Assessing the use of unmodified 40 mer oligonucleotides in barcode microarray technology
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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?.

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Assessing the use of unmodified 40 mer oligonucleotides in barcode microarray technology l.jpg

Assessing the Use of Unmodified 40-mer Oligonucleotides in BarcodeMicroarrayTechnology

Danielle Hyun-jin Choi

Dr. A. Malcolm Campbell

Davidson College, NC


Introduction to genomics and microarrays l.jpg
Introduction to BarcodeGenomics and Microarrays

  • What is a microarray?

  • What is Genomics?


How do you print a microarray l.jpg
How do you print a microarray? Barcode

Printed microarrays

Stored microarrays




Slide6 l.jpg

Cy3 Laser Channel Barcode

Cy5 Laser Channel

Generating Microarray Data

+

=


Introduction to barcode microarrays l.jpg
Introduction to BarcodeBarcode Microarrays


Barcode microarray method l.jpg
Barcode Microarray Method Barcode

  • 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 l.jpg
Synthesizing Probes for Barcode Microarray Barcode

  • 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



My research questions l.jpg
My Research Questions Barcode

  • 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 l.jpg

Probe to Buffer Ratio Barcode

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

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


Hybridization and data analysis l.jpg
Hybridization and Data Analysis Barcode

  • 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 l.jpg
Notable Trends in Data Barcode

  • 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 l.jpg

Methods Barcode

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

Software Dependence

  • Depending on the image and data analysis program (MAGIC Tool and Scanalyze), statistical significance of the ratios varied


Discussion and future directions l.jpg
Discussion and Future Directions Barcode

  • 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 l.jpg
Acknowledgements Barcode

  • Duke Endowment

  • National Science Foundation

  • Davidson College

  • Dr. Daniel S. Yuan, Dr. Jef Boeke, Johns Hopkins University


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