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


RESULTS
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 discrete size fractions and analyzed with different duplex real-time PCR Taqman assays detecting a Y chromosome-specific target and an autosomal target. The real-time PCR research assays used differed significantly in their ability to determine the correct copy number ratio of 0.5 between Y chromosome and autosome targets in DNA of male origin. Longer PCR targets did not amplify quantitatively in circulatory DNA, due to limited presence of full-length target sequence in the sample.


CONCLUSIONS
PCR targets of the same small size are preferred over longer targets when comparing fractional circulatory DNA concentrations by real-time PCR. As an example, a DYS14/18S duplex real-time PCR research assay is presented that correctly measures the fractional concentration of male DNA in a male/female mixture of circulatory, fragmented DNA.
QIAGEN GmbH, R&D Department, Hilden, Germany. martin.horlitz@qiagen.com
Martin
M
Horlitz
Annabelle
A
Lucas
Markus
M
Sprenger-Haussels
D000328:Adult; D002138:Calibration; D041322:Chromosomes, Human, Y / Q000235:genetics; D004247:DNA / Q000097:blood* / Q000378:metabolism; D005260:Female; D005821:Genetic Techniques; D016678:Genome; D006801:Humans; D008297:Male; D044085:Microfluidics; D010949:Plasma / Q000378:metabolism*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D004247:DNA