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Göteborgs universitets publikationer

Multiplex Preamplification of Serum DNA to Facilitate Reliable Detection of Extremely Rare Cancer Mutations in Circulating DNA by Digital PCR

Författare och institution:
J. B. Jackson (-); D. S. Choi (-); J. D. Luketich (-); A. Pennathur (-); Anders Ståhlberg (Sahlgrenska Cancer Center & Institutionen för biomedicin, avdelningen för patologi); T. E. Godfrey (-)
Publicerad i:
Journal of Molecular Diagnostics, 18 ( 2 ) s. 235-243
ISSN:
1525-1578
Publikationstyp:
Artikel, refereegranskad vetenskaplig
Publiceringsår:
2016
Språk:
engelska
Fulltextlänk:
Sammanfattning (abstract):
Tumor-specific mutations can be identified in circulating, cell-free DNA in plasma or serum and may serve as a clinically relevant alternative to biopsy. Detection of tumor-specific mutations in the plasma, however, is technically challenging. First, mutant allele fractions are typically low in a large background of wild-type circulating, cell-free DNA. Second, the amount of circulating, cell-free DNA acquired from plasma is also low. Even when using digital PCR (dPCR), rare mutation detection is challenging because there is not enough circulating, cell-free DNA to run technical replicates and assay or instrument noise does not easily allow for mutation detection <0.1%. This study was undertaken to improve on the robustness of dPCR for mutation detection. A multiplexed, pre amplification step using a high-fidelity polymerase before dPCR was developed to increase total DNA and the number of targets and technical replicates that can be assayed from a single sample. We were able to detect multiple cancer-relevant mutations within tumor-derived samples down to 0.01%. Importantly, the signal/noise ratio was improved for all preamplified targets, allowing for easier discrimination of Low-abundance mutations against false-positive signal. Furthermore, we used this protocol on clinical samples to detect known, tumor-specific mutations in patient sera. This study provides a protocol for robust, sensitive detection of circulating tumor DNA for future clinical applications.
Ämne (baseras på Högskoleverkets indelning av forskningsämnen):
MEDICIN OCH HÄLSOVETENSKAP ->
Klinisk medicin
Nyckelord:
nucleic-acids, plasma, quantification, origin, blood, cells, Pathology
Postens nummer:
233408
Posten skapad:
2016-03-18 12:49
Posten ändrad:
2016-04-29 08:49

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