JP2013505719A - Compositions, methods and kits for nucleic acid isolation and analysis using anion exchange materials - Google Patents

Abstract

The present disclosure isolates, amplifies and / or analyzes nucleic acids in the presence of an anion exchange material by performing isolation, amplification and / or analysis steps in the presence of at least one anionic compound. Related to the method.

Description

Cross-reference to related applications This application includes US Provisional Patent Application No. 61 / 231,371 (filed September 24, 2009), US Provisional Patent Application No. 61 / 295,269 (filed January 15, 2010), and European Patent Application Claim priority based on the number EP 10 006 062 (filed on 11 June 2010). The contents of which are hereby incorporated by reference in their entirety. "Compositions, Methods, and Kits for Isolating and Analyzing Nucleic Acids", filed on August 5, 2010, concurrently with the present application. The US application entitled “Using an Anion Exchange Material” is also incorporated herein by reference in its entirety.

The present invention relates to methods, compositions, and kits for the isolation, elution, and analysis of nucleic acids using anion exchange materials.

Background Ion exchange is a process that allows separation of ions and polar molecules based on the charge characteristics of the molecules. In principle, ion exchange works based on reversible ionic interactions by retaining the target analyte molecule in the stationary phase. Typically, ion exchange compositions include a stationary phase that retains capture molecules. Under appropriate buffer and ionic conditions, the capture molecule retains a net charge and the target molecule retains the opposite net charge, while the contaminant is similar to the net neutral charge or capture molecule. Holds the net charge. As a result, the target molecule is retained by the ion exchange composition, but contaminants are removed. By neutralizing the charge of the capture or target molecule (eg, by altering the pH) or treating the composition with another molecule that displaces the target molecule (eg, by treatment with a high salt solution) The target molecule can be eluted. Such processes are broadly classified as either cation exchange or anion exchange, depending on the net charge of the isolated molecule. Anion exchange is commonly used to purify nucleic acids from complex biological solutions. Nucleic acids can be selectively adsorbed to an anion exchange resin and then eluted due to the negative charge. Elution buffers for low molecular weight nucleic acids typically contain high concentrations of salt. Simple organic and inorganic anions have a lower selectivity factor than nucleic acids, but at high concentrations, they can displace nucleic acids from the resin. High molecular weight nucleic acids cannot be eluted by salts due to the very high selectivity for the resin. In this case, elution is most often performed by increasing the pH, since the weak anion exchange groups of the resin lose charge at high pH.

Thus, elution of nucleic acids from ion exchange resins is usually based on the correlation between pH and salt concentration. However, these approaches present several problems for downstream processing and analysis of target nucleic acids. Since RNA is degraded in an alkaline solution, an alkaline buffer cannot be used for RNA isolation. DNA is also somewhat alkali-sensitive, most notably cytosine deamination (deoxycytosine to deoxyuracil) occurs, particularly when heated. Storage of alkaline buffers is also problematic, and such solutions are often corrosive, toxic and have poor storage stability due to the absorption of CO ₂ from the air. The high salt concentrations often utilized for elution often interfere with downstream enzymatic reactions such as polymerase chain reaction (PCR), ligation reactions, restriction analysis, cDNA production, or isothermal amplification. Thus, the resulting nucleic acid eluate needs to be desalted, which increases the number of handling steps of the nucleic acid isolation procedure, resulting in increased nucleic acid loss due to an increased number of handling steps or possible contamination. There is a possibility to be brought. Since most enzymes are completely denatured under alkaline conditions, DNA cannot be processed directly enzymatically. Therefore, high pH elution also requires an additional handling step since it requires neutralization of the eluate, which further dilutes the eluate, increases work steps, and reduces all enzymatic downstream. May not be appropriate for this assay.

  Furthermore, the presence of commonly used anion exchange materials can interfere with downstream analysis of nucleic acids. Thus, current protocols require that the nucleic acid be separated from the anion exchange material prior to analyzing the purified nucleic acid. Unfortunately, often complete separation of the eluate and anion exchange material cannot be achieved, thus reducing both the efficiency and accuracy of subsequent analysis and amplification. Furthermore, the additional steps of elution and separation of the eluate from the anion exchange material prevent the availability of anion exchange in fully automated nucleic acid purification and analysis procedures.

  Accordingly, materials and methods for isolating nucleic acids are disclosed that do not require additional handling steps prior to further use of the isolated nucleic acids. In particular, an isolated nucleic acid should be directly suitable for any amplification or analysis technique.

  The present disclosure relates to materials and methods for nucleic acid isolation and analysis, including using anion exchange chromatography with anionic compounds. Using an anionic compound, in particular an anionic compound having at least two anionic groups, without using disadvantageously high pH values and / or without using high salt concentrations, Nucleic acids can be eluted from the anion exchange material. Furthermore, it has been found that the presence of an anionic compound having at least two anionic groups allows the amplification of nucleic acids in the presence of an anion exchange material.

  In one aspect, the nucleic acid complexed with the anion exchange material is provided and bound by adding a solution comprising at least one anionic compound comprising at least two anionic groups. Disclosed is a method for isolating a nucleic acid comprising the step of eluting the nucleic acid. In one embodiment, the anionic compound comprising at least two anionic groups is a polyvalent anionic compound, preferably a polyvalent anionic organic compound. In another embodiment, the compound comprising at least two anionic groups is a non-polymeric compound, preferably a non-polymeric organic compound. In yet another embodiment, the elution step is performed without high salt conditions or severe changes in pH. In another embodiment, contacting an anion exchange composition capable of reversibly binding to nucleic acid with a sample comprising nucleic acid; and optionally, an anion exchange composition to remove unbound sample components. A nucleic acid complexed with an anion exchange material is provided by a method comprising a step of washing the product. In the method of the present invention, a compound comprising at least two anionic groups displaces a nucleic acid from an anion exchange material. The use of anionic compounds containing at least two anionic groups for the elution of bound nucleic acids requires these compounds to be removed in order to perform subsequent analytical steps, including amplification steps. Offers the advantage of not. This is because they interfere with such processes at all or very little. In addition, compounds containing at least two anionic groups are required only at low concentrations to effectively elute nucleic acids.

  In another aspect, providing a nucleic acid complexed with an anion exchange material to form a nucleic acid-anion exchange complex, and at least one anionic compound comprising at least two anionic groups There is provided a method of analyzing the nucleic acid in the presence of the anion exchange material comprising the step of analyzing the nucleic acid in the presence of. In one embodiment, the anionic compound comprising at least two anionic groups is a multivalent anionic compound. In another embodiment, the anionic compound comprising at least two anionic groups is a non-polymeric compound. In another embodiment, contacting an anion exchange composition capable of reversibly binding to nucleic acid with a sample comprising nucleic acid; optionally, an anion exchange composition to remove unbound sample components A nucleic acid complexed with the anion exchange material is provided by a method comprising the steps of: washing the nucleic acid-anion exchange complex from other materials.

  Yet another aspect is an elution composition comprising an anionic compound comprising at least two anionic groups and further comprising at least one buffer. One advantage of the provided methods and compositions is that they provide flexibility with respect to the elution conditions and elution buffers used, and can make elution more efficient. Thus, the elution composition of the present invention may comprise any buffer or composition commonly used for elution. For example, without limitation, elution buffers combine anionic compounds with water, low salt buffers, or biological buffers such as CHAPS, MES, HEPES, MOPS, TRIS, TRICINE, and PIPES Can be formed. In one embodiment, the compound comprising at least two anionic groups is a multivalent anionic compound. In another embodiment, the compound comprising at least two anionic groups is a non-polymeric compound.

  Yet another aspect relates to an eluate obtained by the elution method disclosed herein.

Yet another aspect is an amplification composition comprising a polymerase and an anionic compound comprising at least two anionic groups. In another embodiment, the amplification composition comprises an enzyme having reverse transcriptase activity; and an enzyme having helicase activity; a nick inducer; its source such as Mg ²⁺ or MgCl ₂ ; ribonucleotide triphosphate (NTP) Deoxyribonucleotide triphosphate (dNTP); its source such as K ⁺ or KCl; its source such as NH ₄ ⁺ or (NH ₄ ) ₂ SO ₄ ; a buffer such as Tris; and / or 2 -Optionally includes a reducing agent such as mercaptoethanol or dithiothreitol (DTT). In one embodiment, the compound comprising at least two anionic groups is a multivalent anionic compound. In another embodiment, the compound comprising at least two anionic groups is a non-polymeric compound.

  Yet another aspect is a kit for isolating nucleic acids from a sample comprising an anion exchange material and an anionic compound comprising at least two anionic groups.

In another aspect, a kit is provided for amplifying a nucleic acid comprising a polymerase, an anion exchange material, and an anionic compound that includes at least two anionic groups. In another embodiment, the amplification composition comprises a source thereof such as Mg ²⁺ or MgCl ₂ ; a ribonucleotide triphosphate (NTP); a deoxyribonucleotide triphosphate (dNTP); a supply thereof such as K ⁺ or KCl. Source; its source such as NH ₄ ⁺ or (NH ₄ ) ₂ SO ₄ ; buffer such as Tris; and / or optionally a reducing agent such as 2-mercaptoethanol or dithiothreitol (DTT) . In one embodiment, the compound comprising at least two anionic groups is a multivalent anionic compound. In another embodiment, the compound comprising at least two anionic groups is a non-polymeric compound.

  In a further aspect, there is provided the use of an anionic compound comprising at least two anionic groups to displace nucleic acid reversibly bound to an anion exchange material from the anion exchange material. In one embodiment, the compound comprising at least two anionic groups is a multivalent anionic compound. In another embodiment, the compound comprising at least two anionic groups is a non-polymeric compound.

（図２）結合し溶出した核酸のゲル電気泳動分析を示す。プラスミドDNAを、ポリエチレンイミン磁性ビーズに結合させ、デキストラン硫酸、ポリアクリル酸、シュウ酸、メリト酸、またはゲノムDNAを使用して溶出させた。A：pH8.0での結合および溶出。B：pH8.5での結合および溶出。ゲルは以下の通りに負荷された。

（図３）結合し溶出した核酸のゲル電気泳動分析を示す。プラスミドDNAを、スペルミン磁性ビーズに結合させ、カルボキシメチルデキストラン、デキストラン硫酸、ポリアクリル酸、ポリ(4-スチレンスルホン酸マレイン酸)、酢酸、シュウ酸、クエン酸、ピロメリト酸、またはメリト酸を使用して溶出させた。ゲルは以下の通りに負荷された。

（図４）結合し溶出した核酸のゲル電気泳動分析を示す。プラスミドDNAを、スペルミン磁性ビーズまたはポリエチレンイミン磁性ビーズに結合させ、「無塩基」DNAを使用して溶出させた。ゲルは以下の通りに負荷された。

（図５）PCR阻害アッセイ法を示す。βアクチンPCRのct値に対する異なるカルボン酸の影響を試験した。
（図６）PCR阻害アッセイ法を示す。βアクチンPCRのct値に対する異なるクエン酸濃度の影響を試験した。
（図７）結合し溶出した核酸のゲル電気泳動分析を示す。100bp DNA断片を、スペルミン磁性ビーズに結合させ、500bpまたは1000bp DNA断片を使用して溶出させた。ゲルは以下の通りに負荷された。

（図８）結合し溶出した核酸のゲル電気泳動分析を示す。500bpまたは1000bp DNA断片を、スペルミン磁性ビーズに結合させ、200bp DNA断片を使用して溶出させた。ゲルは以下の通りに負荷された。

（図９）結合し溶出した核酸のゲル電気泳動分析を示す。500bp DNA断片を、スペルミン磁性ビーズに結合させ、RNAを使用して溶出させた。ゲルは以下の通りに負荷された。

（図１０）結合し溶出した核酸のゲル電気泳動分析を示す。RNAを、スペルミン磁性ビーズに結合させ、500bp DNA断片を使用して溶出させた。ゲルは以下の通りに負荷された。

（図１１）結合し溶出した核酸のゲル電気泳動分析を示す。ゲノムDNAを、スペルミン磁性ビーズに結合させ、プラスミドDNAを使用して溶出させた。ゲルは以下の通りに負荷された。

（図１２）結合し溶出した核酸のゲル電気泳動分析を示す。プラスミドDNAを、スペルミン磁性ビーズに結合させ、ゲノムDNAを使用して溶出させた。ゲルは以下の通りに負荷された。

（図１３）結合し溶出した核酸のゲル電気泳動分析を示す。siRNAを、ポリエチレンイミン磁性ビーズに結合させ、300bp DNA断片を使用して溶出させた。ゲルは以下の通りに負荷された。

