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Technical Briefs |
1 Department of Biochemistry and Molecular Pharmacology Jefferson Center for Biomedical Research, Thomas Jefferson University, 700 E. Butler Ave., Doylestown, PA 18901
2 Departments of Internal Medicine and Pharmacology, 2150B Cancer and Geriatrics Center, University of Michigan Medical Center, Ann Arbor, MI
aauthor for correspondence: fax 215-489-4920, e-mail Yinghsiu.Su{at}jefferson.edu
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 TGA ACC AGT TA-3'; reverse, 5'-GTC GCC TGT TCA CCA AGG AT-3') at an annealing temperature of 55 °C. As calibrators for quantification, serially diluted genomic DNA was used. The limit of detection with the albumin primers was 0.015 ng.
DNA derived from 50 µL of serum was used to assay for k-ras DNA, mutated in codon 12, by the modified restriction endonuclease-enriched PCR (RE-PCR) assay (7). The PCR product of this modified RE-PCR was 87 bp with an assay detection limit of 15 copies of the mutant k-ras per 100 ng of wild-type DNA per reaction (data not shown). The k-ras mutation was detected in circulating DNA isolated by the G/R method in five of six patients with known k-ras mutations in their diseased tissue. However, when circulating DNA was isolated by the Qiagen method, the k-ras mutation was detected in only one of these six patients (see Table 1
). As controls, circulating DNA was isolated from patients (patients 7–12 in Table 1
) with no detectable mutated k-ras sequences in their diseased tissues, and none scored positive for a k-ras mutation. The failure of the Qiagen method to detect mutated k-ras in 5 of the 6 DNA preparations from patients containing detectable mutated k-ras was not attributable to the low yield of DNA because the Qiagen method gave higher DNA yields in aliquots from 8 of 12 individuals tested (Table 1
). Thus, the G/R method significantly enhanced the sensitivity of detection of the mutated k-ras DNA compared with the Qiagen method, as analyzed by the Fisher exact test (P = 0.0389).
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A recent report (5) suggested that circulating Epstein–Barr virus DNA molecules from patients with nasopharyngeal carcinoma or lymphoma were relatively short, with 87% of them shorter than 181 bp. We therefore hypothesized that the higher detection rate of circulating mutated k-ras DNA by the G/R method compared with the Qiagen method might be attributable to differences in the sizes of DNA isolated. It is known that mutations in the k-ras gene trigger the apoptosis pathway [as reviewed in Ref. (8)] and that DNA fragmentation occurs. It has been shown that serum contains cell-free circulating DNA the size of nucleosomal DNA (3). Mutated k-ras DNA detected in the circulation could be in the form of fragmented DNA. It is possible that DNA isolated by the G/R method is enriched in fragmented DNA the size of nucleosomal DNA, possibly derived from cells undergoing apoptosis.
To investigate this possibility, we isolated DNA from commercially available human serum (Sigma) by the two different methods. The DNA was then separated by polyacrylamide gel electrophoresis and stained with ethidium bromide, as shown in Fig. 1
. As expected, the serum contained DNA fragments the size of nucleosomal DNA, which were resolved in an 8% polyacrylamide gel (as indicated by the arrows in Fig. 1
), and larger DNA, which remained near the sample wells of the gel. Interestingly, much smaller amounts of the low-molecular-weight DNA were observed when DNA was isolated by the Qiagen method (Fig. 1
, lane 4). The only species that was detected was DNA in the size of mononucleosomes. DNA fragments that were in the size of di- and trinucleosomes were not seen. The most abundant DNA detected in the Qiagen preparation was the high-molecular-weight DNA, which remained unresolved and near the sample wells in the gel. In contrast, DNA in the size of mono-, di-, and trinucleosomes was readily detected in the serum DNA isolated by the G/R method (Fig. 1
, lanes 2 and 3) when DNA was resolved in the gel, with very little highmolecular-weight DNA remaining near the well. There was less high-molecular-weight DNA remaining near the sample wells even when DNA was isolated from three times more serum by the G/R method (Fig. 1
, lane 3) compared with the DNA isolated by the Qiagen method (Fig. 1
, lane 4). These data clearly demonstrate that the DNA isolated by the Qiagen method was enriched in higher molecular weight DNA that remained unresolved in an 8% polyacrylamide gel, whereas the DNA isolated by the G/R method was enriched in shorter, fragmented DNA.
