Optimization of transrenal DNA analysis: detection of fetal DNA in maternal urine.

BACKGROUND: Fragments of DNA from cells dying throughout the body are detectable in urine (transrenal DNA, or Tr-DNA). Our goal was the optimization of Tr-DNA isolation and detection techniques, using as a model the analysis of fetal DNA in maternal urine. METHODS: We isolated urinary DNA using a traditional silica-based method and using a new technique based on adsorption of cell-free nucleic acids on Q-Sepharose resin. The presence of Y chromosome-specific SRY (sex-determining region Y) sequences in urine of pregnant women was detected by conventional and real-time PCR using primers/probe sets designed for 25-, 39-, 65-, and 88-bp PCR targets. RESULTS: Method of DNA isolation and PCR target size affected fetal Tr-DNA detection. Assay diagnostic sensitivity increases as the PCR target is shortened. Shorter DNA fragments (50-150 bp) could be isolated by Q-resin-based technique, which also facilitated fetal Tr-DNA analysis. Using DNA isolated by Q-resin-based method and an "ultrashort" DNA target, we successfully detected SRY sequences in 78 of 82 urine samples from women pregnant with male fetuses (positive predictive value 87.6%). Eleven of 91 urine samples from women pregnant with female fetuses produced SRY false-positive results (negative predictive value 95.2%). CONCLUSIONS: Single-copy fetal DNA sequences can be successfully detected in the urine of pregnant women when adequate methods for DNA isolation and analysis are applied. Strong precautions against sample contamination with male cells and DNA are necessary to avoid false-positive results.

Transrenal nucleic acids: from proof of principle to clinical tests.

In spite of numerous publications on potential diagnostic application of circulating DNA and transrenal nucleic acid (Tr-NA) analysis, few, if any, tests based on this technology are available in clinical labs. This delay in test development and implementation is caused, at least in part, by the deficit in robust methods for isolation of short nucleic acid fragments from bodily fluids, as well as in techniques for analyzing these fragments. We have developed a new anion exchanger-based method for the isolation of cell-free nucleic acid fragments from large volumes of bodily fluids, and analyzed these fragments by PCR techniques specially designed to amplify "ultrashort" templates. The combination of these two techniques not only revealed the presence in urine of 10-150 bases or bp DNA and RNA fragments in addition to previously observed 150-200-bp DNA fragments and high molecular weight DNA, but also significantly increased the sensitivity of Tr-DNA detection. Additionally, we detected in urine a variety of miRNAs, including those excreted transrenally, thereby opening new diagnostic possibilities for Tr-NA analysis.

Novel applications of polymerase chain reaction to urinary nucleic acid analysis.

DNA fragments from cells that have died throughout the body not only appear in the bloodstream but also cross the kidney barrier into the urine. The relatively low molecular weight (150-200 bp) of this Transrenal DNA should be considered when deciding on methods of isolation and analysis. In particular, if polymerase chain reaction (PCR) is used for amplification and detection of specific sequences, then the reduction of amplicon size will significantly enhance sensitivity. Detection of DNA mutations is also made more difficult by the presence of a large excess of a wild-type allele. Using K-RAS mutations as an example, two ways around this problem--enriched PCR and stencil-aided mutation analysis-are described, based on selective pre-PCR elimination of wild-type sequences.