（図１４）PCRの増幅曲線が正常であり、PAAがPCRにおけるアーティファクト形成を促進しないことを示す。図14A〜14Bは、TaqMan（登録商標）-MGBプローブを使用して閾値サイクル（Ct）を検出した実験の結果を示す。図14Cは、PCRにおける産物同一性を確認するため融解曲線を記録した実験の結果を提供する。実施例15を参照のこと。
（図１５）異なる溶出：AXpH（商標）ビーズ、アルカリ、またはPAAを比較している、様々な濃度の淋菌（Neisseria Gonorrhoeae）(NG) DNAのリアルタイムPCRの結果を提供する。
（図１６）PCRに対する陰イオン交換材料の阻害効果を証明する。ビーズの非存在下（図16A）、または1：10（図16A）、1：100（図16B）、および1：1000（図16B）希釈のAXpH（商標）ビーズの存在下で、リアルタイムPCR増幅を3通り実施した。Brandis,Dye structure affects Taq DNA polymerase terminator selectivity,Nucl.Acids Res.1999 27(8):1912-1918に記載されたようなカルボキシフルオレセインの異性体である6-FAM（商標）（「FAM」）色素により標識されたレポータープローブを使用して、蛍光シグナルを生成した。阻害は、増幅曲線の直線相の右方シフトにより示される。
（図１７）ウシ血清アルブミンが陰イオン交換材料の存在下でPCRを救済しないことを証明する。1：50希釈のAXpH（商標）ビーズ、ならびに0、1：1、および1：10希釈の1mg/mlストックウシ血清アルブミン（最終濃度0.1mg/mlおよび0.01mg/mlのBSA）の存在下で、リアルタイムPCR増幅を3通り実施した。FAM（上の曲線）または5'-テトラクロロ-フルオレセイン（「TET」）色素（下の曲線）のいずれかにより標識されたレポータープローブを使用して、蛍光シグナルを生成した。
（図１８）担体DNAの添加が陰イオン交換ビーズキャリーオーバーによるPCR反応の阻害を逆転させることを証明する。1：50希釈のAXpH（商標）ビーズ、および0または100ng/mlの担体DNAの存在下で、リアルタイムPCR増幅を3通り実施した。FAM（上の曲線）またはTET（下の曲線）のいずれかにより標識されたレポータープローブを使用して、蛍光シグナルを生成した。見られるように、担体DNAは、FAMにより生成されるシグナルを消光させるようであった。しかしながら、TET色素の使用は、担体DNAがAXpH（商標）ビーズの存在の阻害効果を逆転させることを示した。
（図１９）ポリアクリル酸（「PAA」）も陰イオン交換材料の存在下でPCRを救済することを証明する。25ng/ml PAAの存在下（「PAA+」）または非存在下（「PAA-」）で、1：200（図19A）、1：250（図19A）、1：400（図19B）、および1：500（図19B）希釈のAXpH（商標）ビーズの存在下で、リアルタイムPCR増幅を3通り実施した。FAM（上の曲線）またはTET（下の曲線）のいずれかにより標識されたレポータープローブを使用して、蛍光シグナルを生成した。
（図２０）PAAの救済効果が、高い陰イオン交換濃度の存在下で、低い標的濃度についても維持されることを証明する。1：25希釈のAXpH（商標）ビーズ、および0または25ng/mL PAAの存在下で、10、103、および105コピーの標的核酸に対して、リアルタイムPCR増幅を3通り実施した。FAMまたは5'-テトラクロロ-フルオレセイン色素（「TET」）のいずれかにより標識されたレポータープローブを使用して、蛍光シグナルを生成した。見られるように、対照においては三つの区別される曲線が存在するが（図20A、上の曲線）、それらは、最高濃度の標的核酸を除き、AXpH（商標）ビーズの添加により消失した（図20A、下の曲線）。PAAの添加はこの効果を逆転させた（図20B）。
（図２１）tHDAに対する陰イオン交換材料の阻害効果を証明し、増幅がPAAにより救済され得ることを示す。1：25希釈のAXpH（商標）ビーズ、および0または25ng/mL PAAの存在下で、10、10³、および10⁵コピーの標的核酸に対して、NG DNAのリアルタイム増幅を3通り実施した。標識されたレポータープローブを使用して、蛍光シグナルを生成した。図21Aは対照tHDA活性（PAAもビーズもなし）を証明し、図21Bはビーズの存在下でのtHDA活性を証明し、図21CはビーズおよびPAAの存在下でのtHDA活性を証明する。
（図２２）高分子量PAA（PAA-H）、低分子量PAA（PAA-L）、またはポリメタクリル酸（「+PMA」）の存在が、AXpH（商標）ビーズの存在下での標的NG DNAの増幅を可能にすることを証明する。TET（上の曲線）またはFAM（下の曲線）のいずれかにより標識されたTaqMan（登録商標）プローブを使用して、全ての例について、標準的な3工程リアルタイムPCR反応を3通り実施した。全ての増幅を2.5mM MgCl₂の存在下で実施した。図22A：1は、洗浄緩衝液（10mMトリス（pH8）；0.1％NP-40 alternative）において標的核酸を利用した陽性増幅対照であり；2は、AXpH（商標）ビーズの存在下で洗浄緩衝液において標的核酸を利用したビーズ対照である。図22B：3は、洗浄緩衝液＋PAA-H（0.25％）およびAXpH（商標）ビーズにおいて増幅された標的であり；4は、洗浄緩衝液＋PAA-L（0.25％）およびAXpH（商標）ビーズにおいて増幅された標的である。図22C：5は、洗浄緩衝液＋PMA（0.25％）およびAXpH（商標）ビーズにおいて増幅された標的である。
（図２３）ポリグルタミン酸（「PGA」）の存在が、陰イオン交換材料の存在下でのPCR増幅を可能にすることを証明する。TaqMan（登録商標）プローブを使用して、全ての例について、NG DNAに対して、標準的な3工程リアルタイムPCR反応を3通り実施した。B7-9は、標的核酸およびAXpH（商標）ビーズの両方の非存在下で洗浄緩衝液（10mMトリス（pH8）；0.1％NP-40 alternative）を使用した絶対陰性対照である。Aの曲線は、単独の洗浄緩衝液または洗浄緩衝液＋1.25％PGA、1.25％ポリアデニル酸（「ポリA」）、または1.25％カルボキシメチルデキストラン（「CMD」）のいずれかに溶解させた標的核酸を使用した陽性増幅対照である。見られるように、多価陰イオン性化合物は、いずれも、標的の増幅に有意に影響を与えなかった。B4-6は、標的の増幅に対する陰イオン交換材料の効果を証明するためのビーズ対照である。E4-6は、1.25％CMDおよびAXpH（商標）ビーズの存在下での標的の増幅を示す。D4-6は、1.25％PGAおよびAXpH（商標）ビーズの存在下での標的の増幅を示す。左のプロットは微分曲線として表されたデータを示す。右のプロットは生データを示す。
（図２４）PAAがPCRを阻害し、それが、Mg²⁺の濃度の増加により補正され得ることを証明する。標的NG DNAを、二つの型の陰イオン交換材料（それぞれBCDおよびEFG）、ならびに3、7、または11mM Mg²⁺の存在下で、0.05または0.1％いずれかのPAAの存在下で増幅した。図に見られるように、Mg²⁺の濃度の増加は、PAAの阻害効果を逆転させた。 FIG. 1 shows gel electrophoresis analysis of bound and eluted nucleic acids. The 300 bp DNA fragment was bound to diethylaminopropyl magnetic beads and eluted using the 500 bp DNA fragment. The gel was loaded as follows.

FIG. 2 shows gel electrophoresis analysis of bound and eluted nucleic acid. Plasmid DNA was bound to polyethyleneimine magnetic beads and eluted using dextran sulfate, polyacrylic acid, oxalic acid, melittic acid, or genomic DNA. A: Binding and elution at pH 8.0. B: Binding and elution at pH 8.5. The gel was loaded as follows.

FIG. 3 shows gel electrophoresis analysis of bound and eluted nucleic acid. Plasmid DNA is bound to spermine magnetic beads and using carboxymethyl dextran, dextran sulfate, polyacrylic acid, poly (4-styrenesulfonic acid maleic acid), acetic acid, oxalic acid, citric acid, pyromellitic acid, or melittic acid. And eluted. The gel was loaded as follows.

FIG. 4 shows gel electrophoresis analysis of bound and eluted nucleic acids. Plasmid DNA was bound to spermine or polyethyleneimine magnetic beads and eluted using “basic” DNA. The gel was loaded as follows.

FIG. 5 shows a PCR inhibition assay. The effect of different carboxylic acids on the ct value of β-actin PCR was tested.
FIG. 6 shows a PCR inhibition assay. The effect of different citrate concentrations on the ct value of β-actin PCR was tested.
FIG. 7 shows gel electrophoresis analysis of bound and eluted nucleic acid. A 100 bp DNA fragment was bound to spermine magnetic beads and eluted using a 500 bp or 1000 bp DNA fragment. The gel was loaded as follows.

FIG. 8 shows gel electrophoresis analysis of bound and eluted nucleic acid. 500 bp or 1000 bp DNA fragments were bound to spermine magnetic beads and eluted using 200 bp DNA fragments. The gel was loaded as follows.

FIG. 9 shows gel electrophoresis analysis of bound and eluted nucleic acid. The 500 bp DNA fragment was bound to spermine magnetic beads and eluted using RNA. The gel was loaded as follows.

FIG. 10 shows gel electrophoresis analysis of bound and eluted nucleic acid. RNA was bound to spermine magnetic beads and eluted using a 500 bp DNA fragment. The gel was loaded as follows.

FIG. 11 shows gel electrophoresis analysis of bound and eluted nucleic acid. Genomic DNA was bound to spermine magnetic beads and eluted using plasmid DNA. The gel was loaded as follows.

FIG. 12 shows gel electrophoresis analysis of bound and eluted nucleic acid. Plasmid DNA was bound to spermine magnetic beads and eluted using genomic DNA. The gel was loaded as follows.

FIG. 13 shows gel electrophoresis analysis of bound and eluted nucleic acids. siRNA was bound to polyethyleneimine magnetic beads and eluted using a 300 bp DNA fragment. The gel was loaded as follows.

(FIG. 14) The PCR amplification curve is normal, showing that PAA does not promote artifact formation in PCR. FIGS. 14A-14B show the results of experiments where a threshold cycle (Ct) was detected using the TaqMan®-MGB probe. FIG. 14C provides the results of experiments where melting curves were recorded to confirm product identity in PCR. See Example 15.
(FIG. 15) Different elutions provide real-time PCR results of various concentrations of Neisseria Gonorrhoeae (NG) DNA comparing AXpH ™ beads, alkali, or PAA.
FIG. 16 demonstrates the inhibitory effect of anion exchange material on PCR. Real-time PCR amplification in the absence of beads (Figure 16A) or in the presence of 1:10 (Figure 16A), 1: 100 (Figure 16B), and 1: 1000 (Figure 16B) dilutions of AXpH ™ beads Was carried out in three ways. 6-FAM ™ (“FAM”) dye which is an isomer of carboxyfluorescein as described in Brandis, Dye structure affects Taq DNA polymerase terminator selectivity, Nucl. Acids Res. 1999 27 (8): 1912-1918 A fluorescent signal was generated using a reporter probe labeled with. Inhibition is indicated by a right shift of the linear phase of the amplification curve.
FIG. 17 demonstrates that bovine serum albumin does not rescue PCR in the presence of anion exchange material. In the presence of 1:50 dilution of AXpH ™ beads and 0, 1: 1, and 1:10 dilutions of 1 mg / ml stock bovine serum albumin (final concentrations 0.1 mg / ml and 0.01 mg / ml BSA) Real-time PCR amplification was performed in triplicate. A fluorescent signal was generated using a reporter probe labeled with either FAM (upper curve) or 5′-tetrachloro-fluorescein (“TET”) dye (lower curve).
FIG. 18 demonstrates that the addition of carrier DNA reverses the inhibition of the PCR reaction by anion exchange bead carryover. Real-time PCR amplification was performed in triplicate in the presence of 1:50 dilution of AXpH ™ beads and 0 or 100 ng / ml carrier DNA. Fluorescent signals were generated using reporter probes labeled with either FAM (upper curve) or TET (lower curve). As can be seen, the carrier DNA appeared to quench the signal produced by FAM. However, the use of TET dye has shown that carrier DNA reverses the inhibitory effect of the presence of AXpH ™ beads.
(FIG. 19) Polyacrylic acid (“PAA”) also proves to rescue PCR in the presence of anion exchange material. 1: 200 (Figure 19A), 1: 250 (Figure 19A), 1: 400 (Figure 19B), and 1 in the presence ("PAA +") or absence ("PAA-") of 25ng / ml PAA : Real-time PCR amplification was performed in triplicate in the presence of 500 (FIG. 19B) dilution of AXpH ™ beads. Fluorescent signals were generated using reporter probes labeled with either FAM (upper curve) or TET (lower curve).
FIG. 20 demonstrates that the rescue effect of PAA is maintained even at low target concentrations in the presence of high anion exchange concentrations. Three real-time PCR amplifications were performed on 10, 103, and 105 copies of target nucleic acid in the presence of 1:25 dilution of AXpH ™ beads and 0 or 25 ng / mL PAA. A fluorescent signal was generated using a reporter probe labeled with either FAM or 5′-tetrachloro-fluorescein dye (“TET”). As can be seen, there are three distinct curves in the control (FIG. 20A, upper curve), which disappeared with the addition of AXpH ™ beads, except for the highest concentration of target nucleic acid (FIG. 20A, lower curve). The addition of PAA reversed this effect (Figure 20B).
FIG. 21 demonstrates the inhibitory effect of anion exchange material on tHDA and shows that amplification can be rescued by PAA. Three times real-time amplification of NG DNA was performed on 10, 10 ³ , and 10 ⁵ copies of target nucleic acid in the presence of 1:25 dilution of AXpH ™ beads and 0 or 25 ng / mL PAA. A labeled reporter probe was used to generate a fluorescent signal. FIG. 21A demonstrates control tHDA activity (no PAA or beads), FIG. 21B demonstrates tHDA activity in the presence of beads, and FIG. 21C demonstrates tHDA activity in the presence of beads and PAA.
(FIG. 22) The presence of high molecular weight PAA (PAA-H), low molecular weight PAA (PAA-L), or polymethacrylic acid (“+ PMA”) indicates the presence of target NG DNA in the presence of AXpH ™ beads. Prove that it enables amplification. A standard three-step real-time PCR reaction was performed in triplicate for all examples using TaqMan® probes labeled with either TET (upper curve) or FAM (lower curve). All amplification was carried out in the presence of 2.5 mM MgCl _2. Figure 22A: 1 is a positive amplification control utilizing target nucleic acid in wash buffer (10 mM Tris (pH 8); 0.1% NP-40 alternative); 2 is wash buffer in the presence of AXpH ™ beads 2 is a bead control utilizing a target nucleic acid. Figure 22B: 3 is the target amplified in wash buffer + PAA-H (0.25%) and AXpH ™ beads; 4 is in wash buffer + PAA-L (0.25%) and AXpH ™ beads Amplified target. FIG. 22C: 5 is the target amplified in wash buffer + PMA (0.25%) and AXpH ™ beads.
FIG. 23 demonstrates that the presence of polyglutamic acid (“PGA”) allows PCR amplification in the presence of anion exchange material. A standard three-step real-time PCR reaction was performed in triplicate on NG DNA for all examples using the TaqMan® probe. B7-9 is an absolute negative control using wash buffer (10 mM Tris (pH 8); 0.1% NP-40 alternative) in the absence of both target nucleic acid and AXpH ™ beads. A curve shows target dissolved in either single wash buffer or wash buffer + 1.25% PGA, 1.25% polyadenylic acid ("Poly A"), or 1.25% carboxymethyldextran ("CMD") Positive amplification control using nucleic acids. As can be seen, none of the multivalent anionic compounds significantly affected target amplification. B4-6 is a bead control to demonstrate the effect of anion exchange material on target amplification. E4-6 shows target amplification in the presence of 1.25% CMD and AXpH ™ beads. D4-6 shows amplification of the target in the presence of 1.25% PGA and AXpH ™ beads. The left plot shows the data expressed as a differential curve. The right plot shows the raw data.
(FIG. 24) PAA inhibits PCR, demonstrating that it can be corrected by increasing the concentration of Mg ²⁺ . Target NG DNA was amplified in the presence of two types of anion exchange materials (BCD and EFG, respectively) and either 3, 7 or 11 mM Mg ^{2+ in} the presence of either 0.05 or 0.1% PAA. As can be seen in the figure, increasing the concentration of Mg ²⁺ reversed the inhibitory effect of PAA.

DETAILED DESCRIPTION OF THE INVENTION It has long been known that nucleic acids can be resorbably adsorbed to certain positively ionized materials due to the negatively ionized phosphate of the nucleic acid backbone. This property is frequently manipulated to purify nucleic acids from complex biological materials through a technique termed anion exchange. In a typical scenario, the solid phase is coated with a “capture component” to form an anion exchange material. Under appropriate ionic and pH conditions, the phosphate backbone of the nucleic acid retains a net negative charge and the capture component retains a net positive charge. Thus, the nucleic acid binds to the capture component, but not neutral or positively charged molecules such as proteins. The solid phase to which the nucleic acid is bound can then be separated from the rest by various means such as centrifugation or application of a magnetic field. The anion exchange material is then washed and, in a typical scheme, the nucleic acid is eluted in solution by modifying salt and / or pH conditions to form an eluate. The eluate can then be separated from the anion exchange material and used in subsequent analyses. A number of such methods have been previously described. These methods are clean, easy to perform, provide relatively high nucleic acid yields, and do not require the dangerous and expensive chemicals required by traditional chemical extraction and purification methods. However, they present some problems.

  For example, elution of nucleic acids from anion exchange materials typically requires some combination of pH and / or ionic strength manipulations to elute bound nucleic acids from the anion exchange material. Since the resulting eluate has a sub-optimal pH and / or increased ionic strength, nucleic acids eluted in this way generally cannot be used directly in analytical procedures such as PCR. Accordingly, it would be desirable to develop materials and methods that allow elution of nucleic acids from anion exchange materials at analytically relevant pH and ion concentrations.

