JP2006280277A5 - - Google Patents
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- JP2006280277A5 JP2006280277A5 JP2005104816A JP2005104816A JP2006280277A5 JP 2006280277 A5 JP2006280277 A5 JP 2006280277A5 JP 2005104816 A JP2005104816 A JP 2005104816A JP 2005104816 A JP2005104816 A JP 2005104816A JP 2006280277 A5 JP2006280277 A5 JP 2006280277A5
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- Prior art keywords
- nucleic acid
- adsorbed
- amino group
- phosphate solution
- solution
- Prior art date
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- 150000007523 nucleic acids Chemical class 0.000 claims description 36
- 108020004707 nucleic acids Proteins 0.000 claims description 36
- NBIIXXVUZAFLBC-UHFFFAOYSA-K [O-]P([O-])([O-])=O Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 20
- 239000010452 phosphate Substances 0.000 claims description 20
- 125000003277 amino group Chemical group 0.000 claims description 14
- 239000002245 particle Substances 0.000 claims description 10
- 238000000605 extraction Methods 0.000 claims description 9
- 239000011324 bead Substances 0.000 claims description 4
- 239000000696 magnetic material Substances 0.000 claims description 4
- 241000894006 Bacteria Species 0.000 claims description 2
- 239000011521 glass Substances 0.000 claims description 2
- 229910052751 metal Inorganic materials 0.000 claims description 2
- 239000002184 metal Substances 0.000 claims description 2
- 239000004033 plastic Substances 0.000 claims description 2
- 239000000126 substance Substances 0.000 claims description 2
- 150000002739 metals Chemical class 0.000 claims 1
- 239000000243 solution Substances 0.000 description 24
- 229920003013 deoxyribonucleic acid Polymers 0.000 description 8
- 238000003795 desorption Methods 0.000 description 7
- 239000012064 sodium phosphate buffer Substances 0.000 description 6
- 238000005259 measurement Methods 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 210000004369 Blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 230000001771 impaired Effects 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000004254 Ammonium phosphate Substances 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L Dipotassium phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 102000009645 Mitochondrial Aldehyde Dehydrogenase Human genes 0.000 description 1
- 108010009513 Mitochondrial Aldehyde Dehydrogenase Proteins 0.000 description 1
- 229910000148 ammonium phosphate Inorganic materials 0.000 description 1
- 235000019289 ammonium phosphates Nutrition 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 230000001580 bacterial Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 230000000295 complement Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 235000019797 dipotassium phosphate Nutrition 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 239000010419 fine particle Substances 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000011859 microparticle Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000006011 modification reaction Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000010414 supernatant solution Substances 0.000 description 1
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Description
本 発明者等は、上記課題を解決すべく鋭意研究した結果、粒子の表面に形成されたアミノ基に吸着させた多量の核酸にリン酸塩溶液を特定条件下で反応させ ることにより、該アミノ基に吸着させた核酸を極めて効率良く脱離し、抽出することができることを見出し、本発明を完成するに至った。 As a result of earnest research to solve the above-mentioned problems, the present inventors have made a reaction by reacting a phosphate solution with a large amount of nucleic acid adsorbed on the amino group formed on the particle surface under specific conditions. It has been found that nucleic acids adsorbed to amino groups can be desorbed and extracted very efficiently, and the present invention has been completed.
本発明は以下の事項に関する。すなわち、
（１）粒子の表面に形成されたアミノ基に核酸を吸着させ、該アミノ基に吸着させた核酸をリン酸塩溶液と反応させることにより、該アミノ基に吸着させた核酸を脱離させることを特徴とする核酸抽出方法。
（２）前記粒子は、バクテリア由来の磁性体、人工磁性体、金属、プラスチックビーズ、ガラスビーズ、ゲル状物質の粒子から選ばれる少なくとも１つであることを特徴とする（１）に記載の核酸抽出方法。
（３）前記リン酸塩溶液の濃度は、１．０ｍＭ〜５００ｍＭであることを特徴とする（１）又は（２）のいずれかに記載の核酸抽出方法。
The present invention relates to the following items. That is,
(1) Nucleic acid is adsorbed on the amino group formed on the surface of the particle, and the nucleic acid adsorbed on the amino group is reacted with a phosphate solution to desorb the nucleic acid adsorbed on the amino group. A nucleic acid extraction method characterized by the above.
(2) The nucleic acid according to (1), wherein the particle is at least one selected from bacteria-derived magnetic material, artificial magnetic material, metal, plastic bead, glass bead, and gel substance particle. Extraction method.
(3) The nucleic acid extraction method according to any one of (1) and (2), wherein the concentration of the phosphate solution is 1.0 mM to 500 mM.
