Chemistry: electrical and wave energy – Processes and products – Electrophoresis or electro-osmosis processes and electrolyte...
Reexamination Certificate
1999-12-23
2002-06-18
Warden, Jill (Department: 1743)
Chemistry: electrical and wave energy
Processes and products
Electrophoresis or electro-osmosis processes and electrolyte...
C204S466000, C204S606000, C204S619000, C204S621000, C435S309100, C422S105000
Reexamination Certificate
active
06406602
ABSTRACT:
BACKGROUND OF THE INVENTION
Polyacrylamide gel electrophoresis is one of the most powerful tools used in the field of biotechnology. By passing an electric current through a polyacrylamide gel, the polyacrylamide gel electrophoresis method separates samples, such as nucleic acids, proteins and other biologically relevant molecules, by charge, size, conformation or other characteristics of the sample. One of the critical procedures in performing polyacrylamide gel electrophoresis is gel loading, which involves the addition of samples to the gel. To facilitate the sample loading process, many techniques and devices have been used. However, the loading process still remains one of the most time consuming and technique dependent steps in the polyacrylamide gel electrophoresis process.
One techniques involves using multiple syringes or pipettes to load the sample directly on the gel. Another technique is to use a sample loading device that has a substrate and an absorbent membrane on which a plurality of samples are loaded. A method of “diffusion isolation” is needed to prevent the individual samples from intermixing.
In the use of the substrate/absorbent membrane technique, it is known to use a sample loading device referred to as a “comb” that has a plurality of fingers that can help maintain the integrity of individual samples. For ensuring “diffusion isolation” of each sample within the loading area of the comb, the width of the absorbent material corresponding to the gel loading area is physically separated by cutting the comb into numerous “fingers.” The length of the comb is constrained by the size of the gel and the corresponding “read” area of the apparatus which receives the comb, therefore the number of fingers and the physical separation between the samples is limited by this length of the comb. Furthermore, as the number of fingers and separating cuts is increased, the comb becomes less stable, making it more difficult to handle and place it into the gel.
Another embodiment of the sample loading device has scoring of the absorbent membrane (i.e., removing membrane material from the substrate) to produce barriers between the samples to maintain diffusion isolation. Another technique is creating diffusion isolation by use of hydrophobic ink on the absorbent material to produce barriers between the samples. The use of scoring or hydrophobic ink or a combination thereof is most effective when samples are resuspended in a water soluble solution; however, many loading buffers used for electrophoresis contain organic solvents that are unlikely to be impeded by a scored trough or by hydrophobic ink.
SUMMARY OF THE INVENTION
In the present invention, the properties of the absorbent membrane in a sample loading device are physically altered by pressure, heat, a combination of heat and pressure, or other such treatments, so that the regions between sample loading areas are unable to absorb the sample. The present method produces sample diffusion barriers that prevent sample diffusion and contamination with adjacent samples. The physical stability of the sample loading device is maintained because the membrane material remains continuous and lacks the unsupported “fingers” described in the prior art. Therefore, the present invention is more robust, making it easier to insert the sample loading device into a gel, thereby increasing data quality and resolution and reducing fabrication costs. Additionally due to the inherent and resultant properties of the absorbent and backing materials as a laminate, it is easier and more reliable to fabricate and apply standard quality and redundancy controls to the process/device than with prior art devices.
In one embodiment, the present invention describes a system that allows effective loading and scanning high resolution of about 48 or more samples in a standard polyacrylamide electrophoresis gel. The system produces results that are unexpectedly superior over prior art materials and methods. The sample loading device of the present invention also allows more samples to be loaded in a given area than other sample loading devices.
The sample diffusion barrier(s) of the present invention is formed utilizing the inherent properties of the absorbent membrane. The membrane is altered physically using pressure, conductive heat or convection heat (such as laser energy or RF energy), a combination of heat and pressure, or similar techniques. The material properties of the absorbent membrane are physically altered so that the absorbent membrane is changed into an effective, continuous diffusion barrier that is unable to absorb the sample. Thus, the sample loading device of the present invention with the homogenous altered absorbent membrane as the physical barrier between the sample loading areas, eliminates the need to add extraneous matter, such as hydrophobic inks, or to remove or prevent deposition of an absorbent material.
The present invention describes at least one sample loading device that comprises alternating areas of absorbent membranes and diffusion barriers. The diffusion barriers are formed by physically altering the absorbent membrane using heat, pressure, or combination pressure and heat, or other similar techniques.
At least one method of loading samples into a gel of an electrophoresis gel system is also described. The method includes applying a sample to a sample loading device, where the sample loading device comprises alternating absorbent and diffusion barrier areas of time membrane which areas are created by physically altering the absorbent membrane. The sample loading device is loaded onto the gel electrophoresis apparatus. A voltage is applied across the gel to establish an electrophoretic field which causes the sample to migrate into the gel and is subsequently scanned for analysis.
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Erfle, H., et al., “Simultaneous loading of 200 sample lanes for DNA sequencing on vertical and horizontal, standard and ultrathin gels, ”Nucleic Acids Research, vol. 25, No. 11, 2229-2230 (Oxford University Press, 1997).
Cahill Patrick B.
Montt Jeffrey P.
Genome Therapeutics Corporation
Hamilton Brook Smith & Reynolds P.C.
Noguerola Alex
Pearlmutter, Esq. Nina L.
Warden Jill
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