Abstract

1

• Exploring the Use of Plasmids in DNA Microarray
  Technology
• B. Daniel Pierce and A. Malcolm Campbell
  Biology Department, Davidson
  College, Davidson, NC 28035

11 Probe Ratio Results
Abstract
Chip Design and Sample Scans

• Using microarrays for high-throughput DNA
  detection is one of the fastest growing fields in
  biology today. However, the field is so new that
  most users do not know how to incorporate
  standards and controls into their experiments.
  Furthermore, consistent production of target DNA
  has been problematic and a documented source of
  variability.
• My research centered on three questions
• Can target DNA be cloned into plasmids and used
  for spotting on microarrays?
• Do plasmids work as well as PCR products?
• If you probe with known ratios, does the method
  detect those same ratios?
• The data indicate that plasmid DNA is indeed
  useable as a target in microarray experiments and
  is comparable to PCR product at producing a 11
  Cy5Cy3 dye ratio. However, DNA microarrays
  appear to under-represent the ratios of probes
  used under well-controlled conditions. These
  findings open the way for improved DNA microarray
  experimental design and quality assurance of
  results.

In this experiment, equal amounts of red and
green probes that are complementary to nine
spotted PPP genes. The expected R/G ratio using
the intensities post-scanning would be 1, as red
and green probes should have bound at an equal
rate.
The chip was designed asymmetrically for
orientation purposes. Two
types of negative controls were used spotting
solution alone and unrelated PCR products in
plasmid pCR 2.1.

• Conclusion
• Although there is some inherent variation in the
  system, ratios near 11 were observed.

This pilot study into the possibility of plasmid
use yielded a variety of different scans. Some,
such as the scan above, showed high levels of PCR
product intensity while other, such as the scan
to the left, showed the opposite.
Each bar represents the average of 1408 spots.
Error bars represent 95 confidence intervals.
Exploring Different Concentrations
PCR vs. Plasmid
The Plasmid Advantage
As in the previous experiment, equal
concentrations of red and green probes were used.
Three concentrations of probe were added in
different trials and the resulting R/G ratios
were examined.
The overall intensities of light for both red and
green dyes on three slides were averaged.
Average intensities using plasmid as the spotting
material were higher than those for PCR.
The possibility for the use of plasmids is very
intriguing. The gels to the upper left show the
genes in this experiment after PCR. These
products were individually ligated into the pCR
2.1-TOPO plasmid at left (PCR plus Plasmid
PPP). The gels to the upper right show the
plasmids with the gene insertion after being cut
with EcoRI. The use of plasmids bestows a
precision to the microarray process. Clearly the
gels to the right contain a much cleaner material
than those to the left. Additionally, once genes
are in a plasmid such as this, they are easier,
cheaper, and faster to reproduce.
Picture courtesy Emily Oldham
Insertion Site

• Conclusions
• Plasmid DNA can be used for spotting on
  microarrays
• Plasmid DNA seems to work as well as spotting
  PCR product

• Conclusions
• There are only slight R/G ratio variations when
  different probe concentrations are used
• There is no need to deviate from 1 ng/µL

www.invitrogen.com
Each bar represents the average of 1408 spots.
Error bars represent 95 confidence intervals.
Chip Design and Sample Scan
31 101 Probe Ratio Analysis
Method Oligonucleotide Addition
The GRIDs from the left are oriented vertically
below in a sample scan. Three of the multiple
experiments performed with this chip are
presented below.
Different initial probe ratios were added. Now
the expected R/G ratio using the intensities
post-scanning would be values other than 1 (a R/G
ratio value of 10 would be expected when 10 times
the amount of red probe was added).
When red and green probes bind to the same spot,
the resulting light fluorescence picked up by the
scanner is yellow.
The next chip was designed with multiple PPP as
possible probe binding sites. The PCR products
of eleven genes were inserted into the pCR 2.1
plasmid and spotted in duplicate in three
different concentrations. Two types of negative
controls were used spotting solution alone and
plasmids whose probes were not used.

• Conclusion
• R/G ratio values appear to under represent the
  probe ratios used

Each bar represents the average of 1408 spots.
Error bars represent 95 confidence intervals.
Acknowledgements I give my sincere thanks to
Emily Oldham and Dr. Laurie Heyer.
Helping Students Discover Genomics, Proteomics
Bioinformatics A. Malcolm Campbell, Laurie J.
Heyer, Adam Abele, Brian Akin, Danielle Choi,
Parul Karnik, Peter Lowry, David Moskowitz, Emily
Oldham, Jennifer Madden