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Results

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... designed a stoplight pattern using three S. Cerevisiae genes (Mhp, Idh1, and SHY) ... SHY-O SHY-O Mhp-O. Mhp-O idh1-O idh1-O. RT-C RT-C. Acknowledgements ... – PowerPoint PPT presentation

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Title: Results


1
DNA Microarrays A guide to teaching chips
Allison Amore, Sheena Bossie, Max Citrin, Erin
Cobain, Megan McDonald, Marieta Solé, Emily
Wilson A. Malcolm Campbell
Davidson College Biology Department
3
1
2
Setup
Introduction
The Microarray Process
  • We designed a stoplight pattern using three S.
    Cerevisiae genes (Mhp, Idh1, and SHY).
  • Competent bacterial cells containing plasmids
    with 500bp fragments of genes were obtained from
    a previous study (D. Pierce).
  • Genes were chosen because their probes had been
    previously shown not to bind to either of the
    other two genes (D. Pierce).
  • Design was printed using BioRobotics MicroGrid
    II Compact?
  • We followed the 2-step 3DNA Genisphere? for probe
    hybridization (previously optimized by E. Oldham)
  • DNA microarrays measure expression level changes
    across a genome and have the potential to be a
    powerful diagnostic tool in medicine.
  • Equipment necessary to print DNA microarrays is
    expensive complex
  • Undergraduates have little experience with this
    technique
  • Undergraduates face challenge of learning about
    DNA microarray technology without hands on
    experience
  • PROBLEM TO BE ADDRESSED How can DNA microarray
    technology be introduced at the undergraduate
    level in a laboratory setting?
  • POTENTIAL SOLUTION ? Teaching Chips - provide
    students with thorough understanding of
    microarray methodology but do not necessitate the
    use of expensive RNA samples or genomic DNA.
    Students learn how to design, print (if the
    equipment is available) and process chips that
    are likely to have a high success rate with
    visible data.
  • Essential Steps
  • grow cells containing RNA of interest
  • isolate and extract RNA
  • convert mRNA into cDNA
  • spot cDNA onto slide
  • hybridize with Cy3 and Cy5 tagged probes
  • scan chip to measure fluorescence intensity
  • analyze data to determine repression and
    induction patterns from image profiles.

3DNA dendrimer courtesy of Genisphere, Inc.
Fluorescent labeling process using Cy5 (red) and
Cy3 (green) dyes (Cartoon created by E. Oldham)
Gene Systematic Name Chromosome Brief Description of Function
MHP1 YJL042W X microtubule organization
IDH1 YNL037C XIV Regulatory subunit in cellular energy metabolism
SHY1 YGR112W VII Codes for a mitochondrial protein required for full expression of cytochrome oxidase (COX)
5
4
6
Future Work
Results
Results Continued
Part I Two different plasmid isolation kits
were compared
  • Perform experiment using oligos with correct
    capture sequence.
  • Confirm results using Q-PCR
  • Analyze data using Magic Tool
  • Compose a single detailed protocol
  • Promote the use of teaching chips in
    undergraduate settings
  • Publish work in Cell Biology Education

Part III Prepared test slide to determine
whether all reagents were functioning properly
QIAGEN HiSpeed Plasmid Midi Kit Promega PureYield Plasmid Midiprep kit
List price 250 for 25 preps List price 180 for 25 preps
Requires little centrifugation, Faster Multiple centrifugation steps, Slower
Slightly more consistent results Slightly greater variability
Acknowledgements
  • Part II Chips were scanned to visualize
    stoplight pattern ? no fluorescence was detected
    on any of the slides.
  • Potential Reasons for this Outcome
  • Fluorescent dyes have degraded and lost ability
    to fluoresce
  • Oligos were non-functional or degraded
  • Plasmid DNA may not have been isolated properly

We would like to thank the following people for
their invaluable support Dr. A. Malcolm
Campbell Danielle Choi Emily Oldham Davidson
College Biology Dept. Dan Pierce Various
service technicians Chris Healey
Test Slide Design. Anticipated results if all
reagents were functioning properly. O oligo, RT
Reverse Transcript, C control.
Actual Test Slide Results. Only the two positive
controls fluoresced as expected.
CONCLUSION All proposed reasons for our negative
results were proven false. The unexpected outcome
was due to a capture sequence that was not
complementary to the dendrimer sequence!
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