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