Methylation-sensitive enrichment of minor DNA alleles using a double-strand DNA-specific nuclease
MetadataShow full item record
CitationLiu, Yibin, Chen Song, Ioannis Ladas, Mariana Fitarelli-Kiehl, and G. Mike Makrigiorgos. 2017. “Methylation-sensitive enrichment of minor DNA alleles using a double-strand DNA-specific nuclease.” Nucleic Acids Research 45 (6): e39. doi:10.1093/nar/gkw1166. http://dx.doi.org/10.1093/nar/gkw1166.
AbstractAbstract 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.
Citable link to this pagehttp://nrs.harvard.edu/urn-3:HUL.InstRepos:32630430
- HMS Scholarly Articles