Journal: Cancer cell

Article Title: Comprehensive Molecular Characterization Identifies Distinct Genomic and Immune Hallmarks of Renal Medullary Carcinoma

doi: 10.1016/j.ccell.2020.04.002

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Control DNA was used from Promega (Human Genomic DNA Female N 30742202/Male N 30993901).

Techniques: Control, Virus, Recombinant, Library Quantification, DNA Methylation Assay, Empire Assay, Imaging, Sequencing, Plasmid Preparation, Software

Journal: Nucleic Acids Research

Article Title: Mutation enrichment in human DNA samples via UV-mediated cross-linking

doi: 10.1093/nar/gkab1222

Figure Lengend Snippet: UVME-PCR reaction. ( A ) A UV lamp is attached to a thermal cycler to provide UV irradiation during PCR. The UV lamp can be manually switched on/off any time during PCR cycling to provide UV irradiation to DNA samples. ( B ) CNVK-modified probes are applied in UVME before PCR. Target-specific CNVK-modified probes are designed to match the WT sense strand while forming a mismatch with mutated sense DNA strands. Common CNVK-modified probes matching both WT and mutated DNA are designed for the antisense DNA strand. When UV is applied, the hybridized CNVK-modified probes cross-link with T or C on the opposite DNA strand at the −1 position. ( C ) UVME-PCR is applied in two stages. First, 10–20 cycles of regular closed-lid PCR are performed to build up target DNA copies from genomic DNA. Optionally, the pre-amplification can be performed via COLD-PCR to amplify preferentially mutated DNA, which further enhances the mutation enrichment. In the second stage of UVME-PCR, the lid of the PCR machine is opened, and the UV lamp is attached. Following denaturation, CNVK-modified probes and primers bind to their corresponding sequences during the annealing stage. The target-specific CNVK-modified probes hybridize to the sense strand WT DNA preferentially as compared to the sense strand mutated DNA and application of UV at this stage blocks subsequent polymerization. Meanwhile, the common CNVK-modified probes bind equally to both WT and mutant antisense templates and block antisense strand polymerization. Accordingly, UV-mediated cross-linking reduces amplification of both strands of WT DNA but only one strand of mutated DNA, in each PCR cycle, resulting in robust mutation enrichment during PCR.

Article Snippet: Human male genomic DNA (HMC, Promega Corporation) was used as WT DNA.

Techniques: Irradiation, Modification, Amplification, Co-amplification at Lower Denaturation temperature PCR, Mutagenesis, Blocking Assay

Journal: Nucleic Acids Research

Article Title: Mutation enrichment in human DNA samples via UV-mediated cross-linking

doi: 10.1093/nar/gkab1222

Figure Lengend Snippet: Application of UVME-PCR to enrich KRAS exon 2 mutations (G12S, G12V, G12A and G13D) and combination of pre-PCR-UVME and UVME-PCR. ( A ) UVME-PCR was tested with serial dilutions of genomic DNA from four KRAS -mutated cell lines (A549-G12S, SW480-G12V, H2009-G12A and LOVO-G13D) into WT DNA. A single pair of KRAS target-specific CNVK-modified probes and KRAS common CNVK-modified probes was used for all four mutations. UVME-PCR products screened via ddPCR quantify mutations down to 0.01–0.03% MAF for all four KRAS mutations. ( B ) Pre-PCR-UVME, UVME and combination of pre-PCR-UVME and UVME-PCR were performed on 30 ng sheared gDNA containing 0.1% or 1% KRAS G12V mutation. The final product screened via ddPCR showed that UVME-PCR has somewhat better enrichment efficiency than pre-PCR-UVME, while performing sequential pre-PCR-UVME and UVME-PCR could further boost the enrichment.

Article Snippet: Human male genomic DNA (HMC, Promega Corporation) was used as WT DNA.

Techniques: Modification, Mutagenesis

Journal: Nucleic Acids Research

Article Title: Mutation enrichment in human DNA samples via UV-mediated cross-linking

doi: 10.1093/nar/gkab1222

Figure Lengend Snippet: Application of UVME-PCR to a p53 mutation, C275G. UVME-PCR was tested with serial dilutions of genomic DNA containing a hotspot tp53 mutation (PFSK cell line, C275G) into WT DNA. Serial dilutions of PFSK into HMC DNA down to 0.001% MAF were formed, with the lowest dilutions using an input of 1 μg DNA. ( A ) UVME-PCR followed by Sanger sequencing reveals that mutations in dilutions down to 0.1% were detectable following UV irradiation, while no mutations are evident without UV application. ( B ) When UVME-PCR was followed by HRM, mutations down to 0.001% were discriminated from WT samples following UV irradiation. ( C ) UVME-PCR followed by TaqMan genotyping shows that application of UV increases mutation abundance from 0.01–1% to 3–80%, respectively. ( D ) UVME followed by ddPCR detects mutations down to 0.001% and increases mutation abundance from 0.001–1% to 1–80%, respectively.

Article Snippet: Human male genomic DNA (HMC, Promega Corporation) was used as WT DNA.

Techniques: Mutagenesis, Sequencing, Irradiation

Journal: Nucleic Acids Research

Article Title: Mutation enrichment in human DNA samples via UV-mediated cross-linking

doi: 10.1093/nar/gkab1222

Figure Lengend Snippet: Clinical tissue samples were applied in UVME-PCR. UVME-PCR was applied to DNA extracted from 10 lung tumor tissue samples previously shown to harbor medium to low KRAS G12D, G12V or G13D mutations and four samples with WT KRAS . Human male genomic DNA was also screened as a control. ( A ) ddPCR was first applied to document the mutation abundance in the absence of UVME enrichment and mutations in the 1–40% abundance were demonstrated. ddPCR applied following UVME-PCR resulted in an increase in mutation abundance. No mutations were detected for the four WT samples and HMC DNA. ( B ) UVME-PCR followed by Sanger sequencing depicts mutations for all six samples harboring KRAS mutations; mutations could not be detected in five out of six positive samples in the absence of UV irradiation, using Sanger sequencing.

Article Snippet: Human male genomic DNA (HMC, Promega Corporation) was used as WT DNA.

Techniques: Control, Mutagenesis, Sequencing, Irradiation