Chemistry: electrical and wave energy – Processes and products – Electrophoresis or electro-osmosis processes and electrolyte...
Reexamination Certificate
1998-12-16
2001-02-27
Warden, Jill (Department: 1743)
Chemistry: electrical and wave energy
Processes and products
Electrophoresis or electro-osmosis processes and electrolyte...
C204S600000
Reexamination Certificate
active
06193866
ABSTRACT:
A portion of the disclosure of this patent document contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by any one of the patent disclosure, as it appears in the Patent and Trademark Office patent files and records, but otherwise reserves all copyright rights whatsoever.
1. FIELD OF THE INVENTION
This invention relates to a method and apparatus for the separation of charged particles in a medium according to the differences of the diffusivities of the particles in the medium by use of a spatially and temporally varying electric potential. Particularly, the invention relates to a method and apparatus for separation of charged biopolymers in a liquid medium, and more particularly to a method and apparatus for the separation of single-stranded or double-stranded DNA fragments for DNA sequencing and for general fragment length determination.
This invention further relates to a method and apparatus for using electrode and optical design patterns, and other methods of device construction, to enhance the utility of this diffusivity-based technique to cause the motion or separation of charged species, such as DNA.
2. BACKGROUND OF THE INVENTION
Separations of charged particles, in particular physical mixtures of chemical species, are important analytical operations. Relevant chemical species include non-biological charged species, such as synthetic polymers, and biological charged species, such as DNA, RNA, or proteins (A. J. Kostichka et al., 1992,
Bio/Technology
10:78). Separations of mixtures of DNA fragments are particularly important.
For example, the Human Genome Project demonstrates the need for powerful DNA fragment separation methods and apparatus. This project is an ambitious, international effort to improve human genetic maps, to sequence fully the genomes of humans and several model organisms by 2006, and to develop computational tools for storing and accessing the burgeoning information. This project requires a technological infrastructure capable of supplying high-quality sequence information in a rapid and cost-effective manner.
To sequence fully the human genome, which has approximately 3×10
9
base pairs, by the year 2006 requires roughly 100 times beyond the total, current worldwide DNA sequencing capacity (M. V. Olson, 1993,
Proc. Natl. Acad. Sci. USA
90:4338). Existing DNA sequencing methods, for example, mass spectrometry (T. D. Wood et al., 1995,
Proc. Natl. Acad. Sci. USA
92:11451), sequencing by hybridization (R. Drmanac et al., 1993,
Science
260:1649), chromatography (C. G. Huber et al., 1993,
Nucl. Acids Res.
21:1061), acoustophoresis (J. S. Heyman, U.S. Pat. No. 5,192,450), and electrophoresis, are generally inadequate to meet this sequencing goal.
The above methods have various drawbacks. Mass spectrometry requires an expensive mass spectrometer. Because of this cost, it is unlikely that this method will have widespread applicability. Sequencing by hybridization is still relatively new and untested. Liquid chromatography is capable of performing rapid separation of double-stranded DNA fragments, but is limited by poor resolution. The single-base resolution necessary for sequencing has only been demonstrated for fragments smaller than 150 base pairs. In acoustophoresis, acoustic waves push fragments through a liquid medium. This method is limited by the similarity in the acoustic properties of DNA fragments of similar lengths, preventing effective separation.
Electrophoresis remains the most common method by far for DNA sequencing. All conventional electrophoretic methods are generally similar (F. Sanger et al., 1977,
Proc. Natl. Acad. Sci. USA
74:5463; L. M. Smith, 1993,
Science
262:530). A DNA sample is generally first amplified, that is the DNA chains are made to replicate, usually by the polymerase chain reaction (“PCR”). Next, from the amplified sample, chain terminating DNA polymerase reactions (first described by Sanger et al.) produce nested sets of DNA fragments labeled with one of four unique fluorescent dyes conjugated with one of the four chain terminating bases (either ddATP, ddCTP, ddGTP, or ddTTP). In a related method, the chains are cleaved by chemical means to produce a similar set of labeled fragments (M. Maxam et al., 1977,
Proc. Natl. Acad. Sci. USA
74:560). These fragments are then separated according to their molecular size by a variety of electrophoretic techniques, and the unique dye labeling each chain terminating base is detected by its fluorescence. The DNA base sequence is reconstructed from the detected pattern of chain fragments.
The accuracy required in DNA fragment size determination depends on the application. For example, DNA sequencing reactions produce a mixture, called a “ladder,” of fragments with lengths separated by single bases and require exact length determination. Other applications produce greater differences between the fragment lengths, and methods that provide rapid sizing, but not necessarily exact length information, are valuable. Typical of such applications are the generation of patterns of restriction fragment length polymorphism (“RFLP”), genotyping, linkage analysis, microsatellite analysis and other fragment analysis application.
In an electrophoretic separation, the DNA molecules are separated according to their rates of migration in an electric field. The electric driving force is proportional to the net charge of the molecule. For a uniformly charged biopolymer such as DNA, the driving force is proportional to the number of base pairs in the DNA fragment. Since in a material obeying Stokes' Law, such as a liquid, the friction coefficient is also proportional to the number of base pairs, the DNA fragments have electrophoretic drift velocities that are nearly identical and independent of fragment length. This means electrophoretic separation of DNA fragments is difficult in liquids or other media obeying Stokes' Law.
Therefore, instead of liquid media, cross-linked gels and uncross-linked polymer solutions are universally used in electrophoretic DNA separations. In these media, DNA does not obey Stokes' law, since the electrophoretic drift velocity decreases with increasing length or molecular weight. Thus, electrophoretic separation of biopolymers is ordinarily performed in a polymeric gel, such as agarose or polyacrylamide, in which separation of biopolymers with similar electric charge densities, such as DNA or RNA, depends on molecular weight. The non-Stokes' law dependence of the friction coefficient on the fragment size in a gel permits electrophoretic separation of DNA fragments of different lengths. Biopolymer fragments, therefore, exit the device in size order from small to large.
In a prevalent configuration, the electrophoretic gel is disposed as a thin sheet between two flat, parallel, rectangular glass plates. An electric field is established along the long axis of the rectangular configuration, and molecular migration is arranged to occur simultaneously in several paths, or “lanes,” parallel to the electric field. To ensure high separation resolution, it is advantageous that gel throughout a migration lane be as uniform as possible (or homogeneous like a liquid) and for the lanes to be sufficiently separated to be clearly distinguishable.
It has proven difficult to make, or “to cast,” uniform gels with uniform transport properties. One major problem is uneven gel shrinkage due to cross-linking during gel polymerization. The problems in casting a uniform gel also lead to difficulties in producing a uniform and reproducible loading region, into which sample mixtures are placed prior to separation. It is generally accepted that a separation medium with more reproducible transport properties (i.e., more like a homogeneous liquid) would have great utility.
In addition to high separation resolution, demands for more rapid electrophoresis have created additional problems for gel manipulation. Rapid electrophoresis is desirable for rapid, high capacity biopolymer analysis. This re
Bader Joel S.
Deem Michael W.
Henck Steven
Mulhern Gregory T.
Rothberg Jonathan M.
CuraGen Corporation
Pennie & Edmonds LLP
Starsiak Jr. John S.
Warden Jill
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