Preferential Isolation of Fragmented DNA Enhances the Detection of Circulating Mutated k-ras DNA

Abstract

In the course of studying the incidence of k- ras mutations in DNA in the circulation of patients with colorectal disease, we observed that our ability to detect mutated k- ras DNA in serum varied with the method of DNA isolation. Two methods of DNA isolation were used in this study. One was the QIAamp DNA Blood Midi Kit (Qiagen, Inc.), which has been widely used for isolating circulating DNA from serum or plasma (1)(2)(3)(4)(5). The other is the modified Guanidine/Promega Resin (G/R) method that was developed to isolate DNA from urine (6). To compare the applicability of the Qiagen and G/R methods for isolation of circulating DNA, we purified DNA from aliquots of serum from six patients with known mutant k- ras -positive colorectal disease (colorectal cancer or adenomatous polyps) and analyzed it for the presence of mutated k- ras DNA. As negative controls, serum samples from six patients with no detectable k- ras mutations in their disease tissues were subjected to DNA isolation and subsequently analyzed for k- ras mutations. Briefly, DNA from two aliquots of serum from each patient was isolated by the Qiagen method according to the manufacturer’s instructions or by the G/R method, as follows. Two volumes of 6 mol/L guanidine thiocyanate were mixed with the serum by inverting the mixture eight times. One milliliter of resin (from the Wizard DNA isolation reagent set; Promega) was then added, and the mixture was incubated for 2 h at room temperature with gentle mixing. The resin–DNA complex was transferred to a minicolumn (provided in the reagent set) and washed, and the DNA was eluted with H2O. Isolated DNA was quantified by real-time PCR on a LightCycler (Roche Biochemicals) with human albumin primers (forward, 5′-CCG TGG TCC …