Chemistry: electrical and wave energy – Processes and products – Electrophoresis or electro-osmosis processes and electrolyte...
Reexamination Certificate
1999-11-23
2001-09-25
Whisenant, Ethan (Department: 1655)
Chemistry: electrical and wave energy
Processes and products
Electrophoresis or electro-osmosis processes and electrolyte...
C204S450000, C204S456000, C204S483000, C436S094000, C536S023100, C435S091100
Reexamination Certificate
active
06294064
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Area of the Art
The invention relates generally to capillary electrophoresis of biomolecule analytes, and specifically to methods and reagents for preparing nucleic acid samples for nucleic acid separation with capillary electrophoresis.
2. Description of the Prior Art
Throughout this application various references are referred to within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citation for these references may be found at the end of this application, preceding the claims.
Capillary electrophoresis (CE) is a technique that has been used to separate proteins or nucleic acids such as DNAs from each other. See, for example, Chen, Fu-Tai A. et al., “Capillary Electrophoresis—A New Clinical Tool,”
Clin. Chem.
77/1:14-19 (1991); see, also, U.S. Pat. Nos. 5,120,413 and 5,228,960; see, further, U.S. Pat. No. 5,891,313. These documents are incorporated herein by reference.
In general, CE involves introduction of a sample into a capillary tube, i.e., a tube having an internal diameter of from about 2 to about 2000 microns, and the application of an electric field to the tube. The electric potential of the field both pulls the sample through the tube and separates it into its constituent parts. Each of the sample constituents has its own individual electrophoretic mobility; those having greater mobility travel through the capillary tube faster than those with slower mobility. As a result, the constituents of the sample are resolved into discrete zones in the capillary tube during their migration through the tube. An on-line detector can be used to continuously monitor the separation and provide data as to the various constituents based upon the discrete zones.
The results of CE analysis are typically presented as “electropherograms”, i.e., peaks of various widths and heights which correspond to the constituent parts of the sample. For example, a constituent which is present in a sample in a high concentration may evidence a peak having a large height and wide width compared to a constituent present in a (relatively) low concentration. Typically, the electropherogram is derived by plotting detection units (typically ultraviolet light absorbance) on the vertical axis, and time of constituent traversal through the column to a detection region on the horizontal axis. Results can also be derived in terms of a unit value, typically derived from the peak areas or peak heights.
Electrokinetic loading of a DNA sequencing sample mixture into a capillary electrophoresis tube is a preferred method of introducing a sample of analytes into the capillary electrophoreisis tube. However, DNA sequencing product primarily contains a mixture of labeled DNA sequencing fragments, whose separation and analysis provide sequencing information, and larger DNA template. Therefore, during the electrokinetic injection of the DNA sequencing fragments, the amount of analyte introduced into the capillary is limited by the buildup of nucleic acid template at the injection end of the capillary. This template buildup clogs the end of the capillary and inhibits passage of analyte into the capillary. This phenomenon limits the amount of DNA fragments that can be injected and electrophoresed. In addition, the injection of the large DNA template into a narrow bore capillary is known to reduce the local conductivity of the medium inside the capillary, leading to high electrical resistance and local Joule heat. High Joule heat would deteriorate the separation resolution and generate bubbles inside the capillary. The latter case actually produces a severe current problem with the electrophoretic separation. It is therefore desirable to have a suitable way to suppress the DNA templates entering the capillary to reduce the above-mentioned problems.
Some strategies to solve or remedy the template-clogging issue are: a) thorough purification of sample to remove the template; and b) cutting off the template-clogged end of the capillary end shortly after introduction of the sample. These methods are either time-consuming, inconvenient and/or expensive. None of them are ideal for supporting the high-speed and high-throughput systems as required by the human genome project.
Another known approach to suppress the entrance of nucleic acid sequencing template into the capillary is to dissolve the template-containing sample in a loading solution consisting of linear polymer and denaturant.
(1,2)
The function of the linear polymer is to trap the nucleic acid template to inhibit the entrance of template into the capillary during the sample injection course. While this method is simple and easy, it is found that with this approach, the linear polymer significantly suppresses the mobility of the analyte DNA into the capillary, leading to low sample loading and thus low detection signal. Thus, a need exists to develop a new method that suppresses the entrance of nucleic acid sequencing template into the capillary without compromising the sample loading and detection signal.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide methods and reagents that suppress the entrance of nucleic acid sequencing template into the capillary. It is also an object of the present invention to provide methods and reagents that improve the sample loading efficiency, capillary separation resolution, and detection sensitivity during nucleic acid separations with capillary electrophoresis.
These and other objects and advantages are achieved by the reagents and methods of the present invention. One aspect of the present invention provides a sample loading reagent for loading biomolecule analytes contained in a sample into a capillary electrophoresis tube. The reagent comprises a branched polymer and a solvent. The branched polymer, such as branched polyvinylpyrrolidone, may be dissolved in a solvent such as formamide to form a reagent that can suppress the entrance of macromolecules contained in a sample into a capillary electrophoresis tube during electrokinetic injection of the sample into the tube.
Another aspect of the present invention also provides a method for preparing a sample containing biomolecule analytes and macrobiomolecules for capillary electrophoretic separation of the biomolecule analytes. The method comprises the steps of:
(a) providing a reagent comprising a branched polymer; and
(b) mixing the sample with an amount of the reagent sufficient to suppress the injection of the macrobiomolecules contained in the sample into a capillary electrophoresis tube during electrokinetic injection of the biomolecule analytes.
A further aspect of the present invention provides a method of loading biomolecule analytes into a capillary electrophoresis tube from a sample containing the biomolecule analytes and macrobiomolecules. The method comprises the steps of:
(a) providing a reagent comprising a branched polymer;
(a) mixing the sample with an amount of the reagent to form a mixture;
(b) electrokinetically injecting a portion of the mixture into a capillary electrophoresis tube;
wherein the amount of the reagent is sufficient to suppress the injection of the macrobiomolecules into the capillary electrophoresis tube.
In accordance with embodiments of the present invention, the biomolecule analytes may be DNA sequencing fragments, and the macromolecules may be DNA sequencing templates. The branched polymers may be branched polyvinylpyrrolidone, which may be dissolved in a solvent such as formamide.
The reagents and methods of the present invention provide a number of advantages. They can effectively suppress the entrance of nucleic acid sequencing template into the separation capillary, resulting in stable and reliable electrophoretic separation for DNA analysis. Compared to the commercial loading buffer comprising linear polymers, the reagents and methods of the present invention have demonstrated a better capability of differe
Huang Tung-Liang
Keys Daniel A.
Ratnayake Chitra K.
Reddy M. Parameswara
Beckman Coulter Inc.
Grant Arnold
Lu Frank
May William H.
Whisenant Ethan
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