A Simple, Universal, and Cost-Efficient Digital PCR Method for the Targeted Analysis of Copy Number Variations.
Authors of this article are:
Cassinari K, Quenez O, Joly-Hélas G, Beaussire L, Le Meur N, Castelain M, Goldenberg A, Guerrot AM, Brehin AC, Deleuze JF, Boland A, Rovelet-Lecrux A, Campion D, Saugier-Veber P, Gruchy N, Frebourg T, Nicolas G, Sarafan-Vasseur N, Chambon P.
A summary of the article is shown below:
BACKGROUND: Rare copy number variations (CNVs) are a major cause of genetic diseases. Simple targeted methods are required for their confirmation and segregation analysis. We developed a simple and universal CNV assay based on digital PCR (dPCR) and universal locked nucleic acid (LNA) hydrolysis probes.METHODS: We analyzed the mapping of the 90 LNA hydrolysis probes from the Roche Universal ProbeLibrary (UPL). For each CNV, selection of the optimal primers and LNA probe was almost automated; probes were reused across assays and each dPCR assay included the CNV amplicon and a reference amplicon. We assessed the assay performance on 93 small and large CNVs and performed a comparative cost-efficiency analysis.RESULTS: UPL-LNA probes presented nearly 20 000 000 occurrences on the human genome and were homogeneously distributed with a mean interval of 156 bp. The assay accurately detected all the 93 CNVs, except one ([H11021]200 bp), with coefficient of variation [H11021]10%. The assay was more cost-efficient than all the other methods.CONCLUSIONS: The universal dPCR CNV assay is simple, robust, and cost-efficient because it combines a straight-forward design allowed by universal probes and end point PCR, the advantages of a relative quantification of the target to the reference within the same reaction, and the high flexibility of the LNA hydrolysis probes. This method should be a useful tool for genomic medicine, which requires simple methods for the interpretation and segregation analysis of genomic variations.© 2019 American Association for Clinical Chemistry.
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