Abstract
The emergence of circulating DNA analysis in blood during the past decade has responded to the need for noninvasive alternatives to classical tissue biopsies. This has coincided with the development of techniques that allow the detection of low-frequency allele variants in clinical samples that typically carry very low amounts of fragmented DNA, such as plasma or FFPE samples. Enrichment of rare variants by nuclease-assisted mutant allele enrichment with overlap** probes (NaME-PrO) enables a more sensitive detection of mutations in tissue biopsy samples alongside standard qPCR detection assays. Such sensitivity is normally achieved by other more complex PCR methods, such as TaqMan qPCR and digital droplet PCR (ddPCR). Here we describe a workflow of mutation-specific nuclease-based enrichment combined with a SYBR Green real-time quantitative PCR detection method that provides comparable results to ddPCR. Using a PIK3CA mutation as an example, this combined workflow enables detection and accurate prediction of initial variant allele fraction in samples with a low mutant allele frequency (<1%) and could be applied flexibly to detect other mutations of interest.
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Acknowledgments
The authors’ work develo** these methods was funded by the Chief Scientist Office (ETM/433) and by Medical Research Scotland (PhD-883-2015).
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Keraite, I., Alvarez-Garcia, V., Leslie, N.R. (2023). Nuclease Enrichment and qPCR Detection of Rare Nucleotide Variants. In: Myers, M.B., Schandl, C.A. (eds) Clinical Applications of Nucleic Acid Amplification. Methods in Molecular Biology, vol 2621. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2950-5_4
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DOI: https://doi.org/10.1007/978-1-0716-2950-5_4
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