A novel method for producing partial restriction digestion of DNA fragments by PCR with 5-methyl-CTP |
| |
Authors: | KK Wong LM Markillie JD Saffer |
| |
Affiliation: | Molecular Biosciences Department P7-56, Pacific Northwest National Laboratory, PO Box 999, Richland, WA 99352, USA. kk.wong@pnl.gov |
| |
Abstract: | Partial digestion of DNA fragments is a standard procedure for subcloning analysis and for generating restriction maps. We have developed a novel method to generate a partial digestion for any DNA fragment that can be amplified by PCR. The method involves the incorporation of 5-methyl-dCTP into the PCR product to protect most of the restriction sites. As a result, complete digestion of the modified PCR products with a 5-methyl-dCTP-sensitive enzyme will produce an array of restriction fragments equivalent to a partial restriction enzyme digestion reaction done on unmethylated PCR products. This method reduces the time and material needed to produce partially-digested DNA fragments by traditional methods. Furthermore, using fluorescein-labeled primers in the reaction, we were able to detect the fluorescein-labeled end fragments resulting from the enzyme digestion using a fluorimager or anti-fluorescein-AP antibody and thus determine the restriction maps. |
| |
Keywords: | |
本文献已被 PubMed 等数据库收录! |
|