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Assessing the Use of Unmodified 40-mer Oligonucleotides in
Barcode Microarray Technology
Danielle Hyun-jin Choi
Dr. A. Malcolm Campbell
Davidson College, NC
Introduction to Genomics and Microarrays
• What is a microarray?
• What is Genomics?
How do you print a microarray?
Printed microarrays Stored microarrays
TAS Application Suite and MicroGrid II
MicroGrid II and the Pins
Cy5 Laser Channel Cy3 Laser Channel
+ =
Generating Microarray Data
Introduction to Barcode Microarrays
Barcode Microarray Method
• The red and green arrows represent PCR primers• Unique combination of A, C, G, Ts for each
barcode yeast strain• Barcode microarray uses these unique sequences
as printing material• Each spot on microarray represents a unique
barcode sequence of a deletion strain
Synthesizing Probes for Barcode Microarray
• Allow the mixture of different barcode yeast strains to grow
• Take a sample of the initial population, use the gDNA as templates for PCR using the two pairs of primers, then label the products with red dye
• After the different deletion strains grow more, competing with each other, take another sample and label the products with green dye
Barcode Application
My Research Questions
• Can we make barcode microarrays on glass?
• Is there steric hindrance?
• What is the optimal probe concentration?
• What is the optimal hybridization temperature?
Concentration Dependence
• Suggested concentration of 1:500 in the protocol resulted in no visible spots after scanning
• No significant difference in signal strength was observed between the last three concentrations
Probe to Buffer Ratio
Was signal strong enough to be analyzed?
1:500 No
1:50 No
1:25 No
1:5 Yes
1:3.3 Yes
1:2.5 Yes
Hybridization and Data Analysis
•96 samples of 40-mer barcodes from Johns Hopkins University
•Two identical probes only differing in label colors, prepared by Drs. Daniel S. Yuan and Jef Boeke
Notable Trends in Data
• Because the probes were prepared identically, only differing in the color label, the ratios were expected to be close to 1
• Ratios consistently hovered around 0.5 (Cy5/Cy3) due to weaker Cy5 (Red) signal compared to Cy3 (Green) signal
Software Dependence• Depending on the image and data analysis program
(MAGIC Tool and Scanalyze), statistical significance of the ratios varied
Methods Mean1:5 Mean1:3.3 Mean1:2.5 F variate F critical P value Reject null hypothesis?
MAGIC(total)
0.624 0.627 0.659 1.657 3.013
0.192 No
MAGIC (total-bg)
0.526 0.593 0.565 2.865 3.02 0.058 No
Scanalyze (total)
1.332 0.924 1.033 74.87 3.013 2.27E-29 Yes
MAGIC(avg-bg)
0.546 0.566 0.574 3.917 3.013 0.020 Yes
Scanalyze (avg-bg)
0.463 0.526 0.540 33.75 3.012 1.593E-14 Yes
Discussion and Future Directions
• Signal seems strong enough to generate quality ratio data
• Higher incubation temperatures (40°C and 43°C) at two different probe to buffer ratios have been tested and being analyzed
• Synthesize probes with dendrimer binding sequences and use dendrimers as fluorescent dyes to increase signal
Acknowledgements
• Duke Endowment
• National Science Foundation
• Davidson College
• Dr. Daniel S. Yuan, Dr. Jef Boeke, Johns Hopkins University