Download presentation
Presentation is loading. Please wait.
Published byRocco Luxon Modified over 9 years ago
1
Students:Jaimie Vettichira Araj Sidki Roshni Gandhi Sonia George Diana Barayeva Edward Salib Instructor: Dr. Claude E. Gagna
2
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
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
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
ChIPDNA Microarray ChIP on ChIP
6
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
Result: DNA sequences that can be identified They can be amplified using Polymerase chain reaction (PCR) method
8
Most scientists have stored their tissue samples in 10% neutral buffered formalin have overfixed tissue using aldehyde fixatives
9
To be able to allow scientists to retrieve DNA from overfixed tissue
10
Materials Model 40 GC lab Oven Crude Extract 96 Microwell Formaldehyde Eosin Stain Hemotoxylin Stain Washing Reagent Microplate Reader Control
11
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
12
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
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
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
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
Our results show that overfixed DNA isolated from tissues can still be successfully retrieved
Similar presentations
© 2024 SlidePlayer.com Inc.
All rights reserved.