Electrophoresis gels and gel holders having fiber spacers and me

Details Electrophoresis gels and gel holders having fiber spacers and me Electrophoresis gels and gel holders having fiber spacers and me

1998-12-31

2000-04-25

Warden, Jill

Chemistry: electrical and wave energy

Apparatus

Electrophoretic or electro-osmotic apparatus

204606, G01N 2726

Patent

active

060540362

DESCRIPTION:

BRIEF SUMMARY
BACKGROUND OF THE INVENTION

DNA sequencing may be carried out using automated systems designed for laboratory application. Methods and apparatus for sequencing of DNA are described in U.S. Pat. Nos. 4,811,218; 4,823,007; 5,062,942; 5,091,652; 5,119,316 and 5,122,345, which are incorporated herein by reference.
The general methodology employed in these systems involves breaking up the sample DNA using restriction endonucleases; amplifying (for example with PCR) the restriction fragment of interest; combining the amplified DNA with a sequencing primer which may be the same as or different from the amplification primers; extending the sequencing primer in the presence of normal nucleotide (A, C, G, and T) and a chain-terminating nucleotide, such as a dideoxynucleotide, which prevents further extension of the primer once incorporated; and analyzing the product for the length of the extended fragments obtained. Analysis of fragments may be done by electrophoresis, for example on a polyacrylamide gel.
In performing a nucleic acid sequence analysis on a gel, the characteristics of the gel, including the size and thickness, impact the time and cost required to do the analysis. Since it is desirable to reduce the time and cost of sequencing analyses in order to improve the available of sequencing as a diagnostic tool, it would be advantageous to have a gel which permitted analysis of very small quantities of oligonucleotide fragments in a short period of time. It would further be advantageous to have a disposable, single use gel holder which could be manufactured on a large scale which when filled with a gel would provide these desirable characteristics.
Persons making the very thin gels which can achieve the type of short analysis times and high throughput desired for sequence analysis face several challenges. Significant among these is developing a fabrication technique which defines and maintains a very uniform spacing between the substrates surrounding the gel, so that the gel itself is of uniform thickness. U.S. Pat. Nos. 4,929,329 and 5,164,066, which are incorporated herein by reference, disclose the formation of electrophoresis gels using thin films (on the order of 0.10 to 0.02 inches thick), for example made from mylar, or nylon monofilaments as spacers between front and back plates. The spacers are not adhered to the plates, but are simply placed between the two plates and held in place using clamps while the space between the two plates is filled with gel forming solution. After polymerization, the polymerized gel holds the two plates, as well as the spacers in place.
The method of forming electrophoresis gels disclosed in these patents has several drawbacks. First of all, because the spacers have to be positioned and then held in place during the gel-filling operation, the resulting gels are not well suited to large scale production. Furthermore, because gels have short shelf lives, once prepared, and because it is the gel which holds the plates and the spacers together, assembly of the device must occur at the point of use. This too argues against the use of spacers as disclosed in these patents in the production of significant numbers of gels, or in the production of disposable, single use gel holders.
It an object of the present invention to provide disposable, single use gel holders having a very thin gel chamber of uniform thickness, which can be easily manufactured.
It is a further object of this invention to provide a method of making disposable, single use gel holders having a very thin gel chamber of uniform thickness, which can be easily manufactured.
It is a further object of this invention to provide electrophoresis gels formed within disposable, single use gel holders having a very thin gel chamber of uniform thickness, which can be easily manufactured.


SUMMARY OF THE INVENTION

These and other objects of the invention can be achieved by forming a gel holder for an electrophoresis gel using clad fibers, particularly glass fibers as spacers between the substrates. Thus, one aspect of the

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