Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-12-20
2001-04-17
Horlick, Kenneth R. (Department: 1656)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S006120, C435S091100, C435S091200, C536S025400, C536S025420, C536S025320, C536S023100, C210S656000
Reexamination Certificate
active
06218531
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to the field of materials and methods for isolating biological entities, specifically, to materials and methods for isolating nucleic acids such as ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) from other material in a biological sample, such as cellular debris. More specifically, and without intending to limit the scope hereof, this invention particularly relates to the field of materials and methods for isolating RNA, more particularly to materials and methods for isolating total RNA from biological material such as animal or plant tissue, cultured tissue culture cells, yeast, bacteria, blood cells, viruses, or serum.
Many molecular biological techniques such as reverse transcription, cloning, restriction analysis, and sequencing involve the processing or analysis of biological materials. These techniques generally require that such materials be substantially free of contaminants capable of interfering with such processing or analysis procedures. Such contaminants generally include substances that block or inhibit chemical reactions, (e.g. nucleic acid or protein hybridizations, enzymatically catalyzed reactions, and other types of reactions, used in molecular biological techniques), substances that catalyze the degradation or de-polymerization of a nucleic acid or other biological material of interest, or substances that provide “background” indicative of the presence in a sample of a quantity of a biological target material of interest when the nucleic acid is not, in fact, present in the sample. Contaminants also include macromolecular substances from the in vivo or in vitro medium from which a nucleic acid material of interest is isolated, macromolecular substances such as enzymes, other types of proteins, polysaccharides, or polynucleotides, as well as lower molecular weight substances, such as lipids, low molecular weight enzyme inhibitors, or oligonucleotides. Contaminants can also be introduced into a target biological material from chemicals or other materials used to isolate the material from other substances. Common contaminants of this last type include trace metals, dyes, and organic solvents.
Obtaining nucleic acids, such as RNA or DNA, which are sufficiently free of contaminants for molecular biological applications is complicated by the complex systems in which the nucleic acids are typically found. These systems, e.g., cells from tissues, cells from body fluids such as blood, lymph, milk, urine, feces, semen, or the like, cells in culture, agarose or polyacrylamide gels, or solutions in which target nucleic acid amplification has been carried out, typically include significant quantities of contaminants from which the DNA or RNA of interest must be isolated before being used in a molecular biological technique.
Many different methods have been employed over the past several years to isolate target nucleic acids, such as DNA or RNA or specific types of DNA or RNA, from various different types of biological material. See, e.g. Chapter 2 (DNA) and Chapter 4 (RNA) of F. Ausubel et al., eds.,
Current Protocols in Molecular Biology,
Wiley-Interscience, New York (1993). Conventional nucleic acid isolation protocols begin with the disruption of a sample of biological material under conditions designed to cause any target nucleic acid contained therein to be released into the disruption solution. Cells with a lipid bilayer membrane, such as bacteria cells, eukaryotic tissue culture cells, or blood cells are generally disrupted by being suspended in a solution and by adding a lysis buffer containing enzymes and/or chemicals designed to lyse the cells gently and to release the target nucleic acid into the solution. When RNA is the target nucleic acid to be isolated, biological material disruption is conventionally done under conditions designed to inhibit enzymes such as ribonucleases (RNases) capable of degrading RNA. One conventional way of inhibiting RNases during cell lysis and the initial processing steps is to include a guanidine salt, such as guanidine thiocyanate, and &bgr;-mercaptoethanol in the disruption solution. Chirgwin (2979)
Biochemistry
18:5294. Once the biological material is sufficiently disrupted to release the target nucleic acid material into the disruption solution, the resulting solution is generally spun in a centrifuge to remove at least some of the cell debris and any precipitates formed in the disruption solution during the disruption step. The supernatant is then decanted and processed further to separate the target nucleic acid material from other contaminants in the solution.
Conventional nucleic acid isolation protocols also generally use phenol or an organic solvent mixture containing phenol and chloroform to extract cellular material, such as proteins and lipids, remaining in the disruption solution supernatant produced as described above The phenol/chloroform extraction step is followed by precipitation of the nucleic acid material remaining in the extracted aqueous phase by adding alcohol, such as ethanol or isopropanol, to that aqueous phase. The precipitate is typically removed from the solution by centrifugation, and the resulting pellet of precipitate is allowed to dry before being resuspended in water or a buffer solution for further processing or analysis. The alcohol precipitation step serves two purposes in a conventional nucleic acid isolation procedure. Specifically, it allows one further to isolate the nucleic acid material from contaminants including residual phenol or chloroform remaining in the organic phase, and it allows one to resuspend the resulting precipitated nucleic acid in any solution for any final desired nucleic acid concentration. For examples of conventional lysis and organic extraction methods for isolating total RNA from various types of biological material, see:
Molecular Cloning
by Sambrook et al., 2nd edition, Cold Spring Harbor Laboratory Press, p. 7.3 et seq (1989);
Protocols and Applications Guide
produced by Promega Corporation, 3rd edition, p. 93 et seq. (1996); and by Chirgwin, J. M. et al, 18
Biochemistry
5294 (1979); all of which are incorporated by reference herein. Several different companies, including Promega Corporation (Madison, Wis., USA), have also developed kits which include reagents designed to be used in isolating RNA or mRNA from biological material using such methods of isolation. See, e.g. the RNAgents® Total RNA Isolation Systems and the PolyATract® Systems available from Promega and described in the 1996 Promega Product Catalogue at page 174.
Conventional nucleic acid isolation procedures have significant drawbacks. Among these drawbacks are the time required for the multiple extraction steps needed to isolate any given nucleic acid material from other materials present in a solution produced from disrupting a biological material. For example, multiple extraction steps are required to isolate RNA from proteins, lipids, and chromosomal DNA, all of which are present in a solution produced from disrupting tissue culture cells or plant or animal tissue. Another drawback of conventional nucleic and isolated procedures is the need to use phenol and chloroform. Phenol is a known carcinogen, which causes severe burns on contact with skin. Chloroform is highly volatile, toxic and flammable. Another undesirable characteristic of organic extractions with phenol is that the oxidation products of phenol can damage nucleic acids. Only freshly redistilled phenol can be used, and nucleic acids cannot be left in the presence of phenol. Finally, some of the nucleic acid material is inherently lost at each organic extraction step as well as in the alcohol precipitation stage. Consequently, even under the best circumstances such conventional methods are time consuming, hazardous, and produce relatively low yields of isolated nucleic acid material. Also, the resulting isolated nucleic acid material is frequently contaminated with impurities, particularly with organic solvents, alcohol, and/or non-target nucleic acid material (e.
Frenchick Grady J.
Horlick Kenneth R.
King Karen B.
Michael & Best & Friedrich LLP
Promega Corporation
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