Synthetic resins or natural rubbers -- part of the class 520 ser – Synthetic resins – At least one aryl ring which is part of a fused or bridged...
Reexamination Certificate
2000-06-05
2004-08-03
Egwim, Kelechi C. (Department: 1713)
Synthetic resins or natural rubbers -- part of the class 520 ser
Synthetic resins
At least one aryl ring which is part of a fused or bridged...
C524S500000, C524S501000, C525S903000
Reexamination Certificate
active
06770698
ABSTRACT:
BACKGROUND OF INVENTION
The present invention relates generally to the art of separating charged molecular species, and, in particular, to separation media that are used for capillary electrophoresis.
Gel electrophoresis is one of the most widely used separation techniques in the biologically related sciences. Charged molecular species such as proteins, peptides, nucleic acids and oligonucleotides are separated by causing the species to migrate in a buffer medium under the influence of an applied electric field. The buffer medium normally is used in conjunction with a low to moderate concentration of an appropriate gelling agent, such as for example, agarose or cross-linked polyacrylamide, to promote the separation and to minimize the occurrence of mixing of the species being separated.
Until recently, electrophoretic separations were conducted in gel slabs or open gel beds that were typically fabricated of agarose or cross-linked polyacrylamide material. More recently, capillary electrophoresis (“CE”) using a polymer gel or solution as a separation medium has been used for the separation of DNA. Capillary electrophoresis techniques combined with photometric detection methods have allowed the automation and rapid quantitative analysis of charged molecules. Furthermore, capillary electrophoresis can provide quantitative information about a sample using very small amounts of the sample, gel (or polymer solution) and buffer relative to traditional slab gel processes. Moreover, high-resolution separation of charged macromolecules having different effective charges have been achieved.
Typically, the capillary columns used in capillary gel electrophoresis are fabricated from fused silica tubing having diameters on the order of 25 &mgr;m to 200 &mgr;m and lengths from about 30 cm to about 200 cm. The column interior is filled with buffer and gel separation medium and electrophoretic techniques are used to separate charged molecular species.
Although the pore size of cross-linked polymer gels used for capillary electrophoresis can be controlled by the amount of monomers and cross-linked reagents, polymer gels have been found to be inconvenient as separation media for large scale DNA sequencing analysis due to the instability, irreproducibility and difficulty in controlling the polymerization process inside the capillary tubing.
The inability of many separation media to bind directly to the inner wall of the capillary tubes is a major problem for capillary electrophoresis methods because it creates an electro-osmotic flow when an electric field is applied during electrophoresis. Such migration results in an unsatisfactory separation of the constituent parts of the sample. Traditional methods aimed at preventing electro-osmosis include introducing a compound that binds to the inner surface of a capillary tube wall and that is compatible with the separation medium prior to injecting the separation medium into the tube. For example, U.S. Pat. No. 5,447,617 to Shieh describes covalently bonding polybutadiene to the inner surface of a capillary tube, introducing acrylamide monomers therein and co-polymerizing the acrylamide with the polybutadiene. Such precoating techniques, however, are time consuming, inconvenient and costly.
Another problem encountered in conventional capillary gel electrophoresis results from the use of polyacrylamide-based separation media. Such media are injected into the capillary tube in unpolymerized form. Polymerization of the polyacrylamide is then induced within the capillary tube by well known methods, such as ultraviolet radiation and chemical catalysts. Such methods are characterized by a lack of uniformity in the pore size distribution of the polymer network formed, and by incomplete polymerization.
The irreversible nature of the polymerized polyacrylamide gel also causes numerous problems when a polyacrylamide-based separation medium is used in capillary gel electrophoresis methods. Once the polyacrylamide is polymerized within a capillary electrophoresis tube, the polymerized gel cannot be easily removed from the capillary tube after electrophoresis.
