Transrenal DNA as a Diagnostic Tool: Important Technical Notes

Abstract

A small portion of DNA from apoptotic cells escapes complete degradation, appears in blood as oligonucleosomal-size fragments, is excreted in the urine, and can be used for diagnostic purposes. More detailed study revealed that transrenal DNA (Tr-DNA) belongs to a relatively low molecular-weight (150-250 bp) fraction, thereby requiring more careful attention to methods employed for purification and analysis. For example, here it is demonstrated that the QIAamp blood kit purifies primarily high molecular-weight DNA from serum, whereas the Guanidine/Promega Wizard Resin (GITC/WR) method purifies primarily low molecular-weight DNA. As a result, sensitivity in detection of K-RAS mutations in serum of patients with colorectal tumors is significantly higher with DNA isolated with the GITC/WR method than with the QIAamp kit. Amplicon size is also extremely important in analysis of Tr-DNA, because the shorter the amplicon, the higher is the sensitivity of biomarker detection in Tr-DNA. One hundred fifty-seven and 87 bp amplicons were employed for detection of mutant K-RAS in DNA isolated from 0.1 mL of urine obtained from 15 patients with pancreatic cancer. Mutant K-RAS was found in Tr-DNA of 3 and 10 patients with the long and short amplicons, respectively. The sensitivity and specificity of detection of mutant sequences are reduced in the presence of high excess of a respective wild-type allele, but they can be significantly increased through application of enriched polymerase chain reaction (PCR), peptide nucleic acid (PNA) clamped PCR, and/or stencil-aided mutation analysis (SAMA), based on selective pre-PCR elimination of wild-type sequences.

Evaluation and comparison of in vitro degradation kinetics of DNA in serum, urine and saliva: A qualitative study

“…However, no study has addressed DNA degradation in saliva, and previous works have not systematically researched DNA degradation in serum, urine and saliva. In addition, the reported stability and degradation rates of DNA in bodily fluids vary significantly due to differences in extraction methods, PCR conditions and target sequences (Chiu et al, 2001;Fleischhacker et al, 2011;Su et al, 2004), and the nature of the nucleic acid measured (e.g., cell-free or cellular; mononucleosomes or oligonucleosomes).…”

Other Body Fluids as Non-invasive Sources of Cell-Free DNA/RNA

“…When DNA from paired urine and tissue sections (20 patients with either CRC or adenomatous polyps) were analyzed for the K-RAS mutation, an 83 % concurrence of mutated urine cfDNA and its corresponding disease tissue was obtained. The authors proposed that apoptotic cells were the source of the cfDNA [54]. Moulière and Thierry [65] demonstrated that CRC K-RAS fragments isolated from peripheral blood tended to be smaller than 100 bp, and given that the study of Su et al was based upon fragments of 150-250, it would be of interest to determine if smaller fragments could be present in urine and in high abundance.…”

“…Human urine contains, sub-microgram per milliliter amounts of cfDNA of between 150 and 250 bp [54]. A comparison was made of the mutated K-RAS sequences present in DNA isolated from tumor, blood and urine obtained from a CRC patient with a mutation in codon 12 of the K-RAS proto-oncogene.…”

“…Although there were many problems concerning cfNA isolation from urine, these seem to have been overcome with cfNA extraction kits now available. Much of the DNA so obtained would appear to be genomic DNA (>1 kb) from exfoliate cells present in urine and only low levels of small M Wt fragments (150-250 bp) represent the actual cfNA [54]. Such DNA was considered to be transrenal and to pass from the circulatory system through the kidney barrier to enter the urinary tract, producing so-called transrenal DNA [55].…”

Molecular Triage of Premalignant Lesions in Liquid-Based Cervical Cytology and Circulating Cell-Free DNA from Urine, Using a Panel of Methylated Human Papilloma Virus and Host Genes

“…These fragments are known as transrenal DNA (TrDNA) (47)(48)(49). Recently, a capillary electrophoresis TrDNA test that targets the E1 region of the HPV genome for the detection of hrHPV demonstrated high sensitivity and modest specificity for urine-based detection of cervical precancerous lesions (50).…”