TWI555850B - Composition and method for extracting nucleic acids - Google Patents
Composition and method for extracting nucleic acids Download PDFInfo
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本發明關於快速分離、純化生物樣本中之核酸的組合物及方法。 The present invention relates to compositions and methods for rapidly separating and purifying nucleic acids in biological samples.
習知用於分離核酸的市售試劑，例如Qiagen、Ambion等，存在有磨取組織及純化核酸的時間過長以及因分管操作而可能發生污染的問題。而且，對於新鮮或冷凍樣本中的核酸純化，目前市售的試劑普遍無法分離出高純度的核酸，並且存在鹽類殘留而造成後續核酸分析受阻的困難。目前市售的試劑亦侷限於對特定的生物樣本進行操作。 Commercially available reagents for isolating nucleic acids, such as Qiagen, Ambion, etc., have problems in that the tissue is extracted and the nucleic acid is purified for an excessively long period of time and contamination may occur due to the pipetting operation. Moreover, for the purification of nucleic acids in fresh or frozen samples, currently commercially available reagents are generally unable to isolate high purity nucleic acids, and there are salt residues that cause difficulties in subsequent nucleic acid analysis. Currently commercially available reagents are also limited to manipulation of a particular biological sample.
因此，為了降低操作中的污染風險、提高分離的核酸純度、以及減少操作步驟及時間，有發展新穎方法及試劑的需求。 Therefore, in order to reduce the risk of contamination in the operation, increase the purity of the isolated nucleic acid, and reduce the number of steps and time, there is a need to develop novel methods and reagents.
本發明一實施形態提供一種用於分離核酸的組合物，包括：至少一種鹵烴化合物、至少一種鹽類以及至少一種界面活性劑，其中，該鹵烴化合物的含量為該組合物總重量的1~70重量%。 An embodiment of the present invention provides a composition for separating nucleic acid, comprising: at least one halocarbon compound, at least one salt, and at least one surfactant, wherein the halocarbon compound is present in an amount of 1 of the total weight of the composition. ~70% by weight.
本發明另一實施形態提供一種分離核酸的方法，包括下列步驟：於一生物樣本中添加含有至少一種鹵烴化合物、至少一種鹽類以及至少一種界面活性劑之組合物，形成一均質溶液；以及分離該均質溶液中的核酸；其中該鹵烴化合物的含量為該組合物總重量的1~70重量%。 Another embodiment of the present invention provides a method of isolating a nucleic acid, comprising the steps of: adding a composition comprising at least one halocarbon compound, at least one salt, and at least one surfactant to a biological sample to form a homogeneous solution; The nucleic acid in the homogeneous solution is separated; wherein the content of the halocarbon compound is from 1 to 70% by weight based on the total weight of the composition.
本發明之具體實施詳細說明如下，然而以下的實施例僅用於進一步揭露本發明之技術內容，不應藉以限制本發明的發明範疇。 The specific embodiments of the present invention are described in detail below, but the following examples are only used to further disclose the technical content of the present invention, and should not limit the scope of the invention.
習知核酸分離的步驟，參見第1A圖，需經過樣本的均質化(homogenize)、胞解(lysis)、核酸分子連結(binding)於分離裝置、洗滌、以及將連結於分離裝置的核酸分子沖提(elution)等的各步驟。 The steps of conventional nucleic acid isolation, see Figure 1A, require homogenization, lysis, binding of nucleic acid molecules to the separation device, washing, and rinsing of the nucleic acid molecules attached to the separation device. Each step of the elution and the like.
然而，習知的核酸分離操作流程需分管進行，容易使樣本受到污染，操作時間也因此增長。再者，必須使用酚、氯仿等的有毒溶劑，對操作者及環境並不友善。其次，習知的核酸分離方法侷限於單一類型樣本且樣本量約10~40mg始可得到較佳的純化結果，不利於多種不同樣本類型及微量或大量的樣本體積之操作。 However, the conventional nucleic acid separation operation procedure needs to be carried out in a separate manner, which tends to contaminate the sample and increase the operation time. Furthermore, toxic solvents such as phenol and chloroform must be used, which are not friendly to the operator and the environment. Secondly, the conventional nucleic acid separation method is limited to a single type of sample and a sample amount of about 10 to 40 mg can obtain a better purification result, which is disadvantageous for a plurality of different sample types and a small or large sample volume operation.
