digested products were analyzed by separation on a 0.8% agarose gel.

Article Snippet: phi29-XT and phi29-XT reaction buffer (from E1603: phi29-XT RCA Kit), dNTP solution mix, SacI-HF, XhoI, rCutSmart buffer, T7 endonuclease I, NEBuffer 2, NEBNext FFPE DNA repair mix, NEBNext FFPE DNA repair buffer, NEBNext Ultra II end prep enzyme mix, Next Ultra II end prep reaction buffer, Blunt/TA ligase master mix, NEBNext Quick ligation reaction buffer, Quick T4 DNA ligase were all from New England Biolabs (Ipswich, MA, USA).

Techniques: Amplification, Clone Assay, Agarose Gel Electrophoresis

Journal: Applied and Environmental Microbiology

Article Title: High-throughput nanopore DNA sequencing of large insert fosmid clones directly from bacterial colonies

doi: 10.1128/aem.00243-24

Figure Lengend Snippet: The phiXXer pipeline for de novo assembly of nanopore sequence data for phi29-amplified DNA. The common fosmid vector pCC1 was used in the illustrated protocol. Raw sequencing reads are aligned to the vector sequence, and any matching regions are trimmed out before assembly. If multiple contigs are produced during the assembly, only the contig with the highest coverage is retained. This contig is aligned to the vector, and any matching regions are trimmed away, producing a linear sequence of the cloned environmental or genomic DNA insert (free of the vector backbone). Nucmer is a whole genome sequence aligner ; canu is a long-read de novo assembler .

Article Snippet: phi29-XT and phi29-XT reaction buffer (from E1603: phi29-XT RCA Kit), dNTP solution mix, SacI-HF, XhoI, rCutSmart buffer, T7 endonuclease I, NEBuffer 2, NEBNext FFPE DNA repair mix, NEBNext FFPE DNA repair buffer, NEBNext Ultra II end prep enzyme mix, Next Ultra II end prep reaction buffer, Blunt/TA ligase master mix, NEBNext Quick ligation reaction buffer, Quick T4 DNA ligase were all from New England Biolabs (Ipswich, MA, USA).

Techniques: Sequencing, Amplification, Plasmid Preparation, Produced, Clone Assay

Journal: Nucleic Acids Research

Article Title: Monitoring drug–target interactions through target engagement-mediated amplification on arrays and in situ

doi: 10.1093/nar/gkac842

Figure Lengend Snippet: Principle of TEMA and design of EGFR-directed drug-oligonucleotide conjugates. ( A ) In TEMA, small molecule ligands conjugated to oligonucleotides bind their target proteins in sample matrices such as protein arrays, fixed cells and tissue sections. After washing away excess ligand, any remaining bound conjugate is recognized by padlock probes . These probes serve as substrates for ligation reactions and form oligonucleotide circles that template localized RCA reactions, the products of which are visualized using fluorescent oligonucleotide probes . The illustration of the EGFR kinase domain (light blue) is based on RSCB PDB ( https://www.rcsb.org ) entry 4WKQ (unpublished) using Mol* . ( B ) Illustration of binding by gefitinib (left) and erlotinib (right) in the active site of the EGFR kinase domain . While the morpholine of gefitinib protrudes out of the active site and represents a suitable exit vector for oligonucleotide conjugation (green arrow), the alkyne of erlotinib is buried in the active site such that conjugation causes steric hindrance (red arrow). The illustrations are based on RSCB PDB entries 4WKQ and 1M17 using Mol*. ( C ) Structures of gefitinib, a selective EGFR-targeted drug, and dasatinib, with a broader kinase inhibition profile (erlotinib was included as a control) . The morpholine in gefitinib, and the hydroxyl group of dasatinib were replaced with an alkyne in precursors 1 and 2 and click chemistry afforded conjugation to azide-modified oligonucleotides. The corresponding alkyne precursors of gefitinib (1) and dasatinib (2) were synthesized and used to generate gefitinib- and dasatinib-oligonucleotide conjugates, respectively, while the alkyne in erlotinib (3) allowed direct conjugation to provide erlotinib-oligonucleotide conjugates. Details on synthetic schemes and characterization of the molecules are available is Supplementary Information.

Article Snippet: Next, samples were washed with 1× TBS for 2 × 2 min and again quickly washed with RCA buffer, followed by RCA with 0.5 unit/μl Phi-29 DNA polymerase (Fermentas) in RCA buffer (1× Phi-29 DNA polymerase-buffer (Fermentas), 250 nM detection oligonucleotides (Detection tagD1, ), 7.5 nM polyA (Sigma-Aldrich), 0.25 μg/μl BSA, 0.25 mM dNTP (Thermo Scientific), 0.001% Tween in ddH 2 O) incubated for 90 min at 37°C.

Techniques: Ligation, Binding Assay, Plasmid Preparation, Conjugation Assay, Inhibition, Control, Modification, Synthesized

