Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-05-14
2001-08-07
Jones, W. Gary (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091500, C435S803000, C436S017000, C536S027400, C530S334000
Reexamination Certificate
active
06270970
ABSTRACT:
CROSS-REFERENCE TO RELATED APPLICATIONS
Not Applicable
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
Not Applicable
TECHNICAL FIELD
This invention relates generally to materials and methods for isolating nucleic acids, such as plasmid DNA, chromosomal DNA, total RNA, mRNA, viral DNA, viral RNA, or RNA/DNA hybrids from contaminants, such as proteins, lipids, cellular debris, or other nucleic acids. This invention relates, particularly, to solid phases, including magnetic or non-magnetic matrices and chromatographic stationary phases, which bind to a target nucleic acid under one set of solution conditions and release the target nucleic acid under another set of solution conditions. More particularly, this invention relates to mixed-bed solid phases comprising at least two different solid phases, wherein each solid phase in the mixture binds to and releases a target nucleic acid under different conditions.
BACKGROUND
Many molecular biological techniques such as reverse transcription, cloning, restriction analysis, amplification and sequencing require that nucleic acids used in the techniques 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. substances that hybridize to nucleic acids, or substances that block or inhibit enzymatically catalyzed reactions, and other types of reactions used in molecular biological techniques), substances that catalyze the degradation or depolymerization of a nucleic acid or other biological material of interest, or substances which block or mask detection of the nucleic acid of interest. Substances of this last type can block or mask by providing a “background” indicative of the presence in a sample of a quantity of a nucleic acid of interest, (also referred to herein as a “target nucleic acid”) when the nucleic acid of interest is not, in fact, present in the sample. Contaminants also include macromolecular substances from the in vivo or in vitro medium from which a target nucleic acid 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, oligonucleotides, or non-target nucleic acids. 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 target nucleic acid, which is sufficiently free of contaminants for molecular biological applications, is complicated by the complex systems in which the target nucleic acid is typically found. Such 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 target nucleic acid of interest must be isolated before being used in a molecular biological procedure.
Endotoxins are particularly problematic contaminants in preparations of nucleic acids isolated from gram-negative bacilli. Generally speaking, an endotoxin is a lipopolysaccharide material found in the cell wall of most such bacilli, including
Escherichia coli
(“
E. coli
”). During lysis of bacterial cells, such as is done to release plasmid DNA from
E. coli
transformants, endotoxins are released into the lysate produced thereby. Endotoxin contamination in a nucleic acid sample can adversely limit the utility of the sample, particularly in applications, which are sensitive to such contamination. For example, the transfection efficiency of several different cultured eukaryotic cell lines, including HeLa, Huh7, COS7, and LMH, have been shown to be sharply reduced in the presence of endotoxins. Weber, M. et al. 1995,
BioTechniques
19(6):930-939. Endotoxins have also been found to be toxic to primary human cells, such as primary human skin fibroblasts and primary human melanoma cells, in the presence of entry-competent adenovirus particles. Cotten, M. et al. 1994,
Gene Therapy
1:239-246. Endotoxins have also been shown to produce striking pathophysiological reactions when introduced into animals, including high fever, vasodilation, diarrhea and, in extreme cases, fatal shock. Morrison, David C. 1987,
Ann. Rev. Med.
38:417-32.
Endotoxins are not readily separated from nucleic acids, particularly from plasmid DNA. Endotoxins tend to form micelles, which have a similar density, size, and charge distribution to plasmid DNA on the outer surface of the endotoxin micelles. As a result, endotoxins co-purify with nucleic acids, particularly with plasmid DNA, in most nucleic acid isolation procedures used today. For example, endotoxins appear in the same band as the DNA-ethidium bromide complex in the cesium chloride gradients used to separate plasmid DNA from other materials in a bacterial lysate. Endotoxins also co-migrate and co-elute with plasmid DNA from size exclusion and from anion exchange resins.
Conventional protocols for isolating DNA or RNA from various types of cells, including bacteria, begin with the cell disruption steps. 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 DNA isolation protocols generally entail suspending the cells in a solution and using enzymes and/or chemicals, gently to lyse the cells, thereby releasing the DNA contained within the cells into the resulting lysate solution. For RNA isolation, conventional lysis and solubilization procedures include measures for inhibition of ribonucleases and contaminants, including DNA, to be separated from the RNA.
Many conventional procedures for isolating target nucleic acids from various mixtures of the target nucleic acids and contaminants, including mixtures produced from cells as described above, entail the use of hazardous chemicals such as phenol, chloroform, and ethidium bromide. For example, phenol or an organic solvent mixture containing phenol and chloroform are used in many such conventional procedures to extract contaminants from mixtures of target nucleic acids and various contaminants. Alternatively, cesium chloride-ethidium bromide gradients are used in place of or in addition to phenol or phenol-chloroform extraction. Closed circular DNA, such as plasmid DNA, intercalates with ethidium bromide and forms a band in a cesium chloride gradient formed after several hours of ultracentrifugation. The DNA/ethidium bromide band is extracted therefrom and the plasmid DNA isolated from the ethidium bromide using butanol or other conventional means. See, e.g.,
Molecular Cloning
, ed. by Sambrook et al. (1989), pub. by Cold Spring Harbor Press, pp. 1.42-1.50. The phenol/chloroform extraction step, or cesium chloride banding and ethidium bromide extraction step is generally followed by precipitation of the nucleic acid material remaining in the extracted aqueous phase by adding ethanol 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.
Such conventional nucleic acid isolation procedures have significant drawbacks. Among these drawbacks are the large amount of time required for multiple processing and extraction steps, and the dangers of using phenol and/or chloroform. Phenol causes severe bums on contact. Chloroform is highly volatile, toxic, and carcinogenic. Those characteristics require that phenol be handled and phenol/chloroform extractions be carried out in a fume hood. Another undesirable characteristic of phenol/chloroform extractions is that the oxidation products of phenol can damage nucleic
Bitner Rex M.
Grosch Josephine C.
Holmes Diana L.
Katzenhendler Jehoshua
Simpson Daniel J.
Chakrabarti Arun
Frenchick Grady J.
Jones W. Gary
King Karen B.
Micheal Best & Friedrich LLP
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