PCR amplification following restriction to detect site-specific DNA methylation |
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| Authors: | Shujun Chang Clint W Magill Jane M Magill Franklin Fong Ronald J Newton |
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| Institution: | (1) Department of Forest Science, Texas Agricultural Experiment Station, Texas A&M University, 77843 College Station, Texas;(2) Department of Plant Pathology and Microbiology, Texas Agricultural Experiment Station, Texas A&M University, 77843 College Station, Texas;(3) Department of Biochemistry and Biophysics, Texas Agricultural Experiment Station, Texas A&M University, 77843 College Station, Texas;(4) Department of Biology, University of Portland, Portland, Oregon, USA |
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| Abstract: | A procedure to test for DNA methylation at sites recognized by methylation-sensitive restriction endonucleases is described.
The procedure is based on the assumption that the polymerase chain reaction (PCR) will amplify sequences between two primers
only if the target DNA is intact after digestion. A carrot (Daucus carota) cell line that is heterozygous for two sequenced alleles ofDc8, a gene which is expressed during the later stages of embryogenesis provided an ideal source of DNA for developing and testing
protocols. The promoters of the two alleles differs significantly in length between two sites used for primers, and only one
promoter has a GATC (Sau 3A1 orMbo I) site. This allowed development of a protocol where only the sequence lacking the GATC site was amplified to detectable
levels following digestion of DNA withMbo I which is insensitive to symmetric methylation withm4C orm5C. |
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| Keywords: | DNA methylation restriction isoschizomers PCR amplification |
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