Journal: Nucleic Acids Research

Article Title: Monitoring drug–target interactions through target engagement-mediated amplification on arrays and in situ

doi: 10.1093/nar/gkac842

Figure Lengend Snippet: Schematic of proxTEMA and application in cell lines and fresh frozen tissues sections. ( A ) Using the proxTEMA approach drug molecules with conjugated oligonucleotides were combined with oligonucleotide-conjugated antibodies directed against a protein of interest. In cases where both reagents bound the same or nearby target molecules their attached oligonucleotides could template the formation of oligonucleotide circles by ligating pairs of added oligonucleotides. Once formed, the oligonucleotide circles were replicated in local RCA reactions, primed by the oligonucleotides conjugated to the antibodies. Successful detection depends on proximal binding by oligonucleotide conjugates of both drugs and antibodies. ( B , C ) Example of antibody-guided analysis of drug interactions with specific target proteins via proxTEMA by ABL or EGFR antibodies, combined with dasatinib or gefitinib probes (5 nM). (B) The antibody- and drug-probes were used in isPLA reactions, applied to the cell lines K562 and A431, both positive for ABL and EGFR, and the results were analyzed by flow cytometry. An assay where oligonucleotides having no conjugated drug molecules were combined with anti ABL antibody probes served as a negative control. (C) dasatinib (5 nM) was combined with anti-EGFR for isPLA analysis by microscopy of K562 cells (green signals) and with-ABL antibody probes for analysis of A431 cells (red signals). Similarly, gefitinib probes (5 nM) were applied with anti EGFR antibody probes for analysis of the A431 cells; positive for EGFR, and with CHO-K1 cells negative control for EGFR expression. Cell nuclei were counterstained using DAPI (blue) and images were acquired by fluorescence microscopy. Scale bars represents 20 μm. ( D ) Analysis of breast cancer sections by proxTEMA using anti-EGFR antibody probes together with 10 nM gefitinib probes or inactive erlotinib probes. Signals are shown in red. For comparison a pair of EGFR-specific oligonucleotide-conjugated antibody probes were combined for isPLA to serve as a positive control. The breast cancer tissue sections had been scored as 3+ with respect to HER2 staining using a HercepTest (Dako). The tissues were counterstained using DAPI (blue) and images were acquired by fluorescence microscopy. Scale bar represents 50 μm. (E, F) ProxTEMA was used for detection of the BCR-ABL fusion proteins by flow cytometry using an anti-BCR antibody-oligonucleotide conjugates together either with an anti ABL antibody-oligonucleotide conjugates, serving as a positive control, or with either 2.5 nM of dasatinib or the inactive erlotinib probe. The assays were performed in K562 cells, expressing the BCR-ABL fusion protein, and in U937 cells negative for this fusion protein. The experiment was repeated three times with similar results. K562 and U937 cells were counterstained using DAPI (blue) and images were acquired by fluorescence microscopy for in situ detection of BCR-ABL using an anti-BCR antibody-oligonucleotide conjugates together with 5 nM of drug probe. Scale bar represents 50 μm.

Article Snippet: Next, samples were washed with 1× TBS for 2 × 2 min and again quickly washed with RCA buffer, followed by RCA with 0.5 unit/μl Phi-29 DNA polymerase (Fermentas) in RCA buffer (1× Phi-29 DNA polymerase-buffer (Fermentas), 250 nM detection oligonucleotides (Detection tagD1, ), 7.5 nM polyA (Sigma-Aldrich), 0.25 μg/μl BSA, 0.25 mM dNTP (Thermo Scientific), 0.001% Tween in ddH 2 O) incubated for 90 min at 37°C.

Techniques: Binding Assay, Flow Cytometry, Negative Control, Microscopy, Expressing, Fluorescence, Comparison, Positive Control, Staining, In Situ

Journal: Nucleic Acids Research

Article Title: Monitoring drug–target interactions through target engagement-mediated amplification on arrays and in situ

doi: 10.1093/nar/gkac842

Figure Lengend Snippet: Localization of drug binding in tissues by TEMA. ( A ) Binding of gefitinib probe (at 5 nM; red dots) in the cancer cell line A431, expressing high levels of EGFR, and the azide modified oligonucleotides without conjugated drug molecules, used as a negative control. Cytoplasms and nuclei were stained with phalloidin (green) and DAPI (blue), respectively. The images were acquired by fluorescence microscopy. Scale bars represent 20 μm. ( B ) Flow cytometry analysis by TEMA for quantitative comparisons of target engagement by kinase inhibitor probes (5 nM) in K562 cells, overexpressing the ABL kinase as a fusion protein, and A431 cells, expressing EGFR transcripts at higher levels compared to U937 cells having low/absent EGFR expression. ( C ) Comparison of binding by gefitinib and dasatinib probes at 1 nM in TEMA analyses of fresh-frozen normal human colon tissue sections. TEMA signals are seen in red, while nuclei in the tissues were stained blue using DAPI. ( D ) Investigation of fresh-frozen breast cancer tissue sections, previously scored as 3+ for HER2 protein staining by the HercepTest (Dako), indicating high expression of HER2. TEMA probes at 1 nM signals are seen in red. ( E ) Binding by gefitinib probes at 5 nM added to formalin-fixed paraffin-embedded brain cancer tissue sections and normal brain tissue in a commercial tissue microarray. The numbers of RCA products, representing TEMA gefitinib signals, were quantified per nuclei using CellProfiler software. The pairs of bars in distinct colors show duplicate observation for on average >2000 cells in each sample type. RCA products are seen in red. Scale bars in panels (C)–(E) are 50 μm.

Article Snippet: Next, samples were washed with 1× TBS for 2 × 2 min and again quickly washed with RCA buffer, followed by RCA with 0.5 unit/μl Phi-29 DNA polymerase (Fermentas) in RCA buffer (1× Phi-29 DNA polymerase-buffer (Fermentas), 250 nM detection oligonucleotides (Detection tagD1, ), 7.5 nM polyA (Sigma-Aldrich), 0.25 μg/μl BSA, 0.25 mM dNTP (Thermo Scientific), 0.001% Tween in ddH 2 O) incubated for 90 min at 37°C.

Techniques: Binding Assay, Expressing, Modification, Negative Control, Staining, Fluorescence, Microscopy, Flow Cytometry, Drug discovery, Comparison, Formalin-fixed Paraffin-Embedded, Microarray, Software