PCR amplification in bisulfite methylcytosine mapping in the GC-rich promoter region of amyloid precursor protein gene in autopsy human brain.
Alterations in DNA 5-methyldeoxycytidine pattern influence gene expression for certain mammalian genes in development, differentiation, carcinogenesis, and aging. Detection of DNA methylation at the promoter region, which generally represses transcription activity, is one important element in studying changes in molecular expression with aging and age-associated disorders. Bisulfite genomic sequencing is a useful method for mapping methylated cytosines. However, PCR amplification for bisulfite-treated DNA does not yield a sufficient amount of products that have a sufficient level of specificity, especially in the GC-rich sequences usually seen at the promoter regions of house keeping genes. We present a method for increasing the sensitivity and specificity of PCR amplification in bisulfite methylcytosine mapping in an extremely GC-rich promoter region of amyloid precursor protein (APP) gene from the cerebral cortex of human autopsy brain. The PCR used consists of two cycles using the lower primer alone to amplify the sense sequence, and then eight cycles at a theoretical annealing temperature (60 degrees C) and 30 cycles at a lower annealing temperature (50 degrees C) using both the upper and lower primers. The present method likely can also be applied to other GC-rich genomic sequences.
Nagane
Y
Y
Department of Neurology, Iwate Medical University, 19-1 Uchimaru, Morioka, Japan.
Utsugisawa
K
K
Tohgi
H
H
D000368:Aged; D000369:Aged, 80 and over; D000375:Aging / Q000502:physiology; D016564:Amyloid beta-Protein Precursor / Q000235:genetics*; D001483:Base Sequence / Q000235:genetics; D002102:Cadaver; D002540:Cerebral Cortex / Q000378:metabolism*; D003596:Cytosine / Q000502:physiology*; D019175:DNA Methylation; D016678:Genome; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics*; D012680:Sensitivity and Specificity; D013447:Sulfites / Q000494:pharmacology; D013696:Temperature; D014018:Tissue Distribution
D016564:Amyloid beta-Protein Precursor; D013447:Sulfites; D003596:Cytosine; C042345:hydrogen sulfite