  Accordingly, the present disclosure relates to materials, methods, and kits for isolating nucleic acids using an anion exchange material, wherein an anionic compound containing at least two anionic groups is added during the elution step. .

  In one aspect, (a) providing a nucleic acid-anion exchange complex; and (b) a solution comprising an anionic compound comprising at least two anionic groups, the nucleic acid-anion exchange complex. A method of eluting the nucleic acid from the anion exchange material is disclosed, comprising the step of adding the nucleic acid to the anion exchange material.

  As used herein, the verb “complexing” means that the positively ionized capture component of the anion exchange material is negative of either the phosphate backbone of the target nucleic acid or an anionic compound. A process that directly associates with ionized components. The noun “complex” shall refer to the chemical structure formed by such an association. The term “nucleic acid-anion exchange complex” is intended to refer to the chemical structure formed when a nucleic acid is complexed with a positively ionizable capture component of an anion exchange material.

  In one embodiment, the nucleic acid-anion exchange material (1) contacts the anion exchange material with a sample containing nucleic acid under conditions where a complex is formed between the anion exchange material and the nucleic acid. And (2) by a method comprising isolating the complex from the sample. In a further embodiment, the step of forming a complex between the anion exchange material and the nucleic acid comprises complexing the nucleic acid of interest with the anion exchange material, and contaminants such as proteins, endotoxins, and liposomes. It is carried out under conditions of pH, ionic strength, and / or surfactant so as not to form. The conditions may be further improved so that only certain nucleic acids form a complex with the anion exchange material.

  In another embodiment, the nucleic acid-anion exchange material comprises: (1) contacting a sample containing nucleic acid with the anion exchange material; (2) forming a complex between the anion exchange material and the nucleic acid; Supplied by a method comprising (3) isolating the complex from the sample, and (4) washing the complex to remove impurities. Wash conditions can be selected such that substantially all non-nucleic acid material is removed. Suitable wash buffers are known in the art and include water-containing solutions, alcohols, especially branched or unbranched alcohols having 1 to 5 carbon atoms such as ethanol or isopropanol, polyethylene glycol , Polypropylene glycol, acetone, carbohydrates, aqueous solutions containing salts, and mixtures of the above. For example, without limitation, the wash buffer may include 0.1% NP-40 in 0.1 mM Tris (pH 8.0). Washing conditions may be further improved so that certain nucleic acids are similarly removed as impurities.

  Another problem with nucleic acid isolation using anion exchange materials is that the eluate is often not completely separated from the anion exchange material and is commonly referred to as “bead carryover”. This results in an analysis sample contaminated. This is a problem because the presence of anion exchange material often interferes with subsequent analysis of nucleic acids. Furthermore, molecular biological analysis is increasingly automated. Ideally, it is desirable to combine both isolation and analytical methods into a single automated process involving as few steps and reagents as possible. Predictable and reliable automation of such processes is difficult due to the possibility that anion exchange materials can interfere with the analysis of nucleic acids. Accordingly, it would be desirable to develop materials and methods that allow analysis of nucleic acids in the presence of anion exchange materials.

  Accordingly, the present disclosure relates to materials, methods, and kits for analyzing nucleic acids in the presence of an anion exchange material and an anionic compound comprising at least two anionic groups. Reverses the inhibition of the analytical process caused by the anion exchange material.

  It should be understood that the analytical steps of the methods disclosed herein are for performing in the presence of an anion exchange material. The presence of anion exchange material is unintentional, as in the case of carryover in nucleic acid purification, or when nucleic acid is analyzed directly from an isolated nucleic acid-anion exchange complex. In addition, it may be intentional.

Nucleic acids The nucleic acids according to the present disclosure are not limited and include any nucleic acid. For example, without limitation, nucleic acids can be genomic DNA, mitochondrial DNA, bacterial DNA, viral DNA, plasmids, cosmids, linear oligodeoxynucleotides and polydeoxynucleotides, cDNA, PCR fragments, PCR amplicons, tHDA amplicons DNA including, but not limited to, LCR amplicons, long range PCR amplicons, oligonucleotides, primers, probes, artificial or synthetic DNA; mRNA, tRNA, rRNA, viral RNA, siRNA, miRNA, RNAi, linear oligonucleotides, linear polynucleotides, probes, RNA including but not limited to artificial or synthetic RNA; artificial nucleic acids such as PNA and LNA, and nucleic acids including both DNA and RNA, etc. Their combinations, as well as their hybrids such as RNA: DNA hybrids; nucleic acids and other life It may be a complex with a biological component. Nucleic acids may be single stranded or double stranded. It may contain modifications such as natural and artificial modifications, eg it may contain artificial nucleotides, including artificial bases, artificial sugar components, and / or artificial internucleotide linkages. Good.

  Nucleic acids can include nucleic acids found in specimens or cultures (eg, cell cultures, microbiological cultures, and viral cultures), including but not limited to biological samples and environmental samples. . Ribonucleic acid can be found in any biological sample derived from cell culture, bacteria, viruses, animals including humans, liquids, solids (eg, stool), or tissue samples. The target nucleic acid may further comprise a cervical sample (eg, a sample obtained from a cervical wipe specimen), a blood product such as adenoid cells, anal epithelial cells, blood, serum, plasma, or buffy coat, saliva, cerebrospinal fluid, pleural effusion Can be found in biological samples including, but not limited to, milk, lymph, sputum, urine, and semen.

  In other embodiments, the nucleic acids are other viruses, bacteria, mycobacteria, or malaria parasites such as cytomegalovirus (CMV), herpes, HIV, chlamydia, gonorrhea, Staphylococcus aureus, tubercolis, Sars coronavirus And / or from influenza.

  In one embodiment, the nucleic acid is human papillomavirus (HPV) and includes a genetic variant of HPV. Variants include polymorphs, mutants, derivatives, modified nucleic acids, modified nucleic acids, or other types of nucleic acids. In one embodiment, the nucleic acid is an HPV nucleic acid. In another embodiment, the HPV nucleic acid is high-risk HPV type HPV DNA and / or RNA. In another embodiment, the nucleic acid is a high risk HPV type, such as 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 26, 66, and / or 82. is there.

Samples containing nucleic acid Samples containing nucleic acid include specimens or cultures (eg, cell cultures, microbiological cultures, and virus cultures), including biological samples and environmental samples. However, it is not limited to these. Biological samples include cell cultures, bacteria, viruses, animals including humans, liquid, solid (eg, stool), or tissue samples, and liquid or solid food and feed, and dairy products, vegetables, meat and meat. It can come from any source, such as by-products, as well as elements such as waste. Environmental samples include, for example, environmental materials such as surface materials, soil, water, and industrial samples, and equipment, equipment, equipment, tools, disposable and non-disposable for food and dairy processing Samples obtained from supplies are also included. Exemplary biological samples include cervical epithelial cells (eg, samples obtained from cervical wipes), cell samples such as adenoid cells, anal epithelial cells, blood such as serum, plasma, or buffy coat. Examples include, but are not limited to, preparations, saliva, cerebrospinal fluid, pleural effusions, milk, lymph, sputum, and semen, and can be collected in, for example, Preservcyt, Surepath, and / or Digene Collection Medium (“DCM”). The sample can include deoxyribonucleic acid (DNA) and / or ribonucleic acid (RNA).

  If desired for any reason, the sample can be processed prior to contact with the anion exchange material. For example, a biological sample containing cells containing nucleic acids can be processed to lyse the cells such that the nucleic acids are released. The lysate containing nucleic acid can then be added to the anion exchange material. One skilled in the art will recognize desirable methods for processing samples containing nucleic acids prior to contacting the solution with the anion exchange material. For example, cells can be lysed with a suitable lysis buffer containing 2% Triton X-100, 0.2 M EDTA, 40 mM sodium citrate, 40 mM boric acid in 100 mM Tris HCl, pH 7.0, for example. If alkaline lysis buffer is used to prepare the sample, neutralize the pH of the sample to a pH at which nucleic acids can bind to the anion exchange material before contacting the sample with the anion exchange material. May need to.

  In other embodiments, the sample is other viruses, bacteria, mycobacteria, or malaria parasites such as cytomegalovirus (CMV), herpes, HIV, chlamydia, gonorrhea, Staphylococcus aureus, tubercolis, Sars coronavirus, or influenza The nucleic acid derived from can be included.

  In a further embodiment, the sample is treated such that the nucleic acid is “free”. As used herein, the phrase “free nucleic acid” refers to large molecules such as vesicles, liposomes, micelles, ribosomes, nuclei, mitochondria, viral capsids and / or envelopes, endosomes, or exosomes. It is shown that it is not associated with the polymer structure.

Anion Exchange Materials The present disclosure advantageously utilizes anion exchange materials. The terms “anion exchange material”, “anion exchange material”, “anion exchange matrix”, and “anion exchange resin” are used interchangeably herein, particularly in terms of resin in the chemical sense. It is not limited to certain materials. As used herein, the term “anion exchange material” refers to the removal of nucleic acid from solution through the formation of a complex between the phosphate backbone of the nucleic acid and the positively ionizable capture component of the material. It shall refer to any material that can be used for selective removal. A number of anion exchange materials, including, for example, those described in US Pat. No. 6,914,137, US Pat. No. 5,990,301, US20100009351A1, and EP 0 268 946 B1, the disclosures of which are incorporated herein by reference. Has been previously described and will be readily recognized by those skilled in the art. Any anion exchange material suitable for nucleic acid purification can be used.

  In one aspect, the anion exchange material includes a solid phase and a positively ionizable capture component.

  Silica, borosilicate, silicic acid, non-organic glass, poly (meth) acrylic acid, polyurethane, polystyrene, organic polymers such as agarose, polysaccharides such as cellulose, aluminum oxide, magnesium oxide, titanium oxide, and zirconium oxide Metal oxides, metals such as gold or platinum, agarose, sephadex, sepharose, polyacrylamide, divinylbenzene polymer, styrene divinylbenzene polymer, dextran, and derivatives thereof, and / or silica gel, beads, membranes, and resins Glass or silica surfaces such as beads, plates, and capillary tubes; polystyrene, agarose, polyacrylamide, dextra in which magnetic materials are incorporated or associated with magnetic materials; And / or anionic, including but not limited to magnetizable or magnetic (eg, paramagnetic, superparamagnetic, ferromagnetic, or ferrimagnetic) particles Any solid phase suitable for exchange chromatography can be used. In some embodiments, the capture component may be linked to the surface of a processing vessel such as a microtube, a well of a microplate, or a capillary, and these surfaces are used to isolate nucleic acids on a microscale. be able to. In one embodiment, the solid phase will be processed, manufactured, or otherwise processed so that the nucleic acid of interest does not bind directly to the solid phase in the purification step.

Anion exchange materials include, but are not limited to, materials that are modified with a positively ionizable capture component. Examples of such ionizable groups are monoamines, diamines, polyamines, and nitrogen-containing aromatic or aliphatic heterocyclic groups. In one embodiment, the positively ionizable capture component comprises at least one primary amino group, secondary amino group, or tertiary amino group. In another embodiment, the positively ionizable capture component is selected from the group consisting of a primary amine of formula R ₃ N, a secondary amine of formula R ₂ NH, and a tertiary amine X— (CH ₂ ) _n —Y. ,
Where
X is R ₂ N, RNH, or NH ₂ ,
Y is R ₂ N, RNH, or NH ₂
R is independently of each other preferably a linear, branched or cyclic alkyl substituent, alkenyl substituted, which may contain one or more heteroatoms selected from O, N, S and P A group, an alkynyl substituent or an aryl substituent, and
n is an integer in the range of 0 to 20, preferably 0 to 18.

  Exemplary capture components include aminomethyl (AM), aminoethyl (AE), aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl such as diethylaminoethyl (DEAE), ethylenediamine, diethylenetriamine, triethylenetetraamine, tetraethylene Pentaamine, pentaethylenehexaamine, trimethylamino (TMA), triethylaminoethyl (TEAE), linear or branched polyethyleneimine (PEI), carboxylated or hydroxyalkylated polyethyleneimine, jeffamine, Spermine, spermidine, 3- (propylamino) propylamine, polyamidoamine (PAMAM) dendrimer, polyallylamine, polyvinylamine, N-morpholinoethyl, polylysine, and tetraazacycloalka But it is not limited thereto.

  Biological buffer compounds such as those described in patents or applications, US Pat. No. 6,914,137 and EP 1 473 299 can also be used as functional groups of anion exchange materials. Further examples of positively ionizable groups include polyhydroxylated amines, surfactants, surfactants, heterocycles, dyes, negatively charged groups combined with metal ions or metal oxides, histidine, and poly It is histidine. Such groups are also described, for example, in patent application WO 2003/101494 and patent application EP 09 007 338.8. Zwitterionic groups such as amino acids or betaines may be used. In one embodiment, the anion exchange material comprises magnetic silica beads modified with spermine.

The solid phase can be functionalized for the attachment of functional capture components such as, for example, Si-O-Si, Si-OH, alcohols, diols or polyols, carboxylic acids, amines, phosphates, or phosphonates. Positively ionizable capture components can be incorporated into the solid phase by using, for example, epoxides, (activated) carboxylic acids, acid anhydrides, acid chlorides, formyl groups, tresyl groups, or pentafluorophenyl groups. Can be attached. The functional group can be directly attached to the solid phase or can be a hydrocarbon group such as a (linear or branched) spacer group, eg, a — (CH 2) _n — group, carbohydrate, polyethylene glycol, and polypropylene glycol. It may be attached via. Alternatively, polymers composed of monomers that contain capture components such as amino functional groups can be used as the anion exchange material.

  In some embodiments, ionizable groups may be linked to the surface of a processing vessel such as a microtube, microplate, or capillary, and these surfaces are used to isolate nucleic acids on a microscale. be able to.

  In further embodiments, the solid phase is processed, manufactured, or otherwise processed so that the nucleic acid of interest does not bind directly to the solid phase in the purification step.

  In one embodiment, PEI modified paramagnetic silica beads are used as anion exchange material. Exemplary magnetic silica beads modified with PEI include AXpH ™ beads (commercially available from Qiagen GmbH, Hilden Germany). AXpH ™ beads can be magnetically separated from wash buffers and eluates, which is easier than the filtration required when working with cellulose and other non-magnetic resins.

Nucleic acid binding Ion exchange processes known in the art typically involve two major steps: (1) binding nucleic acid to an anion exchange material at a pH that imparts a positive charge to the material; and (2) a salt. Elution and / or displacement of nucleic acids from the material by increasing the concentration and / or increasing the pH above the pKa of the capture component (“pH shift method”). In the present disclosure, a solution containing at least one compound containing at least two anionic groups is applied to the anion exchange material in order to elute the nucleic acid.

  It will be appreciated by those skilled in the art that the exact ionic and pH conditions necessary to cause complex formation and elution will necessarily depend on both the identity and concentration of both the capture component and the nucleic acid of interest. It will be. Methods for imparting charge to an anion exchange material in preparation for loading a nucleic acid are known to those skilled in the art.

  In one aspect, the positively ionizable capture component retains a first net positive charge at a first pH and ionic strength and a neutral charge or second at a second pH and ionic strength. Retains any of the second net positive charges lower than the one net positive charge, and thus the nucleic acid of interest binds to a positively ionizable capture component at the first pH and ionic strength However, at a second pH and ionic strength, it is released.

  In a further aspect, the positively ionizable capture component retains a first net positive charge at a first pH and ionic strength and a second net positive at a second pH and ionic strength. At the first pH and ionic strength, the first nucleic acid and the second nucleic acid bind to a positively ionizable capture component, and at the second pH and ionic strength, the first nucleic acid is positive. Is released from the ionizable capture component, but the second nucleic acid is not released.

