Minor Allele Enrichment in Liquid Biopsies Using Nuclease-Assisted Elimination of Wild-Type DNA

Circulating DNA analysis presents opportunities in harnessing the potential of tumor biomarkers in a minimally invasive manner but also presents several technical challenges. For example, excess wild-type (WT) DNA often masks low-level DNA alterations containing clinically relevant information for cancerCancer diagnosis or therapy. In this chapter, we describe nuclease-assisted minor allele enrichment with probe overlap (NaME-PrO), an approach to reduce WT-DNA to facilitate detection of rare alterations. NaME-PrO employs a double-strand DNA-specific nuclease and overlapping oligonucleotide probes interrogating multiple DNA targets of interest. Following genomic DNA denaturation, the temperature is lowered so that probes form double-stranded regions with their targets, thereby guiding nuclease digestion to the targeted DNA sites. Mutations create mismatches that inhibit digestion; thus subsequent amplification yields DNA with enhanced mutations at multiple targets. In this manner, WT DNA at numerous DNA regions can be digested simultaneously, thus enabling enhanced detection of mutations. In an adaptation of the same approach in the field of methylation, a methylation-sensitive nuclease-assisted minor allele enrichment, MS-NaME, was developed. Aberrant methylation changes, often present in a minor allelic fraction in clinical samples such as cfDNACell-free DNA (cfDNA), are potentially powerful prognostic and predictive cancer biomarkersBiomarkers. During MS-NaME, oligonucleotide probes targeting unmethylated sequences in bisulfite-treated DNA generate local double-stranded regions resulting in digestion of unmethylated targets and leaving methylated targets intact (or vice versa), thereby leading to selective enrichment of differentially methylated or unmethylated DNA targets that can be used for downstream methylation detection assays. Finally, application of MSI-NaME-PrO to enrich indels at microsatellites and to enhance sensitivitySensitivities for detection of microsatellite instability MSI is also described. NaME-PrO, MS-NaME, and MSI-NaME-PrO provide a simple, low-cost, broad application platform that can be applied at the genomicGenomics DNA level prior to DNA amplification and combines well with existing genotyping approaches. Applications in liquid biopsy-based diagnostics using circulating DNACirculating DNA are presented.