Capillary electrophoresis (“CE”) provides numerous advantages over conventional slab gel electrophoresis for DNA separation. The use of fused silica capillaries with inner diameters of less than 100 &mgr;m enables CE to operate at very high separation voltages and offers fast separation, high efficiency and increased resolution. In addition, rigid gels, which are normally used in slab gel electrophoresis because of their anti-convection ability, are not needed in capillary electrophoresis. Cross-linked polyacrylamide (“PAM”) gels, which are widely used in conventional slab gel electrophoresis, were initially used in capillary electrophoresis. Despite successful results, i.e., 700 bases read lengths with resolution of 0.5 for DNA sequencing in about 230 minutes, PAM gels have encountered problems due to bubble formation, gel inhomogeneity, and short lifetime of the capillary.
Accordingly, attempts have been made to use nonpolymerized separation media for capillary electrophoresis. For example, U.S. Pat. No. 5,468,365 to Menchen et al. describes an electrophoresis medium having a matrix of aggregated copolymers dispersed in an aqueous in medium. The polymer matrix of the '365 patent is described as a dispersion of one substance (micelles) in another substance (water). In such a dispersion, the particles are formed by the association or aggregation of molecules having both hydrophilic and hydrophobic regions. The copolymers of the '365 patent form a polymer matrix having a relatively uniform mesh size which is believed to be related to the regular, i.e., substantially uniform spacing between adjacent hydrophobic polymer segments.
A number of different polymers have been used in CE methods to separate DNA fragments. Many of these polymers are modified polysaccharides, such as, agarose, methylcellulose (“MC”), hydroxypropyl-methyl-cellulose (“HPMC”), hydroxyethylcellulose (“HEC”), hydroxypropylcellulose (“HPC”), glucomannan, galactonmannan, and dextran. Some of them are synthesized polymers, such as, polyethylene glycol (“PEG”), polyethylene oxide (“PEO”), polyvinylpyrrolidone (“PVP”), polyvinylalcohol (“PVA”), polyacrylamide (“PAM”), poly-N-acryloyl-amino-ethoxyethanol (“PAAEE”), polyacryloylaminopropanol (“PAAP”), poly-N,N-dimethylacrylamide (“PDMA”), polyacrylamide-co-allyl-&bgr;-D-glucopyranoside (“P(AM/AG)”), and poly-N-(acryloylaminoethoxyethyl-&bgr;-D-glucopyronoside (“PAEG”). Recently, polymers with viscosity dependent behavior have also been employed. One type of polymer was characterized by the collapse of molecules at high temperature, such as a copolymer of N,N-dimethylacrylamide and N,N-diethylacrylamide (“P(DMA/DEA)”), a copolymer of poly(N-isopropylacrylamide) (“PNIPAM”) densely grafted with short poly(ethylene oxide) (“PEO”) chains (“PNIPAM-g-PEO”), etc. Other polymers involved the formation of micelles, such as fluorocarbon end-capped polyethylene glycols, E
99
P
69
E
99
(with E being polyoxyethylene and P being polyoxyproplene), and n-dodecane-poly(ethylene oxide)-n-dodecane, etc. Each of these polymers has distinct advantages, but each also has inherent problems. For example, only several of them, such as HEC, PEO, PVP, PAM, PDMA, P(DMA/DEA), and fluorocarbon end-capped polyethylene glycols, have ever been used for DNA sequencing; and only HEC and high molecular weight PAM, PEO and PDMA have ever achieved a read length of greater than 500 bases.
Entangled polymer solutions, such as liquefied agarose, poly(acrylamide) (“PAM”), different kinds of cellulose, poly(ethylene oxide) (“PEO”) or poly(dimethylacrylamide), have been widely used as a DNA separation medium in CE with some success. High molecular weight (M
w
) PAM has achieved 1,000-base read length in one run for single-stranded DNA in run times of less than one hour. However, the PAM solution has two disadvantages: the injection is very difficult due to the very high viscosity, and the capillary inner wall has to be coated.
One b
Chu Benjamin
Fang Dufei
Liang Dehai
Liu Tianbo
Song Liguo
Egwim Kelechi C.
Hoffman & Baron LLP
The Research Foundation at State University of New York
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