Bisulfite conversion-specific and methylation-specific PCR: a sensitive technique for accurate evaluation of CpG methylation |
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Authors: | Sasaki Masahiro Anast Jason Bassett William Kawakami Toshifumi Sakuragi Noriaki Dahiya Rajvir |
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Affiliation: | Department of Urology, University of California-San Francisco and Veterans Affairs Medical Center, San Francisco, CA 94121, USA. Urologylab@aol.com |
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Abstract: | 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. |
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Keywords: | Bisulfite conversion-specific MSP Bisulfite conversion-specific restriction analysis Incomplete bisulfite conversion False-positive Methylation status |
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