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

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Circulating Tumor Cells

Part of the book series: Current Cancer Research ((CUCR))

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Abstract

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 cancer 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 cfDNA, are potentially powerful prognostic and predictive cancer biomarkers. 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 sensitivity 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 genomic DNA level prior to DNA amplification and combines well with existing genotyping approaches. Applications in liquid biopsy-based diagnostics using circulating DNA are presented.

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Acknowledgments

The work was partially supported by the National Institutes of Health grants R33 CA217652 and R01 CA221874. The contents of this manuscript do not necessarily represent the official views of the National Cancer Institute or the National Institutes of Health.

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Authors and Affiliations

  1. Department of Radiation Oncology, Dana-Farber Cancer Institute and Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA

    Ka Wai Leong, Fangyan Yu & G. Mike Makrigiorgos

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Correspondence to G. Mike Makrigiorgos .

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  1. Department of Pathology and Immunology, Washington University in St. Louis School of Medicine, St. Louis, MO, USA

    Richard J. Cote

  2. ACTC Lab, Laboratory of Analytical Chemistry, Department of Chemistry, National and Kapodistrian University of Athens, Athens, Greece

    Evi Lianidou

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Leong, K.W., Yu, F., Makrigiorgos, G.M. (2023). Minor Allele Enrichment in Liquid Biopsies Using Nuclease-Assisted Elimination of Wild-Type DNA. In: Cote, R.J., Lianidou, E. (eds) Circulating Tumor Cells. Current Cancer Research. Springer, Cham. https://doi.org/10.1007/978-3-031-22903-9_2

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