JPH01228500A - Method for labeling nucleic acid with biotin using glutaraldehyde as a cross-linking agent - Google Patents
Method for labeling nucleic acid with biotin using glutaraldehyde as a cross-linking agentInfo
- Publication number
- JPH01228500A JPH01228500A JP5432688A JP5432688A JPH01228500A JP H01228500 A JPH01228500 A JP H01228500A JP 5432688 A JP5432688 A JP 5432688A JP 5432688 A JP5432688 A JP 5432688A JP H01228500 A JPH01228500 A JP H01228500A
- Authority
- JP
- Japan
- Prior art keywords
- biotin
- nucleic acid
- glutaraldehyde
- dna
- labeled
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 title claims abstract description 32
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 18
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 18
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 18
- 229960002685 biotin Drugs 0.000 title claims abstract description 17
- 239000011616 biotin Substances 0.000 title claims abstract description 17
- 235000020958 biotin Nutrition 0.000 title claims abstract description 16
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 title claims abstract description 9
- 239000003431 cross linking reagent Substances 0.000 title claims abstract description 5
- 238000000034 method Methods 0.000 title claims description 16
- 238000002372 labelling Methods 0.000 title claims description 9
- HKAVADYDPYUPRD-UHFFFAOYSA-N 1h-pyrazine-2-thione Chemical compound SC1=CN=CC=N1 HKAVADYDPYUPRD-UHFFFAOYSA-N 0.000 claims abstract description 6
- 108020004414 DNA Proteins 0.000 abstract description 18
- 239000000523 sample Substances 0.000 abstract description 10
- 102000053602 DNA Human genes 0.000 abstract description 3
- 239000011541 reaction mixture Substances 0.000 abstract 2
- 108020004682 Single-Stranded DNA Proteins 0.000 abstract 1
- 239000003125 aqueous solvent Substances 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 15
- 238000006243 chemical reaction Methods 0.000 description 11
- 241000701959 Escherichia virus Lambda Species 0.000 description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 238000009396 hybridization Methods 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 238000005259 measurement Methods 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 3
- 239000008363 phosphate buffer Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 108090001008 Avidin Proteins 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 238000004020 luminiscence type Methods 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 230000002285 radioactive effect Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 210000001541 thymus gland Anatomy 0.000 description 2
- BRLRJZRHRJEWJY-VCOUNFBDSA-N 5-[(3as,4s,6ar)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]-n-[3-[3-(4-azido-2-nitroanilino)propyl-methylamino]propyl]pentanamide Chemical compound C([C@H]1[C@H]2NC(=O)N[C@H]2CS1)CCCC(=O)NCCCN(C)CCCNC1=CC=C(N=[N+]=[N-])C=C1[N+]([O-])=O BRLRJZRHRJEWJY-VCOUNFBDSA-N 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 108020003215 DNA Probes Proteins 0.000 description 1
- 239000003298 DNA probe Substances 0.000 description 1
- AHCYMLUZIRLXAA-SHYZEUOFSA-N Deoxyuridine 5'-triphosphate Chemical compound O1[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C[C@@H]1N1C(=O)NC(=O)C=C1 AHCYMLUZIRLXAA-SHYZEUOFSA-N 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- XJLXINKUBYWONI-NNYOXOHSSA-O NADP(+) Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-NNYOXOHSSA-O 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 239000007825 activation reagent Substances 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 239000012736 aqueous medium Substances 0.000 description 1
- 150000001615 biotins Chemical class 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 229960000633 dextran sulfate Drugs 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000007899 nucleic acid hybridization Methods 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000000163 radioactive labelling Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
Landscapes
- Saccharide Compounds (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
【発明の詳細な説明】
［産業上の利用分野コ
本発明は、核酸をビオチンで標識する新規な方法に関す
るものであり、−得られた標識化合物について核酸ハイ
ブリダイゼーションのプローブとして臨床分野における
診断法への利用を容易にしたものである。DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a novel method for labeling nucleic acids with biotin, and uses the resulting labeled compound as a probe for nucleic acid hybridization in diagnostic methods in the clinical field. This makes it easier to use.
