1. Assessing the Use of Unmodified 40-mer Oligonucleotides in Barcode Microarray Technology Danielle Hyun-jin Choi Dr. A. Malcolm Campbell Davidson College, NC

2. Introduction to Genomics and Microarrays What is a microarray? What is Genomics?

3. How do you print a microarray? Printed microarraysStored microarrays

4. TAS Application Suite and MicroGrid II

5. MicroGrid II and the Pins

6. Cy5 Laser Channel Cy3 Laser Channel += Generating Microarray Data

7. Introduction to Barcode Microarrays

8. 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

9. 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

10. Barcode Application

11. 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?

12. 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

13. 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

14. 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

15. Software Dependence Depending on the image and data analysis program (MAGIC Tool and Scanalyze), statistical significance of the ratios varied MethodsMean 1:5 Mean 1:3.3 Mean 1:2.5 F variateF criticalP value Reject null hypothesis ? MAGIC (total) 0.6240.6270.6591.6573.013 0.192No MAGIC (total-bg) 0.5260.5930.5652.8653.020.058No Scanalyze ( total) 1.3320.9241.03374.873.0132.27E-29Yes MAGIC (avg-bg) 0.5460.5660.5743.9173.0130.020Yes Scanalyze (avg-bg) 0.4630.5260.54033.753.0121.593E-14Yes

16. 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

17. Acknowledgements Duke Endowment National Science Foundation Davidson College Dr. Daniel S. Yuan, Dr. Jef Boeke, Johns Hopkins University