Use of magnetic beads for plasma cell-free DNA extraction: toward automation of plasma DNA analysis for molecular diagnostics.

Abstract

Urine, breast milk, plasma, and serum have been shown to contain cell-free DNA (1)(2)(3)(4)(5)(6)(7). For plasma DNA detection, several recent studies addressed the need for careful evaluation and standardization of preanalytical processes (8)(9)(10)(11)(12). Key problems appear, such as possible contamination of plasma by white blood cells; the generally low and variable amount of circulating DNA, making extraction/quantification difficult and time-consuming; poor DNA quality; and the presence of PCR inhibitors. In any case, automation of DNA extraction, which is a prerequisite for introduction of these diagnostic approaches in clinical laboratories, is difficult to achieve because of the volumes of plasma necessary to get sufficient DNA. In this study (summarized in Fig. 1⇓ ), we propose a new semiautomated, time-saving process for extraction of plasma cell-free DNA that provides high yields and is suitable for PCR amplification. Two 5-mL blood samples from each of 23 patients being monitored for lung (n = 19) or colon cancer (n = 4) and 20 healthy controls were collected in EDTA-containing tubes. Informed consent was obtained for each. Blood was centrifuged at 800 g for 10 min, and the collected plasma was transferred to a 15-mL BD FalconTM polypropylene tube for an additional centrifugation step of 10 min at 1500 g to remove any remaining leukocytes and platelets. An equal volume of 2× concentrated proteolytic buffer (2×: 20 mmol/L Tris-HCl, 50 mmol/L EDTA, 200 mmol/L NaCl, 10 g/L sodium dodecyl sulfate, and 400 mg/L proteinase K) was added to the plasma, mixed, and incubated 1 h at 37 °C. After digestion, plasma samples were concentrated by centrifugation at room temperature (to avoid sodium dodecyl sulfate precipitation) for 20 min at 2600 g in Amicon …