Biomedical Engineering Reference
In-Depth Information
Fig. 5. Sequence alignment of stencil oligonucleotide with wild-type and mutated
K
-RAS
DNA.
Alu
I restriction site near codon 12 is shown in bold. H designates any nucleotide
except G.
from codon 12. Six nucleotides flanking both sides of the
Alu
I recognition site
were required for the enzyme to bind and efficiently cleave the target. The DNA
sample (1-1000 ng) in 60
L of
Alu
I restriction endonuclease buffer was mixed
with 20 pmol of plus-stencil, heated at 95°C for 10 min, annealed at 47°C for
10 min and digested by
Alu
I (5 U) at 42.5°C for 2 h.
µ
2.
At the second step, the plus strand of wild-type K
-RAS
was cleaved similarly us-
ing the minus-stencil (5'-GCCACCAGCTCCAACT-3'), the latter being comple-
mentary to the plus-stencil, specified previously. The twofold amount (i.e., 40 pmol)
of minus-stencil in
Alu
I buffer was added to the DNA sample heated at 95°C for
10 min (the excess of the minus-stencil is needed to ensure the complete hybrid-
ization with template DNA in the presence of the complementary plus-stencil).
Denaturation, annealing, and digestion steps were repeated as described.
3.
Finally, the restriction enzyme was inactivated by heating at 95°C for 10 min,
DNA was precipitated with ethanol, washed with 70% ethanol, dissolved in water,
and subjected to PCR.
Figure 6
presents the results of experiments with artificial mixtures of
wild-type and mutant K
-RAS
. After SAMA, the band specific for mutant
K
-RAS
was not reduced, whereas the band of wild-type K
-RAS
disappeared. At
the highest wild-type/mutant sequences ratio (1000/1) the competition of mutant
K
-RAS
with the large excess of wild-type allele in the control sample consider-
ably weakened the mutant signal. However, the stencil-aided pre-PCR diges-
tion of the wild-type sequences made the mutant K
-RAS
band much stronger.
Application of SAMA to three urine samples with undetectable by RE-PCR
levels of mutant K
-RAS
revealed mutation in three urine specimens (
Fig. 7
).
