Chemistry: electrical and wave energy – Processes and products – Electrophoresis or electro-osmosis processes and electrolyte...
Reexamination Certificate
1999-06-18
2001-04-10
Sisson, Bradley L. (Department: 1655)
Chemistry: electrical and wave energy
Processes and products
Electrophoresis or electro-osmosis processes and electrolyte...
C435S006120, C435S091100, C435S287200, C204S456000, C204S466000, C204S467000, C436S094000
Reexamination Certificate
active
06214187
ABSTRACT:
BACKGROUND OF THE INVENTION
Zonal electrophoresis, and particularly gel electrophoresis is one of the best known methods for separation, purification and characterization of charged molecules, particularly macromolecules such as proteins or nucleic acids (Freifelder,
Physical Biochemistry,
2nd ed., (1982) pp. 276-310, Freeman, San Franciso). Electrophoresis can be used to separate molecules based on their size, charge, conformation, and many combinations of these properties.
In most electrophoresis applications, charged molecules migrate through a supporting medium under the influence of an electric field. The supporting medium acts to suppress convection and diffusion, and can be sieving or nonsieving. In affinity electrophoresis, the support medium is also modified with chemical groups (hereinafter “capture ligands”) that interact specifically or nonspecifically with one or more desired targets and, thus, help to accomplish the separation of target and non-target sample components during purification by influencing the mobility of the target.
Affinity electrophoresis has been used to enhance the separtion of nucleic acids. For example, Inami, et al. demonstrated that A:T-rich sequences of nucleic acids have an affinity for malechite green copolymerized with acrylamide and immobilized within an agarose gel (Inami, et al.,
Nucleic Acids Symp. Ser
. (19 ) 29:77-8). In addition, vinyl-adenine modified polyacrylmide media has been used to resolve nucleic acids in capillary electrophoresis (Baba et al.,
Analytical Chemisty
(1992), 64:1920-1924).
Electrophoresis techniques have been used by biochemist to study melting transitions. Electrophoretic mobility is a very sensitive indicator of protein and nucleic acid conformation and, therefore, electrophoresis is a very useful or studying melting induced by chemical or physical denaturants. Gels containing gradients of urea have been used to study the binding of proteins (Creighton,
J Mol. Biol
. (1979) 129:253-264) and melting behavior of nucleic acids (Fischer, et al., Cell (1979)16:191-200).
Temperature gradients have also been used to study these properties with similar results (Thatcher, et al.,
Biochem
. J (1981), 197:105-109), and many methods of producing temperature gradients have been developed (Henco, et al.,
Methods Mol. Biol
. (1994) 31:211-28 and Rosenbaum, et al.,
Biophys. Chem
. (1987) 26:235-246).
While many advances have been made in the resolving power of electrophoresis, nucleic acids that contain only slight structural differences, for example, one or more point mutation in a long nucleic acid sequence, still cannot be successfully separated.
Analytical techniques that improve resolution of biological molecules are needed to provide researchers with the opportunity to further probe and understand biological systems.
SUMMARY OF THE INVENTION
The present invention relates to a method for analyzing, separating or purifying a target nucleic acid. The method uses an electrophoresis medium that has one or more nucleic acid capture ligands (also referred to herein as “capture robes”) immobilized throughout the medium. In addition, a spatial gradient of a nucleic acid denaturant exists within the electrophoresis medium so that a sample migrates from a region of high denaturant activity to a region of lower denaturant activity during electrophoresis. One or more target nucleic acids are electrophoresed through the medium under conditions that allow binding of the target to the immobilized capture ligand at a position within the medium that is related to the sequence complementarity of the target and the capture ligand. For example, if a sample contains a target nucleic acid which has a sequence that has complete complementarity to the capture ligand (i.e., fully complementary to the nucleotide sequence of the capture ligand) and a target nucleic acid sequence that has incomplete complementarity with the capture ligand (e.g., a single nucleotide mismatch), the target nucleic acid that has complete complementarity to the capture ligand will bind to the capture ligand at a position in the medium where denaturant activity is relatively high (e.g., high temperature or high concentration of chemical denaturant). The target nucleic acid that has incomplete complementarity with the capture ligand will migrate further into the medium and bind a capture ligand at a position of lower denaturing activity (e.g., lower temperature or lower concentration of chemical denaturant).
To analyze a target nucleic acid sequence, the location of he target within the medium is determined and compared to the location within the medium of a standard nucleic acid having a predetermined sequence, or binding affinity. The location of the target within the medium is dependent on the nucleotide sequence or structure of the target nucleic acid, and its complementarity to and binding affinity for the immobilized capture ligand. The target nucleic acid will stop migrating at a position within the medium where the denaturing activity is low enough to allow a table binding complex to form. Thus, the target nucleic acid will bind to the immobilized capture ligand at a position within the medium that is relative to the binding affinity between the target and the capture ligand.
Alternatively, a property related to the location of the target within the medium, such as the time it takes for a target to pass a position in the medium that is scanned by a detector can be determined. For example, once nucleic acid sequences in a sample have been separated based on their affinity to a capture ligand using the denaturing gradient affinity electrophoresis method described above, the denaturant (e.g., temperature or chemical denaturant) can be increased to a point where the capture ligand and the target having the highest degree of complementarity to the capture ligand are denatured. For example, the temperature can be raised above the Tm for the capture ligand/target complex. Once the capture ligand/target complex has been denatured, an electric field can be applied and the target nucleic acids in the sample can be electrophoresed under standard conditions for denaturing electrophoresis. If a detector is detecting a position in the medium which is at the opposite end of the medium from the region where the sample was introduced and is at a distance, measured along the path which the target nucleic acids have migrated, further away from where the sample is introduced than any one of the target nucleic acids have migrated during the denaturing gradient affinity electrophoresis step, the target nucleic acids will pass this position at different times because they have already been separated based on their complementarity to the capture ligand in the denaturing gradient affinity electrophoresis step. This embodiment is particularly useful in capillary denaturing gradient affinity electrophoresis where the detector is located at one end of the capillary and the time at which a target nucleic acid passes the position where the detector located is the property which is determined. In addition, this embodiment can be applied to gel electrophoresis systems where a detector scans a line across the gel at the opposite end of the gel from the sample wells to determine the time at which target nucleic acid passes this line. The present invention also encompasses a kit for analyzing the nucleic acid sequence of a test sample for the presence of a degenerate site, or mutation. The kit has an electrophoretic medium that has at least one capture ligand that as a sequence which is complementary to a region of the nucleic acid sequence of the target which contains the degenerate site, or mutation. The kit also has a means for creating a gradient of a nucleic acid denaturant in the electrophoretic medium.
The method of the present invention is operationally simple, and relies on well- understood principles of DNA hybridization. In addition, the use of a gradient of denaturing conditions minimizes the amount of assay optimization required to obtain high quality results. This is par
Boles T. Christian
Hammond Philip W.
Hamilton Brook Smith & Reynolds P.C.
Lu Frank
Mosaic Technologies
Sisson Bradley L.
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