Students:Jaimie Vettichira Araj Sidki Roshni Gandhi Sonia George Diana Barayeva Edward Salib Instructor: Dr. Claude E. Gagna
What are they? Microscopic DNA spots representing single genes Allows scientists to observe the interactions between thousands of genes simultaneously Allows for gene expression studies
How is this done? DNA spots placed on a solid surface (e.g., glass slide) by covalent attachment Observe the whole genome on a single chip Result: DNA microarrays allow scientists the ability to study how specific genes work
What is it? Special type of DNA microarray What does it do? Determines whether DNA-binding proteins including transcription factors bind to a specific region of a gene
ChIPDNA Microarray ChIP on ChIP
How does it work? Uses a gentle formaldehyde fixation Fixation causes DNA-protein complexes to be seen cross-linked together by formaldehyde DNA-protein complex is isolated and sheared into fragments Antibodies specific for the DNA-binding proteins in question are added so the DNA- protein complex can be isolated DNA and proteins are released so that it can be isolated
Result: DNA sequences that can be identified They can be amplified using Polymerase chain reaction (PCR) method
Most scientists have stored their tissue samples in 10% neutral buffered formalin have overfixed tissue using aldehyde fixatives
To be able to allow scientists to retrieve DNA from overfixed tissue
Materials Model 40 GC lab Oven Crude Extract 96 Microwell Formaldehyde Eosin Stain Hemotoxylin Stain Washing Reagent Microplate Reader Control
How is done? First 3 columns of microwell were filled with the control In the 5 th column of microwell put 40µL of high concentration of extract In the 7 th column of microwell put 40µL of low concentration of extract In the 9 th column of microwell put 40µL of washing reagent
With the oven at 50°C, incubated microwell for 20 minutes Stored in the refrigerator at -4°C Staining Chemicals: ◦ Hemotoxylin: stains DNA ◦ Eosin: stains protein
Columns 1, 3, 5-control buffer ◦ Negative control Column 2-High concentration ◦ DNA-protein complex seen ◦ In vivo process: cross- linking by formaldehyde Column 4-Low concentration ◦ Specks of binding seen
Columns 1, 3, 5-control buffer ◦ Negative: no binding Column 2 (top)-eosin stain bound with the protein Column 2 (middle)-controls ◦ Little bit of cross containation Column 2 (bottom)- hemotoxylin stain bound with the DNA Column 4 (top)- dark staining by eosin Column 4 (middle)- control Column 4 (bottom)- light staining by hemotoxylin
Why did column 2 stain darker than column 4? ◦ Column 2 had a higher concentration of the extract while column 4 had a low concentration of the extract Why did eosin give a darker staining than the hemotoxylin in column 4? ◦ Eosin stains protein ◦ When DNA-protein complex made: DNA is surrounded by protein Therefore, eosin staining will show more the hemotoxylin staining
Our results show that overfixed DNA isolated from tissues can still be successfully retrieved