PCR amplification following restriction to detect site-specific DNA methylation
| Autor: | Shujun Chang, Clint W. Magill, Jane M. Magill, Franklin Fong, Ronald J. Newton |
|---|---|
| Rok vydání: | 1992 |
| Předmět: |
Genetics
Inverse polymerase chain reaction Multiple displacement amplification Plant Science Biology Molecular biology Restriction fragment law.invention chemistry.chemical_compound Restriction enzyme chemistry law DNA methylation biology.protein Amplified fragment length polymorphism Molecular Biology DNA Polymerase chain reaction |
| Zdroj: | Plant Molecular Biology Reporter. 10:362-366 |
| ISSN: | 1572-9818 0735-9640 |
| DOI: | 10.1007/bf02668912 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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