Biomedical Engineering Reference
In-Depth Information
16.
Hybridization oven.
17.
Vacuum oven.
3. Methods
Mutant and normal controls as well as a water (no DNA template) control
are always included in the PCR reactions. All control specimens are tested and
analyzed in the same manner as the patient's samples. The DNA template for
each positive control can be the patient's genomic DNA or PCR product con-
taining the mutation. If the positive mutant control is not available, a synthetic
control should be used
(
8
)
.
3.1. Preparation of Synthetic Positive Controls
1.
Synthetic positive controls are generated by PCR using the ASO as one of the
primers, which will produce a mutation-containing synthetic control PCR prod-
uct
(
8
)
. For example, to generate a T3271C-containing positive control, the PCR
reaction in
Table 3
is carried out in a total volume of 100
µ
L.
2.
PCR conditions:
a. 94°C, 4 min.
b. 94°C, 20 s; 45°C, 20 s; 72°C, 30 s, for 30 cycles.
c. 72°C, 5 min.
d. 4°C soak.
3.2. PCR Setup (4)
Three sets of PCR reactions are set up. The first set is a single PCR for
A3243G, T3271C, and G3460A mutations. The second set is a multiplex con-
taining three pairs of primers for the analysis of A8344G, T8356C, G8363A,
T8993G, T8993C, and G11778A. The third set of PCR is also a single PCR for
the analysis of G14459A and T14484C (
see
Note 2
).
1.
Single PCR: two single PCR sets are performed. The PCR setup is similar to the
one described under
Subheading 3.1.
, except that here, each PCR reaction is
25
L, with each reagent reduced accordingly for one-quarter volume, and the prim-
ers used for one set are mtF3130 and mtR3758, for the other set are mtF14437
and mtR15185 (
see
Table 1
). The mutations to be analyzed are A3243G, T3271C,
and G3460A in one set and G14459A and T14484C in the other.
µ
2.
Multiplex PCR
(
4
)
: the multiplex PCR contains three pairs of primers. The PCR
setup is similar to that described under
Subheading 3.1.
, except that here, the
PCR reaction is 50
L, and the primers used are mtF8278 and mtR8475 for
A8344G, T8356C, and G8363A; mtF8768 and mtR9199 for T8993G and
T8993C; and mtF11688 and mtR12360 for G11778A (
see
Table 1
) (
see
Note 3
).
µ
3.
PCR conditions for the multiplex PCR:
a. 94°C, 2 min.
b. 94°C, 1 min; 55°C, 1 min; 72°C, 2 min, for 30 cycles.
