title: Methylation-sensitive enrichment of minor DNA alleles using a double-strand DNA-specific nuclease.
abstract: Aberrant methylation changes, often present in a minor allelic fraction in clinical samples such as plasma-circulating DNA (cfDNA), are potentially powerful prognostic and predictive biomarkers in human disease including cancer. We report on a novel, highly-multiplexed approach to facilitate analysis of clinically useful methylation changes in minor DNA populations. Methylation Specific Nuclease-assisted Minor-allele Enrichment (MS-NaME) employs a double-strand-specific DNA nuclease (DSN) to remove excess DNA with normal methylation patterns. The technique utilizes oligonucleotide-probes that direct DSN activity to multiple targets in bisulfite-treated DNA, simultaneously. Oligonucleotide probes targeting unmethylated sequences generate local double stranded regions resulting to digestion of unmethylated targets, and leaving methylated targets intact; and vice versa. Subsequent amplification of the targeted regions results in enrichment of the targeted methylated or unmethylated minority-epigenetic-alleles. We validate MS-NaME by demonstrating enrichment of RARb2, ATM, MGMT and GSTP1 promoters in multiplexed MS-NaME reactions (177-plex) using dilutions of methylated/unmethylated DNA and in DNA from clinical lung cancer samples and matched normal tissue. MS-NaME is a highly scalable single-step approach performed at the genomic DNA level in solution that combines with most downstream detection technologies including Sanger sequencing, methylation-sensitive-high-resolution melting (MS-HRM) and methylation-specific-Taqman-based-digital-PCR (digital Methylight) to boost detection of low-level aberrant methylation-changes.
authors.affiliation: Department of Radiation Oncology, Dana-Farber Cancer Institute and Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Yibin
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Department of Radiation Oncology, Dana-Farber Cancer Institute and Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Chen
authors.initials: C
authors.lastname: Song
authors.affiliation: Department of Radiation Oncology, Dana-Farber Cancer Institute and Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Ioannis
authors.initials: I
authors.lastname: Ladas
authors.affiliation: Department of Radiation Oncology, Dana-Farber Cancer Institute and Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Mariana
authors.initials: M
authors.lastname: Fitarelli-Kiehl
authors.affiliation: Department of Radiation Oncology, Dana-Farber Cancer Institute and Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: G Mike
authors.initials: GM
authors.lastname: Makrigiorgos
mesh_terms: D000483:Alleles; D004247:DNA / Q000097:blood; D019175:DNA Methylation; D003851:Deoxyribonucleases; D006801:Humans; D008175:Lung Neoplasms / Q000097:blood; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D015345:Oligonucleotide Probes; D060888:Real-Time Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013447:Sulfites
chemical_list: D015345:Oligonucleotide Probes; D013447:Sulfites; D004247:DNA; D003851:Deoxyribonucleases; C042345:hydrogen sulfite