Chemistry: electrical and wave energy – Apparatus – Electrophoretic or electro-osmotic apparatus
Reexamination Certificate
2000-06-30
2003-05-13
Warden, Jill (Department: 1743)
Chemistry: electrical and wave energy
Apparatus
Electrophoretic or electro-osmotic apparatus
C156S300000, C156S301000, C156S299000
Reexamination Certificate
active
06562214
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Area of the Art
This invention relates to a capillary array electrophoresis system and, in particular, to an electrophoresis system with a laminated capillary array assembly.
2. Description of the Prior Art
Electrophoresis has become an indispensable tool of biotechnology, as it is used extensively in a variety of applications, including separation, identification and preparation of pure samples of nucleic acids, proteins and carbohydrates. Traditionally, slab gel electrophoresis has been utilized for DNA sequencing as well as DNA fragment analysis. Recently, capillary gel electrophoresis (CE) has emerged as an attractive alternative to the traditional slab gel method. In CE, an appropriate solution is polymerized or gelled to form a porous matrix in a fused silica capillary tube. An electric field is applied across the matrix. Fragments of sample DNA, injected into one end of the capillary tube, migrate through the matrix under the influence of the electric field at speeds that depend on the length of the fragment. Typically, CE is combined with laser-induced fluorescence (CE-LIF), which provides a high-sensitivity detection. In CE-LIF method, the dideoxynucleotide at one end of a DNA fragment is labeled with a fluorescent marker during a reaction step. When the fragment passes through a beam of light from the laser in the detection zone, the fluorescent marker fluoresces and the fluorescence may be detected. The intensity of the signal depends on the amount of fluorescent marker present in the matrix in the detection zone.
The advantages of CE and CE-LIF arise intrinsically from the use of capillaries with small inner diameters. Since the capillaries have a large surface-to-volume ratio and thin walls, heat generated by Joule heating rapidly dissipates when the capillaries are used in connection with a cooling system. Consequently, high electric fields can be applied along capillaries without a large amount of resistive heating which causes formation of thermal gradients in the gel and impairs separation resolution. Since the electrophoretic velocity of the charged species is proportional to the applied field, CE can achieve rapid, high-resolution separation.
Although the use of CE has greatly improved DNA sequencing rates compared to conventional slab gel electrophoresis, the throughput associated with CE-based DNA sequencing is generally less than that of conventional slab gels when only one capillary is employed in the separation system. In order to overcome this limitation, it has been suggested that a capillary array electrophoresis system, comprising a plurality of spatially organized capillaries, may be used to achieve the desired throughput (
Nature
359, 167-168, 1992;
Nature
361, 565-566, 1993). This approach allows independent manipulation of individual capillaries, thereby facilitating rapid, parallel loading and analysis of multiple samples.
Conventionally, in the capillary array electrophoresis system, the capillaries in the array are aligned and irradiated with light. Fluorescence emitted from the fluorophore-tagged DNA or organic compound is detected by scanning a detector relative to the capillaries (U.S. Pat. No. 5,730,850). Sensitive laser-excited fluorescence detection requires precise alignment of the capillaries in relation to the light source and a photodetector. U.S. Pat. No. 5,730,850 describes stacked capillary array sheets. Each sheet is formed by sandwiching optical-window-facing ends of the capillaries in a capillary holder. The capillary holder is made of a 1.5 mm-thick stainless steel sheet bound to a 0.1 mm-thick polyethylene terephthalate cover. Similarly, in U.S. Pat. No. 5,584,982, the capillaries are closely and evenly spaced, and their end portions are held by a transparent retainer in a fixed position in relation to an optical detection system.
While such capillary holders are useful in retaining one end of the capillaries in a fixed position relative to a light source, they do not protect capillaries from damage caused by their crossing and bending, since large portions of the capillaries are exposed. Bending of the capillaries can result in a loss of the separation efficiency, due to distortions in the gel and multipath effects. Capillary crossover may lead to formation of hot spots. Additionally, mechanical stress, which often leads to capillary breakage, may accumulate at the points where capillaries enter into the capillary holder.
In a typical CE-LIF system, a capillary array is installed in a heater. The heater maintains an elevated temperature along the length of the capillaries to ensure better resolution of DNA fragments. It is, however, difficult to place and retain the capillaries inside the heater. Usually, after installing a capillary array into the heater, a user has to fix each capillary in place with a tape to prevent capillaries from snagging during the operation. Consequently, installation, operation and removal of capillary arrays become laborious. The conventional designs, therefore, fail to provide convenient, stable and sufficiently rigid capillary arrays for CE and CE-LIF.
SUMMARY OF THE INVENTION
Accordingly, the present invention is directed to a laminated capillary array assembly that substantially obviates limitations and disadvantages of the related art.
An object of the present invention is to provide a protection to capillaries forming an array, to prevent their crossover, and to simplify installation and replacement of capillary arrays into a CE instrument.
Additional features and advantages of the invention will be set forth in the descriptions that follow and in part will be apparent from the description, or may be learned by practice of the invention. The objectives and other advantages of the invention will be realized and attained by the structure particularly pointed out in the written description and claims hereof as well as the appended drawings.
To achieve these and other advantages, and in accordance with the purpose of the present invention as embodied and broadly described, the present invention provides a capillary assembly comprising a plurality of capillaries substantially entirely enclosed by a first and a second substrate laminated together.
In another aspect, the present invention provides a method for producing a laminated capillary array assembly, comprising the steps of placing a first substrate on a template having a desired shape, arranging capillaries on the first substrate, placing a second substrate on the capillaries, and laminating the first and second substrates together. The capillaries are substantially entirely enclosed between the two substrates, and the laminated capillary assembly of the desired shape is obtained.
The capillary assembly of the present invention is well suited for use in any system that utilizes a plurality of capillaries. Examples of such systems include, but are not limited to CEQ 2000 DNA Analysis System (Beckman Coulter, Inc., Calif.), Amersham Megabase,, and ABI 310, 377 and 3700.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the invention as claimed.
REFERENCES:
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patent: 5540464 (1996-07-01), Picha
patent: 5584982 (1996-12-01), Dovichi et al.
patent: 5605666 (1997-02-01), Goodale et al.
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patent: 6054032 (2000-04-01), Haddad et al.
patent: 6063251 (2000-05-01), Kane et al.
patent: 6402875 (2002-06-01), Luhmann et al.
Richard A. Mathies, et al., “Capillary Array Electrophoresis: An Approach to High-Speed, High-Throughput DNA Sequencing,” Nature, vol. 359, pp. 167-169, Sep. 10, 1992.
Hideki Kambara, et al., “Multiple-Sheathflow Capillary Array DNA Analyser,” Nature, vol. 361, pp. 565-566, Feb. 11, 1993.
Amrhein Bruce S.
Evans Timothy R.
Hanamoto Barry K.
Platt Clayton R.
Beckman Coulter Inc.
Brown Jennine
Hill D. David
Hogan & Hartson LLP
May William H.
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