Students:Jaimie Vettichira Araj Sidki Roshni Gandhi Sonia George Diana Barayeva Edward Salib...
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Transcript of Students:Jaimie Vettichira Araj Sidki Roshni Gandhi Sonia George Diana Barayeva Edward Salib...
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