然而，根據本發明所提供之組合物，可以單一步驟、單一管操作取代習知核酸分離步驟中所需的均質化、胞解及核酸分子連結於分離裝置之分管操作的步驟(參見第1B圖)，藉此可大幅降低樣本受到污染的機會，並且縮短操作時間及步驟。再者，根據本發明之組合物，不含有酚、氯仿等的有毒溶劑，可減少操作過程中對環境及操作者的毒害。而且，本發明之組合物，如後述之具體實施例，對於各樣本類型，例如各類組織，皆可獲得優異的核酸純化效果，並且可操作的樣本體積擴大至約10μg~約100mg的範圍，可有利於廣泛的生物樣本分析。因此，相較於習知核 酸分離方法，本發明提供純化效果佳、減少操作步驟、縮短純化時間、樣本種類廣泛及無害於人體及環境之組合物及方法，在生物技術領域上具有貢獻。 However, in accordance with the compositions provided herein, the steps of homogenization, cytolysis, and ligation of nucleic acid molecules to the separation device required in conventional nucleic acid isolation steps can be replaced by a single step, single tube operation (see Figure 1B). ), thereby greatly reducing the chance of contamination of the sample and shortening the operation time and steps. Further, the composition according to the present invention does not contain a toxic solvent such as phenol or chloroform, and can reduce the toxicity to the environment and the operator during the operation. Moreover, the composition of the present invention, as described in the specific examples below, can obtain excellent nucleic acid purification effects for each sample type, such as various types of tissues, and the operable sample volume is expanded to a range of about 10 μg to about 100 mg. Can facilitate a wide range of biological sample analysis. Therefore, compared to the conventional core The acid separation method provides the composition and method for improving the purification effect, reducing the operation steps, shortening the purification time, wide variety of samples, and harmless to the human body and the environment, and contributing to the field of biotechnology.
更具體地說，本發明一實施形態所提供之用於分離核酸的組合物，包括至少一種鹵烴化合物、至少一種鹽類以及至少一種界面活性劑。 More specifically, a composition for separating nucleic acids according to an embodiment of the present invention comprises at least one halocarbon compound, at least one salt, and at least one surfactant.
本發明所述之鹵烴化合物可包括氟烴化合物、氯烴化合物、溴烴化合物或碘烴化合物等，其中以全氟化烴為佳。全氟化烴習知係作為電子產品的冷卻液，被廣泛地應用於電子產業。然本發明人等在研發分離核酸之組合物之時，了解到全氟化烴可去除蛋白質的干擾，而且在高溫及低溫環境中具有化學穩定性且不殘留於純化過程中，因此可提高分離的核酸純度。 The halocarbon compound of the present invention may include a fluorocarbon compound, a chlorocarbon compound, a bromine hydrocarbon compound or an iodocarbon compound, and the like, and a perfluorinated hydrocarbon is preferred. Perfluorinated hydrocarbons are widely used in the electronics industry as a coolant for electronic products. However, when the present inventors developed a composition for separating nucleic acids, it was found that perfluorinated hydrocarbons can remove protein interference, and are chemically stable in a high temperature and low temperature environment and do not remain in the purification process, thereby improving separation. The purity of the nucleic acid.
本發明所使用之全氟化烴可包括碳數1~12的全氟烷類，包括四氟甲烷、六氟乙烷、全氟丙烷、全氟丁烷、全氟戊烷、全氟己烷、全氟庚烷或全氟辛烷等；以及碳數3~12的全氟環烷類，例如全氟環丙烷、全氟環丁烷、全氟環戊烷、全氟環己烷、全氟環庚烷或全氟環辛烷等。 The perfluorinated hydrocarbon used in the present invention may include perfluoroalkanes having 1 to 12 carbons, including tetrafluoromethane, hexafluoroethane, perfluoropropane, perfluorobutane, perfluoropentane, perfluorohexane. , perfluoroheptane or perfluorooctane, etc.; and perfluorocycloalkanes having 3 to 12 carbon atoms, such as perfluorocyclopropane, perfluorocyclobutane, perfluorocyclopentane, perfluorocyclohexane, Fluorocycloheptane or perfluorocyclooctane, and the like.
本發明所使用之鹽類可包括鹼金屬鹽、鹼土金屬鹽、胍鹽、或這些的組合。此述之鹼金屬鹽可包括鋰鹽、鈉鹽或鉀鹽等，例如氯化鋰(LiCl)、氯化鈉(NaCl)或氯化鉀(KCl)等。此述之鹼土金屬鹽可包括鎂鹽或鈣鹽等，例如氯化鎂或碳酸鈣等。此述之胍鹽可包括氯化胍(GuCl)、硫化氰胍(GuSCN)等。 The salts used in the present invention may include alkali metal salts, alkaline earth metal salts, phosphonium salts, or a combination of these. The alkali metal salt described herein may include a lithium salt, a sodium salt or a potassium salt, etc., such as lithium chloride (LiCl), sodium chloride (NaCl) or potassium chloride (KCl), and the like. The alkaline earth metal salt described herein may include a magnesium salt or a calcium salt or the like, such as magnesium chloride or calcium carbonate. The phosphonium salts described herein may include ruthenium chloride (GuCl), guanidinium sulfide (GuSCN), and the like.
