Chemistry: electrical and wave energy – Processes and products – Electrophoresis or electro-osmosis processes and electrolyte...
Reexamination Certificate
1999-04-28
2001-01-09
Warden, Jill (Department: 1743)
Chemistry: electrical and wave energy
Processes and products
Electrophoresis or electro-osmosis processes and electrolyte...
C204S466000, C204S467000, C204S470000, C204S606000, C204S616000, C204S618000, C204S620000
Reexamination Certificate
active
06171463
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the electrophoretic separation of analytes of interest, such as bio-organic molecules. In particular, the invention provides an improved slab gel electrophoresis apparatus, and method of use, for electrophoresis of samples using ultra-thin gels.
BACKGROUND OF THE INVENTION
Electrophoresis is a popular method for separating large molecules, such as DNA fragments or proteins, from a mixture of similar molecules. Generally, the method involves passing an electric current through a porous medium containing the mixture. Each kind of molecule travels through the medium at a different rate, depending on its electrical charge and size. This movement produces a series of bands in the medium. Each band corresponds to a molecule or fragment of a particular size, progressively decreasing down the gel. Agarose and acrylamide gels are media commonly used for electrophoresis of proteins and nucleic acids.
Due in large part to its usefulness in nucleic acid sequencing, slab gel electrophoresis has become increasingly important in the field of biotechnology. A slab gel apparatus is typically prepared by disposing two glass plates face-to-face, with spacers interposed at both vertical edges to establish a gap therebetween. In one approach, the plates are clamped together along the vertical edges and a seal is placed along the top or bottom edge. The gap between the plates is then filled with gel solution, and the gel is allowed to set. Next, the top and bottom of the polymerized gel are placed in contact with respective buffer reservoirs. Samples are loaded into sample loading slots at one end of the gel, and an electric field is set up across the gel. The electric field causes the molecules to move from the loading slots into the gel and to separate according to size.
Slab gels of very thin cross-section, in the direction perpendicular to the plane of electrophoresis, are particularly popular for nucleic acid sequencing thanks to a number of advantages they offer over thicker gels. For example, ultra-thin slab gels (e.g., 0.1 mm to 0.3 mm thick) can be electrophoresed at a higher voltage than traditional, thicker gels because of more efficient heat dissipation, thereby permitting a faster electrophoretic run. Ultra-thin gels also offer the advantage of higher resolution, because less sample is required and there is less time for diffusion during electrophoresis. Additionally, owing to their thinness, ultra-thin gels can be fixed for autoradiography with relative quickness and ease.
In using ultra-thin gels, it is desirable to increase lane density to achieve higher sample throughput. The dimensions of the sample lanes in the separation medium will typically depend on the dimensions of the regions into which the samples are loaded, the spacing between sample loading regions, the duration of time needed to transport the samples into the separation medium, and the level of diffusion during electrophoresis, for example.
Two types of sample-holding slots, known as “wells,” are generally used in slab gels. In one approach, the wells are molded directly into the gel material using a template. The template, known as a comb, comprises a plurality of square-ended, evenly spaced protrusions extending off one edge of an elongate support member. To form the wells, the protrusions of the comb are inserted into the top of a gel solution before it has polymerized. The gel is then allowed to solidify. After the template is removed from the polymerized gel, empty wells remain for receiving samples. In such a construction, the loaded samples are separated from each other by finger-like extensions of the polymerized gel. In the other common type of well, often used with ultra-thin gels, sample-holding slots are created by inserting narrow, wedge-shaped, pointed protrusions of a “shark's tooth” comb partially into the top of the gel after it has polymerized. Samples are loaded into empty spaces bounded by adjacent teeth of the comb and the inner surfaces of the glass plates. The teeth of the shark's tooth comb, thus, serve as barriers to prevent the mixing of adjacent samples.
In most slab gel setups, the thickness of each well is determined by the thickness of the gel. Consequently, ultra-thin gels typically have ultra-thin sample wells. As a practical matter, it is difficult to load gels having wells that have a thickness of 0.2 mm or less. In this regard, sample loading is usually attempted by inserting a loader tube, such as a flat pipette tip or tapered glass capillary, having an extremely small outer diameter, between the two plates to deposit samples into the wells. To prevent sample mixing, it is desirable to inject the sample as close to the gel as possible. Under circumstances where the wells are very thin and the lane density is high, however, it can be very difficult to maneuver the tip of the loading implement between the plates and into position proximate the gel. Moreover, the loader tubes themselves can present additional problems. For example, viscous samples can be difficult to deposit with such small loading devices because their narrow bores easily clog and the tapered tips readily break. Also, loader tubes having ultra-small tips can be expensive and difficult to obtain.
As previously discussed, two types of templates, or combs, have traditionally been used for forming sample-loading slots; namely, square-tooth combs and shark's tooth combs. Although satisfactory in certain respects, each type of comb nevertheless presents certain problems.
Often, the bottom of a well formed with a square-tooth comb will have an uneven surface. This can be caused, for example, by unpolymerized gel material remaining in the well and/or by tearing of the gel upon withdrawal of the comb. An uneven gel surface, in turn, can result in crooked bands and/or skewed lanes during electrophoresis.
The projections, or teeth, of a shark's tooth comb tend to be very thin and flexible. This is especially true of combs having a large number of teeth (e.g., 48 teeth or higher). Upon pressing a comb into a polymerized gel, the teeth encounter a slight amount of resistance. Unfortunately, thin teeth sometimes bend as a result of such resistance. Bent teeth can distort the gel, resulting in crooked bands and/or skewed lanes during electrophoresis. Bent teeth can also change the volume between adjacent teeth for accommodating sample. Thus, maximum sample volumes can vary from well to well. Moreover, thin, flexible teeth have also been associated with cross-leakage of samples. Thin teeth also tend to be weak and prone to breakage.
Generally, the teeth of shark's tooth combs are especially thin towards their outer ends, in the region whereat the interface of the well and the gel is defined. For this reason, the barrier between adjacent wells is often not very substantial. Upon electrophoresis, the lanes run very close to one another. That is, there is insufficient separation, or “dark space”, between lanes. As a result, it is sometimes difficult to distinguish the border between adjacent lanes. This can cause misreads by automated sequencing equipment. For example, the reader might interpret two adjacent bands, representing molecules of a particular length or molecular weight, as a single band. Consequently, the reader would report only one band, which it would assign to only one of the two adjacent lanes. Thus, the closely spaced band in the adjacent lane would go unreported. Of course, lane drifting can further complicate this problem, especially where lanes actually converge into one another.
In an effort to accommodate a large number of wells on a single gel, some shark's tooth comb designs have the teeth spaced very close together. With such combs, the apertures between adjacent teeth do not provide much space for accommodating sample. This is particularly true with combs designed for conventional ultra-thin slab gel setups. So, the user is restricted to using only very small sample volumes (e.g., less than 1 microliter).
The widespread use o
Clark David
Goudberg Johan
Sakabe Munechika
Sch{grave over (e)}fer Thomas
Selby Barton G.
Frazier Jeffrey D.
Gabrick Michael T.
Starsiak Jr. John S.
The Perkin-Elmer Corporation
Warden Jill
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