［従来の技術］
遺伝学的な問題の研究にあたって、特定の核酸を標識し
、それをＬ！３識することは非常に重要なことであるが
、従来最も広く使用されている方法は、Ｍｆａを構成し
ている元素に放射性同位元素を使用する方法であるが、
この方法は標識化合物の製造、測定時等において人体へ
の影響を考慮すると、取り扱いに対して厳密な注意が必
要であり面倒であった。[Prior art] When researching genetic problems, specific nucleic acids are labeled and labeled with L! It is very important to know that the most widely used method is to use radioactive isotopes for the elements that make up Mfa.
This method requires strict handling precautions and is troublesome, considering the effects on the human body during the production and measurement of labeled compounds.
そこで、非放射性プローブの使用が研究され始め、最近
ビオチンで核酸を標識する方法が開発された。ビオチン
を核酸に標識する方法としては、２′−デオキシウリジ
ン トリホスフェート ５−アリルアミン−ビオチン（
ビオチン−１１−ｃｌＵＴＰ）を基質とするニックトラ
ンスレーションによる酵素法と５活性化試薬としてフォ
トビオチン［Ｐｈｏｔｏｂｉｏｔｉｎ ：Ｎ−（４−ア
ジド−２−二トロフェニル）−Ｎ−（Ｎｄ−ビオチニル
−３−アミノプロピル）−Ｎ−メチル−１，３−プロパ
ンヂアミン〕を用いて、化学的にｍ３ａする方法が開発
されている。Therefore, the use of non-radioactive probes has begun to be investigated, and a method for labeling nucleic acids with biotin has recently been developed. A method for labeling nucleic acids with biotin is 2'-deoxyuridine triphosphate 5-allylamine-biotin (
An enzymatic method using nick translation using biotin-11-clUTP as a substrate and photobiotin as an activation reagent. A chemical method for m3a has been developed using aminopropyl-N-methyl-1,3-propanediamine].
［発明が解決しようとする問題点コ
フォトビオチンは非放射性の標識試薬としてその利用度
を高めてきているが、光等にたいする安定性に欠け、そ
の取扱が面倒である。[Problems to be Solved by the Invention Cophotobiotin has been increasingly used as a non-radioactive labeling reagent, but it lacks stability against light and is difficult to handle.
またビオチン−１１−ｄｔＪＴＰを用いる酵素法は標：
ａ法が複雑ある。そこで、簡単で安定性のある試薬を用
いて標識する方法の開発が望まれていた。In addition, the enzymatic method using biotin-11-dtJTP is as follows:
The a method is complicated. Therefore, it has been desired to develop a labeling method using a simple and stable reagent.
［問題点を解決するための手段］
本発明者は、前記の要望を満たすべく鋭意研究を重ね、
安定なビオチン誘導体を架橋剤で核酸と結合させること
に想到して本発明を完成した。[Means for Solving the Problems] The present inventor has conducted extensive research in order to satisfy the above-mentioned needs, and
The present invention was completed based on the idea of binding a stable biotin derivative to a nucleic acid using a crosslinking agent.
即ち１本発明の特徴は、ゲルタールアルデヒドの存在下
で核酸をビオチンヒドラジドと反応させることによって
、グルタールアルデヒドを架橋剤として核酸をビオチン
で標識する方法である。That is, one feature of the present invention is a method of labeling a nucleic acid with biotin using glutaraldehyde as a crosslinking agent by reacting the nucleic acid with biotin hydrazide in the presence of glutaraldehyde.
本発明の方法を実施するにあたって１反応は核酸の溶解
性を考慮して水性媒質中で行なうのが好ましく、核酸は
ＤＮＡ、ＲＮＡの何れでもよい。In carrying out the method of the present invention, one reaction is preferably carried out in an aqueous medium in consideration of the solubility of the nucleic acid, and the nucleic acid may be either DNA or RNA.
本発明は、核酸を水に溶がし、これにビオチンヒドラジ
ドとグルタールアルデヒドとを加えて反応させることに
よって、容易に実施できるが、二本鎖のＤＮＡの場合に
は反応に先だって、加熱煮沸し、冷却して一本鎖にし、
しかるのち反応に供せられせられる。The present invention can be easily carried out by dissolving nucleic acids in water, adding biotin hydrazide and glutaraldehyde to the solution, and reacting. However, in the case of double-stranded DNA, prior to the reaction, heating and boiling and cooled to form a single strand.
