Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1997-04-18
2004-11-09
Wilson, James O. (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S025400, C536S025200, C435S091100
Reexamination Certificate
active
06815541
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a coprecipitant and a method of extracting nucleic acids using the coprecipitant. The coprecipitant acts in the same manner as nucleic acids in alcoholic treatment for collecting nucleic acids from biological materials and/or test samples such as blood, urine, medullary fluid, phlegm or sputum, semen, cells, tissue and biopsy samples. The coprecipitant can provide a visible precipitate of the nucleic acids from centrifugal separation, thereby to collect the nucleic acids with high extraction efficiency and good reproducibility.
BACKGROUND ART
Recently, the study or diagnosis using nucleic acid probes has made rapid progress. Thus, it becomes important to realize simplification of handling of the test samples and refine nucleic acids of high purity by elimination of impurities other than the nucleic acids.
For example, in case of effecting nucleic acid hybridization, if refinement of DNA samples are insufficient, protein tends to be bonded with DNA, and if the sample contains carbohydrates, DNA digestion by restriction enzyme is inhibited so that it is impossible to recognize the base sequence of the restriction enzyme and sufficient results are not obtainable. Also, in case of effecting nucleic acid hybridization using RNA samples, insufficient refinement of the RNA samples results in insufficient nucleic acid hybridization. Therefore, it is essential to refine sufficiently and previously the DNA samples in the nucleic acid hybridization or DNA digestion by restriction enzyme.
Moreover, in case of probe detection by the non-radioactive labeling method, if using test samples containing impurities, an antibody or avidin used as reagents may be bonded nonspecifically with the impurities and hence mistakes may happen in the judgment.
It is necessary to basically conduct the following four processes for preparing refined DNA or RNA: (1) lysis of cells, (2) deproteinization and decarbohydration, (3) separation and concentration and (4) washing and refinement.
(1) In the lysis of cells, lytic enzyme such as lysozyme and achromopeptidase, proteolytic enzyme such as proteinase K, and surfactants such as alkali and SDS are used to lyse the cell construction. In case of microbes such as tubercule bacillus and staphylococcus having stable cell walls, the cells may be broken physically using beads or ultrasonic waves. Optionally, together with such physical means lytic enzyme and proteolytic enzyme may be adopted or alkali and surfactants may be added.
(2) In the deproteinization and decarbohydration, extraction in accordance with the conventional phenol-chloroform method is adopted most prevalently. However, this phenol-chloroform method is unadvantageously unhandy because it is attended with strong toxicity and requires much labor. Namely, in this phenol-chloroform method, DNA exists in an aqueous liquid phase (an upper phase), and denatured protein forms a cotton-like white phase in an intermediate phase between the aqueous liquid phase and an organic liquid phase (a lower phase). Therefore, it is necessary to do such a troublesome operation as sucking up quietly the DNA phase alone by the use of a wide-mouth pipette with preventing the white phase from being sucked and then transferring the so-extracted DNA phase into a new microtube. Since the above operation takes much time and requires a great deal of skill, the reproducibility of the DNA collection tends to be degraded and moreover bulk processing is difficult.
(3) In the process of separation and concentration after the process (2), the nucleic acids (DNA or RNA) are precipitated from the aqueous liquid containing the nucleic acids using 100% isopropyl alcohol or 100% ethanol, thereby to be separated and concentrated. However, in the conventional separation and concentration method, as the salt concentration in the aqueous liquid containing the nucleic acids is relatively high, at this stage, when isopropyl alcohol (ultimate concentration: 50%) or ethanol (ultimate concentration: 70%) is added thereto, the precipitate of the nucleic acids is colorless and transparent so that it can not be recognized with eyes. Therefore, the precipitate was discarded and the nucleic acids were insufficiently collected. The efficiency and accuracy of the nucleic acid extraction depends first on the collection of the nucleic acids and coprecipitant without omission in the process of separation and concentration.
(4) In the process of washing and refinement, 70% ethanol is used usually to eliminate impurities from the separated and concentrated nucleic acids.
For simplification and efficiency of the extraction method, the phenol-chloroform method has hitherto been improved, and several methods not based on the phenol-chloroform method have been also developed to evade danger due to phenol.
For example, in case of extracting DNA from blood samples to examine HIV-1 or EB virus infective to lymphocytes, there is known a method wherein blood obtained by the heparin blood gathering is treated with Triton X-100, after the centrifugal separation thereof, guanidine isothiocyanate is added to the separated precipitate, the DNA is deposited by adding isopropanol and then the DNA is washed with ethanol. According to this method, the DNA can be extracted within 2 hours.
This method is very simple but not general because the kind of samples to be examined is limited to blood only.
Also, there is a method which uses a nucleic acid extraction column for rapid separation and extraction of nucleic acids but it is very expensive and requires a high cost. Therefore, this method is not suitable for general application.
Recently, a method utilizing bonding ability to DNA of cationic surfactants such as CTAB (cetyltrimethyl ammonium bromide) was reported as a low cost method for rapidly extracting nucleic acids (Japanese Patent Laid-open Publication No. 2-31696).
In this method, after pretreatment of cell lysis, a complex of nucleic acids and CTAB is formed by adding CTAB in an organic solvent and then the DNA and CTAB are lysed by lysing the complex in an aqueous solution having a high salt concentration and then the DNA is isolated by ethanol or isopropanol. Though this method does not use dangerous phenol, it still requires much labor for many steps of centrifugal and washing operations.
To solve this problem, the so-called agglutination partition method was already proposed (Japanese Patent Laid-open Publication No. 4-220738). This method is applicable to decantation or reversing as separation for extracting and refining rapidly and simply nucleic acids with high purity. In this method, pretreatment for eluting nucleic acids from biological materials is first conducted, and the pretreated materials become a mixture by adding a aqueous liquid and a non-hydrophilic organic liquid having a high specific gravity and containing a thixotropic thickening agent. After centrifugal separation of the mixture a non-flowable agglutinated phase in the boundary interface between the upper and lower phases is formed and the nucleic acids existing in the upper phase is extracted by separation.
On the other hand, the recent development of the polymerase chain reaction (hereinafter, referred to as “PCR” ) and the reverse transcription polymerase chain reaction (hereinafter, referred to as “RT-PCR”) enables to amplify and detect a very small amount of nucleic acids. In the nucleic acid detection according to the PCR or RT-PCR method, a specific gene to: be examined is amplified, and DNA of the amplified gene is then detected by the agarose gel electrophoresis or hybridization method. However, reliable detection greatly depends on the efficiency of extracting nucleic acids from test samples and primers to be used in succeeding cDNA synthesis and PCR.
Especially, in the extraction of a very small amount of nucleic acids, there is needed a method wherein the nucleic acids are extracted at high purity and high yield without influence of qualitative change of sample components due to preservation condition and quantitative change of the nuclei
Aoki Akiji
Kaneshima Motohito
Usui Mitsugu
Yamaguchi Mari
Arent & Fox PLLC
Owens, Jr. Howard V.
Palma Bee'z Research Institute Co., Ltd.
Wilson James O.
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