Biomedical Engineering Reference
In-Depth Information
Fig. 4. Detection of K
-RAS
mutation by enriched polymerase chain reaction in
Tr-DNA of patients with pancreatic cancer.
(A
,
B)
Amplification of 157 and 87-bp
fragments, respectively.
2.
An aliquot of 10
µ
L adjusted to 1X B
st
NI reaction buffer in 25
µ
L was digested
with 10 U of B
st
NI at 60°C for 90 min.
3.
1
µ
L of the digested PCR mixture was transferred to a new tube and a fresh
25-
µ
L reaction mixture was set up for the second amplification stage.
4.
Probes were cycled 35 times with the same thermal profile as the first PCR, but
with different reverse primers (discussed previously).
5.
Finally, diagnostic B
st
NI restriction was performed using 10
µ
L of the second-
step PCR product.
6.
The digestion products were separated by electrophoresis through 7 or 12% poly-
amplicon is much higher. K
-RAS
mutation, undetectable in samples 1-3, 6, and 8
with the 157-bp amplicon, becomes evident with the shorter amplicon.
3.3.2. Pre-PCR Digestion of Wild-Type K-RAS Alleles (SAMA)
Selective digestion of wild-type DNA was achieved by a combination of
high-stringency molecular hybridization of wild-type complementary stencil
oligonucleotide to Tr-DNA with simultaneous restriction endonuclease cleav-
age of hybridized duplexes. Conditions of annealing and nuclease digestion
prevent formation of double-stranded structure with the mutant sequence and
leads to preferential cleavage of wild-type allele (
Fig. 5
). To apply this tech-
nique for testing of codon 12 mutation of K
-RAS
gene, we took advantage of
the presence of
Alu
I digestion site in close vicinity to the site of interest. Treat-
ment of DNA samples was carried out in a two-step reaction.
1. In the first step, the minus strand of wild-type K
-RAS
was cleaved by
Alu
I
using a plus stencil oligonucleotide. Plus-stencil is a 16-bp oligonucleotide
(5'-AGTTGGAGCTGGTGGC-3') spanning the wild-type immediately upstream