  The binding of the nucleic acid to the anion exchange material is usually performed in an aqueous solution, preferably an aqueous solution containing a buffer substance. Suitable biological buffers are CHAPS, MES, HEPES, MOPS, TRIS, TRICINE, and PIPES. Further, the solutions are described in, for example, chaotropic salts such as sodium perchlorate, guanidinium hydrochloride, and guanidinium thiosulfate, as described in US Pat. No. 5,234,809, and / or, for example, in WO 2004/055207. May contain cosmotropic salts such as ammonium sulfate, zinc sulfate, potassium sulfate, and cobalt sulfate. The binding buffer may contain additional salts such as chlorides, sulfates, phosphates, acetates, formates, citrates, azides, and nitrates, alcohols, diols, triols, polyols, Polyethylene glycol, polypropylene glycol, acetone, acetonitrile, urea, guanidine, carbohydrates, and surfactants such as surfactants or surfactants, for example, additional organic compounds such as Tween, Triton, Brigi, Nonidet, or Pluronic You may contain. The salt is preferably present at a concentration of about 1 M or less, preferably 0.5 M or less, more preferably 250 mM or less, and most preferably 100 mM or less. Nucleic acid binding is preferably performed at a pH ranging from about 3 to about 10, preferably from about pH 5 to about 9.

  In addition, the binding of nucleic acids to anion exchange materials can be used to treat samples, such as lysis of cells or tissues in the sample, or digestion of specific compounds such as proteins, DNA, and / or RNA in the sample. They may be combined. For this purpose, the binding buffer is a solubilizing agent such as an enzyme, surfactant, chaotropic salt, or chelating agent (eg EDTA or NTA) and / or a digesting agent such as protease, DNase, and RNase. May further be included. However, these sample treatments may be performed in previous steps prior to binding of the nucleic acid to the anion exchange material (see also above).

  Pretreatment steps such as binding and lysis and / or enzyme pretreatment processes can be performed at elevated temperatures to accelerate each process.

  After adding the sample to the anion exchange material, it may be possible to wash the anion exchange material to remove unbound material in the sample, such as proteins, polysaccharides, and the like. Suitable wash buffers that can be used to remove non-target materials are known in the prior art, solutions containing water, alcohols, in particular branches with 1 to 5 carbon atoms such as ethanol or isopropanol. Including but not limited to type or unbranched alcohols, polyethylene glycols, polypropylene glycols, acetone, carbohydrates, aqueous solutions containing salts, and mixtures of the foregoing. An exemplary wash buffer includes, for example, 0.1% NP-40 in 0.1 mM Tris (pH 8.0).

After binding the eluted nucleic acid to the anion exchange material, the bound nucleic acid is eluted by adding a solution containing an anionic compound containing at least two anionic groups or a mixture of such anionic compounds Can be made.

1. Anionic compounds Anionic compounds displace nucleic acids from anion exchange materials. The method of the present invention does not need to include a change in pH to cause elution, particularly a pH change above the pKa of the capture component, in order to elute the nucleic acid from the anion exchange material to which the nucleic acid is bound. Rely on anionic compounds. Anionic compounds are able to displace nucleic acids from anion exchange materials at relatively low concentrations because of their high selectivity for their own materials.

  The optimal concentration of anionic compound used in the elution should be determined for the particular application; this concentration is high enough to provide high recovery of the nucleic acid, but the subsequent operation and process It should not be too expensive to damage. Anionic compounds can be used at varying concentrations necessary to elute the nucleic acid. For example, the concentration can be from 0.1% to about 2.0%, and in certain embodiments, the concentration is from about 0.5% to about 1.0%. It is understood that the numerical values described herein include all values from lower values to higher values (including lower and higher values). For example, all possible combinations of numerical values between the lowest and highest values listed should be considered explicit in this application. For example, for concentration ranges marked as 0.025% to about 2.5%, values such as 0.05, 0.2, 0.3, 0.4, 1.8, 1.9, etc., or within this range such as 0.3-1.0 or 0.4-2.4, etc. Arbitrary ranges shall be specified herein. These low concentrations have little or no effect on subsequent detection / analysis steps such as amplification or enzymatic reactions. Thus, the eluate can be used directly without the neutralization step typically required when using pH based elution or the desalting step when using salt buffer based elution.

  As used herein, “anionic compound” refers to any compound having a net negative charge at the pH and salt conditions used to elute or analyze nucleic acids. As expected.

  As used herein, the phrase “anionic group” refers to an anion that retains a net negative charge at the pH and salt conditions used to elute or analyze nucleic acids. It shall refer to any functional group covalently bonded to the ionic compound. In all cases, each anionic group of each anionic compound may be the same or different.

  Suitable anionic compounds include multivalent anionic compounds and non-polymeric anionic compounds. Preferably the anionic compound is organic.

  Suitable non-polymeric anionic compounds useful in the methods and compositions disclosed herein include, for example, any non-polymeric organic compound that includes at least two anionic groups. For example, without limitation, the non-polymeric anionic compound may comprise at least 2, at least 3, at least 4, at least 5, or at least 6 anionic groups. In another embodiment, the non-polymeric anionic compound comprises 2 to 6 or 3 to 6 anionic groups. In another embodiment, the non-polymeric anionic compound has no more than 15 anionic groups, no more than 10 anionic groups, no more than 8 anionic groups, or no more than 6. It may contain an anionic group. The non-polymeric organic anionic compound preferably has 2 to 40 carbon atoms, more preferably 2 to 20 carbon atoms, still more preferably 2 to 12 carbon atoms. It can be linear, branched, or cyclic and can be aliphatic (saturated or unsaturated) or aromatic (having one or more conjugated or fused rings).

  In one embodiment, the at least two anionic groups are selected from the group consisting of carboxylic acid groups, sulfonic acid groups, phosphonic acid groups, phosphoric acid groups, carbonic acid groups, and combinations thereof.

  As used herein, the term “acidic group” is intended to encompass both the referenced acid or its conjugate base.

  In one embodiment, the at least two anionic groups are selected from the group consisting of carboxylic acid groups, sulfonic acid groups, phosphonic acid groups, and ionized phosphoric acid groups. In another embodiment, the anionic group is a carboxylic acid / carboxylate group. In another embodiment, the non-polymeric anionic compound is from the group consisting of dicarboxylic acid, tricarboxylic acid, tetracarboxylic acid, pentacarboxylic acid, hexacarboxylic acid, heptacarboxylic acid, octacarboxylic acid, nonacarboxylic acid, and decacarboxylic acid. Selected.

  In another embodiment, the non-polymeric compound comprising at least two anionic groups is oxalic acid, fumaric acid, glutaric acid, maleic acid, malic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelin Acid, suberic acid, azelaic acid, tartronic acid, tartaric acid, citric acid, isocitric acid, citraconic acid, mesaconic acid, itaconic acid, aconitic acid, propane-1,2,3-tricarboxylic acid, aconitic acid, butane-1,2 , 3,4-tetracarboxylic acid, triethyl-1,1,2-ethanetricarboxylic acid, cyclopropanedicarboxylic acid, cyclobutanedicarboxylic acid, cyclobutanetricarboxylic acid, cyclopentanedicarboxylic acid, cyclohexanedicarboxylic acid, cyclohexanetricarboxylic acid, cyclohexanetetracarboxylic Acid, cyclohexane hexacarboxylic acid, cyclooctane di Rubonic acid, phthalic acid, isophthalic acid, terephthalic acid, hemimellitic acid, trimellitic acid, trimesic acid, merophanic acid, prenitic acid, pyromellitic acid, benzenepentacarboxylic acid, and mellitic acid, dierythritol hexacarboxylic acid, trierythritol eight Acetic acid, iminodiacetic acid, nitrilotriacetic acid, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, α-ketoglutaric acid, glutamic acid, aspartic acid, dicarboxymalonic acid, (18-crown-6) -2,3,11,12-tetracarboxylic Carboxylic acids such as 2-10, preferably 2-6 oligomers, of polymerizable, condensable acidic monomers such as acid, acrylic acid, methacrylic acid or vinyl acetic acid.

  In another embodiment, the non-polymeric compound comprising at least two anionic groups is an organic sulfonic acid such as methyl disulfonic acid, ethyl disulfonic acid, benzene disulfonic acid, phenol disulfonic acid, or naphthalene disulfonic acid .

  In another embodiment, the non-polymeric compound comprising at least two anionic groups is an organic phosphonic acid such as hydroxyethanediphosphonic acid.

  In another embodiment, the non-polymeric compound comprising at least two anionic groups is an organophosphate.

  In another embodiment, the non-polymeric compound comprising at least two anionic groups is a carbonate.

  In another embodiment, the non-polymeric compound comprising at least two anionic groups is diacetylacetone.

  In another embodiment, the non-polymeric anionic compound is selected from the group consisting of oxalic acid, mellitic acid, pyromellitic acid, and citric acid.

  The non-polymeric anionic compound may be substituted as long as the substituent does not interfere with and inhibit the ability of the compound to displace the nucleic acid from the anion exchange material. Exemplary substituents include, for example, halogen atoms such as fluorine, chloride, bromide, or iodine, and hydroxyl groups, oxy groups, aldehyde groups, keto groups, alkoxy groups, ether groups, ester groups, amino groups, thiols. Groups, thioether groups, thioester groups, linear or branched alkyl groups, alkenyl groups, alkynyl groups, saturated or unsaturated cycloalkyl groups, and aryl groups. In one embodiment, the anionic compound contains 1 to 6 substituents or 1 to 3 substituents. In another embodiment, these substituents, when present, contain no more than 20, 10, 8, 6, 4, or 2 carbon atoms.

  In one embodiment, the non-polymeric anionic compound is a low molecular weight compound. In another embodiment, the non-polymeric anionic compound has a molecular weight of 5,000 Da or less. In another embodiment, the non-polymeric anionic compound has a molecular weight of 3,000 Da or less. In another embodiment, the non-polymeric anionic compound has a molecular weight of 2,000 Da or less.

  Non-polymeric anionic compounds having at least two anionic groups have the advantage that only a relatively low concentration of anionic compounds is necessary to effectively elute the bound nucleic acids. . Furthermore, the presence of non-polymeric anionic compounds does not interfere with or inhibit subsequent amplification reactions, even at fairly high concentrations.

  In another aspect, the anionic compound comprising at least two anionic groups is a multivalent anionic compound. As used herein, the phrase “multivalent anionic compound” shall refer to a polymeric anionic compound.

  In one embodiment, the polyanionic compound is a polymerized unsaturated carboxylic acid (eg, acrylic acid, methacrylic acid, maleic acid, etc.) or a copolymer of these acids with other monomers such as acrylamide or acrylonitrile. Acidic polypeptides such as polyglutamic acid or polyaspartic acid, or copolymers of acidic polypeptides with other amino acids; modified dextran and other modified polyanionic polycarbohydrates or covalently ionized Multivalent anionic polycarbohydrates that retain a group such as carboxymethyl dextran, dextran sulfate, and dextran phosphate, or mixtures thereof; polystyrene having an anionic group such as polystyrene sulfonate; And other analogs such as double-stranded or single-stranded DNA or RNA, or analogs thereof such as “basic” nucleic acids (nucleic acids that contain only a backbone structure and no base attached to a sugar moiety) Selected from the group consisting of In another embodiment, polyacrylic acid (PAA), polymethacrylic acid (PMA), polyglutamic acid (PGA), and / or dextran sulfate (DS) are selected. In another embodiment, “low molecular weight” polyacrylic acid (weight average Fw about 5,100; approximately 70 residues) is selected. In another embodiment, “high molecular weight” polyacrylic acid (weight average Fw> 200,000; ie, longer than 2,800 residues) is selected.

  In one embodiment, the multivalent anionic compound is a second nucleic acid. When using a second nucleic acid to elute the bound first nucleic acid, the second nucleic acid should not interfere with subsequent processes (eg, if the next step is PCR analysis, the RNA is DNA Can be used to elute The second nucleic acid preferably comprises at least 100 base pairs (bp), more preferably at least 200 bp, at least 500 bp, or at least 1000 bp. It may be, for example, plasmid DNA or genomic DNA, or polydeoxyadenosine (“poly dA”), polydeoxythymidine (“poly dT”), or a copolymer of polydeoxyadenosine and polydeoxythymidine (“poly dA: dT”). Or a carrier nucleic acid such as

  Anionic compounds can be added in the methods disclosed herein as either free acids or salts. Suitable cations for use in such salts are any alkaline cations such as, for example, sodium and lithium.

2. Elution Elution of bound nucleic acid from an anion exchange material is accomplished using an anionic compound comprising at least two anionic groups as defined herein. Preferably, elution is performed using an elution buffer containing an anionic compound. The elution buffer is preferably an aqueous solution, which further contains, for example, a buffer, eg, a buffer, an organic component, and / or a salt as described above for the binding solution. It may be. In one embodiment, the pH used for elution is preferably in the range of about 5 to about 13, about 5 to about 9.5, or about 5 to about 8.5. In another embodiment, the pH is in the range of about 8.2 to about 9.0, especially when the eluate is envisioned to be used directly in an amplification reaction such as a PCR, RT-PCR, or isothermal amplification reaction. It is in.

  The elution of nucleic acids from the anion exchange material can be performed without the need for severe changes in pH. In one embodiment, the pH in the elution step does not impart a neutral or negative charge to the anion exchange material. In another embodiment, the pH at the elution does not significantly reduce the positive charge of the anion exchange material. In yet another embodiment, the pH in the elution step is not above the pKa of the anion exchange material and / or its capture component.

  In another embodiment, the solution used to elute the nucleic acid from the anion exchange material does not contain a high salt concentration. In another embodiment, the total salt concentration in the elution solution does not exceed 1 M, 0.5 M or less, 300 mM or less, 200 mM or less, 150 mM or less, 100 mM or less, 50 mM or less, Or 30 mM or less. In another embodiment, the elution solution does not contain any salts, except for anionic compounds, which contain at least two anionic groups.

  Preferably, the nucleic acid in the biological sample is first bound to an anion exchange material, such as an anion exchange magnetic bead. The material is then washed to remove proteins and other undesirable impurities. The nucleic acid is then eluted with a solution containing at least one anionic compound containing at least two anionic groups as defined herein. This approach does not require a change in pH or at least a severe change for the elution step. Indeed, the pH of the wash buffer and elution buffer can be the same.

  During elution, the anionic compound is present at a concentration high enough to result in elution of at least a portion of the bound nucleic acid. In one embodiment, the anionic compound is present at a concentration high enough to result in a majority elution of bound nucleic acid. In one embodiment, the anionic compound is present at a concentration high enough to result in substantially all elution of bound nucleic acid. By way of example only, the concentration of the non-polymeric anionic compound during elution is about 1 mg / l to about 1 g / l, more preferably about 10 mg / l to about 500 mg / l, more preferably about 20 mg. It is selected within the range of / l to about 100 mg / l. As another example, the amount of the polyanionic compound is about 0.01% to about 5%, more preferably about 0.025% to about 2.5%, more preferably about 0.1% to about 2.0% or about 0.5%. Within the range of ~ 1.0%.

  The optimal concentration of anionic compound used in elution should be determined for any particular application. That is, the concentration is preferably high enough to provide high recovery of the nucleic acid, but should not be too high to impair subsequent operations and processes. On the other hand, if the anionic compound used in the nucleic acid isolation step reduces the efficiency of subsequent procedures such as amplification, such negative effects can be overcome by specific adjustment of the conditions.