本發明所使用之界面活性劑可包括適用於核酸分離的 界面活性劑，沒有特別限制，具體例如聚山梨糖醇酯(polysorbate)，如Tween 20、Tween 80，或聚乙二醇-對(1,1,3,3-四甲基丁基)苯基醚(polyethylene glycol 4-(1,1,3,3-tetramethylbutyl)-phenyl ether)，如Triton X-100，或其組合等。 The surfactant used in the present invention may comprise a nucleic acid separation suitable for use. The surfactant is not particularly limited, and is specifically, for example, a polysorbate such as Tween 20, Tween 80, or polyethylene glycol-p-(1,1,3,3-tetramethylbutyl)phenyl. Polyethylene glycol 4-(1,1,3,3-tetramethylbutyl)-phenyl ether, such as Triton X-100, or a combination thereof.
本發明所述之用於分離核酸的組合物中，該鹵烴化合物的含量較佳為該組合物總重量的1~70重量%，更佳為10~60重量%，再更佳為20~50重量%。本發明所述之界面活性劑的含量，相對於該組合物的總重量，較佳為0.01~10重量%，更佳為0.1~8重量%，再更佳為1~5重量%。本發明所述之鹽類的含量，相對於該組合物的總重量，較佳為5~80重量%，更佳為20~70重量%，再更佳為30~60重量%。本案一實施例中，可使用氯化鋰(LiCl)與硫化氰胍(GuSCN)的混合物作為該組合物中的鹽類，其中相對於鹽類100重量%而言，氯化鋰(LiCl)較佳為14.83~63.58重量%，硫化氰胍(GuSCN)較佳為11.816~70.896重量%。。 In the composition for separating nucleic acids according to the present invention, the content of the halocarbon compound is preferably from 1 to 70% by weight, more preferably from 10 to 60% by weight, even more preferably from 20 to 60% by weight based on the total weight of the composition. 50% by weight. The content of the surfactant in the present invention is preferably from 0.01 to 10% by weight, more preferably from 0.1 to 8% by weight, still more preferably from 1 to 5% by weight, based on the total weight of the composition. The content of the salt of the present invention is preferably from 5 to 80% by weight, more preferably from 20 to 70% by weight, still more preferably from 30 to 60% by weight, based on the total weight of the composition. In an embodiment of the present invention, a mixture of lithium chloride (LiCl) and sulfonium sulfide (GuSCN) may be used as the salt in the composition, wherein lithium chloride (LiCl) is compared with 100% by weight of the salt. Preferably, the amount is from 14.83 to 63.58% by weight, and the bismuth sulfide sulfide (GuSCN) is preferably from 11.816 to 70.896% by weight. .
為了便於分離核酸，本發明所述之用於分離核酸的組合物可更包括磁性粒子。此述的磁性粒子可為適用於分子生物領域中的磁性粒子，沒有特別限定，例如氧化鐵奈米粒子、超順磁氧化鐵奈米粒子(Ultra-small Super Paramagnetic Iron Oxide；USPIO)等。 In order to facilitate the isolation of nucleic acids, the composition for separating nucleic acids of the present invention may further comprise magnetic particles. The magnetic particles described herein may be magnetic particles suitable for use in the field of molecular biology, and are not particularly limited, and examples thereof include iron oxide nanoparticles and ultra-paramagnetic super-paramagnetic iron Oxide (USPIO).
本發明另一實施形態提供一種分離核酸的方法，包括下列步驟：於一生物樣本中添加含有至少一種鹵烴化合物、至少一種鹽類以及至少一種界面活性劑之組合物，形成一均質 溶液；以及分離該均質溶液中的核酸。 Another embodiment of the present invention provides a method for isolating a nucleic acid, comprising the steps of: adding a composition comprising at least one halocarbon compound, at least one salt, and at least one surfactant to a biological sample to form a homogeneous a solution; and isolating the nucleic acid in the homogeneous solution.
