
	1999 Summer Scholars Program

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Study of ddmB, a DNA Methylation Mutation of Arabidopsis

By William Gordon
Mentor: Dr. Eric Richards
Department of Biology, Washington University, St. Louis, MO

This project was a study to begin characterization of a mutant of Arabidopsis titled ddmB (decrease DNA methylation). DNA methylation appears to be important for proper gene regulation and it was hoped that by using Arabidopsis as a model system, genes which are significant for methylation could be identified. The main approach for identification of genes key to specific functions is known as Genetic Dissection, in which mutations are isolated in hopes of seeing which genes cause change and alteration in methylation levels.

Arabidopsis is a quickly growing plant, which is ready for DNA extraction within a month of planting. The DNA was harvested from individual plants via their leaves. Once the DNA was purified and resuspended in a TE buffer, there were two possible paths to follow. The first was a digest, in which the DNA is exposed to a restriction enzyme which cuts specific sequences. The second was to prep the DNA for PCR, which is a thermal cycling that amplifies particular DNA sequences by in vitro polymerization. Both paths required an agarose gel to be run after their completion, to see the DNA size fractioning. The digest gel simply showed that there was DNA, and was then used for a Southern blot, while the PCR gel showed which plants are heterozygous or homozygous for the mutation, identifying possible plants for crossing later. The digest gel was then subjected to a method of DNA transfer called Southern blotting, in which the DNA moves by liquid onto a nylon membrane for radioactive probing. This membrane was hybridized with a random-primed probe labeled with P³², a radioactive isotope, in order to identify how the DNA is methylated, to know what the mutation has changed. Once the blots had incubated for 12 hours, they were placed on film and after about half a day were viewed. The probe identified methylation levels, in order to determine which plants are suitable parents for crossing. With this information, it is possible to cross plants from the Probed group with those from the PCRed group in order to determine if the mutation is dominant or recessive in the next generation. This is as far as my research has progressed this summer, however, there are still two more areas of experimentation to cover, including a complementation analysis of ddmB to ddm1 and ddm2 for similarity analysis and in the long term, gene sequencing and mapping.

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