Multiplexing of E-ice-COLD-PCR Assays for Mutation Detection and Identification

Mauger, Florence; Daunay, Antoine; Deleuze, Jean‐François; Tost, Jörg; How‐Kit, Alexandre

doi:10.1373/clinchem.2016.258830

Cited by 12 publications

(11 citation statements)

References 7 publications

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“…Although E-ice COLD-PCR is emerging as a promising alternative for rare allele quantification, it may also be blind to copy number alterations, and the concurrent screening of multiple exons requires substantial assay optimization. 48,49 Currently available custom or commercial multiplex KRAS ddPCR kits can only simultaneously screen for mutations at a certain exon. Relying on one single screening test may therefore overlook additional mutations underlying treatment resistance, a fact that negatively affects diagnostic sensitivity.…”

Section: R Rmentioning

confidence: 99%

A Novel Multiplex Droplet Digital PCR Assay to Identify and Quantify KRAS Mutations in Clinical Specimens

Alcaide

Cheung

Bushell

et al. 2019

The Journal of Molecular Diagnostics

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Investigating the mutations driving non Hodgkin lymphomas and developing plasma-based assays for tumour detection and monitoring) (R.D.M.); a post-doctoral fellowship from the Michael Smith Foundation for Health Research (H.-L.W.); and a Bio-Rad Droplet Innovation Award (M.A.). M.A., M.C., and K.B. contributed equally to this work. Disclosures: A part of this study was also funded by a Droplet Innovation Award given to M.A. by Bio-Rad.

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Section: R Rmentioning

confidence: 99%

A Novel Multiplex Droplet Digital PCR Assay to Identify and Quantify KRAS Mutations in Clinical Specimens

Alcaide

Cheung

Bushell

et al. 2019

The Journal of Molecular Diagnostics

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“…All molecules different from the blocked pattern will be amplified and the subsequent sequencing-based analysis gives detailed information on the molecules that have been enriched. E-ice-COLD-PCR reaction can be easily multiplexed and the level of mutations or DNA methylation can be quantified using standard curves for the analysis of very low input material such as ccfDNA (Mauger et al, 2016 andSefrioui et al, 2017).…”

Section: Co-amplification At Lower Denaturation Temperature Pcr (Coldmentioning

confidence: 99%

“…1. E-ice-COLD PCR conditions for multiplex assays should be the same as for the simplex assay (Mauger et al, 2016). Prior to the Pyrosequencing reaction on the PyroMark Q96 MD System, a sample preparation step is performed which includes the purification and the denaturation of E-ice-COLD-PCR product to obtain single-stranded product followed by hybridization of the Pyrosequencing primer.…”

Section: Multiplex E-ice-cold-pcrmentioning

confidence: 99%

Enhanced-ice-COLD-PCR for the Sensitive Detection of Rare DNA Methylation Patterns in Liquid Biopsies

Mauger¹,

Tost²

2019

BIO-PROTOCOL

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In the context of precision medicine, the identification of novel biomarkers for the diagnosis of disease, prognosis, predicting treatment outcome and monitoring of treatment success is of great importance. The analysis of methylated circulating-cell free DNA provides great promise to complement or replace genetic markers for these applications, but is associated with substantial challenges. This is particularly true for the detection of rare methylated DNA molecules in a limited amount of sample such as tumor released hypermethylated molecules in the background of DNA fragments from normal cells, especially lymphocytes. Technologies for the sensitive detection of DNA methylation have been developed to enrich specifically methylated DNA or unmethylated DNA using among other methods: enzymatic digestion, methylation-specific PCR (often combined with TaqMan like oligonucleotide probes (MethyLight)) and co-amplification at lower denaturation temperature PCR (COLD-PCR). E-ice-COLD-PCR (Enhanced-improved and complete enrichment-COLD-PCR) is a sensitive method that takes advantage of a Locked Nucleic Acid (LNA)-containing oligonucleotide probe to block specifically unmethylated CpG sites allowing the strong enrichment of low-abundant methylated CpG sites from a limited quantity of input. E-ice-COLD-PCRs are performed on bisulfite-converted DNA followed by Pyrosequencing analysis. The quantification of the initially present DNA methylation level is obtained using calibration curves of methylated and unmethylated DNA. The E-ice-COLD-PCR reactions can be multiplexed, allowing the analysis and quantification of the DNA methylation level of several target genes. In contrast to the above-mentioned assays, E-ice-COLD-PCR will also perform in the presence of frequently occurring heterogeneous DNA methylation patterns at the target sites. The presented protocol describes the development of an E-ice-COLD-PCR assay including assay design, optimization of E-ice-COLD-PCR conditions including annealing temperature, critical temperature and concentration of LNA blocker probe followed by Pyrosequencing analysis.

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“…A number of approaches have been described to reduce excess WT DNA (8), thereby facilitating detection of mutated DNA, via the use of polymerase chain reaction (PCR) (9–19). Use of PCR based mutation enrichment on multiple DNA targets simultaneously has also been reported (20, 21). However, the approach is technically demanding and requires extensive optimization which becomes more difficult as the number of multiplexed targets increases (22).…”

Section: Introductionmentioning

confidence: 99%

Multiplexed Elimination of Wild-Type DNA and High-Resolution Melting Prior to Targeted Resequencing of Liquid Biopsies

et al. 2017

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BACKGROUND The 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 wild-type DNA (WT-DNA). Mutation enrichment is combined with high-resolution-melting (HRM) performed in multiplexed closed-tube reactions, as a rapid, cost-effective screening tool prior to targeted re-sequencing. METHODS We developed a homogeneous, closed-tube approach to utilize a double-strand DNA-specific nuclease (DSN) for degradation of WT-DNA at multiple targets simultaneously. The No-Denaturation Nuclease-assisted Minor Allele Enrichment with Probe-Overlap (ND-NaME-PrO) employs 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 towards WT over mutant DNA. To validate ND-NaME-PrO we employed multiplexed-HRM, digital PCR and Miseq targeted re-sequencing of mutated genomic DNA and cfDNA. RESULTS Serial dilution of KRAS mutation-containing DNA demonstrates mutation enrichment by 10–120-fold and detection of allelic-fractions down to 0.01%. Multiplexed ND-NaME-PrO combined with multiplexed PCR-HRM demonstrated 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 ~100-fold, average ~25-fold, and identified via targeted re-sequencing. CONCLUSIONS Closed-tube homogeneous ND-NaME-PrO combined with multiplexed-HRM is a convenient approach to efficiently enrich for mutations on multiple DNA-targets and enabling pre-screening prior to targeted re-sequencing.

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Multiplexing of E-ice-COLD-PCR Assays for Mutation Detection and Identification

Cited by 12 publications

References 7 publications

A Novel Multiplex Droplet Digital PCR Assay to Identify and Quantify KRAS Mutations in Clinical Specimens

A Novel Multiplex Droplet Digital PCR Assay to Identify and Quantify KRAS Mutations in Clinical Specimens

Enhanced-ice-COLD-PCR for the Sensitive Detection of Rare DNA Methylation Patterns in Liquid Biopsies

Multiplexed Elimination of Wild-Type DNA and High-Resolution Melting Prior to Targeted Resequencing of Liquid Biopsies

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