DNA Sequencing and Genetic Analysis Core Facility

Funded by a Howard Hughes Medical Institute Resources Grant, the Department of Cell & Neurobiology established in 1996 a DNA Sequencing & Genetic Analysis Core Facility under the technical direction of Dr. Hans-Jürgen Fülle. The facility is currently equipped with a Perkin-Elmer Applied Biosystems GeneAmp PCR System 9700 thermal cycler and an ABI PRISM 310 Genetic Analyzer.

DNA samples are fluorescently labeled with multi-colored dye dideoxy terminators (ddNTP’s) using Taq DNA polymerase and a one-tube cycle sequencing protocol. Cycle sequencing is a simple method in which reagents are pipetted into a tube and placed in a thermal cycler. The four sequencing reactions are performed simultaneously in one reaction tube. The dye terminator method has the advantage that any unlabeled sequencing primers provided by the end user can be used. Successive rounds of denaturation, annealing, and extension result in linear amplification and fluorescent labeling of extension products which are then purified and analyzed by the ABI PRISM 310 Genetic Analyzer. The facility accepts double-stranded (ds) plasmid DNA (~400 ng required/reaction) as well as purified PCR products (~20-200 ng required/reaction) for direct sequencing.

With the ABI PRISM 310 Genetic Analyzer the electrophoretic separation of purified labeled extension products is accomplished in a 47 cm x 50 µm i.d. glass capillary filled with a liquid polymer matrix (POP-6). Sample and polymer loading, raw data collection and analysis are highly automated and controlled by a Power Macintosh 7200/120 computer. Optimized run conditions (1 hr. 10 min. @ 50°C) allow a 24 hour throughput of 22 runs at 500-600 base calls/run with a >98.5% base calling accuracy out to 450 bases. Upon request, longer read-outs can be achieved with 61 cm capillaries and different run conditions. A separate set of capillaries and polymer matrix (POP-4) is available for applications requiring high resolution DNA fragment sizing under denaturing electrophoretic conditions such as microsatellite analysis or multiple fluorescent-single strand conformation polymorphism (MF-SSCP) analysis (GeneScan analysis).

So far, the DNA Sequencing & Genetic Analysis Core Facility has performed more than 5,000 DNA sequencing reactions for end-users in the Keck School of Medicine, including researchers in the Departments of Cell & Neurobiology, Ophthalmology, Biochemistry, Medicine, the Institute for Genetic Medicine and in Childrens Hospital Los Angeles.

The DNA Sequencing & Genetic Analysis Core Facility is located in the Mary D. Allen Laboratories for Vision Research, Doheny Eye Institute Vision Research Center, DVRC Room 410, phone 323-442-6686.