Nanopore-Based Assay for Detection of Methylation in Double-Stranded DNA Fragments

DNA methylation is an epigenetic modification of DNA in which methyl groups are added at the 5-carbon position of cytosine. Aberrant DNA methylation, which has been associated with carcinogenesis, can be assessed in various biological fluids and potentially can be used as markers for detection of cancer. Analytically sensitive and specific assays for methylation targeting low-abundance and fragmented DNA are needed for optimal clinical diagnosis and prognosis. We present a nanopore-based direct methylation detection assay that circumvents bisulfite conversion and polymerase chain reaction amplification. Building on our prior work, we used methyl-binding proteins (MBPs), which selectively label the methylated DNA. The nanopore-based assay selectively detects methylated DNA/MBP complexes through a 19 nm nanopore with significantly deeper and prolonged nanopore ionic current blocking, while unmethylated DNA molecules were not detectable due to their smaller diameter. Discrimination of hypermethylated and unmethylated DNA on 90, 60, and 30 bp DNA fragments was demonstrated using sub-10 nm nanopores. Hypermethylated DNA fragments fully bound with MBPs are differentiated from unmethylated DNA at 2.1- to 6.5-fold current blockades and 4.5- to 23.3-fold transport durations. Furthermore, these nanopore assays can detect the <i>CpG</i> dyad in DNA fragments and could someday profile the position of methylated <i>CpG</i> sites on DNA fragments

Transcript levels for CCNB1, DLG7, and HMMR measured in CaP and noncancerous prostate tissue.

<p>Panels on the left compare transcript levels in CaP bulk tissue (full symbols) with the levels measured in benign prostate tissue (open symbols) from men free of CaP (BP) and in benign prostate tissue (BPC) adjacent to CaP of combined Gleason score 6 (gs6). Panels on the right display transcript levels measured in non-neoplastic prostate epithelial cells isolated by laser capture microdissection (LCM) in benign tissues (open symbols): BP, benign prostatic hyperplasia (BPH) and BPC adjacent to CaP of the indicated Gleason score (gs). Full symbols in panels on the right denote transcript levels measured in LCM-isolated CaP cells: high-grade prostatic intraepithelial neoplasia (HGPIN), the cells isolated from areas of combined Gleason scores 6 through 8 and cells isolated from lymph node metastases (met). CCNB1, cyclin B1; DLG7, discs large homolog 7; HMMR, hyaluronan-mediated motility receptor.</p

Three hypoxia-controlled genes associated with Gleason score and prognosis.

<p>Among the hypoxia-regulated genes significantly overexpressed in CaP, cyclin B1 (CCNB1), DLGAP5 and hyaluronan-mediated motility receptor (HMMR) were associated with Gleason score and disease outcome.</p

Receiver operating characteristic (ROC) analysis for DLG7.

<p>The area under the curve was 0.74 (95% CI, 068–0.80). Inset: Scatter plot of the normalized expression values for cases (red) and controls (green).</p

Additional file 1: of Chromoanasynthesis is a common mechanism that leads to ERBB2 amplifications in a cohort of early stage HER2+ breast cancer samples

Figure S1-S17. Genome U plots of all the additional cases. Figure S18 Heat map of the EGFR and ERBB3 expression log2 expression by RNAseq. Dark red indicates low expression where yellow indicates high expression. Table S1 BRISQ summary of tumor specimens. (PPTX 3536 kb