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Transrenal DNA as a Diagnostic Tool: Important Technical Notes

2004; Wiley; Volume: 1022; Issue: 1 Linguagem: Inglês

10.1196/annals.1318.014

1749-6632

Ying‐Hsiu Su, Mengjun Wang, Timothy M. Block, Olfert Landt, I. V. Botezatu, O. I. Serdyuk, A. V. Lichtenstein, Hovsep Melkonyan, Licia Tomei, Samuil R. Umansky,

Prenatal Screening and Diagnostics

A bstract : 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.