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1.  Detection of Mutated K-ras DNA in Urine, Plasma, and Serum of Patients with Colorectal Carcinoma or Adenomatous Polyps 
Our previous studies demonstrated that urine contains DNA derived from the circulation and that this DNA originated, in part, from organ sites and tumors distal to the urinary tract. To explore the potential use of DNA from urine as compared to other body fluids as a source for circulating DNA for cancer detection, the DNA concentration and the frequency of detection of mutated Kristin-ras (K-ras) DNA in serum, plasma, and urine were examined. The concentration of DNA in the urine was similar to that in the serum, but the DNA concentration in plasma was significantly lower than in either urine or serum (P < 0.05). When DNA derived from 10 μL of body fluid was used in each mutation assay, the detection frequency of mutated K-ras DNA was comparable among serum, plasma, and urine. However, when DNA derived from 200 μL of body fluid was used, the incidence of detecting mutated K-ras DNA in urine was significant higher (95%) than in either serum (35%) or plasma (40%) (P < 0.0005), suggesting that inhibitory factors in serum/plasma may be more limiting than in urine. The use and practicality of urine as a source of circulating DNA for cancer detection are discussed.
PMCID: PMC2587049  PMID: 18837947
urine DNA; circulating DNA; cancer biomarkers; K-ras mutations; cancer detection
2.  Removal of High-Molecular-Weight DNA by Carboxylated Magnetic Beads Enhances the Detection of Mutated K-ras DNA in Urine 
We have previously demonstrated that mutated DNA derived from the circulation can be detected in urine and predominantly exists as DNA fragments < 1 kb. To preferentially isolate the trans-renal DNA from urine, we developed a method using carboxylated beads to separate high-MW (1 kb or larger) from low-MW DNA in urine. A primer set for 18s rRNA (generating a PCR product of 872 bp) was designed and optimized for real-time PCR quantification of high-MW DNA templates. To evaluate the method, urine samples from 5 volunteers with no known diseases and 36 patients with various colorectal diseases were collected and tested. It was found that the average removal efficiency of high-MW DNA from total urine DNA using carboxylated beads is 92.72% ± 1.42%. Furthermore, compared with using total urine DNA, our method provides a greater ability to detect mutated K-ras in the urine of colorectal cancer patients. The concurrence of K-ras mutations detected in disease tissue and the corresponding urine specimen is significantly higher (P = 0.0015) when the samples were enriched in low-MW DNA.
PMCID: PMC2572261  PMID: 18837929
circulating DNA; disease biomarker; K-ras mutation; colon cancer

Results 1-2 (2)