Transrenal DNA as a diagnostic tool: important technical notes

Ann N Y Acad Sci. 2004 Jun;1022:81-9. doi: 10.1196/annals.1318.014.

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.

Publication types

  • Comparative Study

MeSH terms

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

Substances

  • Biomarkers, Tumor
  • Codon
  • DNA