bisulfite pcr primer design software  (PrimerDesign Inc)

Journal: Cell Reports Methods

Article Title: Engineering CpG island DNA methylation in pluripotent cells through synthetic CpG-free ssDNA insertion

doi: 10.1016/j.crmeth.2023.100465

Figure Lengend Snippet: CIMR overview (A) Briefly, CG-free DNA is integrated in a target CGI by ssDNA/Cas RNP reaction, cells are clonally isolated after CG-free antibiotic selection, and de novo DNAMe assessed. (B) Schematic of CpG-free plasmid-integrated DNA (PI) and shorter CpG-free ssDNA (ssI), with insert-testing primer sites and expected PCR products displayed. (C) Both NeoR gels indicate successful integration of PI and ssI DNA by PCR, as well pilot Cre mRNA mediated removal 48 h post-transfection (right). (D) The first 6 candidate PI and ssI clones are shown. (E) Visualization of EPIC Illumina Infinium array data and target-region-induced DNAme using IgV Viewer (v.2.3). Individual CpG probes with peak intensity corresponding to DNAme level are displayed at the target CGI (DMR in black box). Blue arrows mark bisulfite (BSF)-PCR primers ( Figure S1 C). (F) Global target DNAme specificity among all probes analyzed. Scatterplots display Infinium probe values for each pairwise comparison (Pearson correlation displayed). Red dots highlight CGs falling within the CIMR DMR (N = 38). (G) The most significant DMR, target MLH1, was specifically and consistently induced across all insert-containing clones ( Table S2 B). DMRs (N = 28) observed between H1 controls and ssI-C1 are displayed by heatmap of mean individual DMR DNAme levels. (H) qRT-PCR analysis of MLH1 expression post CpG-free PI or ssI by the standard curve method. Mean normalized expression ± SE (error bars) to internal control gene TPT1 is displayed (∗p < 0.05, t test).

Article Snippet: To easily visualize strong candidate regions, already available bisulfite PCR primer design software is helpful (e.g., methprimer ), as candidate primers are designed specifically to bind more complex sequences of lower GC content and minimal CpGs.

Techniques: Isolation, Selection, Plasmid Preparation, Transfection, Clone Assay, Comparison, Quantitative RT-PCR, Expressing, Control

Journal: Cell Reports Methods

Article Title: Engineering CpG island DNA methylation in pluripotent cells through synthetic CpG-free ssDNA insertion

doi: 10.1016/j.crmeth.2023.100465

Figure Lengend Snippet: CIMR testing in other PSCs and CIMP cancer lines, and CIMR testing guidelines (A) Visualization of DMRs by heatmap of DNAme levels across samples. Known CIMP lines have higher CGI DNAme and cluster separately from hypomethylated PSCs. For inclusion, a CGI required a DNAme level of >0.5 in ≥2 samples and an SD of >0.25 (N = 6,062 CGIs). (B) CIMR DNAme occurs in multiple PSCs and embryonal carcinoma Nt2d1 cells but not CIMP cancer lines. See Figure 1 E for IgV. (C) CIMR DNAme testing guidelines. Briefly, after CGI selection, gRNAs can be ranked for cutting specificity, prevalidated by Surveyor or T7E1 assays, and then paired with a candidate ssDNA repair template. CIMR RNP/ssDNA reactions are followed by antibiotic selection, FACS isolation of single-cell clones as desired, inserts validated by PCR, and DNAme assessed by Infinium analysis or another suitable technique. (D) ONECUT1 CIMRs were tested at 2 different CGIs ( Figure S4 G). (E) Specificity of the edited ONECUT1 CGI is shown by x/y scatterplot of all tested CGs, with 8 altered CGs highlighted in red. (F) TP53 CGI CIMR testing. 4/12 TP53 clones harbored full-length CG-free inserts. (G) Global specificity is shown as in (E), with the 4 CGI spanning probes marked in red. (H and I) TP53 CGI DNAme clones have repressed TP53 (H) and antisense WRAP53 (I) expression (mean normalized expression ± SE (error bars) to internal control TPT1 , ∗p < 0.05, t test).

Article Snippet: To easily visualize strong candidate regions, already available bisulfite PCR primer design software is helpful (e.g., methprimer ), as candidate primers are designed specifically to bind more complex sequences of lower GC content and minimal CpGs.

Techniques: Selection, Isolation, Clone Assay, Expressing, Control