此述之鹵烴化合物、鹽類及界面活性劑與上述定義相同。如上所述，根據本發明之分離核酸的方法，可提供純化效果佳、減少操作步驟、縮短純化時間、樣本種類廣泛及無害於人體及環境等之優於習知技術的效果。 The halocarbon compounds, salts and surfactants described herein are the same as defined above. As described above, the method for isolating nucleic acid according to the present invention can provide an effect superior to the conventional technique in that the purification effect is good, the number of steps are shortened, the purification time is shortened, the sample is extensive, and the human body and the environment are harmless.
根據本發明之分離核酸的方法，可廣泛分析各類型的生物樣本，包括細胞、組織、血液、尿液、羊膜液、淋巴液、組織液、或這些的組合。 According to the method of isolating nucleic acids of the present invention, various types of biological samples can be widely analyzed, including cells, tissues, blood, urine, amniotic fluid, lymph, tissue fluid, or a combination of these.
本發明之分離核酸的方法中，從該均質溶液分離核酸之步驟，可採用離心、層析法等方法分離，也可進一步使用磁性粒子進行核酸的分離。更具體地說，本發明所述之含有至少一種鹵烴化合物、至少一種鹽類及至少一種界面活性劑之組合物，可進一步地包含磁性粒子。藉由混合胞解的生物樣本與含有磁性粒子的本發明組合物，可使得生物樣本中的核酸分子與磁性粒子連結。再透過磁性收集裝置，例如磁台，收集所有磁性粒子，經由特定溶液沖提(elute)磁性粒子，可獲得連結於磁性粒子表面的核酸分子，藉此獲得分離、純化的核酸。 In the method for isolating nucleic acid of the present invention, the step of isolating the nucleic acid from the homogeneous solution may be carried out by a method such as centrifugation or chromatography, or the separation of the nucleic acid may be further carried out using magnetic particles. More specifically, the composition of the present invention comprising at least one halocarbon compound, at least one salt, and at least one surfactant may further comprise magnetic particles. The nucleic acid molecules in the biological sample can be linked to the magnetic particles by mixing the cell-decomposed biological sample with the composition of the present invention containing the magnetic particles. Further, all the magnetic particles are collected by a magnetic collecting device such as a magnetic table, and magnetic particles are eluted through a specific solution to obtain a nucleic acid molecule attached to the surface of the magnetic particle, thereby obtaining a separated and purified nucleic acid.
此述的磁性粒子可包含適用於分子生物領域中的磁性粒子，沒有特別限定，例如氧化鐵奈米粒子、超順磁氧化鐵奈米粒子(Ultra-small Super Paramagnetic Iron Oxide；USPIO)等。磁性分離裝置及沖提的條件可根據所選用的磁性粒子特性、欲分離的核酸分子特性等條件而適當選用。可使用的磁性分離裝置可例如磁盤(magnetic plate)、磁環(ring magnet)、或類似的磁性裝置等。沖提的溶液可包括市 售之沖提緩衝液(elution buffer)，例如磷酸二氫鈉(NaH2PO3)、尿素(urea)、硫化尿素(thiourea)、鹽酸胍(GuCl)、三(羥甲基)胺甲烷(Tris Cl)、或其組合等。 The magnetic particles described herein may include magnetic particles suitable for use in the field of molecular biology, and are not particularly limited, and are, for example, iron oxide nanoparticles or ultra-paramagnetic iron oxide particles (UPtra-Small Super Paramagnetic Iron Oxide; USPIO). The conditions of the magnetic separation device and the elution can be appropriately selected depending on the characteristics of the magnetic particles to be used, the characteristics of the nucleic acid molecules to be separated, and the like. A magnetic separation device that can be used may be, for example, a magnetic plate, a ring magnet, or the like. The rinsed solution may include a commercially available elution buffer such as sodium dihydrogen phosphate (NaH 2 PO 3 ), urea (urea), thiourea, guanidine hydrochloride (GuCl), tris (hydroxyl) Methyl)amine methane (Tris Cl), or a combination thereof.
為了提高核酸的純度，本發明所提供之分離核酸的方法可於在分離均質溶液中的核酸之前，更包括一洗滌步驟，去除均質溶液中的蛋白雜質。此述洗滌步驟可使用市售之洗滌緩衝液(washing buffer)，例如包括去離子水(ddH2O)、乙醇、或其組合等。 In order to increase the purity of the nucleic acid, the method for isolating the nucleic acid provided by the present invention may further comprise a washing step to remove protein impurities in the homogeneous solution before separating the nucleic acid in the homogeneous solution. The washing step can be carried out using a commercially available washing buffer, for example, including deionized water (ddH 2 O), ethanol, a combination thereof, or the like.