It is then subjected to a reaction.
反応終了後、目的とする標識されたプローブは常法によ
って採取し保存される。例えば反応終了後、冷却した反
応液中に沈殿した標識プローブを遠心等の分離方法で単
離し、洗浄し乾燥後保存される。また、迅速に行うとき
は、セファデックスＧ−２５カラム等により分離精製で
きる。After the reaction is completed, the labeled probe of interest is collected and stored by a conventional method. For example, after the reaction is completed, the labeled probe precipitated in the cooled reaction solution is isolated by a separation method such as centrifugation, washed, dried, and stored. Moreover, when carrying out quickly, separation and purification can be performed using a Sephadex G-25 column or the like.
こうして得られたプローブは、ハイブリダイゼーション
後、アビジンを結合した被測定物質例えば酵素等と反応
させることによって定量することができる。［実施例］
以下に実施例を挙げて本発明をさらに具体的に説明する
が、本発明はこれによって限定されるものではない。After hybridization, the thus obtained probe can be quantified by reacting with an avidin-bound analyte, such as an enzyme. [Example] The present invention will be described in more detail with reference to Examples below, but the present invention is not limited thereto.
実施例１ プローブの調製法（ビオチンによるＤＮＡの
標識）
ＤＮＡの水溶液（１μｇ／μ１）２５μｌを９５℃で５
分間煮沸し、次いで５分間水中冷却する。Example 1 Probe preparation method (labeling of DNA with biotin) 25 μl of an aqueous DNA solution (1 μg/μ1) was incubated at 95°C for 5 minutes.
Boil for minutes, then cool in water for 5 minutes.
これにビオチンヒドラジドの水溶液（ＩＯＢ／ｌｌ１ｌ
）１２．５μｌ及びグルタールアルデヒドの水溶液（５
％）１５μｌを加え、３７℃で１０分間反応させた。次
に３Ｍ酢酸ソーダ５．８３μｌとエタノール１４６μｌ
を加え、−４０℃で一夜放置した。生じた沈殿を遠心分
離（小型卓上遠心器１０．００Ｏｒρｌ。Add to this an aqueous solution of biotin hydrazide (IOB/11
) 12.5 μl and an aqueous solution of glutaraldehyde (5
%) was added and reacted at 37°C for 10 minutes. Next, 5.83 μl of 3M sodium acetate and 146 μl of ethanol.
was added and left at -40°C overnight. The resulting precipitate was centrifuged (small tabletop centrifuge 10.00 Orρl).
１０分）Ｌ、、８０％エタノールで洗浄し、減圧デシケ
ータ−中で真空乾燥して保存するか、若しくは０．０℃
Ｍ ＥＤＴＡ水溶液５０μｍを加えて溶かし、４℃で保
存した。10 minutes) Wash with 80% ethanol, vacuum dry in a vacuum desiccator and store, or at 0.0°C.
50 μm of M EDTA aqueous solution was added to dissolve and stored at 4°C.
上記方法に従ってビオチン標識λ−ファージ（ＤＮＡ）
を製造した。Biotin-labeled λ-phage (DNA) according to the above method
was manufactured.
参考例 λ−ファージＤＮＡのハイブリダイゼーション
ハイブリダイゼーションは１１１便で定量化ができるマ
イクロタイタープレート法で行った・
ｌ）プレハイブリダイゼーション溶液の調製１０ＸＳＳ
Ｃｓ、ｚ＝ｔ、０．０６ｘＰＶＰ ＆ ０．０６％Ｆｉ
ｃｏＬｌ（膨潤剤） ２．６ｍｌ、０．３２％ウシ血清
アルブミン２．６ｍｌ、水２．４７ ｍｌ、及び変性ウ
シ胸腺ＤＮＡ（Σ社Ｄ−１５０１） ０．１３ｍ１（最
終濃度５０ ｉＬ ｇ／ｍ１）を混合した。Reference example Hybridization of λ-phage DNA Hybridization was performed using a microtiter plate method that allows quantification in 111 stools. l) Preparation of prehybridization solution 10XSS
Cs, z=t, 0.06xPVP & 0.06%Fi
coLl (swelling agent) 2.6 ml, 0.32% bovine serum albumin 2.6 ml, water 2.47 ml, and denatured bovine thymus DNA (Sigma D-1501) 0.13 ml (final concentration 50 iL g/ml) were mixed.
