Background: Cell-free DNA from dying cells recently has been discovered in human blood plasma. In experiments performed on animals and humans, we examined whether this cell-free DNA can cross the kidney barrier and be used as a diagnostic tool. Methods: Mice received subcutaneous injections of either human Raji cells or purified 32P-labeled DNA. DNA was isolated from urine and analyzed by measurement of radioactivity, agarose gel electrophoresis, and PCR. In humans, the permeability of the kidney barrier to polymeric DNA was assessed by detection in urine of sequences that were different from an organism bulk nuclear DNA. Results: In the experiments on laboratory animals, we found that ∼0.06% of injected DNA was excreted into urine within 3 days in a polymeric form and that human-specific Alu sequences that passed through the kidneys could be amplified by PCR. In humans, male-specific sequences could be detected in the urine of females who had been transfused with male blood as well as in DNA isolated from urine of women pregnant with male fetuses. K-ras mutations were detected in the urine of patients with colon adenocarcinomas and pancreatic carcinomas. Conclusions: The data suggest that the kidney barrier in rodents and humans is permeable to DNA molecules large enough to be analyzed by standard genetic methodologies.

Isotachophoresis is an electrophoretic method of separation of charged substances. The method is characterized by a discontinuous buffer system, constant velocity of separated molecules, and the distribution of separated components in the form of narrow concentrated bands located one right after another. As a rule, isotachophoresis is not used for the separation of nucleic acids because the mobility of polynucleotides in this system does not depend on their size. However, this circumstance proved to be very useful for the quantitative isolation of heterogeneous DNA fragments from biological fluids, for gene diagnostics of cancer in particular. The proposed method of agarose gel isotachophoresis of DNA has been used for the isolation of blood DNA and its successful PCR analysis.

No abstract

The data in this article are related to the research article entitled “Optimization of melting analysis with TaqMan probes for detection of KRAS, NRAS, and BRAF mutations” Botezatu et al. [1]. Somatic mutations in the PIK3CA gene (“hot spots” in exons 9 and 20) are found in many human cancers, and their presence can determine prognosis and a treatment strategy. An effective method of mutation scanning PIK3CA in clinical laboratories is DNA Melting Analysis (DMA) (Vorkas et al., 2010; Simi et al., 2008) [2], [3]. It was demonstrated recently that the TaqMan probes which have been long used in Real Time PCR may also be utilized in DMA (Huang et al., 2011) [4]. After optimization of this method Botezatu et al. [1], it was used for multiplex scanning PIK3CA hotspot mutations in formalin-fixed paraffin-embedded (FFPE) samples from patients with colorectal and lung cancer.

No abstract