本発明の核酸の抽出方法は、粒子の表面に形成されたアミノ基に多量の核酸を吸着させ、該アミノ基に吸着させた核酸をリン酸塩溶液と反応させることにより、核酸を脱離し、抽出することを特徴としている。 In the nucleic acid extraction method of the present invention, a large amount of nucleic acid is adsorbed on the amino group formed on the surface of the particle, and the nucleic acid adsorbed on the amino group is reacted with a phosphate solution, thereby desorbing the nucleic acid, It is characterized by extracting.
＜核酸の脱離＞
[リン酸塩溶液について]
本 発明において、粒子表面に形成されたアミノ基と静電相互作用による相補的結合により吸着した多量の核酸を効率よく脱離するためにリン酸塩溶液を使用 する。リン酸塩溶液中の陰イオンがアミノ基と吸着している核酸に置き換わり、これによって吸着していた核酸の脱離が起こる。
<Desorption of nucleic acid>
[About phosphate solution]
In the present invention, a phosphate solution is used in order to efficiently desorb a large amount of nucleic acid adsorbed by complementary binding due to electrostatic interaction with amino groups formed on the particle surface. The anion in the phosphate solution is replaced with the nucleic acid adsorbed to the amino group, and this causes the desorption of the adsorbed nucleic acid.
リン酸塩としては、アミノ基との静電相互作用が核酸よりも大きいものであれば良く、特に制限されるものではないが、具 体的にはリン酸水素アンモニウム、リン酸水素カリウム等を例示することができ る。これらの中でもリン酸ナトリウム塩緩衝溶液が特に好ましい。なお、上記緩衝溶液には、少量の塩化ナトリウム等の電解質を添加することもでき、溶液のイ オン強度を調整することができる。 The phosphate is not particularly limited as long as the electrostatic interaction with the amino group is larger than that of the nucleic acid. Specifically, ammonium phosphate, potassium hydrogen phosphate and the like are used. It can be illustrated. Among these, a sodium phosphate buffer solution is particularly preferable. A small amount of an electrolyte such as sodium chloride can be added to the buffer solution, and the ionic strength of the solution can be adjusted.
上記リン酸塩溶液の濃度は、１．０ｍＭ〜５００ｍＭであることが必要である。濃度が１．０ｍＭ未満であると、核酸の脱離が不十分となり、一方、濃度が５００ｍＭを超えると、分離、抽出後の核酸に支障をきたし、ＰＣＲ法による核酸増幅に障害が生じる。 The concentration of the phosphate solution needs to be 1.0 mM to 500 mM. When the concentration is less than 1.0 mM, nucleic acid is not sufficiently desorbed. On the other hand, when the concentration exceeds 500 mM, the nucleic acid after separation and extraction is impaired, and nucleic acid amplification by the PCR method is impaired.
多 量の核酸が吸着した粒子上に上記リン酸塩溶液を加え、所定の温度にてインキュベートした後、核酸を脱離する。インキュベートする温度としては、 １０℃〜９０℃、好ましくは２０℃〜８０℃である。インキュベートする温度が１０℃未満であると核酸の脱離が起こりにくく、９０℃以上であると脱離した核 酸に障害を与えることとなり好ましくない。なお、リン酸塩溶液の濃度とインキュベートする温度の最適化、ＰＣＲ法の適用を考えると、核酸の脱離する 割合が同程度ならば、核酸脱離用リン酸塩溶液は低塩下であることが好ましいので、リン酸塩の濃度を低く設定するとよい。 The phosphate solution is added onto the particles to which a large amount of nucleic acid is adsorbed, and incubated at a predetermined temperature, and then the nucleic acid is desorbed. The incubation temperature is 10 ° C to 90 ° C, preferably 20 ° C to 80 ° C. If the incubation temperature is less than 10 ° C., nucleic acid is hardly detached, and if it is 90 ° C. or higher, the detached nucleic acid is undesirably damaged. Considering the optimization of the phosphate solution concentration and incubation temperature and the application of the PCR method, the nucleic acid desorption phosphate solution should be under low salt if the nucleic acid desorption rate is comparable. It is preferable to set the phosphate concentration low.
本発明において、吸着した核酸をリン酸塩溶液によって脱離した核酸等の定量は、核酸が吸着した微粒子にリン酸塩溶液を加えた後の上澄み溶液に遊離している核酸を測定することにより行う。 In the present invention, quantification of nucleic acid or the like obtained by desorbing adsorbed nucleic acid with a phosphate solution is performed by measuring the nucleic acid released in the supernatant solution after adding the phosphate solution to the fine particles adsorbed with nucleic acid. Do.
