Denaturation-Enhanced Droplet Digital PCR for Liquid Biopsies.
BACKGROUND: Although interest in droplet-digital PCR technology (ddPCR) for cell-free circulating DNA (cfDNA) analysis is burgeoning, the technology is compromised by subsampling errors and the few clinical targets that can be analyzed from limited input DNA. The paucity of starting material acts as...
| Publicado en: | Clinical Chemistry Vol. 64; no. 12; pp. 1762 - 1772 |
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| Autores principales: | , , , , , , , , , , |
| Formato: | Journal Article |
| Publicado: |
Oxford University Press / USA
Dec2018
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| Acceso en línea: | Ver este registro en EBSCOhost |
| fields | @attributes: recordID: 1 pdfLink: plink: https://search.ebscohost.com/login.aspx?direct=true&db=ccm&AN=134362643&site=ehost-live header: @attributes: shortDbName: ccm uiTerm: 134362643 longDbName: CINAHL Complete uiTag: AN controlInfo: bkinfo: dissinfo: jinfo: jid: 00099147 10CS jtl: Clinical Chemistry issn: 00099147 maglogo: N pubinfo: dt: Dec2018 vid: 64 iid: 12 pid: 622 pub: Oxford University Press / USA artinfo: ui: 134362643 10.1373/clinchem.2018.293845 134362643 ppf: 1762 ppct: 10 formats: fmt: @attributes: type: P tig: atl: Denaturation-Enhanced Droplet Digital PCR for Liquid Biopsies. aug: au: Fitarelli-Kiehl, Mariana Fangyan Yu Ashtaputre, Ravina Ka Wai Leong Ladas, Ioannis Supplee, Julianna Paweletz, Cloud Mitra, Devarati Schoenfeld, Jonathan D. Parangi, Sareh Makrigiorgos, G. Mike affil: Department of Radiation Oncology, Dana-Farber Cancer Institute and Brigham and Women’s Hospital, Harvard Medical School, Boston, MA. sug: ab: BACKGROUND: Although interest in droplet-digital PCR technology (ddPCR) for cell-free circulating DNA (cfDNA) analysis is burgeoning, the technology is compromised by subsampling errors and the few clinical targets that can be analyzed from limited input DNA. The paucity of starting material acts as a “glass ceiling” in liquid biopsies because, irrespective how analytically sensitive ddPCR techniques are, detection limits cannot be improved past DNA input limitations. METHODS: We applied denaturation-enhanced ddPCR (dddPCR) using fragmented genomic DNA (gDNA) with defined mutations. We then tested dddPCR on cfDNA from volunteers and patients with cancer for commonly-used mutations. gDNA and cfDNA were tested with and without end repair before denaturation and digital PCR. RESULTS: By applying complete denaturation of double-stranded DNA before ddPCR droplet formation the number of positive droplets increased. dddPCR using gDNA resulted in a 1.9–2.0-fold increase in data-positive droplets, whereas dddPCR applied on highly-fragmented cfDNA resulted in a 1.6–1.7-fold increase. End repair of cfDNA before denaturation enabled cfDNA to display a 1.9–2.0-fold increase in data-positive signals, similar to gDNA. Doubling of data-positive droplets doubled the number of potential ddPCR assays that could be conducted from a given DNA input and improved ddPCR precision for cfDNA mutation detection. CONCLUSIONS: dddPCR is a simple and useful modification in ddPCR that enables extraction of more information from low-input clinical samples with minor change in protocols. It should be applicable to all ddPCR platforms for mutation detection and, potentially, for gene copy-number analysis in cancer and prenatal screening. pubtype: Academic Journal doctype: Journal Article ougenre: Article language: English refInfo: holdings: @attributes: islocal: N |
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