Optimized quantification of fragmented, free circulating DNA in human blood plasma using a calibrated duplex real-time PCR.

ABSTRACT:

Background

Duplex real-time PCR assays have been widely used to determine amounts and concentrations of free circulating DNA in human blood plasma samples. Circulatory plasma DNA is highly fragmented and hence a PCR-based determination of DNA concentration may be affected by the limited availability of full-length targets in the DNA sample. This leads to inaccuracies when counting PCR target copy numbers as whole genome equivalents.

Methodology/principal findings

A model system was designed allowing for assessment of bias in a duplex real-time PCR research assay. We collected blood plasma samples from male donors in pools of 6 to 8 individuals. Circulatory plasma DNA was extracted and separated by agarose gel electrophoresis. Separated DNA was recovered from the gel in discr

SUBMITTER: Horlitz M 

PROVIDER: S-EPMC2746311 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature