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Nuclease enrichment and qPCR detection of rare nucleotide variants

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 overlapping 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

 Fuente: Methods in Molecular Biology, 2023, 2621, 41-56

 Editorial: Springer Nature

 Año de publicación: 2023

 Nº de páginas: 16

 Tipo de publicación: Artículo de Revista

 DOI: 10.1007/978-1-0716-2950-5_4

 ISSN: 1064-3745,1940-6029

 Url de la publicación: https://doi.org/10.1007/978-1-0716-2950-5_4

Autoría

KERAITE, IEVA

LESLIE, NICHOLAS R.