Transrenal DNA as a diagnostic tool: important technical notes.
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.
Su
Ying-Hsiu
YH
Department of Biochemistry and Molecular Pharmacology, Jefferson Center for Biomedical Research, Thomas Jefferson University, Doylestown, Pennsylvania 18901, USA.
Wang
Mengjun
M
Block
Timothy M
TM
Landt
Olfert
O
Botezatu
Irina
I
Serdyuk
Ol'ga
O
Lichtenstein
Anatoly
A
Melkonyan
Hovsep
H
Tomei
L David
LD
Umansky
Samuil
S
D017209:Apoptosis; D014408:Biomarkers, Tumor; D003062:Codon; D015179:Colorectal Neoplasms / Q000175:diagnosis / Q000235:genetics / Q000652:urine; D004247:DNA / Q000097:blood* / Q000302:isolation & purification / Q000378:metabolism / Q000652:urine*; D004252:DNA Mutational Analysis; D004591:Electrophoresis, Polyacrylamide Gel; D011905:Genes, ras; D006801:Humans; D007668:Kidney / Q000378:metabolism; D008970:Molecular Weight; D009154:Mutation; D010190:Pancreatic Neoplasms / Q000175:diagnosis / Q000235:genetics / Q000652:urine; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D014408:Biomarkers, Tumor; D003062:Codon; D004247:DNA