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