JP2014525750A - 核酸を精製及び/又は単離するマトリックス及び方法 - Google Patents
核酸を精製及び/又は単離するマトリックス及び方法 Download PDFInfo
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- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 39
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 38
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 38
- 238000000034 method Methods 0.000 title claims abstract description 31
- 229910052732 germanium Inorganic materials 0.000 claims abstract description 16
- GNPVGFCGXDBREM-UHFFFAOYSA-N germanium atom Chemical group [Ge] GNPVGFCGXDBREM-UHFFFAOYSA-N 0.000 claims abstract description 16
- 150000003839 salts Chemical class 0.000 claims abstract description 15
- 239000012472 biological sample Substances 0.000 claims abstract description 8
- ATJFFYVFTNAWJD-UHFFFAOYSA-N Tin Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 claims abstract description 7
- YBMRDBCBODYGJE-UHFFFAOYSA-N germanium oxide Inorganic materials O=[Ge]=O YBMRDBCBODYGJE-UHFFFAOYSA-N 0.000 claims description 21
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- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 8
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- 238000006243 chemical reaction Methods 0.000 claims description 5
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- 229910010272 inorganic material Inorganic materials 0.000 claims description 3
- MHCFAGZWMAWTNR-UHFFFAOYSA-M lithium perchlorate Chemical compound [Li+].[O-]Cl(=O)(=O)=O MHCFAGZWMAWTNR-UHFFFAOYSA-M 0.000 claims description 3
- 229910001486 lithium perchlorate Inorganic materials 0.000 claims description 3
- 239000000843 powder Substances 0.000 claims description 3
- -1 sodium iodide Chemical class 0.000 claims description 3
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/101—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
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- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
【選択図】図1
Description
生物学的試料から核酸を精製及び単離することは、分子診断学、疫学、食品分析学、法医学及び生物科学における主要な技術である。最も普及している手法の1つは、核酸をカオトロピック剤の存在下でシリカ表面に結合することを含む。この手法の原理は、例えば、Boomら(1990)の臨床微生物学ジャーナル(J Clin Microbiol.)1990年3月;28(3):495〜503に記載されている。この技術を使用するキットは、例えば、BioMerieux、Qiagen又はPromegaにより市販されている。
●反応容器被覆、
●粒子、
●粉末、
●繊維、
●膜
からなる群より選択される少なくとも1つの形状で提供されることが提供される。
●球状、
●≧0.01μm〜≦100μmの直径
の群から選択される少なくとも1つの特徴を有することが提供される。
●酸化鉄、
●磁性ポリマー、
●金
からなる群より選択される磁性又は常磁性材料を含むことが特に好ましい。
●新鮮な組織試料、
●凍結組織試料、
●固定組織試料、
●法医学(Forensic)若しくは古生物学的(paleontologic)試料
●糞便、乾燥生物学的材料、ミイラ、剥製化(taxidermized)生物から得られる試料、
●食品試料及び/又は
●植物試料
からなる群より選択される少なくとも1つである。
