Bisulfite conversion-specific and methylation-specific PCR: a sensitive technique for accurate evaluation of CpG methylation.
Methylation-specific PCR (MSP) is frequently used to distinguish methylated alleles in the genome. Sequences that have been incompletely converted during bisulfite treatment are frequently co-amplified during MSP. For accurate MSP, it is important to detect methylated sequences in a background of unconverted DNA with a high level of sensitivity. We report here sensitive techniques, bisulfite conversion-specific MSP (BS-MSP) to accurately evaluate CpG methylation. BS-MSP provides accurate results across a wide spectrum of bisulfite conversion levels. BS-MSP is also confirmed to be a useful technique for the routine analysis of clinical tumor specimens that were paraffin-embedded and microdissected. BS-MSP thus provides the powerful features of ease of use and compatibility with paraffin sections. We recommend that methylation analysis should include a step to eliminate unconverted DNA to avoid overestimation of the DNA methylation level in the samples.
Sasaki
Masahiro
M
Department of Urology, University of California-San Francisco and Veterans Affairs Medical Center, San Francisco, CA 94121, USA. Urologylab@aol.com
Anast
Jason
J
Bassett
William
W
Kawakami
Toshifumi
T
Sakuragi
Noriaki
N
Dahiya
Rajvir
R
D015820:Cadherins / Q000235:genetics* / Q000378:metabolism; D018899:CpG Islands / Q000235:genetics*; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012016:Reference Values; D015203:Reproducibility of Results; D015183:Restriction Mapping / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods; D013447:Sulfites; D001749:Urinary Bladder Neoplasms / Q000235:genetics / Q000378:metabolism*
D015820:Cadherins; D004273:DNA, Neoplasm; D013447:Sulfites; C009279:sodium bisulfite