Biomedical Engineering Reference
In-Depth Information
13
Novel Applications of Polymerase Chain Reaction
to Urinary Nucleic Acid Analysis
Anatoly V. Lichtenstein, Hovsep S. Melkonyan, L. David Tomei,
and Samuil R. Umansky
Summary
DNA fragments from cells that have died throughout the body not only appear in the
bloodstream but also cross the kidney barrier into the urine. The relatively low molecular
weight (150-200 bp) of this Transrenal DNA should be considered when deciding on
methods of isolation and analysis. In particular, if polymerase chain reaction (PCR) is
used for amplification and detection of specific sequences, then the reduction of amplicon
size will significantly enhance sensitivity. Detection of DNA mutations is also made
more difficult by the presence of a large excess of a wild-type allele. Using K-
RAS
muta-
tions as an example, two ways around this problem—enriched PCR and stencil-aided
mutation analysis—are described, based on selective pre-PCR elimination of wild-type
sequences.
Key Words:
Transrenal DNA (Tr-DNA); urine; cancer; DNA markers; K-
RAS
oncogene; mutations.
1. Introduction
Each day, 10
11
-10
12
cells in the human body die as a result of physiological
and pathological processes. Cell death is normally accompanied by DNA deg-
radation, and it is currently well established that a portion of resulting DNA
fragments appears in the bloodstream as so-called cell free circulating DNA (for
review,
see
refs.
1-7
). It has also been found that circulating DNA fragments
from blood cross the kidney barrier into the urine
(
8
)
. This Transrenal DNA
(Tr-DNA) contains sequences from cells that have died throughout the body,
including tumor cells
(
8
,
9
)
, developing fetus cells
(
8
,
10
)
, or cells from trans-
planted organs
(
8
,
11
,
12
)
. The range of potential diagnostic applications of cir-
culating and Tr-DNA is very broad and includes tumor diagnostics and
