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Compositions and methods for nucleic acid extraction from biological samplesRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Nucleic AcidCompositions and methods for nucleic acid extraction from biological samples description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070166751, Compositions and methods for nucleic acid extraction from biological samples. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application is a divisional and claims the benefit of U.S. patent application Ser. No. 10/322,103, filed Dec. 17, 2002, the content of which is hereby incorporated herein by reference. BACKGROUND OF THE INVENTION [0002] 1. Field of the Invention [0003] This invention relates generally to methods and compositions for the extraction of nucleic acids from biological samples, and, more particularly, to methods and compositions for rapid extraction of nucleic acids from tissue samples using an alkaline solution containing proteinase K. The extraction solution is suitable for further processing of the extracted nucleic acid using PCR. [0004] 2. Description of the Related Art [0005] With the advent of modern molecular biology, the ability to study nucleic acids in biological samples has allowed many significant advances in biological and biochemical research. One method that has provided such advances has been the polymerase chain reaction (PCR) which allows the rapid amplification of target nucleic acid from as little starting material as a single molecule (for review see Baumforth et al, J. Clin. Pathol. Mol. Pathol. 52:1-10, 1999; Rapley et al, Medical Laboratory Sciences 49:119-128, 1992). [0006] The application of PCR and other methods in molecular biology require the extraction of nucleic acid from biological samples and a number of approaches have been devised for performing such extraction. Various approaches have included treatment with a surface active agent such as sodium dodecyl sulfate and proteinase K to lyse cells and release the nucleic acid along with extraction using phenol and/or chloroform (see, for example, Sambrook et al., Molecular Cloning A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). [0007] In recent years, a number of approaches have been developed for rapid extraction of nucleic acids from biological samples. The methods not only provide an ease and convenience of tissue processing, they allow the processing of a high volume of samples (see for example, Steiner et al, Nucleic Acids Research 23:2569-2570, 1995). Nevertheless, efficient extraction of biological samples has not always been achieved. [0008] Both animal and plant tissues have been studied and approaches for nucleic acid extraction have been developed for both. For mammalian tissue extraction, some studies have reported on the digestion of the tissue by incubation with proteinase K for hours at elevated temperatures (see for example, Goldenberger et al., PCR Methods and Applications 4: 368-370, 1995; Zimmermann et al., Comparative Medicine 50:314-316, 2000) Such methods can form the basis for kits, which are commercially available (see for example GenElute.TM. Mammalian Genomic DNA Miniprep kit available from Sigma-Aldrich, St. Louis Mo.). [0009] Other studies have reported shorter incubation times, which are more applicable for use in high throughput assays. For example, U.S. Pat. No. 6,469,159 discloses an extraction method using a buffer, a non-ionic surfactant and heating at alkaline pH. Heating was to 70.degree. C. to 100.degree. C. for 5 minutes to 3 hours. This reference, however, did not disclose extraction at temperatures less than 70.degree. C., which could have been more conveniently performed or the use of an extraction solution, which did not contain a surfactant. [0010] Drews et al. reported on a 15 minute procedure for extraction of mouse tail sections at 55.degree. C. The procedure used a Tris-HCl buffer at pH 8.0 and containing the ionic surfactant, sodium dodecyl sulfate (SDS), and Proteinase K (Drews et al., BioTechniques 17:866-867, 1994). Similarly, Chen et al. reported on a 30 min procedure for extracting mouse ear-punch tissues at 55.degree. C. using a Tris-HCl buffer at pH 8.0, SDS and Proteinase K (Chen et al., BioTechniques 8:32-33, 1990). Although SDS is known to inhibit Taq polymerase in PCR reactions (e.g. see Gelfand, in PCR Technology, H. A. Erlich, Ed., Stockton Press, N.Y., 1989 pp. 17-22), these groups provided no suggestion that extraction could be carried out without surfactant. In addition, there was no suggestion in these references that extraction of the mouse tissues might have been carried out at room temperature, which would have been more convenient. [0011] Ren et al, however, reported on the extraction of mouse ear-punch tissue using a detergent-free, proteinase K solution in sterile water at room temperature for 30 min (Ren et al., Contemp. Top. Lab. Anim. Sci. (US) 40:27-30, 2001). This group, however, did not use a Tris-HCl buffer and particularly noted the absence of strong bases and acids in their extraction solution. [0012] Methods for rapid extraction of plant tissues have also been reported. Thomson et al. reported on the extraction of DNA from ground leaf, seeds and embryos using a Tris-HCl buffer at pH 9.5 and incubation at 95.degree. for 5-60 min and at 65.degree. for 10-60 min (Thomson et al, BioTechniques 19:394-400, 2002). McCarthy et al. reported on the extraction of DNA from ground transgenic wheat seeds at room temperature using an extraction buffer containing urea, SDS and EDTA. A 1:1 phenol:chloroform mixture was then added to the extraction buffer (BioTechniques 32:560-564, 2002). Steiner et al. reported on the extraction of lyophilized and ground leaf tissue at 90.degree. for 20 min using an extraction buffer containing Tris-HCl at pH 8, sodium lauryl sarkosyl and polyvinylpolypyrrolidone. None of these groups reported on the use of a protease enzyme such as Proteinase K in the extraction buffer. [0013] Guidet reported incorporating Proteinase K into the extraction buffer to extract DNA from lyophilized and crushed leaf samples (Guidet, Nucleic Acids Res. 21:4153-4154, 1994). The buffer contained Tris-HCl at pH 8, EDTA, sodium lauryl sarkosyl and Proteinase K and the extraction was at 50.degree. C. for 1 hour. The Proteinase K, however, may not have significantly contributed to the extraction since the EDTA, which is known to be a Ca.sup.2+ chelator, was present at a concentration of 450 mM. This is because Proteinase K is Ca.sup.2+-dependent in that the enzyme is unstable at the high temperatures used by Guidet as well as exhibiting a decrease in enzyme activity in the absence of Ca.sup.2+ (Bajorath et al., Nature 337:481-484, 