本發明所述之磁性粒子、沖提緩衝液及洗滌緩衝液也可依欲處理的生物樣本選用合適的市售磁性粒子套組，以提高純化效率。 The magnetic particles, the elution buffer and the washing buffer of the present invention may also be selected from suitable biological particle sets according to the biological sample to be treated to improve the purification efficiency.
根據本發明之分離核酸的組合物及方法，可簡化從生物樣本中分離包括例如單股核酸、雙股核酸、核酸片段或其組合之核酸分子，並對廣範圍的樣本型態及樣本體積皆可達成優良的核酸分離、純化效果。 The composition and method for isolating nucleic acids according to the present invention can simplify the isolation of nucleic acid molecules including, for example, single-stranded nucleic acids, double-stranded nucleic acids, nucleic acid fragments, or a combination thereof from biological samples, and for a wide range of sample types and sample volumes. Excellent nucleic acid separation and purification effects can be achieved.
[實施例1][Example 1]
1.利用本案組合物進行組織核酸純化1. Using the composition of the present invention for tissue nucleic acid purification
(1)組合物(1)之製備(1) Preparation of composition (1)
取500μl的全氟己烷(FluorinertTM)、190.75μl的4.5MLiCl/5MGuCl及1μl的TritonX-100均勻混合，形成組合物(1)，作為下述使用之胞解緩衝液(lysis buffer)。 Take 500μl of perfluorohexane (Fluorinert TM), TritonX-100 is uniformly mixed 190.75μl 4.5MLiCl / 5MGuCl and 1μl form composition (1), as the following cell lysis buffer (lysis buffer) use it.
(2)組織核酸純化(2) Tissue nucleic acid purification
取小鼠肝組織1mg加入含有上述組合物(1)之胞解緩衝液的研磨管中研磨約5分鐘。以1000μl緩衝液(300μl 的3.5M LiCl與700μl的70%的乙醇的混合液)清洗10次後，加入20μl的磁珠(Ambion Magmax total RNA pure beads)後均勻混合。 1 mg of mouse liver tissue was taken and ground in a grinding tube containing the cellulolytic buffer of the above composition (1) for about 5 minutes. In 1000 μl buffer (300 μl After washing 10 times with a mixture of 3.5 M LiCl and 700 μl of 70% ethanol, 20 μl of magnetic beads (Ambion Magmax total RNA pure beads) were added and uniformly mixed.
將此溶液以磁台(Ambion)分離出結合RNA的磁珠，以焦碳酸乙二酯(DEPC)水溶液、沖提速度20次/分鐘沖提磁珠，獲得純化的生物樣本(稱為ITRI樣本)，上述以組合物(1)進行純化的時間約20分鐘。 The magnetic beads bound to the RNA were separated by a magnetic plate (Ambion), and the magnetic beads were extracted with an aqueous solution of ethylene carbonate (DEPC) at a rinsing speed of 20 times/min to obtain a purified biological sample (referred to as an ITRI sample). The above-mentioned purification with the composition (1) was carried out for about 20 minutes.
2.利用市售商品進行組織核酸純化2. Purification of tissue nucleic acids using commercially available products
另一方面，於一試管內加入市售Qiagen RNAeasy®緩衝液1mg以及RNA穩定液600μl，於震盪器(Vortex)震盪約20~40秒。 On the other hand, was added commercially available Qiagen RNAeasy ® 1mg buffer and 600μl RNA stabilizing solution in a test tube, to a shaker (Vortex) from about 20 to 40 seconds shaking.
於該試管中加入1mg小鼠肝組織，繼續震盪10分鐘。之後加入市售Qiagen RNAeasy®的胞解緩衝液(lysate buffer)500μl，均勻混合後，將Qiagen RNAeasy®的胞解緩衝液分批放入離心管柱內，全速離心5分鐘。離心後之Qiagen RNAeasy®的胞解緩衝液移至新的試管，加入350μl的市售RLT緩衝液，震盪後以300g離心5分鐘以打破細胞。試管內的溶液經抽吸20次，重複上述胞解步驟5次。 1 mg of mouse liver tissue was added to the tube and shaking was continued for 10 minutes. After the addition of commercially available Qiagen RNAeasy ® cell lysis buffer (lysate buffer) 500μl, homogeneously mixing the cell lysis buffer Qiagen RNAeasy ® batch into the column was centrifuged at full speed for 5 minutes. Qiagen RNAeasy ® after centrifugation of the cell lysis buffer, transferred to a new tube and add 350μl RLT buffer commercially available, after the shock was centrifuged at 300g for 5 minutes to break the cells. The solution in the test tube was aspirated 20 times, and the above-mentioned cytolysis step was repeated 5 times.
