Direct Quantification of Cell-Free, Circulating DNA from Unpurified Plasma

Abstract

Cell-free DNA (cfDNA) in body tissues or fluids is extensively investigated in clinical medicine and other research fields. In this article we provide a direct quantitative real-time PCR (qPCR) as a sensitive tool for the measurement of cfDNA from plasma without previous DNA extraction, which is known to be accompanied by a reduction of DNA yield. The primer sets were designed to amplify a 90 and 222 bp multi-locus L1PA2 sequence. In the first module, cfDNA concentrations in unpurified plasma were compared to cfDNA concentrations in the eluate and the flow-through of the QIAamp DNA Blood Mini Kit and in the eluate of a phenol-chloroform isoamyl (PCI) based DNA extraction, to elucidate the DNA losses during extraction. The analyses revealed 2.79-fold higher cfDNA concentrations in unpurified plasma compared to the eluate of the QIAamp DNA Blood Mini Kit, while 36.7% of the total cfDNA were found in the flow-through. The PCI procedure only performed well on samples with high cfDNA concentrations, showing 87.4% of the concentrations measured in plasma. The DNA integrity strongly depended on the sample treatment. Further qualitative analyses indicated differing fractions of cfDNA fragment lengths in the eluate of both extraction methods. In the second module, cfDNA concentrations in the plasma of 74 coronary heart disease patients were compared to cfDNA concentrations of 74 healthy controls, using the direct L1PA2 qPCR for cfDNA quantification. The patient collective showed significantly higher cfDNA levels (mean (SD) 20.1 (23.8) ng/ml; range 5.1–183.0 ng/ml) compared to the healthy controls (9.7 (4.2) ng/ml; range 1.6–23.7 ng/ml). With our direct qPCR, we recommend a simple, economic and sensitive procedure for the quantification of cfDNA concentrations from plasma that might find broad applicability, if cfDNA became an established marker in the assessment of pathophysiological conditions.

Circulating Tumor DNA as a Liquid Biopsy for Cancer

“…This is especially true for the quantification of ctDNA, because a variety of copy number changes occur in a tumor (38 ). Other approaches rely on the quantification of cfDNA directly from plasma without any prior DNA isolation (39 ). Third, less is known about the origin and the detailed mechanism of cfDNA release, and in most studies confounding events that might also contribute to the release of cfDNA, e.g., nonmalignant diseases, heavy smoking, pregnancy, exercise, and heart dysfunction, have not been taken into account.…”

A field guide for cancer diagnostics using cell‐free DNA: From principles to practice and clinical applications

“…Since interaction effects between the method for cfDNA extraction and the method applied for mutation detection may lead to increased artifacts, careful comparison during establishing liquid biopsy procedures is warranted . Different abilities to recover longer or shorter fragments have been described in the literature . Besides biological and pathological parameters reviewed above in chapter 1, the concentration of isolated cfDNA is influenced by the efficiency of the DNA extraction method and the final elution volume of the respective method.…”

Circulating tumor-derived DNA is shorter than somatic DNA in plasma

“…Long fragments may reach circulation through nonapoptotic mechanisms and may prove to be informative or biologically active. Size profiles of cfDNA in plasma, without hybridization-based extraction, showed very different patterns of fragmentation (19). Direct visualization of DNA fragments, PCRbased approaches, and animal model systems, can be used as a complementary tool to study smaller DNA fragments (3,20).…”