For example, DNA elution from AXpH beads, a specific magnetic silica bead bearing a polyethyleneimine group available from QIAGEN, Germany, was performed using 1% polyacrylic acid as an example of a polyvalent anionic compound. If it did, it completely inhibited PCR when the eluate constituted one-tenth of the PCR volume. However, increasing the MgCl ₂ concentration from 5 mM to 11 mM completely restored the PCR efficiency. Thus, in certain embodiments, after isolation, the isolated nucleic acid can be amplified in the presence of Mg ²⁺ at a concentration of about 10 to about 40 mM. Multivalent anionic compounds can be used at varying concentrations necessary to elute nucleic acids. For example, the concentration can advantageously be 0.1% to about 2.0%, and in certain embodiments, the concentration is about 0.5% to about 1.0%. It is understood that any numerical value described herein includes all values from lower values to higher values (including lower and higher values). For example, all possible combinations of numerical values between the lowest and highest values listed should be considered explicit in this application. For example, for concentration ranges marked as 0.025% to about 2.5%, values such as 0.05, 0.2, 0.3, 0.4, 1.8, 1.9, etc., or within this range such as 0.3-1.0 or 0.4-2.4, etc. Arbitrary ranges shall be specified herein. These low concentrations have little or no effect on subsequent detection / analysis steps such as amplification or enzymatic reactions. Thus, the eluate can be obtained directly without neutralization (typically required when using pH-based elution) and / or desalting steps (when using salt buffer-based elution). Can be used.

  For example, PAA elutes DNA from anion exchange magnetic beads at a concentration that does not significantly affect isothermal amplification such as PCR or tHDA (thermophilic helicase dependent amplification). The concentration of PAA is typically 0.025 to 2.5%, but is not limited thereto. In one embodiment, PAA is present at a concentration of 0.1% to 2.0%, in another embodiment, more preferably 0.5 to 1.0%, the exact concentration also depending on the volume of sample included in the PCR. For example, higher PAA concentrations provide better nucleic acid recovery from AXpH beads, but tend to slow amplification somewhat. Thus, if higher PAA concentrations are used for elution, it may be preferable to dilute the sample in the downstream reaction or use the sample in a larger overall volume in the downstream reaction. As an example, when DNA is eluted with 1% PAA, loading 8 μL of such eluate per 25 μL of PCR reaction can lead to poor results. However, if the volume of the eluate is smaller (eg 2 μL of such eluate per 25 μL of PCR) or if the total PCR volume is higher (eg 100 μL of PCR reaction is loaded with 8 μL of eluate) The eluate will give the expected results. Therefore, the concentration of PAA in the elution buffer, sample volume per PCR, and individual PCR volume should be found experimentally as a reasonable compromise between the required assay sensitivity and the PCR volume limit. It is. Alternatively, applications where the eluate does not require amplification; for example, when used in nucleic acid hybridization techniques, the concentration of multivalent anionic compound that can be inhibitory to PCR or other nucleic acid amplification techniques is Can be tolerated. Thus, the eluate can be used directly for nucleic acid detection.

  According to one embodiment, the elution is performed at an elevated temperature, such as a temperature of ≧ 50 ° C., ≧ 60 ° C., or even ≧ 70 ° C.

  The present disclosure also provides a method for nucleic acid isolation, analysis and in particular amplification that allows automation utilizing anion exchange materials suitable for the mild isolation of DNA and RNA. Furthermore, the eluted DNA or RNA generally does not require additional handling steps such as neutralization or desalting steps because it is fully compatible with downstream reactions.

Eluate, analysis, and amplification compositions One advantage of the isolation and elution methods disclosed herein is that an eluate containing the nucleic acid of interest can be generated without resorting to pH switch or high salt methods. It can be done. Thus, eluates generated according to the methods described herein can vary, including, for example, amplification methods such as PCR, RT-PCR, helicase-dependent amplification, hybrid capture assays, sequencing, or transfection. Can be used directly in various analytical methods. If the eluted nucleic acid is used in an amplification method, any anionic compound, as defined herein, comprising at least two anionic groups can be used. However, in certain embodiments involving subsequent amplification reactions, particularly PCR, the anionic compound is not dextran sulfate.

Accordingly, in one aspect, an eluate obtained according to any of the above methods is provided. In one embodiment, the eluate is directly compatible with subsequent enzymatic reactions. “Direct compatibility” means that the resulting eluate does not require further processing steps (such as desalting, neutralization, etc.) before being included in the enzyme reaction mixture. In another embodiment, the eluate comprises (a) providing a nucleic acid-anion exchange complex; and (b) a composition comprising an anionic compound comprising at least two anionic groups, the nucleic acid- Adding to the anion exchange complex, wherein the anionic compound displaces a nucleic acid from the anion exchange material and the composition further comprising the anionic compound further comprises an amplification component. It is obtained by a method including: For example, without limitation, amplification components, an enzyme having polymerase activity; nucleotides; primer nucleic acid; enzyme having helicase activity; enzyme having reverse transcriptase activity amplification buffer; nick-inducing agent; Mg ²⁺ and / or A source of Mn ²⁺ , eg, MgCl ₂ and / or MnCl ₂ ; Ribonucleotide triphosphate (NTP); Deoxyribonucleotide triphosphate (dNTP); A source of K ⁺ , eg, KCl; NH ₄ ⁺ Source, such as (NH ₄ ) ₂ SO ₄ ; or a reducing agent such as 2-mercaptoethanol and / or dithiothreitol (DTT).

  In another aspect, (a) providing a nucleic acid-anion exchange complex; (b) a composition comprising an anionic compound comprising at least two anionic groups, the nucleic acid-anion exchange complex And (c) comprises analyzing the nucleic acid in the presence of the anionic compound, or consists essentially of (a); (b); and (c), or (a) A method for analyzing said nucleic acid, comprising: (b); and (c).

  In one embodiment, the analytical method includes nucleic acid amplification. Nucleic acid amplification can be broadly divided into two categories: temperature cycle amplification and isothermal amplification. Either class of amplification can be used. Amplification in the disclosed methods can be either temperature cycle amplification or isothermal amplification. Exemplary amplification methods include polymerase chain reaction (“PCR”), reverse transcriptase (“RT”) reaction, thermophilic helicase-dependent amplification (“tHDA”), whole genome amplification, and ligase chain reaction (“LCR”). ”), But is not limited to. Exemplary analytical methods including amplification steps include PCR, RT-PCR, real-time PCR, real-time RT-PCR, quantitative real-time PCR, quantitative real-time RT-PCR, multiplex analysis, melting curve analysis, high resolution melting curve analysis , THDA, RT-tHDA, real-time tHDA, quantitative real-time tHDA, quantitative real-time RT-tHDA, NIA, RT-NIA, real-time NIA, quantitative real-time NIA, and quantitative real-time RT-NIA It is not limited.

  In temperature cycle amplification, such as PCR, typically, to “melt” any double stranded portion, the temperature is raised above the melting point of the target nucleic acid, and then the oligonucleotide primer is single stranded on the target nucleic acid. Decrease to the point where the part anneals, and then raise again to a temperature where the primer continues to anneal and the polymerase is active.

  In isothermal amplification, such as tHDA and whole genome amplification, an agent is added to the reaction mixture to allow amplification without temperature cycling. For example, in tHDA, an enzyme having helicase activity is added to the amplification mixture. As used herein, “helicase” or “enzyme comprising or having helicase activity” refers to any enzyme capable of unwinding a double-stranded nucleic acid. The helicase functions to unwind the double stranded nucleic acid, thus avoiding the need to repeat the melting cycle. Exemplary helicases include E. coli helicases I, II, III and IV, Rep, DnaB, PriA, PcrA, T4 Gp41 helicase, T4 Dda helicase, T7 Gp4 helicase, SV40 large T antigen, yeast RAD. Additional helicases that may be useful are derived from RecQ helicase, T. tengcongensis and thermostable UvrD helicase from T. thermophilus, T. aquaticus Included are thermostable DnaB helicases, and MCM helicases from archaea and eukaryotes. As another example, in nick-initiated amplification (“NIA”), a nick inducer is used to induce phosphodiester bond breakage of the nucleic acid backbone. A polymerase with strand displacement activity can then initiate amplification at the nick site using one strand of the nucleic acid as a primer and the other strand as a template. As used herein, a “nick inducer” is any enzymatic or chemical that introduces a break in the phosphodiester bond between two adjacent nucleotides in one strand of a double-stranded nucleic acid. Refers to a reagent or physical treatment. Examples of nick-inducing enzymes include Bpu10 I, BstNB I, Alw I, BbvC I, BbvC I, Bsm I, BsrD, and E. coli endonuclease I.

Analytical anion exchange materials in the presence of anion exchange materials are known to interfere with analysis of nucleic acids. Apart from their availability as eluents, anionic compounds as described throughout the present specification also have the unexpected property of allowing analysis of nucleic acids in the presence of anion exchange materials. . Accordingly, the present disclosure relates to materials and methods for analyzing nucleic acids in the presence of an anion exchange material where an anionic compound is added during the analysis process.

  In one embodiment, (a) purifying a nucleic acid of interest from a sample using an anion exchange material; and (b) analyzing the nucleic acid in a first solution comprising at least one anionic compound. Disclosed is a method of analyzing the nucleic acid of interest in the presence of the anion exchange material, wherein the anionic compound comprises at least two anionic groups . In one embodiment, the anionic compound is non-polymeric. In another embodiment, the anionic compound is a multivalent anionic compound.

  In another embodiment, the purification step comprises (1) contacting a sample containing nucleic acid with an anion exchange material, and (2) forming a nucleic acid-anion exchange complex between the anion exchange material and the nucleic acid. And (3) isolating the nucleic acid-anion exchange complex from the sample.

  In a further aspect, the purification step comprises (1) contacting a sample containing nucleic acid with an anion exchange material, (2) forming a complex between the anion exchange material and the nucleic acid, and (3) from the sample. Isolating the complex, and (4) washing the complex to remove impurities. Wash conditions can be selected such that substantially all non-nucleic acid material is removed. Washing conditions may be further improved so that certain nucleic acids are similarly removed as impurities.

  In another embodiment, the nucleic acid is eluted from the nucleic acid-anion exchange complex before the anionic compound is added. For example, nucleic acids can be eluted using a pH switch or a high salt solution. In such cases, the resulting eluate is likely to need to be further processed before being used in the analytical process. As another example, nucleic acids can be eluted using a solution containing an anionic compound, as described above. The eluate can then be separated from a substantial portion of the anion exchange material prior to analysis (as in the case of bead carryover) or the eluate can be analyzed in the presence of a complete portion. Good.

  In another embodiment, the analytical solution containing the anionic compound is added directly to the nucleic acid-anion exchange complex. For example, without limitation, if the analytical method is amplification, a nucleic acid amplification composition comprising an anionic compound comprising at least two anionic groups can be added directly to the nucleic acid-anion exchange complex. .

As used herein, the term “nucleic acid amplification composition” includes an anionic compound as described above and further includes appropriate components and physical properties such that nucleic acid amplification can be performed. It shall refer to any composition it has. Such components include, in appropriate situations, buffers; salt solutions; primers; nucleotide triphosphates; polymers such as RNases, RNase inhibitors, coenzymes, and / or catalysts; polyethylene glycol; sorbitol; As well as DMSO. Physical properties include, but are not limited to, pH, ionic strength, and Mg ²⁺ concentration. The exact identity of the additional components and physical properties depends on the particular amplification method used; identity of the nucleic acid being amplified; desired amplicon length and G / C content; and Will depend on a number of factors, including but not limited to certain polymerases and / or helicases. These additional components and physical properties will be readily apparent to those skilled in the art. For example, without limitation, amplification components, an enzyme having polymerase activity; nucleotides; primer nucleic acid; enzyme having helicase activity; enzyme with reverse transcriptase activity amplification buffer; nick-inducing agent; Mg ²⁺ and / Or a source of Mn ²⁺ , eg, MgCl ₂ and / or MnCl ₂ ; ribonucleotide triphosphate (NTP); deoxyribonucleotide triphosphate (dNTP); a source of K ⁺ , eg, KCl; NH ₄ ⁺ It can be a source, for example (NH ₄ ) ₂ SO ₄ ; or any combination of reducing agents, for example 2-mercaptoethanol and / or dithiothreitol (DTT).

  The nucleic acid amplification composition may be provided at a working (1 ×) concentration or in an enriched format. For example, without limitation, the concentrated format can be provided as an aqueous solution at 5 ×, 10 ×, 20 ×, or 50 × concentration, or as a lyophilized concentrate.

  In one embodiment, the nucleic acid amplification composition comprises (a) at least one target nucleic acid; (b) an anion exchange material; (c) at least one anionic compound comprising at least two anionic groups; and (D) including at least one protein having polymerase activity.

  Another embodiment includes (a) at least one target nucleic acid; (b) an anion exchange material; (c) at least one anionic compound comprising at least two anionic groups; (d) having polymerase activity. A nucleic acid amplification composition comprising at least one protein; and (e) at least one protein having helicase activity.

  Such materials and methods are particularly suitable for use in automated methods.

The present disclosure provides an elution buffer comprising an anion exchange material, preferably an anion exchange material as described above; and at least one anionic compound comprising at least two anionic groups as described above. Kits for isolating nucleic acids from samples, such as biological samples, including fluids are also provided. Kits include wash buffer, loading buffer, equilibration buffer, lysis buffer (eg for lysis of samples such as cell or tissue samples), nucleases such as RNase and / or DNase, inhibitors of RNase or DNase , As well as one or more components selected from the group consisting of instructions for use. Kits for isolating nucleic acids can also include additional components suitable for analyzing and / or amplifying the isolated nucleic acids.

Furthermore, the present disclosure provides a nucleic acid comprising an enzyme having polymerase activity and at least one anionic compound comprising at least two anionic groups, preferably a non-polymeric anionic compound as described above. A kit for amplifying is also provided. The kit comprises an enzyme having reverse transcriptase activity; an enzyme having helicase activity; a nucleotide; a primer nucleic acid; an amplification buffer; a nick inducer; a source of Mg ²⁺ and / or Mn ²⁺ , eg, MgCl ₂ and / or MnCl ₂ ; Ribonucleotide triphosphate (NTP); Deoxyribonucleotide triphosphate (dNTP); K ⁺ source, eg KCl; NH ₄ ⁺ source, eg (NH ₄ ) ₂ SO ₄ ; Reducing agent One or more additional components may also be included, such as, for example, 2-mercaptoethanol and / or dithiothreitol (DTT), and instructions for use. The kit for amplifying a nucleic acid may further contain any of the components of the kit for isolating the nucleic acid.

  The various components of the kits disclosed herein may be provided at a working (1 ×) concentration or in a concentrated format. For example, without limitation, the concentrated format can be provided as an aqueous solution at 5 ×, 10 ×, 20 ×, or 50 × concentration, or as a lyophilized concentrate.

Use The present disclosure relates to an anionic compound, preferably at least two, comprising at least two anionic groups to displace a nucleic acid reversibly bound to an anion exchange material from the anion exchange material. The use of non-polymeric compounds containing anionic groups is also provided. The anionic compound, non-polymeric compound, nucleic acid, and / or anion exchange material are preferably as described above.

