PCR amplification in bisulfite methylcytosine mapping in the GC-rich promoter region of amyloid precursor protein gene in autopsy human brain.
Brain Res Brain Res Protoc
; 5(2): 167-71, 2000 Apr.
Article
en En
| MEDLINE
| ID: mdl-10775837
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.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Sulfitos
/
Corteza Cerebral
/
Reacción en Cadena de la Polimerasa
/
Regiones Promotoras Genéticas
/
Precursor de Proteína beta-Amiloide
/
Metilación de ADN
/
Citosina
Tipo de estudio:
Diagnostic_studies
Límite:
Aged
/
Aged80
/
Humans
/
Male
Idioma:
En
Revista:
Brain Res Brain Res Protoc
Asunto de la revista:
CEREBRO
Año:
2000
Tipo del documento:
Article
País de afiliación:
Japón
Pais de publicación:
Países Bajos