Biomedical Engineering Reference
In-Depth Information
Table 5
Preparation of Hybridization
Membrane
Hot ASO
Cold ASO
3243nl
A3243
A3243G
A3243G
A3243G
A3243
T3271C
T3271C
T3271
G3460A
G3460A
G3460
8344nl
A8344
A8344G
A8344G
A8344G
A8344
T8356C
T8356C
T8356
G8363A
G8363A
G8363
8993nl
T8993
T8993C, T8993G (10
µ
L each)
T8993C
T8993C
T8993, T8993G (10
µ
L each)
T8993G
T8993G
T8993, T8993C (10
µ
L each)
11778nl
G11778
G11778A
G11778A
G11778A
G11778
14459nl
G14459
G14459A
G14459A
G14459A
G14459
14484nl
T14484
T14484C
T14484C
T14484C
T14484
ASO, allele-specific oligonucleotide.
4. Notes
1.
The ASO probe is usually designed as a 19-mer with the point mutation located
at the middle of the ASO. If the nucleotide is a G in the mutant sequence, an
antisense ASO is made to reduce the nonspecific basepairing. For example, the
antisense probes are used for A3243G and A8344G. In the case of T8993G and
T8993C, because both G and C mutants are to be analyzed, the sense sequence of
the mutation is used.
2.
It is possible to run one single multiplex PCR reaction ( 4 ) containing all five
pairs of primers, but the background may be higher. In order to detect a low
percentage of mutant heteroplasmy, the background should be minimized.
3.
Conditions for multiplex PCR should always be optimized before ASO analysis
is carried out. Try to design the primers such that the PCR products can be easily
separated and identified on agarose gel.
4.
The reason for using a cold ASO counter part is to reduce the background. There-
fore, if the background is high, the amount of the cold ASO counter part can be
increased. The optimal concentration of the cold ASO to be used should be tested
out for each probe.
5.
The washing temperature depends on the melting temperature (T m ) of the ASO.
A temperature of 34°C for washing works for most of the ASOs. Occasionally,
 
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