Biomedical Engineering Reference
In-Depth Information
monitoring, prenatal genetic testing, transplantation monitoring, analysis of age-
related accumulation of DNA mutations, and so on.
There are several features of Tr-DNA that should be taken into account when
developing diagnostic applications based on Tr-DNA analysis. First, the rela-
tively low concentration of Tr-DNA in the urine must be considered
(
4
,
8
)
. In
many cases, sensitive methods such as multi-round polymerase chain reaction
(PCR) should be used for detection of specific sequences. Second, Tr-DNA
fragments are relatively short, having an average molecular weight of approx
150-200 bp
(
8
)
, which dictates use of amplicons that are as short as possible.
Third, if a target is a specific DNA mutation, one should consider whether a
great excess of wild-type alleles is present. In the presence of a large excess of
wild-type alleles, a significantly lower sensitivity of mutant DNA detection
will likely result because of competition of wild-type and mutant sequences for
primers. Furthermore, the presence of a great excess of wild-type sequences
leads to the increased frequency of false-positive results owing to
Taq
poly-
merase errors
(
13
,
14
)
.
Here, we describe our experiences and provide recommendations in several
specific areas of Tr-DNA analysis including preparation of urine samples
designed to protect DNA from additional degradation, methods of Tr-DNA
isolation, and detection of a mutant DNA sequence. As a model application,
we used the detection of mutant K-
RAS
in the urine of cancer patients. Muta-
tions in codon 12 or 13 of K-
RAS
occur in approximately half of all colorectal
cancers
(
15
,
16
)
, 90% of pancreatic cancer
(
16
)
, and approx 30-40% of lung
adenocarcinoma
(
17
)
. In these studies, two approaches were used for detection
of mutant K-
RAS
in a great excess of wild-type allele: (1) stencil-aided muta-
tion analysis (SAMA) as a pre-PCR procedure aimed at reducing the concen-
tration of wild-type alleles directly in the DNA sample
(
18
)
; and (2) enriched
PCR, which includes selective restriction-enzyme digestion of wild-type
sequences after several initial PCR cycles and subsequent amplification of frag-
ments containing the mutant codon
(
19
)
.
2. Materials
1.
Restriction enzymes and buffers.
2.
Oligonucleotide primers.
3.
Reagents for PCR:
a.
Taq
polymerase.
b. Mixture of deoxynucleotide triphosphates.
c. 10X PCR Buffer.
4.
Polyacrylamide gel electrophoresis equipment.
5.
Tris-borate-ethylenediamine tetraacetic acid (EDTA) buffer for electrophoresis
(5X TBE).