Students: Jaimie VettichiraAraj Sidki

Roshni GandhiSonia George

Diana BarayevaEdward 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

ChIP DNA 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 usingPolymerase 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 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

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 proteinTherefore, eosin staining will show

more the hemotoxylin staining

Our results show that overfixed DNA isolated from tissues can still be successfully retrieved