Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-12-28
2001-07-24
Fredman, Jeffrey (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S005000
Reexamination Certificate
active
06265164
ABSTRACT:
FIELD OF THE INVENTION
The invention is directed to novel compositions and methods for the rapid and direct analysis of biochemicals from microorganisms. The compositions and methods of the present invention obviate the need for extracting and purifying the biochemicals prior to analysis.
BACKGROUND OF THE INVENTION
Microorganisms have long been studied and characterized by analysis of their biochemical constituents. Prior to analysis, the biochemical(s) of interest, such as DNA, RNA, lipids, proteins, carbohydrates or inorganic molecules, are often extracted and purified. However, extraction and purification procedures are time-consuming, labor-intensive, and are often the rate-limiting step, especially when multiple samples are being processed. Extraction and purification procedures also are expensive and often produce excessive amounts of material in comparison to the amounts required for analysis. Furthermore, these procedures often utilize hazardous chemicals and produce toxic waste that requires the implementation of expensive handling and disposal procedures.
As an example, bacteria harboring a plasmid with a cloned insert are commonly screened and identified by miniprep analysis (Ausubel et al. (1989)
Current Protocols in Molecular Biology,
Vol. 2, John Wiley & Sons, New York). This procedure requires culturing individual, transformed bacteria overnight in liquid media. The following day, plasmid DNA is extracted and purified from an aliquot of the overnight culture. Miniprep analysis involves multiple centrifugation steps, phenol and chloroform extractions, and precipitation steps. Once purified, the plasmid DNA is in most instances analyzed by gel electrophoresis. Often the plasmid DNA is restriction enzyme digested prior to electrophoresis to facilitate the identification of plasmids containing the appropriate insert. The miniprep procedure is time-consuming, requiring at least two days, utilizes toxic organic chemicals, such as phenol and chloroform, and is labor-intensive. In addition, only about 10% of the purified plasmid DNA is required for analysis.
Recently, a miniprep procedure has been reported that uses a modified alkaline lysis of the transformed bacteria to release the plasmid DNA with analysis of the supercoiled plasmid at the midway point of the purification procedure. Although this method is a modest improvement, its disadvantages again include the preparation of an overnight culture, plasmid extraction and purification, and restriction enzyme analysis to confirm the results (Biao et al.,
Bio Techniques
23:601-607 (1997)).
Another approach to screening and identifying recombinant bacteria employs the polymerase chain reaction (PCR) (Costa and Weinter,
Strategies
7:35-37 (1994)). This procedure does not require culturing the bacteria overnight in liquid media. The plasmid DNA is bacterial colonies from transformation plates is directly analyzed by PCR using primers that flank and amplify the insert, if present. Plasmids containing the cloned insert yield a PCR product of the appropriate size. Although this procedure appears to be convenient and rapid, it has the disadvantage that the PCR conditions and primer sets for each plasmid must be optimized before it can be used routinely. Additionally, synthesis and testing of primer pairs and PCR conditions is expensive and time-consuming. Moreover, when screening large inserts, PCR products are not as reliably produced. Under these circumstances, background amplification and the production of spurious PCR fragments may be problematic.
Large numbers of recombinant bacterial colonies can also be screened using other methods, such as colony hybridization and autoradiography (Sambrook et al. (1989)
Molecular Cloning: A Laboratory Manual,
2nd ed. CSH Laboratory Press. Cold Spring Harbor, N.Y.). However, this procedure is no longer routinely performed because many investigators prefer to avoid radioisotopes for safe, environmental, and financial reasons.
Much like the techniques for analyzing DNA molecules described above, techniques for analyzing RNA, protein or other non-nucleic acid components from microorganisms are also quite labor intensive and require elaborate extraction and/or purification steps prior to analysis of the component of interest.
Therefore, there is a significant interest in the development of novel methods for rapidly analyzing the biochemical components of microorganisms, wherein those method may be performed rapidly and directly without extraction and purification of the component of interest prior to analysis. There is also an interest in the development of novel methods for the analysis of biochemical components of microorganisms without the use of toxic or unsafe materials.
The present invention is based upon the fact that a single colony of a microorganism of interest has enough of a biochemical molecule of interest, such as DNA, RNA or protein, to detect using techniques available in the art, such as gel electrophoresis. As such, procedures and solutions have been developed such that these molecules may be detected and analyzed directly from the microorganism itself and without the need for prior extraction or purification of the molecule from other contaminating cellular components. These novel methods greatly enhance the ability to quickly and unambiguously detect and analyze cellular components of interest over those techniques that are currently available.
SUMMARY OF THE INVENTION
The present invention is directed to novel compositions and methods for analyzing biochemical components of microorganisms in a rapid and direct manner. The presently described compositions and methods allow for the analysis of a biochemical component of interest from a microorganism without prior extraction and/or purification of that component prior to analysis. In this way, biochemical components of a microorganism may be analyzed in a high throughput manner which provides unambiguous results.
One embodiment of the present invention is directed to a method for detecting the presence of a nucleic acid molecule in a microorganism without prior separation of that nucleic acid molecule from other cellular components, wherein the method comprises:
(a) suspending the microorganism in a first solution comprising from about 0.1% (v/v) to about 5% (v/v) of a nonionic detergent, wherein the detergent causes the lysis of the microorganism and the liberation of the nucleic acid molecule from the microorganism so as to provide a first suspension;
(b) optionally heating the first suspension for at least about 10 seconds at at least about 65° C.;
(c) combining the first suspension with a second solution comprising a restriction endonuclease so as to provide a second suspension, wherein the nucleic acid molecule is digested by the restriction endonuclease; and
(d) detecting the presence of the nucleic acid molecule in the second suspension.
An additional embodiment of the present invention is directed to a method for detecting the presence of a nucleic acid molecule in a microorganism without prior separation of that nucleic acid molecule from other cellular components, wherein the method comprises:
(a) suspending the microorganism in a solution comprising (i) from about 0.1% (v/v) to about 5% (v/v) of a nonionic detergent and (ii) a restriction endonuclease, wherein the detergent causes the lysis of the microorganism and the liberation of the nucleic acid molecule from the microorganism and wherein the restriction endonuclease digests the liberated nucleic acid molecule, the step of suspending providing a suspension; and
(b) detecting the presence of the nucleic acid molecule in the suspension.
Yet another embodiment of the present invention is directed to a method for detecting the presence of a biochemical component in a microorganism without prior separation of that biochemical component from other cellular constituents, wherein the method comprises:
(a) suspending the microorganism in a first solution comprising a component that substantially prevents DNase activity so as to provide a first suspension;
Han Sean Xiaoliang
Wang Wan-Heng
Biochain Institute, Inc.
Einsmann Juliet C.
Flehr Hohbach Test Albritton & Herbert LLP
Fredman Jeffrey
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