ChIP on ChIP

1. ChIP on ChIP Students: Jaimie Vettichira Araj Sidki Roshni Gandhi Sonia George Diana Barayeva Edward Salib Instructor: Dr. Claude E. Gagna

2. DNA Microarrays 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

3. DNA Microarrays (cont.) 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

4. Chromatin Immunoprecipitation(ChIP) Assay 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

5. What is “ChIP on ChIP”? ChIP DNA Microarray ChIP on ChIP

6. 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

7. ChIP on ChIP (cont.) Result: • DNA sequences that can be identified They can be amplified using Polymerase chain reaction (PCR) method

8. The Problem • Most scientists have stored their tissue samples in 10% neutral buffered formalin have overfixed tissue using aldehyde fixatives

9. Purpose of this Project • To be able to allow scientists to retrieve DNA from overfixed tissue

10. Procedure Done Materials • Model 40 GC lab Oven • Crude Extract • 96 Microwell • Formaldehyde • Eosin Stain • Hemotoxylin Stain • Washing Reagent • Microplate Reader • Control

11. Procedure Done (cont.) How is done? • First 3 columns of microwell were filled with the control • In the 5th column of microwell put 40µL of high concentration of extract • In the 7th column of microwell put 40µL of low concentration of extract • In the 9th column of microwell put 40µL of washing reagent

12. Procedure Done (cont.) • 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

13. Results: Before Staining • 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

14. Results: After Staining • 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

15. Conclusion • 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

16. Overall Conclusion • Our results show that overfixed DNA isolated from tissues can still be successfully retrieved