1989; Muller et al, J. Biol. Chem. 269:23108-23111; Kolvenbach et al., Int. J. Pept. Protein Res 36:387-391, 1990). Moreover, the detergent, sodium lauryl sarkosyl is present which may affect subsequent PCR amplification (Gelfand, supra, 1989). [0014] Thus, in view of the deficiencies of earlier methods and compositions, there remains a continuing need for improved methods and compositions for extracting nucleic acids from plant and animal samples. BRIEF SUMMARY OF THE INVENTION [0015] Accordingly, the inventors herein have succeeded in discovering that an extraction composition which comprises a protease enzyme and which contains a buffering component to buffer the composition to an alkaline pH of 7.5 or greater, can be used to efficiently extract nucleic acids from animal or plant samples. One protease enzyme suitable for use in the present invention is Proteinase K. By extraction of nucleic acids from a sample it is meant that the release of nucleic acid from tissue components is effected so that subsequent procedures such as PCR can be performed. In the subsequent PCR procedures, the threshold cycle value (C.sub.t) is detectable over background. The C.sub.t value will depend upon instrumentation and detection system used and can be readily ascertained by the skilled artisan. Typical C.sub.t values are less than or equal to about 20, less than or equal to about 35 or less than or equal to about 50 or less than or equal to any value therebetween. Nucleic acids are, thus, extracted in such a manner that the nucleic acids can be subsequently amplified by PCR. It is possible that PCR inhibitors which would prevent amplification could be present in the extraction solution, whether obtained as part of the extraction process from the biological sample or from some other source. The presence of PCR inhibitors in the extraction solution would result in little or no amplification of nucleic acids and this would be deemed to constitute absence of effective extraction from the sample. Because surfactants can sometimes interfere with subsequent PCR amplification, the extraction composition, in certain embodiments, preferably, does not contain surfactants. The term "surfactant" as used herein is intended mean a surface active substance, which is used herein interchangeably with the term "surfactant". Surfactants include anionic surfactants, cationic surfactants, non-ionic surfactants and zwitterionic surfactants. Although surfactants are not included in the extraction compositions of certain embodiments, surfactants may, nevertheless, be included in the extraction compositions of certain other embodiments. [0016] In addition, because an absence of Ca.sup.2+ can, in some instances, decrease protease activity either directly or indirectly (Id., Muller et al., supra, 1994; Bajorath et al, supra, 1989), the extraction composition, preferably, does not contain a high concentration of a Ca.sup.2+-chelator such as EDTA. Preferably, if present at all, the Ca.sup.2+-chelator is at a concentration of not more than about 100 mM, not more than about 50 mM, not more than about 25 mM or not more than about 10 mM in the extraction composition. [0017] Samples can be from any of a wide variety of biological sources including the non-limiting examples of species of animals such as, mammals including humans, fish, birds, insects such as drosophila, nematodes such as C. elegans and the like; plants including moss, ferns, trees, bushes, flowering plants and the like; fungi including mushrooms, molds, yeast and the like; protista including amea, paramecium, algae, seaweed, diatoms and the like; or monera including species of bacteria or cyanobacteria. Samples of tissues are considered to be distinct from samples of isolated cells such as, for example, blood cells, cells from tissue culture preparations or cells isolated by any process from a tissue or biopsy sample. The term tissue as used herein includes plant seeds and seed tissues. Generally, it is more difficult to extract nucleic acids from tissues than to extract nucleic acids from cells although this is not always the case. [0018] Samples such as tissues or cells from a human or other mammal can be extracted at room temperature upon incubation for not more than about 30 minutes, and in certain aspects of the invention, not more than 20 minutes, and in other aspects, not more than about 10 minutes or not more than about 5 minutes. A preferred incubation time is about 10 minutes. Reference to room temperature or ambient temperature which may be used interchangeably herein, is intended to mean a laboratory temperature in which humans can perform tissue extractions, in absence of applied heat to the extraction mixture. Room temperature is typically about 20.degree. C. or less, about 22.degree. C. or less, up to about 25.degree. C. or less, or, in some instances, up to even higher temperatures as would be readily understood by the skilled artisan. [0019] Samples from plant tissues, such as portions of leaf or seed can be extracted at room temperature or at an increased temperature above room temperature. Extraction temperatures for plant tissues are sufficiently high to facilitate extraction, but not so high as to cause denaturation of the protease enzyme. In certain embodiments, extraction of plant tissue is carried out at an increased temperature of at least about 37.degree. C., at least about 45.degree. C., at least about 50.degree. C., at least about 55.degree. C., or at least about 60.degree. C. or higher, depending upon the heat stability of the particular protease enzyme. [0020] Thus in certain aspects, the present invention is directed to a method for extracting nucleic acid from a biological sample. The method comprises incubating the sample in an extraction composition, which is buffered to an alkaline pH of about 7.5 or greater. The extraction composition comprises a protease enzyme and does not contain a surface active agent. Incubation time for extraction of nucleic acids from samples are, preferably for sufficient time to release the nucleic acid, but not more than about 60 minutes. Incubation time, i.e. extraction time can be, in certain instances, about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes or about 45 minutes. [0021] In another aspect, the present invention provides a method for extracting nucleic acid from a biological sample obtained from a mammal. The method comprises incubating the sample at room temperature in an extraction composition, which is buffered to a pH of 7.5 or greater for an incubation time of not more than 30 minutes. Continue reading about Compositions and methods for nucleic acid extraction from biological samples... 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