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

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Presentation transcript:

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 microarraysStored 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 MethodsMean 1:5 Mean 1:3.3 Mean 1:2.5 F variateF criticalP value Reject null hypothesis ? MAGIC (total) No MAGIC (total-bg) No Scanalyze ( total) E-29Yes MAGIC (avg-bg) Yes Scanalyze (avg-bg) E-14Yes

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