  The present disclosure also provides a method for isolating nucleic acid from a sample containing nucleic acid. The method includes the step of (a) contacting a sample containing nucleic acid with an anion exchange material to allow binding of the nucleic acid. The anion exchange material can reversibly bind to the nucleic acid. The method includes (b) optionally washing the anion exchange material to remove unbound sample components; and (c) adding at least one compound comprising at least two anionic groups. This further includes the step of eluting the bound nucleic acid, wherein the anionic compound can displace the nucleic acid from the anion exchange material.

  Details regarding compounds containing at least two anionic groups, and anion exchange materials, and methods are detailed above. See the disclosure above.

  As described above, the present disclosure provides a step of complexing a nucleic acid with an anion exchange material to form a nucleic acid-anion exchange complex; optionally separating the nucleic acid-anion exchange complex from other materials. And in the presence of the anion exchange material, comprising analyzing the nucleic acid in solution, preferably amplifying the nucleic acid, comprising at least one compound comprising at least two anionic groups Also provided are methods of analyzing the nucleic acid, preferably amplifying the nucleic acid. The anionic compound preferably does not inhibit or interfere with the amplification reaction in each analytical step. Furthermore, the anionic compound preferably reduces or eliminates any inhibitory effect that the anion exchange material has on the amplification reaction or analytical process. That is, an anion exchange material, if present, can reduce the effectiveness of the analysis, particularly the amplification process. This decrease in effectiveness is at least partially neutralized by the presence of the anionic compound used in accordance with the present invention.

As mentioned above
(A) contacting the sample with an anion exchange material that reversibly binds to a nucleic acid;
(B) optionally washing the anion exchange material to remove unbound sample components; and (c) adding at least one non-polymeric compound comprising at least two anionic groups. Thereby eluting the bound nucleic acid, wherein the compound comprises a step of substituting the bound nucleic acid from the anion exchange material to obtain an isolated nucleic acid. A method for isolating nucleic acid from the sample is also provided.

According to one embodiment, the anion exchange material comprises a solid phase modified with a positively ionizable capture component. A positively ionizable capture component may have one or more of the following characteristics.
(A) selected from the group consisting of primary amines, secondary amines, or tertiary amines having the formula R ₃ N, R ₂ NH, RNH ₂ , and / or X— (CH ₂ ) _n —Y;
Where
X is R ₂ N, RNH, or NH ₂ ,
Y is R ₂ N, RNH, or NH ₂
R is independently of each other preferably a linear, branched or cyclic alkyl substituent, alkenyl substituted, which may contain one or more heteroatoms selected from O, N, S and P A group, an alkynyl substituent, or an aryl substituent, and
n is an integer in the range of 0-20, preferably 0-18,
(B) From the group consisting of aminomethyl, aminoethyl, diethylaminoethyl, trimethylamino, triethylaminoethyl, spermine, spermidine, 3- (propylamino) propylamine, polyamidoamine dendrimer, polyethyleneimine, N-morpholinoethyl, and polylysine And / or (c) selected from the group consisting of spermine, spermidine, and polyethyleneimine.

The non-polymeric compound can have one or more of the following characteristics.
(A) contains at least 2 anionic groups, preferably 3-6 anionic groups;
(B) an organic compound, preferably an organic compound having 2 to 20 carbon atoms, more preferably 2 to 12 carbon atoms;
(C) Oxalic acid, fumaric acid, glutaric acid, maleic acid, malic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberin optionally substituted by one or more substituents Acid, azelaic acid, tartronic acid, tartaric acid, citric acid, isocitric acid, citraconic acid, mesaconic acid, itaconic acid, aconitic acid, propane-1,2,3-tricarboxylic acid, aconitic acid, butane-1,2,3, 4-tetracarboxylic acid, triethyl-1,1,2-ethanetricarboxylic acid, cyclopropanedicarboxylic acid, cyclobutanedicarboxylic acid, cyclobutanetricarboxylic acid, cyclopentanedicarboxylic acid, cyclohexanedicarboxylic acid, cyclohexanetricarboxylic acid, cyclohexanetetracarboxylic acid, cyclohexane Hexacarboxylic acid, cyclooctane dicarboxylic acid, phthalic acid , Isophthalic acid, terephthalic acid, hemimellitic acid, trimellitic acid, trimesic acid, merophanic acid, prenitic acid, pyromellitic acid, benzenepentacarboxylic acid, and mellitic acid, dierythritol hexacarboxylic acid, trierythritol octaacetic acid, iminodiacetic acid Nitrilotriacetic acid, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, alpha ketoglutaric acid, glutamic acid, aspartic acid, dicarboxymalonic acid, (18-crown-6) -2,3,11,12-tetracarboxylic acid, acrylic acid, A carboxylic acid such as methacrylic acid or 2-10, preferably 2-6, oligomers of polymerizable or condensable acidic monomers such as vinyl acetate;
(D) organic sulfonic acids, such as methyl disulfonic acid, ethyl disulfonic acid, benzene disulfonic acid, phenol disulfonic acid, and naphthalene disulfonic acid, optionally substituted by one or more substituents;
(E) an organic phosphonic acid such as hydroxyethanediphosphonic acid;
(F) an organophosphate; and / or (g) carbonate or diacetylacetone.

  As described above, anion exchange material magnetic beads, preferably magnetic silica beads or magnetic polymer beads, functionalized with a positively ionizable capture component can be used, and non-polymeric compounds include oxalic acid, melitto Carboxylic acids selected from acids, pyromellitic acid, and citric acid can be used.

The elution step of the method may have one or more of the following characteristics.
(A) The pH during elution is not above the pKa of the positively ionizable group of the capture component;
(B) the pH during elution is in the range of 5 to 13, preferably 5 to 8.5;
(C) The total salt concentration in the solution does not exceed 1M, preferably 0.5M.
The method may further comprise the step of amplifying the isolated nucleic acid, preferably without performing an intermediate purification step, buffer step, or desalting step.

As mentioned above
(A) complexing a nucleic acid with an anion exchange material to form a nucleic acid-anion exchange complex;
(B) separating the nucleic acid-anion exchange complex from other materials; and (c) at least one non-polymeric compound comprising at least two anionic groups, There is also provided a method of analyzing, preferably amplifying said nucleic acid in the presence of said anion exchange material comprising the step of analyzing, preferably amplifying said nucleic acid.

  Analytical steps include PCR, RT-PCR, quantitative real-time PCR, quantitative real-time RT-PCR, multiplex analysis, melting curve analysis, high resolution melting curve analysis, tHDA, RT-tHDA, quantitative real-time tHDA, or quantitative Real-time RT-tHDA, or a combination thereof may be included.

  According to one embodiment of the method, the nucleic acid is not eluted from the anion exchange material prior to the analysis step.

  According to one embodiment, anion exchange materials as defined above and / or non-polymeric compounds as defined above are used.

Kits for isolating nucleic acids from a sample are also provided, including:
An elution buffer comprising (a) an anion exchange material; and (b) at least one non-polymeric compound comprising at least two anionic groups.

  According to one embodiment, anion exchange materials as defined above and / or non-polymeric compounds as defined above are used.

I. Material
1. Anion exchange modification of magnetic silica gel
In a 100 ml 1-neck flask with a two-neck cap, supply 23 ml of deionized water, 3 ml of 250 mM NaPO ₄ (pH 6.0), 100 μl of 3-glycidoxypropyl-trimethoxysilane, and 91.7 μl of diethylaminopropyl-triethoxysilane . The solution is adjusted to pH 5.50 with 2M NaOH. Prior to addition, 2 g of MagAttract beads “G” is placed in a Nalgen flask containing 15 ml of deionized water, shaken for 5 minutes on a shaker at level 5 and magnetically separated (3 minutes), and the supernatant water is drained . The suspension is replenished to a total of 7 g and added to the reaction flask. The Nalgen flask is washed once with 1 ml of deionized water. The flask is then added to the KPG stirrer and reflux condenser. The stirring speed is 500 / min. The temperature of the suspension is increased to 90 ° C. using an oil bath (heater without magnet). Maintain 90 ° C for 4 hours. The oil bath is then removed and stirred for 1 hour to cool the suspension. The flask volume is converted to a 60 ml Nalgen flask and the stirrer is washed with a small amount of deionized water. To convert the rest of the beads, the suspension in the Nalgen flask is magnetically separated for 3 minutes and the flask is washed once with liquid. The beads are separated by a magnet at the mouth of the bottle, and the liquid is discharged. For washing, the following amount of liquid is added to the beads; the suspension is shaken on a shaker at level 5 for 5 minutes, then the beads are separated by a bottle mouth magnet and the liquid is drained. Washing was performed twice with 25 ml Tris / NaCl buffer, once with 25 ml deionized water, and three times with ethanol 25. Subsequently, three washes with 25 ml deionized water were performed to convert to water.

2. Synthesis of “basic” nucleic acid Under a cover gas, 530 mg (2.2 mmol) of 2-cyanoethyl-N, N-diisopropylchlorophosphoramidite is dissolved in 15 ml of dichloromethane and, in parallel, in a 100 ml three-necked flask, 146 μl of 1,3-propanediol (154 mg, 2 mmol) is dissolved in anhydrous dichloromethane. The solutions are combined in a 3-neck flask and allowed to react for 3 hours. Then 16 ml of “activation solution” (0.25 M 4,5-dicyanoimidazole in CH ₃ CN) was added and shaken for 1 hour at room temperature, then “oxidation solution” (H ₂ O / pyridine / THF ( Add 4 ml of 1M iodine) in 2:21:77 v / v / v) and allow to react overnight. After fading, several iodine balls are added until the yellow dye no longer disappears. The flask is then placed in a cooling device. If no sediment is indicated, no sediment has appeared and 50 ml of hexane is added again and then condensed. The residue is then washed twice with 40 ml hexane, decanted and resuspended in 50 ml RNase-free water. Dialyze against 5 l of 50 mM NaH ₂ PO ₄ (pH 7.0) using a dialysis tube (MWCO 5,000) and dialyze twice against 5 l of VE water. Thereafter, the solvent is removed by lyophilization. The yield for this experiment was 40 mg.

3. Synthesis of amino-functional magnetic carboxylic acid beads 500 mg of magnetic particles (Carboxyl-Adembeads, Ademtech, order number 02111) are resuspended in 10 ml of 50 mM MES buffer (pH 6.1) and 50 mg / ml N-hydroxysulfosuccinimide Add 11.5 ml of solution. Mix with a mini shaker. Add 10 ml of 52 μmol / l 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide solution and mix again. Then, it is made to react for 30 minutes on a rotary shaker. The supernatant is then removed. Resuspend in 50 ml of 50 mM MES buffer (pH 6.1) and make a 10 ml aliquot. After magnetic separation, the supernatant is removed. Then resuspend in 1 ml of 50 mM MES buffer (pH 6.1) and add 2 ml of amine at a pH of 8.5 at a concentration of 500 mg / ml in 50 mM MES and mix well. After 10 minutes of sonication, react for 1 hour on a rotary shaker. Then, it is washed twice with 10 ml of 50 mM MES buffer (pH 6.1), magnetically separated, and the supernatant is discarded. Resuspend the particles in 2 ml of MES buffer having a pH of 4.5-7.0.

4. Synthesis of magnetic carboxylic acid beads modified with polyethyleneimine
Resuspend 500 mg of magnetic particles (Estapor) in 10 ml of 50 mM MES buffer (pH 6.1), add 11.5 ml of 50 mg / ml 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide solution, and mix again . Then, it is made to react for 30 minutes on a rotary shaker. The supernatant is then removed. Resuspend in 50 ml of 50 mM MES buffer (pH 6.1) and make a 10 ml aliquot. After magnetic separation, the supernatant is removed. It is then resuspended in 1 ml of 50 mM MES buffer (pH 6.1) and 2 ml of polyethyleneimine at a concentration of 500 mg / ml in 50 mM MES and a pH of 8.5 is added and mixed well. After 10 minutes of sonication, react for 1 hour on a rotary shaker. Then, it is washed twice with 10 ml of 200 mM NaCl and 50 mM MES buffer (pH 7.0), and washed four times with 10 ml of 50 mM MES buffer (pH 6.1). After magnetic separation, the supernatant is removed. The particles are resuspended in 2 ml of MES buffer having a pH of 4.5-7.0.

II. Examples Example 1: Nucleic acid elution using a second nucleic acid and magnetic anion exchange silica particles
1 μg of a 300 bp DNA fragment is dissolved in 100 μl of a buffer (25 mM MES (pH 7.0)). Then 1 mg of magnetic silica gel prepared according to Example I.1 is added and incubated at RT and 1,000 rpm. After magnetic separation, the supernatant is discarded. The beads are then washed twice by incubation for 5 minutes in 100 μl of deionized water at 1,000 rpm on a thermal shaker, magnetic separation, and discarding the supernatant. 50 μl of elution buffer containing 1-3 μg of 500 bp DNA fragment is added to the beads and after 5 minutes incubation on a magnetic shaker and magnetic separation, the supernatant is obtained. The elution is then repeated using 50 μl of elution buffer containing 50 mM Tris, 50 mM NaCl, pH 8.5.

  FIG. 1 shows that no DNA was present in the remaining sample and magnetic particles were bound to the total DNA. In the eluate with the second DNA 1, 2, or 3 μg and pH 7, 300 bp and 500 bp DNA fragments are visible, indicating that the second DNA (500 bp) is bound to the bound first DNA (300 bp). ) Is replaced at least partially. Residual DNA fragments were eluted in the second eluate at pH 8.5.

Example 2: Nucleic acid elution with different anionic compounds and slightly alkaline buffer 2 μg of plasmid DNA (pUC21) dissolved in 25 mM MES (pH 8.0 or 8.5), 0.25 mg of magnetic particles coated with polyethyleneimine (50 mg / ml in deionized water) is added. The dispersion is mixed for 5 minutes at RT and 1,000 rpm. After magnetic separation, the supernatant is removed. The beads are then washed twice by RT on a thermal shaker and 5 minutes incubation in 100 μl deionized water at 1,000 rpm, magnetic separation, and discarding the supernatant. Using 100 μl of elution buffer (25 mM MES (pH 8.0 or 8.5)) additionally containing 2,000 ng of dextran sulfate (Mw 9,000-25,000), polyacrylic acid, oxalic acid, or melittic acid Perform elution. A second elution is then performed using 50 μl of 50 mM MES, 50 mM NaCl, pH 8.5.

  FIG. 2 shows that 2,000 ng dextran sulfate, polyacrylic acid, oxalic acid, or mellitic acid is used to elute most of the pDNA from the magnetic particles. The second elution at pH 8.5 elutes only the remainder of the plasmid.

Example 3: Elution using carboxylic acid 2 μg of plasmid DNA (pUC21) is dissolved in 25 mM MES (pH 7.0) and 0.125 mg of spermine-coated magnetic particles (50 mg / ml in deionized water) are added. The dispersion is then incubated for 5 minutes at RT and 1,000 rpm. After magnetic separation, the supernatant is removed. The beads are then washed twice by RT on a thermal shaker and 5 minutes incubation in 100 μl deionized water at 1,000 rpm, magnetic separation, and discarding the supernatant. Carboxymethyl dextran, dextran sulfate (Mw 6,500-10,000), dextran sulfate (Mw 9,000-25,000), polyacrylic acid, poly (4-styrenesulfonic acid maleic acid), acetic acid, oxalic acid, citric acid, pyromellitic acid, Alternatively, elution is performed using 100 μl of elution buffer (25 mM MES, pH 7.0) additionally containing 2,000, 5,000, or 10,000 ng of melittic acid. A second elution is then performed using 50 μl of 50 mM MES, 50 mM NaCL (pH 8.5).