２）ハイブリダイゼーション溶液の調製１０ＸＳＳＣ５
，２ｎ＋１．０．０６ｘＰＶＰ ＆ ０．０６％Ｆｉｃ
ｏｌ１２．６ｎ＋１．０．３２％ウシ血清アルブミン２
．６Ｉ１１．５部デキストラン硫酸２．６ｍ１．変性ウ
シ胸腺ＤＮＡ０．１３ｍ１（最終濃度５０μｇ／ｍｌ）
、及び実施例１で得られたビオチン標識λ−ファージＤ
ＮＡプローブ溶液２５μｌを混合した。2) Preparation of hybridization solution 10XSSC5
,2n+1.0.06xPVP & 0.06%Fic
ol12.6n+1.0.32% bovine serum albumin 2
．． 11.5 parts of 6I 2.6 ml of dextran sulfate. Denatured calf thymus DNA 0.13ml (final concentration 50μg/ml)
, and biotin-labeled λ-phage D obtained in Example 1.
25 μl of NA probe solution was mixed.
４）ハイブリダイゼーション
λ−ファージＤＮＡを水に溶かし、５分間煮沸して一本
鎖とし、０．１Ｍ塩化マグネシウムを含むリン酸緩衝液
で希釈し、２００μｍづつウェルに加え、−夜４℃に放
置することによリウエルにファージを固定した。4) Hybridization λ-Phage DNA was dissolved in water, boiled for 5 minutes to make it a single strand, diluted with phosphate buffer containing 0.1M magnesium chloride, added to wells of 200 μm each, and left at 4°C overnight. The phages were immobilized on Rewell by this method.
ウェルから未固定のλ−ファージを取り除き、これに上
記プレハイブリダイゼーション溶液２００μｍを加え、
プレートシールでプレートをカバーし、プラスチックバ
ッグに入れ６５℃の水浴上に浮かべ１時間反応させる。Remove unfixed λ-phage from the wells, add 200 μm of the above prehybridization solution,
Cover the plate with a plate seal, place it in a plastic bag, float it on a 65°C water bath, and incubate for 1 hour.
反応後プレハイブリダイゼーション溶液を吸引除去し、
ハイブリダイゼーション溶液２００μｍを加え、−夜６
５℃で反応させた。洗浄は２ Ｘ５ＳＣ２００μｌで６
５℃、３０分で１回、及び０．０５Ｍ リン酸緩衝液２
００μｍで３回行った。After the reaction, remove the prehybridization solution by suction,
Add 200μm of hybridization solution - night 6
The reaction was carried out at 5°C. Wash 6 times with 200μl of 2X5SC.
5°C, 30 min once, and 0.05M phosphate buffer 2
The test was carried out three times at 00 μm.
５） Ｄ Ｎ Ａの定量
上記のようにして得られたハイブリダイズしたビオチン
標識ＤＮＡにアビヂン結合アルカリホスファターゼ溶液
（パイオーイエダ社製） （２００００倍希釈溶液）１
００μｍを加え、室温で２時間反応させた後、生理食塩
水で３回洗浄した。これにＮＡＤＰ溶液（３Ｂを０．０
５ＭＤＥＡ緩衝液２０ｍ１に溶解）１００μｌを加え室
温で１時間反応させた。次に酵素溶液［０，２５Ｍ リ
ン酸緩衝液中ＡＤＨ０，０８ｍｇ／＋Ｉｌｌ、エタノー
ル０．３７５％、ｌ−ＭＰＭＳ １．２５Ｘ１０−５Ｍ
）ヲ加えて室温で２Ｇ分間反応させた。反応停止は０．
２Ｍ硫Ｍ ５０μｍで行い、その反応液ｌＯμｍを使用
して化学発光ｉｌｌり定した。5) Quantification of DNA Avidin-conjugated alkaline phosphatase solution (manufactured by Paio Ieda) (20,000-fold diluted solution) 1 to the hybridized biotin-labeled DNA obtained as above
After adding 00 μm and reacting at room temperature for 2 hours, the plate was washed three times with physiological saline. Add NADP solution (3B to 0.0
100 μl of 5MDEA buffer (dissolved in 20 ml) was added and reacted at room temperature for 1 hour. Then the enzyme solution [0.08 mg/+ Ill of ADH in 0.25 M phosphate buffer, 0.375% ethanol, l-MPMS 1.25X10-5 M
) was added and reacted for 2G minutes at room temperature. Reaction termination is 0.
