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ChIP on ChIP. Students:Jaimie Vettichira Araj Sidki Roshni Gandhi Sonia George Diana Barayeva Edward Salib Instructor: Dr. Claude E. Gagna. DNA Microarrays. What are they? Microscopic DNA spots representing single genes

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ChIP on ChIP

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Chip on chip l.jpg

ChIP on ChIP

Students:Jaimie Vettichira

Araj Sidki

Roshni Gandhi

Sonia George

Diana Barayeva

Edward Salib

Instructor: Dr. Claude E. Gagna


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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


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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


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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


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What is “ChIP on ChIP”?

ChIPDNA Microarray

ChIP on ChIP


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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


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ChIP on ChIP (cont.)

Result:

  • DNA sequences that can be identified

    They can be amplified usingPolymerase chain reaction (PCR) method


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The Problem

  • Most scientists have stored their tissue samples in 10% neutral buffered formalin

    have overfixed tissue using aldehyde fixatives


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Purpose of this Project

  • To be able to allow scientists to retrieve DNA from overfixed tissue


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Procedure Done

Materials

  • Model 40 GC lab Oven

  • Crude Extract

  • 96 Microwell

  • Formaldehyde

  • Eosin Stain

  • Hemotoxylin Stain

  • Washing Reagent

  • Microplate Reader

  • Control


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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


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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


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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


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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


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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


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Overall Conclusion

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


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