Chip Asbury, David Basiji, Kelly Dillon, Rich Esposito, Curran Fey, Rene Gelderman, Steve Knowles, David Makihara, Dennis Siemer,[1] Todd Smith, Barb Trask, Ger van den Engh
Department of Molecular Biotechnology, University of Washington, Seattle, WA,
We are developing automated instruments for several stages of the mapping and sequencing process.
One project concerns the sub-cloning of a BAC or cosmid into M13. The aim is an automated process that will select and expand M13 sub-clones from a library. The daily capacity of the instrument will be sufficient to completely sequence a BAC or cosmid. Individual transformed bacteria are selected by fluorescence-activated cell sorting. Bacteria are deposited into individual culture wells. The turbidity of the wells is continuously monitored providing quality criteria for the growth conditions. The bacterial cultures are automatically harvested when the cultures reach an optimum density within a preset time window. Cultures that do not meet the growth criteria are discarded.
We are also developing an electrophoresis-based extraction technique that will yield DNA of sufficient quality for a sequencing reaction. Two different isolation methods are under investigation. One of them uses dipole induction to separate the DNA from other organic components. The DNA extraction machine connects to the clone isolation and expansion machine. The samples that come out of the extraction process are stored in a sealed plastic ribbon.
We have developed a gel loader that automatically injects DNA samples into an electrophoresis gel. The prototype reproducibly loads samples onto an agarose gel with a density of one lane per 2 mm. A version that is capable of loading samples onto thin polyacrylamide gels is under construction.
A gel scanner for evaluating restriction fragment gels has been developed. The scanner evaluates that gels that have been prepared in the automatic sample loader. Present activities concentrate on reducing the background signal from the gel.
In all these instruments, the samples are transported in wells in a sealed plastic ribbon. This system will form the basis for a DNA sample repository that can store and access a large number of samples in a small space. The advantages of storing DNA and bacteria samples in plastic ribbons will be discussed.
* Supported by a grant from the Director, OER/OHER of the U. S. Department of Energy under contract DE-FG06 93ER61662 and DE-FG06-93ER61553.
[1] V-TEK, Mankato, MN.