Qualitative and Quantitative Polymerase Chain Reaction-Based Methods for DNA Methylation Analyses - Clinical Applications of PCR - page 41

4. Notes
1.

The best design of primers and probes is crucial for virtually all qualitative or
quantitative methods for DNA methylation analyses.

2. The methods described in this chapter are most widely applied for DNA methyla-

tion analyses. Other methods have also been developed for high-throughput DNA
methylation analyses ( 2 ) ; however, these are not described in this chapter for
simplicity.

3. MSP is one of the most common methods for detecting DNA methylation because

of its high sensitivity and specificity. Another advantage of MSP is that it enables
qualitative analysis and can be easily upgraded for quantitative analysis using
real-time PCR.

4. The potential problem of obtaining false-negativity in different sample sources

can be avoided by using MSP and choosing various DNA regions for the DNA
methylation analysis.

5. Bisulfite conversion of all unmethylated cytosines to uracils in DNA has been

incorporated into many techniques for DNA methylation analyses ( 12 ) . These
methods include MSP, quantitative real-time MSP, COBRA, MsSNuPE, and
bisulfite sequencing. However, bisulfite modification of DNA must be complete;
otherwise, false-positive results may be obtained.

6. MSP offers high specificity and has the advantage over methylation-sensitive

restriction enzyme PCR, which can give rise to false-positivity as a result of
incomplete restriction enzyme digestion.

7. It has been difficult to measure accurately the extent of DNA methylation at speci-

fied CpG sites within target sequences. Previously, methylation analysis has been
based on methylation-sensitive restriction enzyme digestion, which allows the
analysis of CpG dinucleotides limited within the restriction sites of a particular
region. Also, this method does not allow an accurate analysis of DNA methyla-
tion in formalin-fixed material in which the DNA quality is sometimes not good
enough.

8. Quantitative real-time MSP, COBRA, and MsSNuPE allow the quantitative

assessment of DNA methylation changes at multiple or individual CpG dinucle-
otides.

References
1. Herman, J. G. and Baylin, S. B. (2003) Gene silencing in cancer in association
with promoter hypermethylation. N. Engl. J. Med. 349, 2042-2054.
2. Ushijima, T. (2005) Detection and interpretation of altered methylation patterns
in cancer cells. Nat. Rev. Cancer 5, 223-231.
3. Herman, J. G., Graff, J. R., Myohanen, S., Nelkin, B. D., and Baylin, S. B. (1996)
Methylation-specific PCR: a novel PCR assay for methylation status of CpG
islands. Proc. Natl. Acad. Sci. USA 93, 9821-9826.
4. Dupont, J.M., Tost, J., Jammes, H., and Gut, I.G. (2004) De novo quantitative
bisulfite sequencing using the pyrosequencing technology. Anal. Biochem. 333,
119-127.