DNA Microarray Automation and Analysis
EMSL Project ID
1710
Abstract
The objective of this project is to automate the molecular biology and reagent manipulations associated with the hybridization and detection of nucleic acid binding events on the surface of a DNA microarray. The research team is developing microarrays for the detection and characterization of microorganisms from environmental samples, and is on track to fluidically link a nucleic acid microarray with automated sample preparation components (including cell lysis, nucleic acid purification, and PCR amplification modules). For this proposal, a reusable microfluidic flow cell is under construction that will allow the team to insert custom-printed microarrays, introduce molecular biology reagents, and detect hybridization via fluorescence detection on an inverted microscope. Microarrays will be printed on glass slides with oligonucleotide probes specific for E. coli. Target DNA will be generated by PCR amplification of the eae locus from E. coli genomic DNA, using biotinylated primers. Hybridization will proceed at room temperature under variable salt conditions, times and target concentrations. Unbound and non-specifically bound target will be washed away, and hybrids developed with a streptavidin-alkaline phosphatase system and ELF-97 substrate (Molecular Probes). Detection will occur on an inverted microscope with CCD image capture capability. The expected results of this research are a) demonstration of automated microarray analysis, b) computer algorithms and programs for automating said analysis that are simultaneously compatible with other microfluidic devices under construction by the research team, and c) proof-of-principle extraction, purification, amplification and microarray detection of microorganisms from environmental samples using microfluidic systems.
Project Details
Project type
Exploratory Research
Start Date
1999-10-31
End Date
2002-06-01
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members