ArticleClinical chemistry2017
Multiplexed Elimination of Wild-Type DNA and High-Resolution Melting Prior to Targeted Resequencing of Liquid Biopsies.
Article in Clinical chemistry, 2017. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 13 papers.
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Who cites it
13 citing papers in PubMed, 26 citations in OpenAlex.
- Enzymatic Methods for Mutation Detection in Cancer Samples and Liquid Biopsies.International journal of molecular sciences · 2023Review
- Review
- Nuclease-Assisted, Multiplexed Minor-Allele Enrichment: Application in Liquid Biopsy of Cancer.Methods in molecular biology (Clifton, N.J.) · 2022Article
- Detection of Microsatellite Instability by High-Resolution Melting Analysis in Colorectal CancerIranian biomedical journal · 2022Article
- Argonaute integrated single-tube PCR system enables supersensitive detection of rare mutations.Nucleic acids research · 2021Article
- Sensitive detection of microsatellite instability in tissues and liquid biopsies: Recent developments and updates.Computational and structural biotechnology journal · 2021Review
- PIK3CA mutation enrichment and quantitation from blood and tissue.Scientific reports · 2020Article
- SLAM-MS: Mutation scanning of stem-loop amplicons with TaqMan probes by quantitative DNA melting analysis.Scientific reports · 2020Article
- Cell-free DNA in cancer: current insights.Cellular oncology (Dordrecht, Netherlands) · 2019Review
- Extreme PCR Meets High-Speed Melting: A Step Closer to Molecular Diagnostics "While You Wait".Clinical chemistry · 2019Article
- Denaturation-Enhanced Droplet Digital PCR for Liquid Biopsies.Clinical chemistry · 2018Article
- The Omics Revolution Continues: The Maturation of High-Throughput Biological Data Sources.Yearbook of medical informatics · 2018Review
- Enhanced detection of microsatellite instability using pre-PCR elimination of wild-type DNA homo-polymers in tissue and liquid biopsies.Nucleic acids research · 2018Article
Corrections and comments
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Authors and funding
8 authors at 4 institutions in 1 country.
Funding
Abstract
backgroundThe use of clinical samples and circulating cell-free DNA (cfDNA) collected from liquid biopsies for diagnostic and prognostic applications in cancer is burgeoning, and improved methods that reduce the influence of excess wild-type (WT) portion of the sample are desirable. Here we present enrichment of mutation-containing sequences using enzymatic degradation of WT DNA. Mutation enrichment is combined with high-resolution melting (HRM) performed in multiplexed closed-tube reactions as a rapid, cost-effective screening tool before targeted resequencing.
methodsWe developed a homogeneous, closed-tube approach to use a double-stranded DNA-specific nuclease for degradation of WT DNA at multiple targets simultaneously. The No Denaturation Nuclease-assisted Minor Allele Enrichment with Probe Overlap (ND-NaME-PrO) uses WT oligonucleotides overlapping both strands on putative DNA targets. Under conditions of partial denaturation (DNA breathing), the oligonucleotide probes enhance double-stranded DNA-specific nuclease digestion at the selected targets, with high preference toward WT over mutant DNA. To validate ND-NaME-PrO, we used multiplexed HRM, digital PCR, and MiSeq targeted resequencing of mutated genomic DNA and cfDNA.
resultsSerial dilution of KRAS mutation-containing DNA shows mutation enrichment by 10- to 120-fold and detection of allelic fractions down to 0.01%. Multiplexed ND-NaME-PrO combined with multiplexed PCR-HRM showed mutation scanning of 10-20 DNA amplicons simultaneously. ND-NaME-PrO applied on cfDNA from clinical samples enables mutation enrichment and HRM scanning over 10 DNA targets. cfDNA mutations were enriched up to approximately 100-fold (average approximately 25-fold) and identified via targeted resequencing.
conclusionsClosed-tube homogeneous ND-NaME-PrO combined with multiplexed HRM is a convenient approach to efficiently enrich for mutations on multiple DNA targets and to enable prescreening before targeted resequencing.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.