Biomedical Engineering Reference
In-Depth Information
Fig. 2. Schematic view of the K-
RAS
fragment amplified by forward (F) and reverse
primers producing 87-bp (RS) and 157-bp (RL) DNA fragments, respectively.
Fig. 3. Dependence of polymerase chain reaction sensitivity on amplicon size.
Different lines illustrate short DNA fragments (the 157-bp fragment is given as an
example) generated by random cleavage of K
-RAS
in the area of codon 12. Bold solid
line represents the only fragment that is amplified by primers designed for the 157-bp
amplicon. Thin solid lines represent subset of DNA fragments amplified by the pair of
primers targeting the 87-bp amplicon. Dashed lines are DNA fragments that are not
amplified by either set of primers.
(5'-gtccacaaaatgattctgaattagc-3') amplifying 157- and 87-bp DNA fragments,
respectively. The first primer, which is immediately upstream of codon 12, was
modified at nucleotide 28 (G to C) to create a restriction enzyme site specific
for B
st
NI in wild-type K
-RAS
DNA. Any mutations of the first or second nucle-
otide of codon 12 destroy this restriction site that can be seen by mapping of
the DNA fragment by B
st
NI cleavage sites. For the second stage, reverse
primers are also modified to introduce additional B
st
NI cleavage site for con-
trol of the completeness of restriction: K
-RAS
-RL' (5'-tcaaagaatggtcctggacc-3')
and K-
RAS
-S' (5'-gtccacaaaatgatcctggattagc-3'). The analysis of K
-RAS
muta-
tions was carried out as follows.
1.
One-fiftieth of DNA purified from 10 mL urine was subjected to the first step of
PCR. 25-
L reaction mixtures were cycled 20 times at 94°C for 30 s, 56°C for
30 s, and 72°C for 30 s.
µ