（比較例１）
実施例１で使用したリン酸ナトリウム緩衝溶液に代えて、比較例として塩化ナトリウム溶液を使用した以外は実施例１と同様にしてλＤＮＡの脱離、抽出を行い、同様に抽出したλＤＮＡの割合を測定し、比較例１とした。測定結果を図２に示す。
(Comparative Example 1)
Instead of the sodium phosphate buffer solution used in Example 1, λDNA was desorbed and extracted in the same manner as in Example 1 except that a sodium chloride solution was used as a comparative example. Measurement was made to be Comparative Example 1. The measurement results are shown in FIG.
＜リン酸ナトリウム緩衝溶液の濃度について＞
（実施例２〜実施例５）
リン酸塩溶液として、リン酸ナトリウム緩衝溶液を使用し、ＤＮＡ脱離の温度を２５℃とし、その濃度を変化させた以外は、実施例１と同様にしてλＤＮＡの脱離、抽出を行った。λＤＮＡの抽出量の測定結果を図３に示す。
（実施例６〜実施例９）
λＤＮＡ脱離の温度を８０℃とした以外は実施例４と同様にしてλＤＮＡの脱離、抽出を行った。λＤＮＡの抽出量の測定結果を図３に示す。
<Concentration of sodium phosphate buffer solution>
(Example 2 to Example 5)
ΛDNA was desorbed and extracted in the same manner as in Example 1 except that a sodium phosphate buffer solution was used as the phosphate solution, the temperature of DNA desorption was 25 ° C., and the concentration was changed. . The measurement results of the amount of λDNA extracted are shown in FIG.
(Example 6 to Example 9)
λDNA was desorbed and extracted in the same manner as in Example 4 except that the temperature of λDNA desorption was 80 ° C. The measurement results of the amount of λDNA extracted are shown in FIG.
図１〜図３からも明らかなように、リン酸塩溶液として、リン酸ナトリウム緩衝溶液を使用し、温度を８０℃に設定することにより、アミノ基修飾磁性細菌粒子に吸着した多量のλＤＮＡをほぼ１００％という極めて高効率で脱離し、抽出することができることが理解される。 As is clear from FIGS. 1 to 3, by using a sodium phosphate buffer solution as the phosphate solution and setting the temperature to 80 ° C., a large amount of λDNA adsorbed on the amino group-modified magnetic bacterial particles can be obtained. It is understood that desorption and extraction can be performed with extremely high efficiency of almost 100%.
Claims (3)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2005104816A JP2006280277A (en) | 2005-03-31 | 2005-03-31 | Method for extracting nucleic acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2005104816A JP2006280277A (en) | 2005-03-31 | 2005-03-31 | Method for extracting nucleic acid |
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| Publication Number | Publication Date |
|---|---|
| JP2006280277A JP2006280277A (en) | 2006-10-19 |
| JP2006280277A5 true JP2006280277A5 (en) | 2008-04-24 |
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| JP2005104816A Pending JP2006280277A (en) | 2005-03-31 | 2005-03-31 | Method for extracting nucleic acid |
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Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010110435A1 (en) * | 2009-03-27 | 2010-09-30 | 国立大学法人岡山大学 | Organic-inorganic composite material and process for producing same |
| JP4857373B2 (en) * | 2009-09-09 | 2012-01-18 | 国立大学法人東京農工大学 | Dendrimer-coated magnetic fine particles, production method and use thereof |
| JP5914338B2 (en) * | 2009-09-24 | 2016-05-11 | キアジェン ゲイサーズバーグ インコーポレイテッド | Compositions, methods and kits for nucleic acid isolation and analysis using anion exchange materials |
| WO2011074587A1 (en) | 2009-12-15 | 2011-06-23 | 国立大学法人岡山大学 | Magnetic ceramic and process for production thereof |
| CN115041143A (en) * | 2022-04-02 | 2022-09-13 | 中国医学科学院基础医学研究所 | Magnetic polymer, preparation method, kit and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US7393698B2 (en) * | 2000-08-21 | 2008-07-01 | National Institute Of Advanced Industrial Science And Technology | Magnetic fine particles and process for producing the same |
| JP3756477B2 (en) * | 2002-09-17 | 2006-03-15 | 横河電機株式会社 | Method for extracting nucleic acid or protein with dendrimer and dendrimer composition |
| JP4078247B2 (en) * | 2003-05-02 | 2008-04-23 | キヤノン株式会社 | Magnetic substance-biological substance complex type structure, peptide fragment having amino acid sequence capable of binding to magnetic substance and gene thereof, and method for producing magnetic substance-biological substance complex type structure |
| JP4422982B2 (en) * | 2003-06-24 | 2010-03-03 | 日立マクセル株式会社 | Magnetic carrier for binding biological materials |
| WO2005012522A1 (en) * | 2003-07-23 | 2005-02-10 | Cyclops Genome Sciences Limited | Clean-up beads |
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2005
- 2005-03-31 JP JP2005104816A patent/JP2006280277A/en active Pending
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