Compositions and methods for extracting a nucleic acid

Details Compositions and methods for extracting a nucleic acid Compositions and methods for extracting a nucleic acid

2000-11-28

2003-01-07

Horlick, Kenneth R. (Department: 1656)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C536S023100

Reexamination Certificate

active

06503716

ABSTRACT:

FIELD OF THE INVENTION
This invention relates in general to methods, compositions and kits for rapidly and efficiently purifying nucleic acids from a biological sample.
BACKGROUND
Many of the techniques of modem molecular biology and molecular medicine begin with the isolation of a nucleic acid from a biological source. Typically, the nucleic acid is extracted from a cell or virus and then modified or manipulated with one or more enzymes. In order to be useful, the extraction process must meet at least three criteria. First, it must make the nucleic acid available for manipulation by the operator by removing it from the cell or virus that contains it. Second, it must remove inhibitors of enzymes what would otherwise interfere with the manipulation. Third, it must remove nucleases that would otherwise destroy the nucleic acid. Each of these criteria is especially difficult to satisfy when the source of the nucleic acid is not a relatively pure culture of cells or viruses, but instead contains other contaminants. These problems are especially great when the source of the nucleic acid is itself a minor component of the starting material. Such is the case, for example, when a nucleic acid is extracted from a food pathogen that is part of a food sample.
Most methods of DNA extraction comprise at least two steps. In the first step, the cell or virus is lysed by chemical treatment, boiling, enzymatic digestion of the cell wall, or mechanical forces. Lysis releases the DNA from the cell or virus and makes it available for manipulation. Centrifugation or filtration separates cell or viral debris from a crude fraction comprising the DNA and impurities such as inhibitors of enzymes and nucleases. In the second step, the DNA is purified by removing the inhibitors, nucleases and other unwanted proteins from the crude fraction. Traditionally, this has been accomplished by extracting the crude fraction with phenol and precipitating the DNA with ethanol or isopropanol. The phenol extraction removes protein contaminants. Unfortunately, phenol is a highly toxic and corrosive chemical, requiring the operator to wear protective clothing, gloves and safety glasses and to use a chemical hood. Before it can be used to extract DNA, the phenol must be equilibrated to a pH of greater than 7.8. The equilibration process is time consuming and dangerous, as it requires the phenol to be heated to 68° C. The phenol extraction step is made more efficient by combining the equilibrated phenol with chloroform and isoamyl alcohol in a ratio of 25:24:1. However, the mixture is stable at 4° C. for no more than a month, and chloroform is highly toxic and a suspected carcinogen. The alcohol precipitation is necessary to remove contaminants, including traces of phenol and chloroform. As a single phenol extraction or ethanol precipitation is typically not completely effective at removing impurities from the DNA, they often must be repeated several times in order to obtain DNA of acceptable purity. However, with each extraction and precipitation, a portion of the DNA is lost, resulting in lower yields. Each precipitation step also requires a drying step to remove all traces of alcohol from the DNA. The alcohol can be evaporated at ambient temperature and pressure, which is time consuming, or at elevated temperature and reduced pressure in a heated, vacuum-sealed centrifuge, which is not as slow but requires an expensive and complicated apparatus and a significant amount of operator time.
More recently, alternatives to the traditional method of DNA isolation have been developed that do not use phenol or chloroform. These alternative methods typically involve removing inhibitors, nucleases and other proteins by binding the DNA to a solid substrate such as a column, resin, filter or slurry. The DNA is washed one or more times to remove impurities, then eluted from the substrate. While these alternatives offer some advantages over the traditional methods, the binding substrates required are expensive and cannot be reused. Moreover, these methods require the operator to invest significant time and energy. Also, substrate-bound DNA can be susceptible to destruction by shearing.
The isolation of RNA presents even greater difficulties. Trace amounts of RNAse present during isolation can quickly destroy all of the RNA in a sample. The operator must both inactivate the RNAse that is originally present in the sample and prevent RNAse from outside sources being introduced into the sample. This is a difficult task because RNAses are ubiquitous, abundant and hardy enzymes. Most methods of isolating RNA are complicated and involve many time consuming steps, each step being an opportunity for the contamination of the sample with an RNAse that will destroy the desired RNA.
The shortcomings of the nucleic acid extraction methods described above are greatly multiplied when the starting material is not a relatively pure laboratory-grown culture, but instead is a crude sample. Examples of crude samples that have thwarted existing methods of nucleic acid isolation include food samples, clinical samples, forensic samples, agricultural samples and environmental samples. Making matters worse, the cell or virus that is the source of the nucleic acid often is a tiny fraction of the total mass of the sample. The nucleic acid must be separated from both the cell or viral debris and from the other material in the sample, and from any nucleases or inhibitors of enzymes that it contains. The problem is particularly acute when the nucleic acid is RNA, because RNAs are acutely sensitive to RNAse-catalyzed hydrolyis, or DNA that is to be amplified using the Polymerase Chain Reaction (“PCR”) or another amplification technique. PCR requires only minute amounts of substrate DNA, but the polymerase enzyme used to amplify the DNA is sensitive to even trace amounts of inhibitors.
Accordingly, there is a need in the art for fast and efficient methods for isolating nucleic acids from biological samples. The present invention meets this need. The methods of the invention allow total nucleic acid to be isolated from virtually any biological source. The methods of the invention are especially useful under conditions where previous methods are ineffective or impractical: the biological sample contains large amounts of contaminating material, the source of the nucleic acid is a small fraction of the total biological sample, the isolation is large-scale or automated, or electricity or laboratory equipment are not available.
SUMMARY OF THE INVENTION
In one aspect, the present invention provides a simple, fast and efficient method for isolating nucleic acids from samples, typically from biological samples. According to the method, a biological sample is contacted with a nucleic acid extraction reagent for a period of time and at a temperature sufficient to lyse cells in the biological sample. Following lysis, the nucleic acids are recovered from the cell debris, typically by centrifuging the sample to pellet the cell debris and recovering the supernatant, which comprises the nucleic acids.
Nucleic acid extraction reagents useful in the methods of the invention are typically aqueous compositions comprising about 0.1% (w/w) to about 18% (w/w) sodium metasilicate and about 0.05% (w/w) to about 80% (w/w), and preferably about 0.5% (w/w) to about 40% (w/w) of a substituted ether. The weight ratio of the metasilicate to substituted ether is typically in the range of about 1:0.5 to about 1:2. In a preferred embodiment, the weight ratio of sodium metasilicate to substituted ether is about 1:1.3. Typical substituted ethers include, but are not limited to, alkoxy alkyl alcohols, aryloxy alkyl alcohols and alkyloxy aryl alcohols comprising from 2 to 12 carbon atoms, more typically from 3 or 4 to 8 carbon atoms. Preferred substituted ethers are unbranched primary alkoxy alkanols according to the formula CH
3
(CH
2
)
m
—O—(CH
2
)
n
CH
2
OH, where m and n are each, independently of one another, integers between 0 and 6. Examples of preferred alkylated alkyl alcohols include 2-butox

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