Plasma DNA restoration for PCR applications

Plasma DNA is a valuable source of a wide range of molecular indicators that relate to the general condition and stage of cancer patients. However, continual patient monitoring and/or long-term studies require the use of archived plasma samples, frequently yielding a poor quality DNA upon storage. A restoration treatment was developed for plasma DNA that includes a pre-PCR incubation with Taq polymerase, dNTPs and 1× PCR buffer that improves the efficiency of subsequent PCRs. Pre-PCR treatment of isolated DNA resulted in superior PCR efficiency and sensitivity of detection both on a fresh plasma DNA isolate and on the plasma DNA isolates from 12 long-term archived plasma samples from lung cancer patients. This procedure allowed the recovery of a group of plasma samples considered otherwise useless. It is concluded that the quality of archived plasma DNA can be remarkably improved by subjecting the samples to a pre-PCR restoration treatment.

Human blood plasma from both healthy and sick individuals contains variable quantities of nucleic acids. The majority of the mechanisms proposed for its release from cells are related to cell death by necrosis or apoptosis, though active release has also been suggested.^1,2 Patients suffering from different diseases where there is a clear increase in cell death have comparatively higher plasma DNA levels than healthy individuals.^3,4 This phenomenon has been consistently observed in patients diagnosed with different cancers, whose plasma DNAs exhibit many genetic and epigenetic tumour DNA alterations, such as Ras and p53 mutations^5–8 or microsatellite instability.^9,10 This trait make plasma DNA a valuable diagnostic source for the assessment of different genetic alterations in tumours and is probably correlated with the general condition of the patient and the treatment response.^4,7,11

Large clinical studies must often be performed on archived plasma samples …