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.

• Authors: Ying-Hsiu Su, Mengjun Wang, Timothy M Block, Olfert Landt, Irina Botezatu, Ol'ga Serdyuk, Anatoly Lichtenstein, Hovsep Melkonyan, L David Tomei, Samuil Umansky
• Journal: Annals of the New York Academy of Sciences (Ann N Y Acad Sci) Vol. 1022 Pg. 81-9 (Jun 2004) ISSN: 0077-8923 [Print] United States
• PMID: 15251944 (Publication Type: Comparative Study, Journal Article)

Chemical References

• Biomarkers, Tumor
• Codon
• DNA

Topics

• Apoptosis
• Biomarkers, Tumor
• Codon
• Colorectal Neoplasms (diagnosis, genetics, urine)
• DNA (blood, isolation & purification, metabolism, urine)
• DNA Mutational Analysis
• Electrophoresis, Polyacrylamide Gel
• Genes, ras
• Humans
• Kidney (metabolism)
• Molecular Weight
• Mutation
• Pancreatic Neoplasms (diagnosis, genetics, urine)
• Polymerase Chain Reaction
• Sensitivity and Specificity