sanger sequencing analysis  (Fasmac Co Ltd)

Journal: International Journal of Oral Science

Article Title: Soluble Notch agonist enables human ameloblast maturation and enamel-like tissue formation for tooth regeneration

doi: 10.1038/s41368-026-00429-4

Figure Lengend Snippet: Notch pathway activation is required for ameloblast maturation in co-culture with odontoblasts. a TopPath pathway analysis of single-cell sequencing data from fetal tissues, identifying key signaling pathways involved in odontoblast (OB) to ameloblast (AM) communication. The Notch pathway (10.6%) is highlighted as a major contributor to AM maturation, alongside other pathways such as FGF, WNT, EGF, and BMP. b Schematic model depicting Notch signaling interactions between OBs and AMs, showing ligand-receptor interactions focusing on incoming signal to eAM/sAM (red arrows). The width of the arrows is proportional to the number of interactions. Black arrow indicates other possible interaction not considered in the analysis of TopPath. c Heatmap representation of Notch ligands (DLL1, DLL4) and Notch receptors (NOTCH1, NOTCH2, NOTCH3, NOTCH4), illustrating ligand expression in OBs and receptor expression in AMs, supporting OB-to-AM signaling. d Differentiation and co-culture model for generating induced ameloblasts (iAMs) from human iPSCs. Induced odontoblasts (iOBs) are co-cultured with induced early ameloblast (ieAM) organoids to promote their maturation into secretory ameloblasts (isAMs), with and without DAPT-mediated Notch inhibition. e 3D confocal images of co-cultured iAM organoids showing ENAM (yellow) and DSPP (red) expression, confirming ameloblast maturation in co-culture. Merged images include DAPI (nuclei, blue) and Phalloidin (cytoskeleton, purple). f Schematic representation summarizing the observed expression patterns of DSPP (red) and ENAM (yellow) in differentiated OB-like and AM-like cells within the co-culture. g Comparative immunofluorescence staining of ENAM in Control (top) vs. DAPT-treated (bottom) conditions, revealing reduced ENAM expression and altered cellular morphology upon Notch inhibition. Scale bar = 100 μm. h Quantification of ENAM fluorescence intensity, normalized to control, showing a significant reduction in ENAM expression in the DAPT-treated group ( **** P < 0.000 1)

Article Snippet: The PCR product was purified using ExoSap-IT (Thermofisher) and sent for Sanger sequencing analysis (Genewiz, Azenta Life Sciences) to identify potential KO clones. gRNA sequence: TAGCTGGAGTAGATCGTACG.

Techniques: Activation Assay, Co-Culture Assay, Single Cell, Sequencing, Protein-Protein interactions, Expressing, Cell Culture, Inhibition, Immunofluorescence, Staining, Control, Fluorescence

Journal: Nature Communications

Article Title: Programmable Argonaute-mediated single-nucleotide variant sequencing of cell-free DNA for multi-cancer early detection

doi: 10.1038/s41467-025-66893-4

Figure Lengend Snippet: a Schematic illustration of the EC-SNV-Seq assay process. First, the cfDNA, including both wild-type and mutated cfDNA, undergoes pre-amplification by hot-start PCR. Then, the PCR pre-amplification products are incubated with the TtAgo protein/guide DNA complex, resulting in the specific cleavage of mutant sequences while sparing the wild-type DNA. Next, in the presence of stem-loop DNA and spacer oligo, the cleaved mutant allele strongly hybridizes with the stem-loop DNA, initiating a stem-loop DNA-initiated cascade-PCR reaction for signal amplification. In contrast, due to the higher affinity of the spacer oligo for the non-cleaved wild-type allele, PCR amplification of the wild-type DNA is inhibited. b Workflow of the EC-SNV-Seq assay for programmable TtAgo -mediated cfDNA mutation profiling in multi-cancer early detection. The 500 µL pooled plasma sample is collected from ten patients, and cfDNA is extracted from the pooled sample. After the EC-SNV-Seq assay, we can accurately detect and sequence the mutant nucleotide, enabling same-day clinical diagnosis and screening of multiple cancers. Created in BioRender. Guo, C. (2026) https://BioRender.com/d4zbt0w .

Article Snippet: Finally, 10 μL of purified PCR products were subjected to Sanger sequencing analysis by Genewiz (USA).

Techniques: Amplification, Hot Start PCR, Incubation, Mutagenesis, Biomarker Discovery, Clinical Proteomics, Sequencing

Journal: Nature Communications

Article Title: Programmable Argonaute-mediated single-nucleotide variant sequencing of cell-free DNA for multi-cancer early detection

doi: 10.1038/s41467-025-66893-4

Figure Lengend Snippet: a Gel electrophoresis analysis of the TtAgo -mediated cleavage reaction showing cleavage of KRAS G12D while leaving KRAS WT intact. The experiment was independently repeated twice with similar results. b Sanger sequencing analysis of TtAgo -mediated cleavage of mixed KRAS G12D and KRAS WT targets at equal molar concentrations. c , Comparative blocking efficiency of ΔCt values and RFU ratios using three spacer oligos. Data are presented as mean ± s.d. from three independent experiments. Statistical analysis was performed using Multiple Student’s t -tests ( P < 0.05), indicating a statistically significant difference. d EC-SNV-Seq employed to analyze samples containing varying concentrations of synthetic 1% KRAS G12D DNA. Both the ΔCt values and the RFU ratios were calculated as differences relative to the 0% KRAS G12D control at each tested concentration. EC-SNV-Seq exhibited an LOD of 0.1 fM, each data point is a mean value from technical triplicates. e EC-SNV-S e q assay for KRAS G12D detection in synthetic DNA, with varying VAFs (e.g., 1% VAF (1 µM in total) = 0.99 µM KRAS WT and 0.01 µM KRAS G12D ). NTC, no template DNA control group. Panel Ⅰ: Triplicate stem-loop DNA-initiated cascade-PCR results for various VAFs. The Ct values (Panel Ⅱ) and the RFU (Panel Ⅲ) were collected from stem-loop DNA-initiated cascade-PCR results for mixtures of synthetic DNA with different VAFs of KRAS WT and KRAS G12D . All data points ranging from 0.01% to 100% exhibited a linear fit. Data are presented with three independent experiments.