之後將經過上述處理的Qiagen RNAeasy®胞解緩衝液更換至新的試管，全速離心2分鐘，去除上清液。之後再震盪(vortex)30秒，加入700μl的70%乙醇清洗，移至旋轉柱(spin column)離心。之後，重複上述步驟2次。再加入700μl的市售RW1緩衝液，全速離心2分鐘。之後加入500μl的市售RPE緩衝液，移至旋轉柱(spin column)離心。之後，重複上述步驟2次。最後更換下管以去除上清液， 重複離心動作。再加入50μl的RNA DEPC水，獲得Qiagen純化的生物樣本。上述以市售商品Qiagen RANassay®進行純化的時間約120分鐘。 After the above-described processing through Qiagen RNAeasy ® cell lysis buffer exchanged to a new tube, centrifuged for 2 minutes at full speed, the supernatant was removed. Then vortex for another 30 seconds, add 700 μl of 70% ethanol, and transfer to a spin column for centrifugation. After that, repeat the above steps twice. Another 700 μl of commercially available RW1 buffer was added and centrifuged at full speed for 2 minutes. 500 μl of commercially available RPE buffer was then added and transferred to a spin column for centrifugation. After that, repeat the above steps twice. Finally, the lower tube is replaced to remove the supernatant, and the centrifugation is repeated. Further, 50 μl of RNA DEPC water was added to obtain a Qiagen-purified biological sample. In the above-described commercially available Qiagen RANassay ® purifying time of about 120 minutes.
3.分析純化的RNA樣本3. Analysis of purified RNA samples
之後，將上述ITRI樣本、Qiagen純化的生物樣本溶液進行75V、TAE膠的電泳，獲得第2圖所示之電泳圖，其中第L欄表示樣品條帶(ladder)，第1欄表示上述所得的ITRI樣本，第2欄表示上述所得的Qiagen純化的生物樣本。 Thereafter, the above ITRI sample and the Qiagen purified biological sample solution were subjected to electrophoresis of 75 V and TAE gel to obtain an electrophoresis pattern shown in FIG. 2, wherein the column L indicates a sample strip, and the first column indicates the above-mentioned result. The ITRI sample, column 2 represents the Qiagen purified biological sample obtained above.
以RNA檢測儀(Nanodrop/Tecane)全光譜掃描檢測ITRI樣本、Qiagen純化的生物樣本溶液中的RNA總量以及在波長OD260、OD280及OD230的吸光度，計算OD260/OD280、OD260/OD230的比值，結果如下表1。 The total amount of RNA in the ITRI sample, Qiagen-purified biological sample solution, and the absorbance at wavelengths OD 260 , OD 280, and OD 230 were measured by a full-spectrum scan of an RNA detector (Nanodrop/Tecane) to calculate OD 260 /OD 280 , OD 260 . /OD 230 ratio, the results are shown in Table 1.
由第2圖及表1之結果可知，使用實施例1所製備之組合物(1)所得的RNA純度不低於使用市售Qiagen RNAassay®試劑所分離的RNA純度，但其所需實驗時間大幅縮短，且整個操作流程簡化，更能突顯本發明組合物應用於核酸純化之效果。 From the results of Table 2 and FIG. 1, the compositions prepared in Example 1 (1) RNA was obtained using a commercial purity is not less than Qiagen RNAassay ® reagent purity of RNA isolated using the embodiment, but the time required to test a substantial Shortening, and the entire process flow is simplified, and the effect of the composition of the present invention on nucleic acid purification is more prominent.
[實施例2]各種組織類型的核酸純化[Example 2] Nucleic acid purification of various tissue types
1.組合物(2)之製備1. Preparation of composition (2)
取500μl的全氟己烷(FluorinertTM)、169.5μl的4M的LiCl與700μl的乙醇，均勻混合後，形成組合物(2)作為下述使用之胞解緩衝液(lysis buffer)。 Take 500μl of perfluorohexane (Fluorinert TM), 4M LiCl and 700μl of ethanol 169.5μl, and uniformly mixed to form a composition (2) as described below using the cell lysis buffer (lysis buffer).
