This application for funds is submitted to produce a comprehensive system that allows for analysis of nucleic acids from a macrolevel through individual base analysis. The Nucleic Acid System involves two separate instruments: The Phospho-Imager from Fuji and the 373 Gene Scanner and DNA Sequencer from Applied Biosystems. The Applied Biosystems 373 consists of electrophoresis and detection unit, a Mac II computer, a Tektroxin II printer and software for both DNA fragment quantitation and sizing as well as DNA sequencing. The new automated DNA sequencer allows for automated DNA fragment analysis using an internal-lane standard. This instrument analyzes microsatellites fast and reliably using simple sequence tandem repeat polymorphisms for linkage mapping of genes. Length polymorphism of microsatellites are visualized by analysis of PCR products which are fluorescent tagged in the oligonucleotide primer. The PCR products are loaded directly on the DNA sequencer. The same principle applies to quantitative PCR analysis for any gene of interest. Standard COP-PCR (competitive oligonucleotide primers can also be performed with the gene scan features of the Applied Biosystems 373. The fluorescent detection system is linear over the three orders of magnitude and quantitative data has a 0.5% coefficient of variation. In addition because three differently labelled primers, reliability can be checked. The 373 accepts 3 different gel sizes for optimization of sample migration differences. Advantages to this include is flexible software available for analysis. The 373 can detect ethidium bromide stained DNA fragments on the GENE Scanner system which is 20 times more sensitive than detection with UV. DNA samples as small as 0.2ng per band can be analyzed. The same molecules. The PhosphorImager will be used for quantitative analysis of DNA protein interactions, Northern and Southern analysis. The primary advantage is that it eliminates the need for conventional autoradiography, allowing for increased efficiency and cost reduction and increased sensitivity. The proposed instruments will comprise an integrated system that will be housed in the Nudeic Acid Analysis Facility at UMDN. Over 18 NIH funded researchers will utilize the requested equipment which will allow for types of analysis not currently present at UMDNJ.
