Ip. Pogribny et Sj. James, A METHOD TO ESTIMATE THE PERCENT LOSS OF CYTOSINE METHYL-GROUPS AT DEFINED CPG SITES IN LIVER DNA FROM METHYL-DEFICIENT RATS, Journal of nutritional biochemistry, 8(6), 1997, pp. 355-359
Dietary methyl deficiency provides an ideal in vivo model system in wh
ich to study progressive alterations in DNA methylation patterns as th
ey occur during multistage hepatocarcinogenesis. Weanling male F344 ra
ts were given a semipurified diet deficient in the methyl-donors choli
ne, methionine, and folic acid for a 36-week period with sampling inte
rvals at 3,9,24, and 36 weeks. Using a genomic sequencing procedure ba
sed on the PCR amplification of bisulfite-modified DNA, the methylatio
n status of individual CpG sites within exons 6 and 7 of the p53 gene
in liver samples from control and deficient rats was assessed. Treatme
nt of denatured nuclear DNA with sodium bisulfite converts unmethylate
d cytosine residues to uracil, which are then amplified as thymine in
the PCR reaction. In contrast, methylated cytosines are resistant to b
isulfite deamination under these reaction conditions and are amplified
as cytosine. In this report, we describe a novel application of autom
ated sequencing technology to estimate the proportion of methylated cy
tosines present at defined CpG sites within the total population of DN
A molecules extracted. Using the bisulfite conversion-PCR genomic sequ
encing method, we demonstrate the validity of peak height analysis of
co-eluting peaks in the autosequencer electropherogram to estimate the
percent methylation at a defined CpG site. The sensitivity of this me
thod is demonstrated by the progressive loss of methyl groups at a def
ined CpG site in the methyl-deficient rats after 9, 24, and 36 weeks.
The application of this sequence-specific technology will allow site-s
pecific definition of the methylation status of each CpG site within a
coding sequence or promoter region and should provide new insights in
to mechanisms and consequences of methylation dysregulation as a resul
t of dietary deprivation of methyl donors. (C) Elsevier Science Inc. 1
997.