2.不同小鼠臟器的RNA純化2. RNA purification of different mouse organs
取小鼠肝、心、脾、腎的組織各0.1mg，加入含有上物組合物(2)之胞解緩衝液的研磨管中研磨約5分鐘。之後以1000μl緩衝液(300μl的4M LiCl與700μl的70~90%的乙醇)清洗10次後，加入磁珠20μl(Ambion)後均勻混合。之後將此溶液以磁台(Ambion)分離出結合RNA的磁珠，以DEPC水、沖提速度20次/分鐘沖提磁珠，收集純化後的RNA溶液，進行75V、TAE膠的電泳，獲得第3圖所示之電泳圖。 0.1 mg of each of the liver, heart, spleen and kidney tissues of the mice was taken and ground in a grinding tube containing the cell buffer of the top composition (2) for about 5 minutes. Thereafter, after washing 10 times with 1000 μl of a buffer (300 μl of 4 M LiCl and 700 μl of 70 to 90% ethanol), 20 μl of magnetic beads (Ambion) was added and uniformly mixed. Then, the solution was separated into magnetic beads bound by RNA on a magnetic table (Ambion), and the magnetic beads were eluted with DEPC water at a rinsing speed of 20 times/min. The purified RNA solution was collected and subjected to electrophoresis of 75 V and TAE gel. The electropherogram shown in Figure 3.
第3圖中，第L欄表示樣品條帶(marker)，第1、2、3欄表示小鼠心組織，第4、5、6欄表示小鼠脾組織，第7、8、9、10欄表示小鼠肝組織，第11、12欄表示小鼠腎組織。 In Fig. 3, column L indicates a sample marker, columns 1, 2, and 3 indicate mouse heart tissue, and columns 4, 5, and 6 indicate mouse spleen tissue, 7th, 8th, 9th, and 10th. The column indicates mouse liver tissue, and the columns 11 and 12 indicate mouse kidney tissue.
另一方面，將上述所得RNA溶液分別以Angilent 2100®檢測RNA完整度指標(RNA integrity number；RIN)，得到如下表2之結果。 On the other hand, the RNA solution obtained above was examined for RNA integrity number (RIN) by Angilent 2100 ® to obtain the results shown in Table 2 below.
由第3圖及表2之結果可知，本案之組合物可廣泛地 用於分離各組織類型之核酸，並達到優良的純化效果。 From the results of Fig. 3 and Table 2, the composition of the present invention can be widely used. It is used to separate nucleic acids of various tissue types and achieve excellent purification results.
[實施例3]離心純化與磁性純化[Example 3] Centrifugal purification and magnetic purification
1.組合物(3)之製備1. Preparation of composition (3)
取500μl的全氟己烷(FluorinertTM)、169.5μl的4M的LiCl與700μl的乙醇，均勻混合後，形成組合物(3)作為下述使用之胞解緩衝液(lysis buffer)。 Take 500μl of perfluorohexane (Fluorinert TM), 4M LiCl and 700μl of ethanol 169.5μl, and uniformly mixed to form a composition (3) as described below using the cell lysis buffer (lysis buffer).
2.離心純化2. Centrifugal purification
取小鼠肝、肺、胸腺、脾、股骨的組織各0.1mg，加入含有上述組合物(3)之胞解緩衝液的研磨管中研磨約5分鐘。之後以1000μl緩衝液(300μl的4M LiCl與700μl的70~90%的乙醇)清洗10次後，將所得溶液進行離心(轉速10000rpm，離心機：eppendoff)，獲得離心分離之RNA溶液。 0.1 mg of each of the liver, lung, thymus, spleen and femur tissues of the mice was taken and ground in a grinding tube containing the cell buffer of the above composition (3) for about 5 minutes. Thereafter, the mixture was washed 10 times with 1000 μl of a buffer solution (300 μl of 4 M LiCl and 700 μl of 70 to 90% ethanol), and the resulting solution was centrifuged (rotation speed: 10,000 rpm, centrifuge: eppendoff) to obtain a centrifuged RNA solution.
檢測離心分離所得的RNA溶液中的RNA總量，如第4圖所示。 The total amount of RNA in the RNA solution obtained by centrifugation was measured as shown in Fig. 4.
另以Angilent 2100®檢測離心分離所得的RNA溶液中的RIN值，得到如第6圖之結果。 In another RIN value resulting solution Angilent 2100 ® RNA detection centrifugation to obtain the results of FIG. 6.
