کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
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1174999 | 961782 | 2010 | 5 صفحه PDF | دانلود رایگان |
![عکس صفحه اول مقاله: Fluorescence-based high-throughput assay for human DNA (cytosine-5)-methyltransferase 1 Fluorescence-based high-throughput assay for human DNA (cytosine-5)-methyltransferase 1](/preview/png/1174999.png)
We have developed the first economical and rapid nonradioactive assay method that is suitable for high-throughput screening of the important pharmacological target human DNA (cytosine-5)-methyltransferase 1 (DNMT1). The method combines three key innovations: the use of a truncated form of the enzyme that is highly active on a 26-bp hemimethylated DNA duplex substrate, the introduction of the methylation site into the recognition sequence of a restriction endonuclease, and the use of a fluorogenic read-out method. The extent of DNMT1 methylation is reflected in the protection of the DNA substrate from endonuclease cleavage that would otherwise result in a large fluorescence increase. The assay has been validated in a high-throughput format, and trivial changes in the substrate sequence and endonuclease allow adaptation of the method to any bacterial or human DNA methyltransferase.
Journal: Analytical Biochemistry - Volume 401, Issue 1, 1 June 2010, Pages 168–172