●尿素、
●チオ尿素、
●塩化グアニジニウム、
●グアニジニウム塩酸塩、
●グアニジニウムチオシアネートのようなチオシアネート、
●過塩素酸リチウム又は過塩素酸ナトリウムのような過塩素酸塩、
●トリクロロ酢酸ナトリウムのようなトリクロロ酢酸塩、
●ヨウ化ナトリウムのようなヨウ化物、
●バリウム塩
からなる群より選択される少なくとも1つのカオトロピック剤を含む。
●分解酵素、
●界面活性剤、
●アルコール
からなる群より選択される少なくとも1つの試薬を更に含む。
●法医学、
●分子診断学、
●食品分析学及び/又は
●植物分析学
からなる群より選択される少なくとも1つの目的のために、本発明のキット、方法又はマトリックスの使用が提供される。
1.本発明のマトリックス材料の生成
Na2GeO3は、以下のスキームに従って溶融したとき、炭酸ナトリウムと二酸化ゲルマニウムの反応により生成される。
Na2CO3+GeO2→Na2GeO3+CO2
本発明の核酸精製キットの非限定例は、少なくとも以下の項目を含む。
結合緩衝液(500μl):5Mのグアニジニウムイソチオシアネート、10mMのトリスHCl、20%のトリトン、pH8.8、場合によりNaCl及び/又はKClが高濃度で添加されうる。
洗浄緩衝液:50容量%のエタノール、20mMのNaCl、10mMのトリス−HCl、pH7.5
低塩濃度緩衝液(50μl):50ulのTE緩衝液(10mMのトリス、1mMのEDTA、pH7.0)
GeO2被覆粒子(任意):3mg
RNAを、ホルマリン固定パラフィンパラフィン包埋(「FFPF」)腫瘍組織薄片試料から単離する。FFPF薄片を、振とうしながら、プロテイナーゼKにより55℃で2時間溶解及び処理する。結合緩衝液(高塩濃度+カオトロピック塩)及びGeO2被覆磁性粒子を加えた後、核酸は粒子に室温で15分以内に結合する。磁気スタンドにより上澄みを取り出し、ビーズを洗浄緩衝液で数回洗浄する。溶出緩衝液(低塩濃度)を添加し、70℃で10分間インキュベートした後、上澄みを、ビーズに接触することなく磁気スタンドで取り出す。
Claims (18)
- 生物学的試料から核酸を精製及び/又は単離するための使用に適したマトリックス材料であって、ゲルマニウム、スズ及び/若しくは鉛又は少なくとも1つのそれらの塩からなる群より選択される少なくとも1つの元素を含む表面を含む、マトリックス材料。
- 前記マトリックスが、ゲルマニウム又はゲルマニウム塩、好ましくは酸化ゲルマニウムを含む、請求項1に記載のマトリックス材料。
- 前記マトリックス材料が、下記:
●反応容器被覆、
●粒子、
●粉末、
●繊維、
●膜
からなる群より選択される少なくとも1つの形状で提供される、請求項1〜2のいずれか1項に記載のマトリックス材料。 - 前記粒子が、下記:
●球状、
●≧0.01μm〜≦100μmの直径
からなる群より選択される少なくとも1つの特徴を有する、請求項1〜3のいずれか1項に記載のマトリックス材料。 - 前記材料が、少なくとも部分的に磁気応答性である、請求項1〜4のいずれか1項に記載のマトリックス材料。
- 前記材料が、少なくとも部分的に無機性材料を含む又はからなる、請求項1〜5のいずれか1項に記載のマトリックス材料。
- 前記磁気応答性マトリックス材料が、下記:
●酸化鉄、
●磁性ポリマー、
●金
からなる群より選択される磁性又は常磁性材料を含む、請求項1〜6のいずれか1項に記載のマトリックス材料。 - 生物学的試料から核酸を精製及び/又は単離する方法であって、請求項1〜7のいずれか1項に記載のマトリックス材料が使用される方法。
- 精製及び/又は単離される核酸が、下記:
●DNA、
●RNA
からなる群より選択される、請求項8に記載の方法。 - 生物学的試料が、下記:
●新鮮な組織試料、
●凍結組織試料、
●固定組織試料、
●法医学若しくは古生物学的(paleontologic)試料
●糞便、乾燥生物学的材料、ミイラ、剥製化生物から得られる試料、
●食品試料及び/又は
●植物試料
からなる群より選択される少なくとも1つである、請求項8〜9のいずれか1項に記載の方法。 - 核酸がカオトロピック剤の存在下で精製及び/又は単離される、請求項8〜10のいずれか1項に記載の方法。
- 精製及び/又は単離が、磁気応答性マトリックス材料に磁場を集中させる工程を含む、請求項8〜11のいずれか1項に記載の方法。
- 請求項8〜12のいずれか1項に記載の方法における使用に適したパーツのキットであって、カオトロピック剤及び場合により本発明のマトリックス材料を含むキット。
- 前記キットが結合緩衝液及び低塩濃度緩衝液を更に含む、請求項13に記載のキット。
- カオトロピック剤が、下記:
●尿素、
●チオ尿素、
●塩化グアニジニウム、
●グアニジニウム塩酸塩、
●グアニジニウムチオシアネートのようなチオシアネート、
●過塩素酸リチウム又は過塩素酸ナトリウムのような過塩素酸塩、
●トリクロロ酢酸ナトリウムのようなトリクロロ酢酸塩、
●ヨウ化ナトリウムのようなヨウ化物、
●バリウム塩
からなる群より選択される少なくとも1つである、請求項1〜14のいずれか1項に記載のキット又は方法。 - 前記キットが磁気分離器を更に含む、請求項13〜15のいずれか1項に記載のキット。
- キット又は方法が、下記:
●分解酵素、
●界面活性剤、
●アルコール
からなる群より選択される少なくとも1つの作用物質を更に含む、請求項1〜16のいずれか1項に記載のキット又は方法。 - 下記:
●法医学、
●分子診断学、
●食品分析学及び/又は
●植物分析学
からなる群より選択される少なくとも1つの目的のための、請求項1〜17のいずれか1項に記載のキット、方法又はマトリックスの使用。
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