  Figure 3 shows the elution of bound nucleic acids using dextran sulfate, polyacrylic acid, poly (4-styrene sulfonic acid maleic acid), oxalic acid, citric acid, pyromellitic acid, or 2,000-10,000 ng of mellitic acid. It is shown that. Acetic acid eluted only a small amount of plasmid. In that case, most of the plasmid is eluted in the second elution step. However, other compounds can elute bound nucleic acids.

Example 4: Elution using “Non-basic” nucleic acid 2 μg of plasmid DNA (pUC21) was dissolved in 25 mM MES (pH 7.0), and spermine (pH 7.0, 7.5) or polyethyleneimine (pH 8.0, 8.5) Add 0.25 mg of each coated magnetic particle (50 mg / ml each in deionized water). The dispersion is then mixed for 5 minutes at RT and 1,000 rpm. After magnetic separation, the supernatant is removed. The beads are then washed twice by RT for 5 minutes in 100 μl deionized water at 1000 rpm on a thermal shaker, magnetic separation, and removal of the supernatant. Elution is performed using 100 μl of elution buffer (25 mM MES (pH 7.0, 7.5, 8.0, or 8.5)) additionally containing 2,000, 5,000, or 10,000 ng of “basic” DNA. A second elution is then performed using 50 μl of 50 mM MES, 50 mM NaCl, pH 8.5.

  FIG. 4 shows that plasmid DNA was bound to the solid phase because no DNA could be detected in the remaining sample. It is impossible to remove DNA from spermine beads at pH 7.0 and 7.5. However, at pH 8.0 and 8.5, the plasmid elutes from the polyethyleneimine beads.

Example 5: PCR inhibition with various anions To a β-actin PCR reaction mixture, add 5, 10, or 20 ng of genomic DNA and 20 ng of acetic acid, oxalic acid, citric acid, polyacrylic acid or melittic acid each. In parallel, PCR results without addition of carboxylic acid as a control and blank values are determined.

The results of the inhibition test (FIG. 5) show that PCR works well in the presence of any of the carboxylic acids tested. In particular, the addition of acetic acid, oxalic acid, or citric acid has no effect on PCR. Although a slight increase in ct value is detectable, the addition of polyacrylic acid and melittic acid does not inhibit PCR in these examples. However, as shown in later examples, polyacrylic acid can inhibit PCR under some conditions. For example, the inhibitory effect of polyacrylic acid can be reversed / rescued by increasing the Mg ²⁺ concentration, as demonstrated in FIG.

Example 6: PCR inhibition assay using citric acid 5 or 10 ng of genomic DNA and 30, 60, 120 or 240 citrate are added to the β-actin PCR reaction mixture. In parallel, PCR results without addition of carboxylic acid as a control and blank values are determined. Β-actin PCR is then performed to determine the Ct value. The Ct value represents the number of PCR reaction cycles until the amount of nucleic acid produced exceeds a certain threshold level (used to distinguish between the signal of the nucleic acid product and the background signal).

  As shown in FIG. 6, the Ct value decreases rather as the amount of citric acid increases. One possible explanation for this is that citric acid is complexed with the cationic constituents of the PCR buffer, thereby increasing the specificity of the PCR reaction. Even up to 360 ng of gDNA, no inhibitory effect of citrate can be detected.

Example 7: DNA binding and release using a second DNA
Dissolve 1 μg of DNA fragment (100 bp) in 100 μl of 25 mM MES buffer (pH 7.0), add 15 μl of a suspension of magnetic particles modified with spermine (26.4 mg / ml), and suspend the suspension at RT and Mix at 1,000 rpm for 5 minutes. After magnetic separation, the supernatant is removed. The particles are then washed twice by incubation for 5 minutes in 100 μl deionized water at RT and 1,000 rpm, magnetic separation, and removal of the supernatant. The first DNA is then eluted using 50 μl of elution buffer (25 mM MES, pH 7.0) containing 500 bp or 1000 bp DNA 1, 2, or 3 μg. Elution is performed by incubation for 5 minutes in elution buffer at RT and 1,000 rpm, magnetic separation, and obtaining supernatant. The supernatant sample after binding, washing and elution is then separated by agarose gel electrophoresis.

  FIG. 7 shows that the shorter first DNA binds to the magnetic particles and is effectively eluted by the longer second DNA.

Example 8: DNA binding and release using a second DNA having a shorter length
1 μg of a 500 bp or 1000 bp DNA fragment is dissolved in 100 μl of 25 mM MES buffer (pH 7.0). 15 μl of a suspension of magnetic particles modified with spermine (17 mg / ml) is added and the suspension is mixed for 5 minutes at RT and 1,000 rpm to bind to the magnetic particles modified with spermine. After magnetic separation, the supernatant is removed. The particles are then washed twice by incubation for 5 minutes in 100 μl deionized water at RT and 1,000 rpm, magnetic separation, and removal of the supernatant. The elution is then performed using 50 μl of elution buffer (25 mM MES, pH 7.0) containing 200 bp DNA 1, 2, or 3 μg. Elution is performed by incubation for 5 minutes in elution buffer at RT and 1,000 rpm, magnetic separation, and obtaining supernatant. For control, a second elution is performed using 50 μl of 50 mM Tris, 50 mM NaCl, pH 8.5. The supernatant samples after binding, washing, first elution and second elution are then separated by agarose gel electrophoresis.

  FIG. 8 shows that a shorter second DNA can be used to effectively elute the longer first DNA. Subsequent control elution showed only a small amount of residual first DNA.

Example 9: Binding and release of DNA using RNA for elution 0.125 mg of spermine modified beads are suspended in 100 μl of 25 mM MES buffer (pH 7.0) and 1 μg of 500 bp DNA fragment is added . The suspension is incubated at RT and 1,000 rpm for 5 minutes, magnetically separated and the supernatant removed. The beads are then washed twice by incubation in 100 μl deionized water for 5 minutes, magnetic separation, and removal of the supernatant. The elution is then carried out using 50 μl of elution buffer (25 mM MES, pH 7.0) containing 1, 2, or 3 μg of RNA. Elution is performed by incubation for 5 minutes in elution buffer, magnetic separation, and obtaining supernatant. As a control, a second elution is performed using 50 μl of 50 mM Tris, 50 mM NaCl, pH 8.5. The supernatant samples after binding, washing, first elution and second elution are then separated by agarose gel electrophoresis.

  FIG. 9 shows that significant amounts of bound DNA are eluted using 3 μg RNA.

Example 10: Binding and release of RNA using DNA for elution 0.125 mg of spermine modified beads are suspended in 100 μl of 25 mM MES buffer (pH 7.0) and 2 μg of RNA are added. The suspension is incubated at RT and 1,000 rpm for 5 minutes, magnetically separated and the supernatant removed. The beads are then washed twice by incubation in 100 μl deionized water for 5 minutes, magnetic separation, and removal of the supernatant. The elution is then performed using 50 μl of elution buffer (25 mM MES, pH 7.0) containing 500 bp DNA 1, 2, or 3 μg. Elution is performed by incubation for 5 minutes in elution buffer, magnetic separation, and obtaining supernatant. As a control, a second elution is performed using 50 μl of 50 mM Tris, 50 mM NaCl, pH 8.5. The supernatant samples after binding, washing, first elution and second elution are then separated by agarose gel electrophoresis.

  FIG. 10 shows that bound RNA is eluted from the beads using DNA. Using 3 μg of DNA, the bound RNA is almost completely eluted.

Example 11: Binding and release of genomic DNA using plasmid DNA for elution 0.125 mg or 0.25 mg of spermine modified beads are suspended in 100 μl of 25 mM MES buffer (pH 7.0) and 1 μg of genomic DNA Add. The suspension is incubated at RT and 1,000 rpm for 5 minutes, magnetically separated and the supernatant removed. The beads are then washed twice by incubation in 100 μl deionized water for 5 minutes, magnetic separation, and removal of the supernatant. Elution is then performed using 50 μl of elution buffer (12.5 mM MES, pH 7.0) containing 1, 2, or 3 μg of plasmid DNA. Elution is performed by incubation for 5 minutes in elution buffer, magnetic separation, and obtaining supernatant. As a control, a second elution is performed using 50 μl of 50 mM Tris, 50 mM NaCl, pH 8.5. The supernatant samples after binding, washing, first elution and second elution are then separated by agarose gel electrophoresis.

  FIG. 11 shows the elution of genomic DNA when 3 μg of plasmid DNA is used.

Example 12: Binding and release of plasmid DNA using genomic DNA for elution 0.25 mg of spermine modified beads were suspended in 100 μl of 25 mM MES buffer (pH 7.0) and 1 μg of plasmid DNA (pUC21) Add. The suspension is incubated at RT and 1,000 rpm for 5 minutes, magnetically separated and the supernatant removed. The beads are then washed twice by incubation in 100 μl deionized water for 5 minutes, magnetic separation, and removal of the supernatant. Elution is then performed using 50 μl of elution buffer (12.5 mM MES, pH 7.0) containing 1, 2, or 3 μg genomic DNA. Elution is performed by incubation for 5 minutes in elution buffer, magnetic separation, and obtaining supernatant. As a control, a second elution is performed using 50 μl of 50 mM Tris, 50 mM NaCl, pH 8.5. The supernatant samples after binding, washing, first elution and second elution are then separated by agarose gel electrophoresis.

  FIG. 12 shows that plasmid DNA can be effectively eluted using genomic DNA. Subsequent control elution showed only a small amount of residual plasmid DNA.

Example 13: Binding and release of siRNA using short DNA fragments for elution 0.125 mg of polyethyleneimine modified beads prepared according to Example I.4 in 100 μl of 25 mM MES buffer (pH 8.0) Suspend and add 1 μg of siRNA (Qiagen, order number SI00300650). The suspension is incubated at RT and 1,000 rpm for 5 minutes, magnetically separated and the supernatant removed. The beads are then washed twice by incubation in 100 μl deionized water for 5 minutes, magnetic separation, and removal of the supernatant. The elution is then performed using 50 μl of elution buffer (25 mM MES, pH 8.0) containing 2 or 3 μg of 300 bp DNA. Elution is performed by incubation for 5 minutes in elution buffer, magnetic separation, and obtaining supernatant. As a control, a second elution is performed using 50 μl of 50 mM Tris, 50 mM NaCl, pH 8.5. The supernatant samples after binding, washing, first elution and second elution are then separated by agarose gel electrophoresis.

  FIG. 13 shows that bound siRNA can be effectively eluted using short DNA fragments. Subsequent control elution showed only a small amount of residual RNA.

Example 14: Analysis of the inhibitory effect of plasmid DNA on PCR using genomic DNA To analyze the effect of plasmid DNA used for elution on PCR using the first DNA, different amounts of genomic DNA and Perform a continuous assay of β-actin PCR with added plasmid DNA. For PCR, Applied Biosystems 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA), TaqMan β-Actin Control Reagents (PE Applied Biosystems, order number 401846), QuantiTect Probe PCRMastermix (nucleotides, polymerase, and salts) Qiagen, order number 127 137 815), as well as the fluorescent dye FAM (from β-Actin Control Reagents). Genomic DNA is obtained from a nucleic acid preparation derived from human blood using the kit QIAamp Blood (Qiagen, order number 51106), and plasmid DNA (pUC21) is used using the Qiagen Plasmid Mega Kit (Qiagen, order number 12183) And prepared by plasmid isolation from E. coli. The amounts of genomic and plasmid DNA used and the recorded Ct values are shown in Table 1.

  The inhibitory effect indicated by the higher Ct value is only seen when a very excess of plasmid DNA is used. As shown in other examples, only up to a 5-fold excess of the second nucleic acid is required for efficient elution of the bound nucleic acid, and the inhibitory effect of the second nucleic acid is a 25-fold excess. Or it can only be detected above. Thus, the use of a second nucleic acid to elute the first nucleic acid from the anion exchange material under the conditions used herein does not interfere with subsequent PCR reactions.

Example 15: Elution with polyacrylic acid (PAA) A DNA sample containing 5 × 10 ⁵ copies of Neisseria gonorrhoeae (NG) DNA was added to 1 mL of PreserCyt® medium. Then 0.5 mL of lysis buffer (2% Triton X-100, 0.2 M EDTA, 40 mM sodium citrate, 40 mM boric acid in 100 mM Tris HCl, pH 7.0) is added to bring the sample pH to about 7.8. did. The sample was then mixed with 30 μL of AXpH ™ bead suspension and incubated at 60 ° C. for 10 minutes. AXpH ™ beads were magnetically separated and washed once with 500 μl of wash buffer (0.1% NP-40 in 0.1 mM Tris, pH 8.0). Elution was then performed by adding either (a) 25 μL wash buffer or (b) a solution of PAA, Na salt (Fw>200,000; adjusted to pH 8.0 with Tris base) in wash buffer. Carried out. The eluate (sample 2.5 μL) was then compared to an equal volume of control DNA in the same elution buffer that was not processed according to the above procedure in real-time PCR performed in triplicate 25 μL reactions for each eluate. . Thus, control DNA dilutions will mimic 100% recovery of DNA from the beads and will also reveal whether PAA has a negative effect on PCR. A threshold cycle (Ct) was detected using the TaqMan-MGB probe (TET channel, see FIGS. 14A-14C). To confirm product identity, EvaGreen fluorescent dye was also added to the PCR to record post-PCR melting curves (FIG. 14C).

  The results are summarized in Table 2. The “eluent” column shows the results of using the indicated buffer to elute nucleic acids from AXpH ™ beads. The “Control” column shows the results of direct dilution of the nucleic acid in the indicated buffer composition. “ΔCt rec.” Is calculated by subtracting the average value of the “control” column replicates from the average value of the “eluate” column replicates for each buffer composition. “ΔCt eff.” Is calculated by subtracting the average value of the “control” column replicates for each buffer from the average value of the “control” column replicates for the wash buffer. Therefore, “ΔCt rec.” Reflects the efficiency of DNA elution from AXpH ™ beads, and “ΔCt eff.” Is a possible PCR efficiency caused by the presence of PAA in the amplification reaction. Shows some loss. Ideally, both parameters should be close to zero, reflecting that the recovery is highly efficient and that there is no inhibition of the PCR reaction.

  The data in Table 2 shows that the recovery of DNA from AXpH ™ beads in the PAA eluate is close to 100%. The pH of the elution buffer containing the wash buffer and PAA are all the same at pH 8.0, thus eliminating the effect of any pH on DNA elution. FIGS. 14A-14C show that the amplification curve in PCR is normal and PAA does not promote artifact formation in PCR.

Example 16: Comparison of DNA elution with PAA and "pH shift" elution Experiments were performed essentially the same as Example 15 with the following differences. (A) Each set of 3 samples contained NG DNA 10 ⁵ , 10 ³ , and 10 copies; (b) One sample set was eluted with 25 μL of 0.5% PAA; (c ) One sample set must be eluted with 15 μL of alkaline solution (and then neutralized with 10 μL of 170 mM Tris HCl (pH 8.0)); and (d) The control set of NG DNA is washed buffer Prepared to simulate 100% recovery of samples from AXpH ™ beads by diluting the appropriate amount of NG DNA with 1. Elution with alkali and PAA as shown in Figure 15. The results for are comparable.