Article Snippet: Finally, 10 μL of purified PCR products were subjected to Sanger sequencing analysis by Genewiz (USA).

Techniques: Nucleic Acid Electrophoresis, Sequencing, Blocking Assay, Control, Concentration Assay

Journal: Nature Communications

Article Title: Programmable Argonaute-mediated single-nucleotide variant sequencing of cell-free DNA for multi-cancer early detection

doi: 10.1038/s41467-025-66893-4

Figure Lengend Snippet: a Workflow of multiplex mutation detection. Synthesized oligos or pre-amplified cfDNA are first mixed in a reaction tube. Then, TtAgo /guide DNA complexes specific to each variant are respectively added to the reaction mixture in individual reaction tubes. After TtAgo -mediated DNA cleavage, the cleaved product undergoes stem-loop DNA-initiated cascade-PCR for signal amplification and detection. b Panel Ⅰ: multiplex DNA mutation detection with synthetic nucleotides. The synthetic mutation sequence pool contains six mutation types, each at a concentration of 1 µM, with a 1% VAF for the mutant allele. Multiple TtAgo /guide DNA complexes are applied to the mutation pool, and the cleaved products are analyzed using the EC-SNV-Seq assay to detect mutations. c Panel Ⅰ: multiplex mutation detection and classification with NSCLC cfDNA reference standards. The NSCLC ctDNA reference standard includes mutations at 0.8% EGFR L858R , 0.8% EGFR E746_A750 , 0.5% KRAS G12V , and 1% KRAS G12D . A total of 15 ng of cfDNA reference standard is amplified by multiplex PCR, and the amplified product undergoes a cleavage reaction with various combinations of the corresponding guide DNAs. d Panel Ⅰ: multiplex mutation detection and classification with clinical plasma samples from tumor patients. The plasma sample is a combination of samples from six cancer patients diagnosed with mutations in PIK3CA E545K , EGFR L858R , EGFR E746_A750 , KRAS G12A , KRAS G12V , and KRAS G12D . cfDNA extracted from the plasma mixture undergoes multiplex amplification, and the amplified product is subjected to a TtAgo -mediated cleavage reaction with specific guide DNAs. b–d Panel Ⅱ: the diluted cleavage products are analyzed using the EC-SNV-Seq assay for mutation detection. In the guide DNA panel, plus signs indicate where the corresponding guide DNA mediated a cleavage reaction with samples from the mutation sequence pool, while minus signs indicate the absence of a corresponding guide DNA for cleavage. In the mutation panel, each reaction includes the corresponding stem-loop DNA and spacer oligo. Blue rectangles indicate a negative signal, red rectangles indicate a positive signal, orange rectangles represent accurately classified mutations, and purple rectangles denote inaccurately classified mutations in the multiplex mutation detection. Created in BioRender. Created in BioRender. Guo, C. (2026) https://BioRender.com/d4zbt0w .

Article Snippet: Finally, 10 μL of purified PCR products were subjected to Sanger sequencing analysis by Genewiz (USA).

Techniques: Multiplex Assay, Mutagenesis, Synthesized, Amplification, Variant Assay, Sequencing, Concentration Assay, Clinical Proteomics

Journal: Nature Communications

Article Title: Programmable Argonaute-mediated single-nucleotide variant sequencing of cell-free DNA for multi-cancer early detection

doi: 10.1038/s41467-025-66893-4

Figure Lengend Snippet: a Workflow representation of single-mutant nucleotide sequencing. The extracted cfDNA from cancer patients’ plasma was first amplified by hot-start PCR. Then, the amplicons were selectively cleaved by TtAgo in the presence of four pairs of guide DNA. Finally, the EC-SNV-Seq assay quantified the cleaved products using either Ct or RFU values. b Denaturing PAGE gel analysis of selected guide DNA specifically cleaving the target DNA of KRAS single-mutant nucleotide missense substitutions. The experiment was independently repeated twice with similar results. c Ct values and RFU observed from the EC-SNV-Seq detection of mutations in the NSCLC panel ctDNA reference standard with TtAgo -mediated DNA cleavage using four different pairs of guide DNAs at cleavage times of 40 min. The reference standard contains 1% KRAS G12D and 0.5% KRAS G12V in cfDNA. Data are presented as mean ± s.d. from three independent experiments. d Validation of single-nucleotide variant sequencing in clinical cancer patient plasma samples with KRAS G12V/D/A mutations using the EC-SNV-Seq assay. Patients No. 1, 19, and 20 were misidentified. e Confusion matrices representing the accuracy of predicted mutant single-nucleotide classifications in the clinical sample validation set. The precision of KRAS G12D and KRAS G12A by EC-SNV-Seq is 100%, while KRAS G12V achieves a 75% identification precision. Created in BioRender. Guo, C. (2026) https://BioRender.com/d4zbt0w .

Article Snippet: Finally, 10 μL of purified PCR products were subjected to Sanger sequencing analysis by Genewiz (USA).

Techniques: Mutagenesis, Sequencing, Clinical Proteomics, Amplification, Hot Start PCR, Biomarker Discovery, Variant Assay