کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
1176030 | 961828 | 2006 | 8 صفحه PDF | دانلود رایگان |
![عکس صفحه اول مقاله: Methylation-dependent fragment separation: Direct detection of DNA methylation by capillary electrophoresis of PCR products from bisulfite-converted genomic DNA Methylation-dependent fragment separation: Direct detection of DNA methylation by capillary electrophoresis of PCR products from bisulfite-converted genomic DNA](/preview/png/1176030.png)
Fundamental to understanding the role of cytosine (C) methylation in genomic DNA (gDNA) is the need for robust analysis methods to determine the location and degree of this modification. We report a novel method for methylation detection by denaturing capillary electrophoresis (CE) using standard fragment analysis conditions. Bisulfite treatment of gDNA will selectively deaminate C but not 5-methylcytosine (5mC). Amplicons generated from bisulfite-converted gDNA are analyzed immediately after PCR using a 6-carboxy fluorescein (6-FAM) dye-labeled primer. The amplicons from methylated and unmethylated gDNA separate based solely on base composition due to the presence of multiple C versus thymine (T) differences. By direct detection of PCR amplicons following PCR using primers that anneal independent of methylation status, the overall workflow from gDNA sample input to data analysis is relatively simple. Furthermore, the same PCR product is suitable for additional analyses such as direct sequencing, cloning and sequencing, single-base extension, and post-PCR incorporation of a modified dCTP, the latter of which allows resolution of amplicons with as little as a single C/T difference. We show the utility of this novel CE detection assay by analyzing the hypermethylated region of the fragile-X FMR1 locus.
Journal: Analytical Biochemistry - Volume 354, Issue 2, 15 July 2006, Pages 266–273