Biomedical Engineering Reference
In-Depth Information
2.2. Enzymes
1.
GeneAmp DNA amplification kit including Ampli
Taq
Gold polymerase (Applied
Biosystems, Foster City, CA).
2.
Methylation-sensitive restriction enzymes such as
Sma
I,
Eag
I, and
Sac
II, which
recognize specific CpG-rich sequences for digestion (New England Biolabs,
Beverly, MA).
2.3. Radioisotopes for MsSNuPE
[
32
P] dCTP, [
32
P] dTTP, [
32
P] dATP, and [
32
P] dGTP.
2.4. Quantitative Real-Time MSP
Fluorescent probes (
see
Note 1
).
2.5. Equipment and Apparatus
1.
Heat block or water bath.
2.
PCR thermocycler.
3.
Vertical or horizontal gel electrophoresis apparatus.
4.
Gel documentation system.
5.
PhosphorImager.
6.
Applied Biosystems
7700 Sequence Detector.
3. Methods (see Note 2)
3.1. Methylation-Specific PCR (MSP)
Based on DNA methylation abnormalities, MSP (
see
Note 3
) is particularly
useful for detecting circulating tumor DNA in the plasma/serum and circulat-
ing tumor cells in the blood of cancer patients
(
9
,
13
)
. Aberrant promoter
methylation has increasingly become a fundamental molecular abnormality
leading to transcriptional silencing of tumor suppressor genes, DNA repair
genes, and metastasis inhibitor genes
(
14
)
. Of significance, DNA hyper-
methylation of multiple genes successfully detected in circulating tumor DNA
or cells from cancer patients may prove valuable for cancer detection and moni-
toring
(
15
)
. A number of methylation markers may allow the detection of
circulating tumor DNA or cells from patients with different cancer types
(
16
)
.
The methylated and unmethylated DNA sequences are different after
bisulfite modification, by which cytosine residues are deaminated to uracil resi-
dues. MSP primers are designed so that different primer sets can specifically
anneal to sequences that contain methylated cytosines or modified thymines in
the CpG sites within the target DNA sequence
(
3
)
. Careful design of primers is
very important for eliminating or reducing the possibility of false-positivity or
false-negativity (
see
Note 4
). Incomplete bisulfite modification of DNA can
give rise to false-positivity for methylated cytosines, which are actually
unmodified cytosines. The sensitivities of MSP for various genes are very
