Biomedical Engineering Reference
In-Depth Information
3.1.4. Mutant DNA Detection in the Presence of a Large Excess
of Wild-Type DNA
On at least two occasions, the robust detection of mutant DNA in the pres-
ence of a large excess of wild type DNA may be of clinical and biological
significance. One involves cancer mutant DNA in plasma
(
12
)
. The other
involves fetal-specific DNA in maternal plasma
(
13
)
. In both cases, it is
extremely difficult to detect single-base mutations when 99% or more of the
DNA is normal. A method called single allele base extension reaction (SABER)
was recently developed
(
14
)
. The SABER technique uses a termination mix-
ture (in the primer extension) that will only extend the mutant DNA (e.g., only
ddATP is added for the primer extension in
Fig. 3
). This selective extension
step results in robust detection of mutant DNA with potentially 100% specific-
ity and sensitivity by effectively eliminating the background signal from the
normal DNA (
Fig. 3
). The SABER technique may be useful for early cancer
diagnosis and noninvasive diagnosis of genetic diseases such as
β
-thalassemia
and cystic fibrosis
(
14
)
.
3.2. rcPCR Experimental Procedure
For gene expression analysis, it is assumed that cDNA samples have already
been obtained through reverse transcription using either random hexamers,
poly-T primers, or gene-specific primers (
see
Note 3
).
3.2.1. Polymerase Chain Reaction
A protocol for carrying out PCR reactions in a 384-well microplate is pro-
vided in this section. Generally, one can scale up or down.
1.
Prepare a PCR cocktail as specified in
Table 1
. For the SABER method, no com-
petitor is needed; thus, 1
µ
L DNA is used.
2.
Add 5
L of PCR cocktail into each well of a 384-well microplate. Seal the plate
and centrifuge it for 3 min at 600
g
.
µ
3.
Perform PCR as follows:
a. 95°C, 15 min.
b. 95°C, 20 s.
c. 56°C, 30 s.
d. 72°C, 1 min.
e. Perform
step b
44 times.
f. 72°C, 3 min.
g. 4°C, forever.
3.2.2. Shrimp Alkaline Phosphatase Treatment
This step is used to neutralize remaining dNTPs from the PCR reactions into
dNDPs so that they will not interfere with the subsequent primer extension
reaction.