Example 17: Elution with different PAA size fractions, polymethacrylic acid, and polyglutamic acid This experiment consists of two different polyacrylic acid samples (heavy PAA “PAAH” and light PAA “PAAL”) and polymethacrylic acid (PMA). ) Proved that the DNA eluted from the AXpH ™ beads but not with the wash buffer. Each DNA sample contained 5 × 10 ⁵ copies of Neisseria gonorrhoeae (NG) DNA in 1 mL of PreserCyt® medium. After adding 0.5 mL of lysis buffer (2% Triton X-100 in 100 mM Tris HCl, pH 7.0, 0.2 M EDTA, 40 mM sodium citrate, 40 mM boric acid) (the final pH of the sample was about 7.8) The sample was mixed with 30 μL of AXpH ™ bead suspension and incubated at 60 ° C. for 10 minutes. AXpH ™ beads were magnetically separated and washed once with 500 μl of wash buffer (0.1% NP-40 in 0.1 mM Tris pH 8.0). (B) 0.25% PAAH in washing buffer (polyacrylic acid, Na salt, Fw>200,000); (c) 0.1% PAAL in washing buffer (polyacrylic acid, Na salt) Elution was performed by adding any of 0.1% PMA (polymethacrylic acid, Na salt, Fw approximately 483,000) in wash buffer. The eluate (2 μL of sample) was then compared to an equal amount of control DNA that had not been processed according to the above procedure in real-time PCR performed in triplicate 25 μL reactions for each eluate. EvaGreen fluorescent dye was used to detect the threshold cycle (Ct). Mean Ct was 27.2 (PAAH), 24.4 (PAAL), 26.1 (PMA), and 23.5 (control DNA), respectively. The eluate with wash buffer gave no amplification product. These results indicate that elution of Neisseria gonorrhoeae (NG) DNA was caused by the presence of PAA and PMA rather than a “pH shift”.

Furthermore, this experiment demonstrated that lower formula amounts of PAA, PMA, and polyglutamic acid (“PGA”) also eluted DNA from AXpH ™ beads. Each DNA sample contained ⁵ μl of 105 copies of NG DNA in PreserCyt® medium. Lysis buffer (as described in Example 15) was added and the sample vortexed and incubated at 60 ° C. for 10 minutes. Each sample was shaken once after 5 minutes. AXpH ™ beads were separated magnetically, the supernatant was aspirated and washed with 500 μl of wash buffer (as described in Example 15), then the beads were separated and the wash buffer was aspirated. Then (a) a wash buffer; (b) an alkaline solution; (c) a wash buffer containing PAAH; (d) a wash buffer containing PAAL; (e) containing PMA Elution was performed by adding either wash buffer; or (f) wash buffer containing PGA. As can be seen in Table 3, the wash buffer control demonstrated good wash removal of DNA in a single step. Alkaline wash buffer was ineffective. PAAH gave very good recovery. PAAL worked even better than PAAH. PMA provided good recovery results comparable to PAAL, but the efficiency was somewhat lower.

Example 17: Elution of RNA with PAA This example demonstrates that RNA can also be eluted from AXpH ™ beads with polyacrylic acid. The RNA used in this example was a 7,983 nt transcript of the cloned HPV16 sequence. Add 25 μL of RNA solution (7.5 × 10 ⁴ copies / μL) in 1 mL PresevCyt medium to 60 μL of a suspension of Qiagen AXpH ™ beads in 1 mL of lysis buffer (as described in Example 15) Incubated at 60 ° C. and washed as described in Example 15. After the washing step, the beads were resuspended in 500 μL of wash buffer and bisected into 250 μl. After magnetic separation of the beads, one part was eluted with 25 μL of 0.5% PAA (> 200,000) containing 0.1% NP-40 for 10 minutes at 60C; the other part of the beads was subjected to the same conditions Elute with 2.5% PAA containing 0.1% NP-40. After separating the beads, the eluate from the first part was loaded directly into reverse transcription / PCR, and the eluate from the second part was diluted 5-fold with water. In either case, 2.5 μL of eluate volume, one-step reverse transcription using Qiagen 1-Step QuantiTech Virus Kit, containing primers and TaqMan® probe for the HPV16 6467-6590 region Added to 25 μL of PCR reaction. RNA controls were prepared by diluting the RNA solution appropriately with the corresponding elution buffer (ie 0.5% and 2.5% PAA) to mimic 100% elution of RNA from AXpH ™ beads. For the eluate, a 7.5 × 10 ⁴ copy / μL RNA stock solution, 10-fold and 50-fold dilution, respectively). One-step RT / PCR was performed in triplicate for each eluate / control according to the manufacturer's protocol. The results are shown in Table 4. The resulting difference in Ct between the control dilution and the eluate is within experimental error, thus suggesting that RNA elution was close to 100%.

Example 18: Effect of anion exchange material on PCR and reversal by multivalent anionic compounds Anecdotally observed that anion exchange material can interfere with effective amplification of purified nucleic acids Has been. To demonstrate this effect, real-time PCR was performed in the absence of AXpH beads or in the presence of 1: 10,000, 1: 1,000, or 1: 100 dilutions of AXpH beads. The results are shown in FIG. Inhibition is indicated by a right shift of the linear phase of the amplification curve.

  Carrier DNA was tested to determine if this inhibitory effect could be reversed. Bovine serum albumin (“BSA”) was tested as a control. 1:50 dilution of AXpH ™ beads and the presence of either 1 mg / ml BSA (1: 1 and 1:10 dilutions for final concentrations of 0.1 and 0.01 mg / ml BSA) or 100 ng / ml carrier DNA Below, real-time PCR amplification was performed in triplicate. A fluorescent signal was generated using a reporter probe labeled with either FAM (upper curve) or 5′-tetrachloro-fluorescein (“TET”) dye (lower curve). The results are shown in FIG. 17 and FIG.

  Polyacrylic acid was then tested to determine if this effect was specific for nucleic acids or could be replicated by other multivalent anionic compounds. Real-time in the presence of 1: 200, 1: 250; 1: 400; and 1: 500 dilutions of AXpH beads in the presence (“PAA +”) or absence (“PAA-”) of 25 ng / ml PAA PCR amplification was performed in triplicate. A fluorescent signal was generated using a reporter probe labeled with either FAM (upper curve) or 5′-tetrachloro-fluorescein dye (“TET”) (lower curve). As can be seen in FIG. 18, polyacrylic acid was similarly able to reverse PCR inhibition by anion exchange material. The results are shown in FIG.

In addition, PAA was tested at a range of target concentrations to determine whether this effect was maintained for low target concentrations in the presence of high anion exchange concentrations. ^Three real-time PCR amplifications were performed on 10, 10 ³ , and 10 ⁵ copies of the target nucleic acid in the presence of 1:25 dilution of AXpH beads and 0 or 25 ng / mL PAA. A fluorescent signal was generated using a reporter probe labeled with either FAM (upper curve) or 5′-tetrachloro-fluorescein dye (“TET”) (lower curve). The results are shown in FIG.

Example 19: Effect of anion exchange material on tHDA and reversal by polyanionic compounds
PCR is the most commonly used nucleic acid amplification method, but other methods are also known. To determine if the effect is specific for PCR, tHDA amplification was performed in the presence of anion exchange material with or without polyacrylic acid.

  As seen in FIG. 21, the presence of polyacrylic acid rescues amplification using tHDA, both at low and high target nucleic acid concentrations. Thus, multivalent anionic compounds are effective to rescue amplification using either PCR or tHDA.

Example 20: Amplification of nucleic acids in the presence of anion exchange materials Due to the inhibitory effect of anion exchange materials, nucleic acids are generally eluted and separated from the anion exchange material prior to analysis. . However, it would be advantageous to be able to analyze nucleic acids without having to separate from the beads. Therefore, an anionic compound disclosed herein was tested to determine whether it would allow real-time PCR amplification of nucleic acids in the presence of an anion exchange material.

  In one example, three different polyanionic compounds: polyacrylic acid, polymethacrylic acid, and polyglutamic acid were tested in real-time PCR. Target NG DNA was added to AXpH beads, vortexed and incubated for 10 minutes. The beads were then magnetically separated from the supernatant and washed with a wash buffer (10 mM Tris (pH 8); 0.1% NP-40 alternative) by vortexing. The beads were magnetically separated from the wash buffer and resuspended by vortexing the beads in either wash buffer or wash buffer + 0.25% of each multivalent anionic compound. A portion of these suspensions were then added to the plate wells for analysis. The results are shown in FIG.

  As another example, polyglutamic acid (“PGA”) was compared to polyadenylic acid (“Poly A”) and carboxymethyldextran (“CMD”). Where appropriate, this analysis was performed substantially as described above, except that 1.25% PGA, poly A, or CMD was used. The results are shown in FIG.

Example 22: Effect of polyanionic compounds on PCR and reversal by Mg ²⁺
PAA inhibits PCR, which can be corrected by increasing the concentration of Mg ²⁺ . Target NG DNA was amplified in the presence of either 0.05 or 0.1% PAA in the presence of two types of anion exchange material and 3, 7, or 11 mM Mg2 ⁺ . As can be seen in the figure, increasing the concentration of Mg ²⁺ reversed the inhibitory effect of PAA. The results are shown in FIG.

Claims (31)

(A) supplying a nucleic acid-anion exchange complex; and (b) adding a solution containing the anionic compound to the nucleic acid-anion exchange complex, wherein the anionic compound comprises: A method for eluting nucleic acids from an anion exchange material comprising the step of comprising at least two anionic groups comprising:
A method wherein the anionic compound disrupts the nucleic acid-anion exchange complex. (A) supplying a sample containing nucleic acid; and (b) adding the sample to the anion exchange material under pH and salt conditions where the anion exchange material is reversibly complexed with the nucleic acid. 2. The method of claim 1, wherein the nucleic acid-anion exchange complex is provided by a method comprising the step of: The anion exchange material is
(A) an amine of the formula R ₃ N;
(B) an amine of formula R ₂ NH;
(C) an amine of formula RNH ₂ ; and (d) a positively ionizable capture component selected from the group consisting of amines of formula X- (CH ₂ ) _n -Y;
Where
X is R ₂ N, RNH, or NH ₂ ;
Y is R ₂ N, RNH, or NH ₂ ;
A linear, branched or cyclic alkyl substituent, alkenyl substituted, wherein R may contain one or more heteroatoms independently of each other, preferably selected from O, N, S, and P A group, an alkynyl substituent or an aryl substituent, and
n is an integer ranging from 0 to 20,
The method of claim 1.   The method of claim 1, wherein the anion exchange material comprises magnetic silica beads modified with polyethyleneimine (PEI). At least one of the anionic compounds is
(A) a non-polymeric compound selected from the group consisting of carboxylic acids, oligomers of polymerizable or condensable acidic monomers, organic sulfonic acids, organic phosphonic acids, organophosphates, carbonates, and diacetylacetone, and (b) Polymerized unsaturated carboxylic acid; copolymer of unsaturated carboxylic acid and at least one other monomer; polypeptide of acidic amino acid; copolymer of acidic amino acid and at least one other amino acid; carboxylic acid, sulfonic acid, phosphonic acid, A polycarbohydrate having a covalently bonded ionizable group selected from the group consisting of phosphate and carbonate; a covalent selected from the group consisting of carboxylic acid, sulfonic acid, phosphonic acid, phosphate, and carbonate Combined ionization Polystyrene holding the ability group; second nucleic acid; and is selected from the group consisting of nucleic acid analogs, multivalent is selected from the group consisting of anionic compounds, the process of claim 1.   The method of claim 1, wherein the anionic compound comprises at least 3 anionic groups.   The method of claim 1, wherein the anionic compound is a non-polymeric compound containing 2 to 20 carbon atoms.   The anionic compound is selected from the group consisting of oxalic acid, mellitic acid, pyromellitic acid, citric acid, polyacrylic acid (PAA), polymethacrylic acid (PMA), polyglutamic acid (PGA), and dextran sulfate (DS). 2. The method of claim 1, wherein:   2. The method of claim 1, wherein the solution has a pH such that the nucleic acid-anion exchange complex is not disrupted in the absence of the anionic compound.   The method of claim 1, wherein the solution has a pH that does not exceed the pKa of the positively ionizable group of the capture component.   The method of claim 1, wherein the solution has a pH in the range of 5-13.   The method of claim 1, wherein the solution has a pH in the range of 5 to 8.5.   The method of claim 1, wherein the solution has a total salt concentration of approximately 1 M or less.   The method of claim 1, wherein the solution has a total salt concentration of about 0.5 M or less.   The method of claim 1, wherein the solution has a pH. The method of claim 1, wherein the solution further comprises an enzyme having polymerase activity and optionally a source of Mg ²⁺ .   3. The method of claim 2, wherein the solution has a pH and salt concentration that does not exceed pH and salt conditions at which the nucleic acid-anion exchange complex is formed. (A) providing a nucleic acid-anion exchange complex;
(B) adding a solution comprising an anionic compound to the nucleic acid-anion exchange complex, wherein the anionic compound comprises at least two anionic groups; and (c) A method of analyzing the nucleic acid in the presence of the anion exchange material comprising the step of analyzing the nucleic acid in the presence of the anionic compound and the anion exchange material.   19. The method of claim 18, wherein analyzing the nucleic acid comprises amplifying the nucleic acid.   Amplifying said nucleic acid is PCR, reverse transcriptase (“RT”) reaction, RT-PCR, quantitative real-time PCR, quantitative real-time RT-PCR, multiplex analysis, melting curve analysis, high resolution melting curve analysis, tHDA 19. The method of claim 18, comprising a procedure selected from the group consisting of: RT-tHDA, quantitative real-time tHDA, and quantitative real-time RT-tHDA. (A) comprises at least two anionic groups; and (b) comprises an anionic compound present in a concentration sufficient to disrupt the nucleic acid-anion exchange complex; and Having a pH that does not disrupt the nucleic acid-anion exchange complex in the absence of,
Elution composition.   The elution composition of claim 21, wherein the elution buffer has a pH and salt concentration suitable for performing a nucleic acid amplification reaction. 22. The elution composition of claim 21, further comprising an enzyme having polymerase activity and optionally including Mg ²⁺ or a source thereof. (A) nucleic acid;
(B) anion exchange material;
(C) an enzyme having polymerase activity;
(D) comprising an anionic compound comprising at least two anionic groups, and (e) optionally comprising Mg ²⁺ or a source thereof.
Amplification composition.   18. An eluate produced according to the method of any one of claims 1 to 17, which can be used directly in an enzymatic reaction without the need for further purification of the nucleic acid.   The enzyme reaction is PCR, reverse transcriptase (“RT”) reaction, RT-PCR, quantitative real-time PCR, quantitative real-time RT-PCR, multiplex analysis, melting curve analysis, high resolution melting curve analysis, thermophilic helicase dependence 26. The eluate of claim 25, wherein the eluate is a nucleic acid amplification selected from the group consisting of sex amplification ("tHDA"), RT-tHDA, quantitative real-time tHDA, and quantitative real-time RT-tHDA.   26. The eluate according to claim 25, wherein the nucleic acid is RNA. (A) an anion exchange material; and (b) a kit for isolating a nucleic acid from a biological sample comprising an anionic compound comprising at least two anionic groups.   30. The kit of claim 28, wherein the anionic compound is included in an elution composition. (A) anion exchange material;
(B) an enzyme having polymerase activity;
(C) comprises an anionic compound comprising at least two anionic groups, and (d) optionally further comprises Mg ²⁺ or a source thereof.
Kit for analyzing nucleic acids. Use of Mg ²⁺ to reverse PCR inhibition caused by anionic compounds containing at least two anionic groups.

Images