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.
Department of Microbiology and Immunology, College of Medicine, Drexel University, Doylestown, Pennsylvania, USA. ys98@drexel.edu
Ying-Hsiu
YH
Su
Janet
J
Song
Zhili
Z
Wang
Xiao-He
XH
Wang
Mengjun
M
Wang
Dean E
DE
Brenner
Timothy M
TM
Block
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000235:genetics / Q000652:urine; D015179:Colorectal Neoplasms / Q000235:genetics / Q000652:urine; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000652:urine*; D005260:Female; D006801:Humans; D008280:Magnetics; D008875:Middle Aged; D008970:Molecular Weight; D009154:Mutation; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics / Q000652:urine*
D014408:Biomarkers, Tumor; D004247:DNA; D016283:Proto-Oncogene Proteins p21(ras)