Methyl CpG binding protein 2 binding sites on chromosome 22 in hepatocellular
carcinoma B cells
Cancer cells often have wide spread methylation across their genes. This
methylation, especially on gene promoters, can result in gene repression. When tumor
suppressor genes are methylated, they may be turned off resulting in a cancer phenotype.
The methylation recruits methyl CpG binding proteins (MBDs) to bind to the nucleotides
and recruit other proteins to deactivate the gene. By identifying the location of bound
MBDs we can hypothesize which genes have been repressed. The purpose of this lab is to
determine the binding sites of MBD2 on chromosome 22 in hepatocellular carcinoma B
cells. To accomplish this, cells are cultured and harvested in Eagle’s medium and
trypsinized. The nuclei are extracted, sonicated, and MBD2 antibody is introduced. The
bound fragments are precipitated, purified and amplified with PCR. Finally, the amplified
DNA is fluorescently labeled with Cy3 and hybridized to a microarray of chromosome
22. Unfortunately during the purification step during labeling the hybridization buffer
was not given adequate time to dissolve the DNA. This prevented successful completion
of the entire protocol, including the hybridization. The experiment should be repeated
with the aforementioned warning, and possibly with others methyl CpG binding proteins.
Degree project in Biology. Spring 10 p
Anish Chari