Unknown

Dataset Information

0

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.

SUBMITTER: Liu Y 

PROVIDER: S-EPMC5389605 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

altmetric image

Publications

Methylation-sensitive enrichment of minor DNA alleles using a double-strand DNA-specific nuclease.

Liu Yibin Y   Song Chen C   Ladas Ioannis I   Fitarelli-Kiehl Mariana M   Makrigiorgos G Mike GM  

Nucleic acids research 20170401 6


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 rem  ...[more]

Similar Datasets

| S-EPMC5100565 | biostudies-literature
| S-EPMC5031800 | biostudies-literature
| S-EPMC4654060 | biostudies-literature
| S-EPMC2681233 | biostudies-literature
| S-EPMC3909494 | biostudies-literature
| S-EPMC4824098 | biostudies-literature
| S-EPMC9712704 | biostudies-literature
| S-EPMC2966045 | biostudies-literature
| S-EPMC6326791 | biostudies-literature
| S-EPMC6208412 | biostudies-literature