Redefining DNA Microarrays

Tiffany McAninchAdvised by:Dr. Frederick Haselton, PhDMark McQuain, 2BPhD

Purpose

If humanity was able to understand genetics more completely and able to determine what the causes were for many disorders, there would be a huge sweep for this information. DNA microarrays are the most recent tool to put this information to use in a large scale way. This project has been to design a system similar to microarrays, but one in which the information can be obtained faster, more accurately, and more easily.

Northern Blotting

Microarray Procedure

Target genes prepared and hyb. onto slides

Culture cellsIsolate mRNAPrepare labeled cDNA2 cDNAs competitively hybridizedAnalyzed by fluorescent signature

Microarray Schematic

mRNA from Sample 1 mRNA from Sample 2

Reverse transcribe each sampleusing a different fluorescent

nucleotide (CY3 or CY5)

Mix equal amounts of eachcomplex probe together and

hybridize overnight

Scan and quantitate gene expression levels

(Green Fluorescence) (Red Fluorescence)

Streptavidin Beads

Flow Cytometer

PMT

PMT

PMT

Dichroic Filters

BandpassFilters

Laser

Flow Manifold

SSC

FSC

Cells

Raman Spectroscopy

Trp-Phe

0.00E+00

2.00E+03

4.00E+03

6.00E+03

8.00E+03

1.00E+04

0 500 1000 1500 2000

Rel cm

Ram

an I

nte

nsi

ty

Phe-Gly-Phe-Gly

0.00E+00

5.00E+02

1.00E+03

1.50E+03

2.00E+03

2.50E+03

0 500 1000 1500 2000

Rel cm

Ram

an I

nte

nsi

ty

Trp-Trp

0.00E+00

5.00E+02

1.00E+03

1.50E+03

2.00E+03

0 500 1000 1500 2000

Rel cm

Ram

an I

nte

nsi

ty

Comparison

Microbeads Easier to store Do chemically Hold patent! Less Reagents Speed Flow Cytometer 2 million signatures

Microarrays Don’t need Raman

tag No flow cyt. for

Raman 50,000 signatures

Conclusion

The design has been formulated, which was the purpose of this project. There has also been much research that will aid in future use of narrowing the design further. Having found the beads with suitable surface chemistries and measured the Raman of the tags, the project is ready to be further tested.

Thanks!!

Professor HaseltonMark McQuainProfessor Mahadevan-JansenChad LieberTodd Monroe