Chemistry: analytical and immunological testing – Heterocyclic carbon compound – Hetero-o
Reexamination Certificate
2000-09-11
2001-09-18
Sisson, Bradley L. (Department: 1655)
Chemistry: analytical and immunological testing
Heterocyclic carbon compound
Hetero-o
C435S287200, C536S023100, C536S024300
Reexamination Certificate
active
06291248
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to methods for nucleic acid purification and in particular to purification methods wherein nucleic acid is removed from a solution by binding it to an inert carrier.
BACKGROUND OF THE INVENTION
It is well known that the purity of DNA and RNA used in scientific and medical research is a critical factor in the success of such research. For example, gene transfer experiments in cell cultures, immunization using DNA-based vaccines and DNA sequencing projects are a few of the research areas where DNA of high purity is essential for project success. Sufficiently pure DNA is not normally attainable using traditional methods of DNA isolation. Furthermore, the process of isolating and purifying DNA is a major rate-limiting step in molecular biology. Traditional methods of isolating pure DNA have typically involved toxic chemicals such as phenol/chloroform extraction procedures, and very long centrifugation times such as in Cesium Chloride banding procedures. Not only are such procedures slow and costly, they also represent a health risk to laboratory staff and demand the usually expensive disposal of hazardous chemicals. Furthermore, despite careful preparation, DNA prepared using these methods may not always be free of RNA, proteins and chromosomal DNA.
As a result, the demand for safe, high quality and rapidly obtainable DNA preparations grown in bacteria such as
E. coli
has risen steadily and a variety of commercial kits have become available to meet a wide range of DNA purification needs. However, a number of these kits suffer from disadvantages, ranging from high price to failing to produce the desired quality and yield.
It is known that DNA will bind to silicon-containing materials such as glass slurries and diatomaceous earth. In fact, DNA purification kits are available which make use of these silicon-containing substances. For example, Bio 101 offers the GENECLEAN™ kit which makes use of a glass slurry and sodium iodide (binding buffer) and BioRad™ offers a plasmid purification kit using diatomaceous earth (Celite™) suspended in guanidine hydrochloride buffer.
U.S. Pat. Nos. 5,503,816 and 5,525,319 and 5,693,785 by Woodard et al. describe the use of silicate compounds for DNA purification. The disadvantage with these materials is that the required silicate material is not readily commercially available in the appropriate form, and typically must be prepared requiring additional time for DNA isolation procedures.
U.S. Pat. Nos 5,438,129 and 5,625,054 describe DNA isolation procedures which utilize flourinated Celite™, flourinated silicon dioxide or flourinated aluminum hydroxide. These inventions require the use of toxic chemicals to create the flourinated surfaces to which the DNA will bind. In addition, chaotropes which are also toxic, are still required for these procedures.
U.S. Pat. Nos. 5,534,054 and 5,705,628 both disclose methods for isolating DNA which do not require the use of toxic chaotropic agents. The former patent discloses the use of silicon tetrahydrazide for the purification of DNA, but the preparation of the binding material requires the use of toxic chemicals which can lead to conditions such as nausea and temporary blindness. The latter of the patents relates to a non-specific, reversible binding of DNA particles using magnetic microparticles.
SUMMARY OF THE INVENTION
It is now an object of the present invention to provide a method for the purification of DNA using an inexpensive commercially available material, that does not require the use of chaotropic agents, and rapidly yields high quality preparations of DNA.
Accordingly, the invention provides the use of silicon carbide for binding a nucleotide polymer.
The invention further provides a method for purifying nucleic acid from a sample including the steps of
providing a silicon carbide carrier for binding nucleic acids;
adding the nucleic acid containing sample to the carrier for binding nucleic acid in the sample to the silicon carbide;
separating the silicon carbide from the liquid;
eluting from the silican carbide the nucleic acid bound thereto.
The present invention provides an economical nucleic acid purification method using silicon carbide compounds, preferably commercially available industrial quality silicon carbide. A typical industrial preparation of silicon carbide (SiC) which is applicable for use in the purification is composed of 97.8% silicon carbide and small amounts of silicon dioxide, silicon, iron, aluminum and carbon. This substance is affordable and readily available as a DNA-binding material. Silicon carbide is available in a variety of grit sizes or grades, and each grade has a different capacity for binding nucleic acids (all obtained through Ritchey Supply Ltd. Mississauga, Ontario). Any grade of SiC used in the method according to the present invention is preferably suspended as a 15% (w/v) slurry, preferably in either distilled water or a solution of guanidine hydrochloride.
Another preferred process of the invention is used for the purification of plasmid DNA from a sample and involves the following steps:
1. Immobilizing the DNA in the sample onto silicon carbide 1000 in the presence or absence of a binding buffer;
2. Separating the silicon carbide with the DNA immobilized thereon from the sample;
3. Washing the subsequent silicon carbide-bound DNA with an ethanol-containing buffer;
4. Removing the ethanol-containing buffer; and
5. Eluting the DNA in a low salt buffer (TE) or in water.
REFERENCES:
patent: 5464765 (1995-11-01), Coffee et al.
Fulbright & Jaworski LLP
Norgen Biotek Corporation
Sisson Bradley L.
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