Chemiluminescence illumination was determined using 10 μm of the reaction solution.
反応液１０μ）をガラスチューブにとり、これに比／ｍ
−ＰＯＤ混液（２，４Ｘ １０−’Ｍ／Ｉ Ｘ １０−
’Ｍ、 ０．４Ｍ炭酸緩衝液ｐＨ９，５） ２００μｌ
を加え、生じた発光をアロカルミネッセンスリーダー（
待ち時間１５秒、積算時間６秒）で測定した。Transfer 10μ of the reaction solution to a glass tube and add the ratio/m
-POD mixture (2,4X 10-'M/I X 10-
'M, 0.4M carbonate buffer pH 9,5) 200μl
was added, and the resulting luminescence was converted into an alloluminescence reader (
The measurement was performed with a waiting time of 15 seconds and an integration time of 6 seconds).
このようにして得られたλ−ファージＤＮＡの検量線は
第１図に示した通りであり、検量域はｔｏｐｇ〜５ｎ匹
であった。The calibration curve of the λ-phage DNA thus obtained was as shown in FIG. 1, and the calibration range was from topg to 5n mice.
［発明の効果］
本発明は、核酸をビオチンで標識する新規な方法であり
、これにより安定なビオチン標識プローブが得られた。[Effects of the Invention] The present invention is a novel method for labeling nucleic acids with biotin, and thereby a stable biotin-labeled probe was obtained.
このプローブを使用し、化学発光測定法によって核酸の
定量すると、非常に感度の高い測定を行うことができた
。When this probe was used to quantify nucleic acids by chemiluminescence measurement, very sensitive measurements were possible.
第１図は本発明の方法によって得られたビオチン標識λ
−ファージＤＮＡプローブを使用して検出されたえ一フ
ァージＤＮＡの検量線であり、縦軸は発光強度（カウン
ト／６秒）を示し、横軸はλ−ファージＤＮＡの量（ｐ
ｇ／ウェル）を示す。Figure 1 shows biotin-labeled λ obtained by the method of the present invention.
- A standard curve of phage DNA detected using a phage DNA probe, where the vertical axis shows the luminescence intensity (counts/6 seconds) and the horizontal axis shows the amount of λ-phage DNA (p
g/well).
Claims (1)
ドラジドと反応させることを特徴とするグルタールアル
デヒドを架橋剤とする核酸へのビオチンの標識法。1. A method for labeling a nucleic acid with biotin using glutaraldehyde as a crosslinking agent, which comprises reacting the nucleic acid with biotin hydrazide in the presence of glutaraldehyde.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP5432688A JP2647676B2 (en) | 1988-03-08 | 1988-03-08 | Labeling of biotin to nucleic acids using glutaraldehyde as a cross-linking agent |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP5432688A JP2647676B2 (en) | 1988-03-08 | 1988-03-08 | Labeling of biotin to nucleic acids using glutaraldehyde as a cross-linking agent |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH01228500A true JPH01228500A (en) | 1989-09-12 |
| JP2647676B2 JP2647676B2 (en) | 1997-08-27 |
Family
ID=12967466
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP5432688A Expired - Lifetime JP2647676B2 (en) | 1988-03-08 | 1988-03-08 | Labeling of biotin to nucleic acids using glutaraldehyde as a cross-linking agent |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2647676B2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5700935A (en) * | 1994-12-22 | 1997-12-23 | Nisshinbo Industries, Inc. | Carbodiimide derivative |
-
1988
- 1988-03-08 JP JP5432688A patent/JP2647676B2/en not_active Expired - Lifetime
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5700935A (en) * | 1994-12-22 | 1997-12-23 | Nisshinbo Industries, Inc. | Carbodiimide derivative |
| US5789588A (en) * | 1994-12-22 | 1998-08-04 | Nisshinbo Industries, Inc. | Carbodiimide derivative |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2647676B2 (en) | 1997-08-27 |
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