pK-matched running buffers for gel electrophoresis

Details pK-matched running buffers for gel electrophoresis pK-matched running buffers for gel electrophoresis

2000-03-28

2003-06-24

Lovering, Richard D. (Department: 1712)

Chemistry: electrical and wave energy

Processes and products

Electrophoresis or electro-osmosis processes and electrolyte...

C252S189000, C204S461000, C435S967000, C436S018000

Reexamination Certificate

active

06582574

ABSTRACT:

BACKGROUND
Since Tiselius pioneered electrophoretic separation of human serum albumin, &agr;-, &bgr;- and &ggr;- globulin in 1937, electrophoresis of biological molecules has been critical to biomedical research (1). Electrophoretic analysis has become more sophisticated, specialized and useful as new types of electrophoresis are developed (2,3). McDonell et al. and Southern offered detailed descriptions of standard agarose gel electrophoresis and its use for DNA analysis (4,5). Pulsed-field agarose gel electrophoresis is an alternative for separation of very large DNA fragments up to 2000 kb (6). Another important application is polyacrylamide gel electrophoresis for separation of small DNA segments, such as dideoxy sequencing analysis (7,8) and SSCP analysis (9-11).
Electrophoresis of nucleic acids in agarose and polyacrylamide gels is generally performed with TAE or TBE buffers. These buffers perform well in many applications, but certain limitations exist. A key limitation is buffering capacity which determines the working concentration and, in turn, determines the rate at which electrophoresis can occur without distortions due to heating. Limiting buffer capacity may require a change of buffer when long electrophoresis times are required, e.g., in mutation scanning using restriction endonuclease fingerprinting or SSCP. TAE buffer cannot be used for sequencing gels because of its low buffering capacity and TAE has a relatively low solubility, such that the maximal stock solution is 20X (2,3). Typically, laboratories that perform sequencing or SSCP-type mutation scanning prepare large volumes of stock solution.
SUMMARY OF THE INVENTION
This invention provides pK-matched buffers comprising a mixture of a weak acid and a weak base which have pKa values at 25° C. within about 0.3 units of one another. The buffers are useful as running buffers for gel electrophoresis of nucleic acids or polypeptides. These pK-matched buffers have the following advantages relative to standard TBE and TAE electrophoresis buffers: 1) high resolution, 2) high electrophoretic stability, 3) low working concentration, and 4) a wide range of pH values for selection.
The invention includes an improved gel electrophoresis method of separating nucleic acids or polypeptides. The improvement comprises running the electrophoresis in a pK-matched buffer of the invention.


