Nuclease Enrichment and qPCR Detection of Rare Nucleotide Variants

Ieva Keraite, Virginia Alvarez-Garcia, Nicholas R. Leslie

Research output: Chapter in Book/Report/Conference proceedingChapter (peer-reviewed)peer-review

1 Citation (Scopus)


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.

Original languageEnglish
Title of host publicationClinical Applications of Nucleic Acid Amplification
PublisherHumana Press
Number of pages16
ISBN (Electronic)9781071629505
ISBN (Print)9781071629499
Publication statusPublished - 11 Apr 2023

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029


  • Breast cancer
  • Mutant allele enrichment
  • PIK3CA
  • qPCR
  • Quantification
  • Tissue biopsy
  • VAF

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics


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