3.磁珠純化3. Magnetic bead purification
另一方面，取小鼠肝、心、脾、腎的組織各0.1mg，加入含有上述組合物(3)之胞解緩衝液的研磨管中研磨約5分鐘。之後以1000μl緩衝液(300μl的4M LiCl與700μl的70~90%的乙醇)清洗10次後，加入磁珠20μl(Ambion)後均勻混合。之後將此溶液以磁台(Ambion)分離出結合RNA的磁珠，以DEPC水、沖提速度20次/分鐘沖提磁珠， 獲得磁性純化的RNA溶液。 On the other hand, 0.1 mg of each of the liver, heart, spleen, and kidney tissues of the mice was taken and ground in a grinding tube containing the cell buffer of the above composition (3) for about 5 minutes. Thereafter, after washing 10 times with 1000 μl of a buffer (300 μl of 4 M LiCl and 700 μl of 70 to 90% ethanol), 20 μl of magnetic beads (Ambion) was added and uniformly mixed. Then, the solution was separated into magnetic beads bound to RNA by a magnetic table (Ambion), and the magnetic beads were eluted with DEPC water at a rinsing speed of 20 times/min. A magnetically purified RNA solution was obtained.
檢測磁性純化的RNA溶液中的RNA總量，如第5圖所示。 The total amount of RNA in the magnetically purified RNA solution was measured as shown in Figure 5.
另以Angilent 2100®檢測磁性純化的RNA溶液中的RIN值，得到如第7圖之結果。 In another RIN value Angilent 2100 ® RNA was detected in the purified magnetic, 7 give the results of FIG.
由第4~7圖之結果顯示，透過本案之組合物分離組織中的核酸，不論使用離心或磁性純化的方法，皆可獲得良好的純化效果。 From the results of Figures 4 to 7, it was revealed that the nucleic acid in the tissue was separated by the composition of the present invention, and a good purification effect was obtained regardless of the method of centrifugation or magnetic purification.
[實施例4][Example 4]
根據下表3所示之體積配製實施例4之組合物(4)~(10)，重複前述實施例2之核酸純化程序及實施例1之吸光度分析，獲得OD260/OD280、OD260/OD230的比值，其結果如下表3所示。 The composition (4) to (10) of Example 4 was prepared according to the volume shown in Table 3 below, and the nucleic acid purification procedure of the above Example 2 and the absorbance analysis of Example 1 were repeated to obtain OD 260 /OD 280 and OD 260 / The ratio of OD 230 is shown in Table 3 below.
由表3之結果可看出，以不同比例配製之本案組合物，亦可達到在短時間內完成高純度核酸純化之效果。 As can be seen from the results of Table 3, the compositions of the present invention prepared in different ratios can also achieve the effect of purifying high purity nucleic acids in a short period of time.
雖然本發明已以較佳實施例揭露如上，然其並非用以限定本發明，任何熟悉此項技藝者，在不脫離本發明之精神和範圍內，當可做些許更動與潤飾，因此本發明之保護範圍當視後附之申請專利範圍所界定者為準。 While the present invention has been described in its preferred embodiments, the present invention is not intended to limit the invention, and the present invention may be modified and modified without departing from the spirit and scope of the invention. The scope of protection is subject to the definition of the scope of the patent application.
第1A圖為顯示一般習知核酸分離流程之示意圖。 Figure 1A is a schematic diagram showing the flow of a conventional nucleic acid separation process.
第1B圖為顯示本發明一實施例之操作流程之示意圖。 Fig. 1B is a schematic view showing the operational flow of an embodiment of the present invention.
第2圖為顯示本發明一實施例之萃取組織中RNA的電泳圖，圖中包括與使用市售試劑(Qiagen)者的比較。 Fig. 2 is a chart showing the electrophoresis of RNA in the extracted tissue according to an embodiment of the present invention, which includes comparison with those using a commercially available reagent (Qiagen).
第3圖為顯示本發明一實施例之萃取多種組織中RNA的電泳圖。 Fig. 3 is an electropherogram showing the extraction of RNA in various tissues according to an embodiment of the present invention.
第4圖為顯示本發明一實施例之以離心純化方法萃取多種組織中RNA的RNA總量之柱狀圖。 Fig. 4 is a bar graph showing the total amount of RNA extracted from RNA in various tissues by a centrifugal purification method according to an embodiment of the present invention.
第5圖為顯示本發明一實施例之以磁性純化方法萃取多種組織中RNA的RNA總量之柱狀圖。 Fig. 5 is a bar graph showing the total amount of RNA extracted from RNA in various tissues by a magnetic purification method according to an embodiment of the present invention.
第6圖為顯示本發明一實施例之以離心純化方法萃取多種組織中RNA的RIN值之柱狀圖。 Fig. 6 is a bar graph showing the RIN value of extracting RNA in various tissues by a centrifugal purification method according to an embodiment of the present invention.
第7圖為顯示本發明一實施例之以磁性純化方法萃取多種組織中RNA的RIN值之柱狀圖。 Fig. 7 is a bar graph showing the RIN value of extracting RNA in various tissues by a magnetic purification method according to an embodiment of the present invention.
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