A real-time PCR assay for DNA-methylation using.
A procedure for the analysis of the methylation status of imprinted genes is described. The method offers a rapid and reliable alternative to conventional methods such as Southern blots and methylation-specific polymerase chain reaction (PCR) (i.e., allele-specific methylation-specific PCR).
Figure 4: Methylation-specific PCR is a sensitive method to discriminately amplify and detect a methylated region of interest using methylated-specific primers on bisulfite-converted genomic DNA. Such primers will anneal only to sequences that are methylated, and thus containing 5-methylcytosines that are resistant to conversion by bisulfite.
To analyze the methylation status of two differentially inherited and methylated loci (the human androgen receptor (HUMARA) and the small nuclear ribonucleoprotein-associated polypeptide N (SNRPN) gene) in testicular biopsy samples, and to compare the results with microscopic evaluation.
Kubota T, Das S, Christian SL, Baylin SB, Herman JG, Ledbetter DH. Methylation specific PCR simplifies imprinting analysis. Nat Genet 1997;16:16-17. Mascari MJ, Gottlieb W, Rogan PK, Butler MG, Waller DA, Armour JA, et al. The frequency of uniparental disomy in Prader-Willi syndrome. Implications for molecular diagnosis. N Engl J Med. 1992;326.
Study of DNA methylation patterns of imprinted genes in children born after assisted reproductive technologies reveals no imprinting errors: a pilot study.
The human androgen receptor gene locus (HUMARA) assay using methylation-specific PCR was performed as described previously. Briefly, the assay uses a bisulfite-treatment followed by PCR; the inactive X pattern is based on the methylated allele at the HUMARA locus and the active X pattern is based on the unmethylated allele at the same locus.
Methylation Specific PCR (MSP). Over the years, bisulfite conversion has become the most widely used method for DNA methylation analysis. It is the most convenient and effective way to map DNA methylation to individual bases. As the first step in numerous downstream analysis techniques, it is absolutely the bisulfite conversion process is.