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It has been suggested that there are two categories of circulating DNA (3). One is large, genomic DNA, mostly derived from necrotic or hematopoietic cells; the other species is fragmented, cell-free DNA the size of nucleosomal DNA, mostly derived from cells undergoing apoptosis. As discussed earlier, the source of mutated k-ras DNA in the circulation could be apoptotic cells. Thus, mutated k-ras DNA should be enriched in the fragmented, nucleosomal DNA species relative to the large species found near the sample wells of the gel. Therefore, the DNA isolation method that favors isolation of fragmented DNA should produce a better ratio of k-ras-mutated DNA to wild-type DNA. As shown in Table 1
, mutated k-ras was readily detected in the DNA isolated by the G/R method but was detected less frequently in DNA isolated by the Qiagen method from the specimen from the same patient, although more DNA was recovered by the Qiagen method. Another possible contributor to this discrepancy in assay results is the nature of the RE-PCR assay. This is a PCR-based assay, and with more wild-type genomic DNA in the assay, the sensitivity of detection of the mutated DNA is reduced because of interference with the PCR.
This study clearly demonstrates that the method chosen for isolation of DNA can contribute significantly to the outcome of mutation detection (in this case, k-ras mutations). It has been suggested that malignant, benign, and even preneoplastic cells often proliferate at abnormal rates, accompanied by an increase in apoptotic cell death (9)(10)(11), and that this small, fragmented DNA may accumulate in the circulation. These results further suggest that to enhance assay sensitivity for detection of somatic mutations or epigenetic modifications in circulating DNA for cancer detection, monitoring, or prognosis, a method that can preferentially isolate small DNA should be used.
Acknowledgments
We thank Drs. Hovsep Melkonyan and Samuil Umansky (Xenomics, Richmond, CA) for technical support and the critical reading of the manuscript. This work was supported in part by the Early Detection Research Network of the National Cancer Institute, the Hepatitis B Foundation of America, and an appropriation from the Commonwealth of Pennsylvania. We acknowledge the Great Lakes New England Clinical Epidemiological Centers (within the Early Detection Research Network; NCI Grant CA86400) for providing clinical specimens.
References
The following articles in journals at HighWire Press have cited this article:
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I. Muller, C. Beeger, C. Alix-Panabieres, X. Rebillard, K. Pantel, and H. Schwarzenbach Identification of Loss of Heterozygosity on Circulating Free DNA in Peripheral Blood of Prostate Cancer Patients: Potential and Technical Improvements Clin. Chem., April 1, 2008; 54(4): 688 - 696. [Abstract] [Full Text] [PDF]
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J. Mora, E. Urgell, A. Farre, L. Comas, E. Montserrat, and F. Gonzalez-Sastre Agreement between K-ras Sequence Variations Detected in Plasma and Tissue DNA in Pancreatic and Colorectal Cancer. Clin. Chem., July 1, 2006; 52(7): 1448 - 1449. [Full Text] [PDF]
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S. Trombino, M. Neri, R. Puntoni, C. Angelini, M. Loprevite, A. Cesario, P. Granone, A. Imperatori, L. Dominioni, A. Ardizzoni, et al. Mutations in K-ras Codon 12 Detected in Plasma DNA Are Not an Indicator of Disease in Patients with Non-Small Cell Lung Cancer Clin. Chem., July 1, 2005; 51(7): 1313 - 1314. [Full Text] [PDF]
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