Novel Applications of Polymerase Chain Reaction to Urinary Nucleic Acid Analysis - Clinical Applications of PCR - page 148

Fig. 1. Polyacrylamide gel electrophoresis of cell-free DNA isolated from human
urine.
Urine samples were centrifuged for 10 min at 800 g . DNA was isolated from the
supernatants and subjected to agarose gel electrophoresis. Lanes 1 and 2, DNA from
the urine of two volunteers; lane 3, molecular weight markers.
a heater set at 56°C. The yield of DNA purified according to this protocol varied
significantly among different specimens providing a final range of 5 to 200 ng
DNA/mL of urine. Figure 1 illustrates that DNA isolated from cell-depleted urine
specimens consists of two major fractions: a low-molecular-weight DNA whose
size is close to that of mononucleosomal DNA, and high-molecular-weight DNA
that most likely originates from residual cells that were not eliminated by mild
centrifugation of urine samples.
3.3. Determination of K-RAS Mutations
3.3.1. Enriched PCR
K- RAS mutations were detected by restriction-enriched PCR (RE-PCR), a
method developed for detection of a mutant codon 12 K- RAS allele in the pres-
ence of as many as 10 4 copies of the wild-type codon 12 allele ( 19 ) . This method
employs a two-stage PCR. During the first stage, both mutant and wild-type
alleles are amplified and then wild-type sequences are selectively digested with
restriction endonuclease at an artificially created site. In the second stage, undi-
gested PCR products enriched in mutant codon 12 sequences are amplified.
This technique was originally developed for the analysis of K -RAS mutation in
DNA extracted from tumor cells. Therefore, the amplicon size of 157 bp
proposed by the authors ( Fig. 2 ) was not well suited for analysis of
Tr-DNA, because these fragments themselves are about 150 bp in size ( see
Note 5 ). To adjust RE-PCR for analysis of Tr-DNA, we modified the existing
protocol by designing a new reverse primer targeting a shorter (87 bp) DNA
fragment ( see Figs. 2 and 3 and refs. 20 , 21 ). For comparison, PCR were carried
out with two sets of primers in parallel: K- RAS -F (5'-actgaatataaacttgtggtagtt
ggacct-3') paired with K- RAS -RL (5'-tcaaagaatggtcctgcacc-3') or with K- RAS -RS