REFERENCES:
patent: 5174872 (1992-12-01), Scott
patent: 5227305 (1993-07-01), Manzoni et al.
patent: 5284771 (1994-02-01), Fan et al.
patent: 5651876 (1997-07-01), West et al.
patent: 5998216 (1999-12-01), O'Donnell
patent: 6056920 (2000-05-01), Lepre
Voytas, D., et al., “Current Protocols in Molecular Biology,” (1988), pp. 2.5.1-2.5.9.
Sambrook, J., et al., “Molecular Cloning: A Laboratory Manual,”Cold Spring Laboratory Press,, (1989), pp. 6.1-6.62.
McDonnel, M. et al., “Analysis of Restriction Fragments of T7 DNA and Determination of Molecular Weights by Electrophoresis in Neutral and Alkaline Gels,”J. Mol. Biol., (1977), vol. 110, pp. 119-146.
Southern, E., “Gel Electrophoresis of Restriction Fragments,”Methods in Enzymology, (1979), vol. 68, pp. 152-176.
Schwartz, D., et al., “Separation of Yeast Chromosome-Sized DNAs by Pulsed Field Grad ient Gel Electrophoresis,”Cell, (1984), vol. 37, pp. 67-75.
Sanger, F., et al., “DNA sequencing with Chain-terminating Inhibitors,”Proc. Natl. Acad. Sci. U.S.A., (1977), vol. 74, No. 12, pp. 5463-5467.
Innis, M., et al., “DNA Sequencing withThermus aquaticusDNA polymerase and Direct Sequencing of Polymerase Chain Reaction-Amplified DNA,”Proc. Natl. Acad. Sci. U.S.A., (1988), vol. 85, pp. 9436-9440.
Orita, M. et al. “Detection of Polymorphisms of Human DNA by Gel Electrophoresis as Single-strand Conformation Polymorphisms,”Proc. Natl. Acad. Sci. USA, (1989), vol. 86, pp. 2766-2770.
Sarkar, G., et al., “Dideoxy Fingerprinting (ddF): A Rapid and Efficient Screen for the Presence of Mutations,”Genomics, (1992), vol. 13, pp. 441-443.
Liu, Q., et al., “Restriction Endonuclease Fingerprinting (REF): A Sensitive Method for Screening Mutations n Long, Contiguous Segments of DNA,”BioTechniques, (1995), vol. 18, pp. 470-477.
Kuhn, R., et al., “Capillary Electrophoresis: Principles and Practice,” (1993), pp. 37-101.
Yoshitake, S., et al., “Nucleotide Sequence of the Gene for Human Factor IX (Antihemophilic Factor B),”Biochemistry, (1985), vol. 24, pp. 3736-3750.
Sarkar, G., et al., “Access to a Messenger RNA Sequence or Its Protein Product is not Limited by Tissue or Species Specificity,”Science, (1989), vol. 244, pp. 331-334.
Liu, Q., et al., “Parameters Affecting the Sensitivities of Dideoxy Fingerprinting and SSCP,”PCR Methods and Applications, (1994), vol. 4 pp. 97-108.
Helling, R., et al., “Analysis of Endonuclease R-EcoRI Fragments of DNA from Lambdoid Bacteriophages and Other Viruses by Agarose-Gel Electrophoresis,”Journal of Virology, (1974), vol. 14, pp. 1235-1244.
Yarmola, E., et al., “The Relative Contributions of Dispersion and Diffusion to Band Spreading (resolution) in Gel Electrophoresis,”Electrophoresis, (1996), vol. 17, pp. 1416-1419.
Good, N., et al., “Hydrogen Ion Buffers for Biological Research,”Biochemistry, (1966), vol. 5, No. 2, pp. 467-477.
Stoll, V., et al., “Buffers: Principles and Practice,”Methods Enzymol., (1990), vol. 182, pp. 24-38.
Ellis, K., et al., “Buffers of Constant Ionic Strength for Studying pH-Dependent Processes,”Methods of Enzymol., (1982), vol. 87, pp. 405-426.
Ganguly, A., et al., “Conformation-sensitive Gel Electrophoresis for Rapid Detection of Single-base Differences in Double-stranded PCR Products and DNA Fragments: Evidence for Solvent-Induced Bends in DNA Heteroduplexes,”Proc. Natl. Acad. Sci. USA, (1993), vol. 90, pp. 10325-10329.
Kohn, J., et al. “A Cellulose Acetate Immunofixation Technique,”J. Immunol. Methods, (1978), vol. 20, pp. 325-331.
Ambler, J., et al., “Two New Non-Barbiturate Buffers for Electrophoresis of Serum Proteins on Cellulose Acetate Membranes,”Clin. Chem.(1980), vol. 26, No. 8, pp. 1221-1223.
Liu, Q., et al., “The SSCP Phenomenon: Addition of HEPES Buffer Dramatically Affects Electrophoretic Mobility,”BioTechniques, (1998), vol. 25, pp. 50-56.
Kukita, Y., et al., “SSCP Analysis of Long DNA Fragments in Low pH Gel,”Hum. Mutat., (1997), vol. 10, pp. 400-407.
Sasaki, T., et al., “ATM Mutations in Patients with Ataxia Telangiectasia Screened by a Hierarchical Strategy,”Hum. Mutat., (1998), vol. 12, pp. 186-195.
Orban, L., et al., “Quantitative Gel Electrophoresis of Polystyrene Particles with 20-60nm Radii on 30% Crosslinked Polyacrylamide Gel,”Electrophoresis, (1987), vol. 8, pp. 465-471.
Chramback, A., et al., “Selected Buffer Systems for Moving Boundary Electrophoresis on Gels at Various pH Values, Presented in a Simplified Manner,”Electrophoresis, (1983), vol. 4, pp. 190-204.

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