WO2019023937A1 - 一种高效的内切酶缓冲液体系 - Google Patents

一种高效的内切酶缓冲液体系 Download PDF

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WO2019023937A1
WO2019023937A1 PCT/CN2017/095470 CN2017095470W WO2019023937A1 WO 2019023937 A1 WO2019023937 A1 WO 2019023937A1 CN 2017095470 W CN2017095470 W CN 2017095470W WO 2019023937 A1 WO2019023937 A1 WO 2019023937A1
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mass
buffer system
endo
surfactant
endonuclease buffer
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PCT/CN2017/095470
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French (fr)
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张长领
刘二凯
陈奥
章文蔚
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深圳华大智造科技有限公司
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Priority to CN201780090482.6A priority Critical patent/CN110869501B/zh
Priority to PCT/CN2017/095470 priority patent/WO2019023937A1/zh
Publication of WO2019023937A1 publication Critical patent/WO2019023937A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • the invention relates to the field of enzyme technology, in particular to a highly efficient endonuclease buffer system.
  • Endo IV is an endonuclease that acts on a variety of DNA oxidative damage and hydrolyzes intact depurination/depyrimidine (AP) sites.
  • the first phosphodiester bond attached to the AP site can be cleaved leaving an -OH at the 3' end and a deoxyribose 5' phosphate at the 5' end.
  • the Endo IV enzyme also has 3'-diesterase activity, which releases glyceraldehyde phosphate, intact deoxyribose 5' phosphate, and phosphate from the 3' end of DNA (David J. Hosfield 1999).
  • Endo IV enzymes can be applied to DNA damage and repair studies (Demple & Harrison 1994), DNA structure studies (Hosfield et al. 1999), antitumor drug studies (Levin & Demple 1996), and SNP analysis (Kutyavin et al. 2006).
  • Endo IV enzyme buffer is NEB's buffer 3, including 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl. 2 , 1 mM DTT (dithiothreitol), pH 7.9 (25 ° C).
  • NEB's buffer 3 including 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl. 2 , 1 mM DTT (dithiothreitol), pH 7.9 (25 ° C).
  • Endo IV enzyme has a relatively low resection efficiency, and the excision time is more than 60 minutes (Wouter Meuleman 2013), which cannot meet the timely repair of DNA damage and efficient excision of some blocking groups (Backman et al) .1996), affecting the practical application of Endo IV enzyme.
  • the invention provides a high efficiency endonuclease buffer system, and solves the problem that the Endo IV enzyme has a long reaction time and low resection efficiency in the existing buffer solution by changing the reaction buffer system of the enzyme.
  • a highly efficient endonuclease buffer system comprising 20 to 200 mM Tris-HCl, 0.05 to 0.2 M NaCl, 2 to 20 mM MgSO 4 , 0.2 to 10 mM DTT, 0.05 to 5% by mass of a surfactant,
  • the pH is 7-8.
  • Tris-HCl Preferably, 50 mM Tris-HCl, 0.1 M NaCl, 2 mM MgSO 4 , 2 mM DTT, 1% by mass surfactant, pH 7.5 are included.
  • the above surfactant is selected from the group consisting of a cationic surfactant, an anionic surfactant, a nonionic surfactant, and a zwitterionic surfactant.
  • the above surfactant is Tween-80.
  • Tris-HCl Preferably, 50 mM Tris-HCl, 0.1 M NaCl, 2 mM MgSO 4 , 2 mM DTT, 1% by mass Tween-80, pH 7.5 are included.
  • the concentration of the above short-chain alcohol is 0.5% by mass.
  • the short chain alcohols are selected from the group consisting of ethanol, propanol, isopropanol, n-butanol and tert-butanol.
  • the above short-chain alcohol is 0.5% by mass of t-butanol.
  • Tris-HCl 0.1 M NaCl, 2 mM MgSO 4 , 2 mM DTT, 1% by mass of Tween-80, 0.5% by mass of t-butanol, pH 7.5 are included.
  • the original Endo IV enzyme buffer can optimize the excision efficiency of the Endo IV enzyme to the 3'-terminal phosphate and phosphate monoester nucleotide blocking groups in the DNA double strand, shorten the excision time and broaden the Endo The application of IV enzyme in practice.
  • FIG. 1 is a schematic diagram of a chip and a process according to an embodiment of the present invention
  • 2 is a signal picture of L01 and L02 on a chip in an embodiment of the present invention.
  • the present invention provides an endonuclease buffer system which enables the Endo IV enzyme to efficiently perform excision of the 3' phosphate and the phosphate monoester nucleotide blocking group in the polynucleotide chain.
  • the Endo IV enzyme is capable of cleavage of the position of the phosphodiester bond leaving an -OH at the 3' end.
  • the Endo IV enzyme is not efficient at excising the 3' phosphate group in the NEB buffer system.
  • An embodiment of the invention replaces a new Endo IV enzyme Working buffer, mainly including: Tris-HCl buffer at different pH and concentration, sodium chloride, magnesium sulfate, dithiothreitol (DTT), and preferably various surfactants and short-chain alcohols.
  • Working buffer mainly including: Tris-HCl buffer at different pH and concentration, sodium chloride, magnesium sulfate, dithiothreitol (DTT), and preferably various surfactants and short-chain alcohols.
  • a highly efficient endonuclease buffer system comprises 20 to 200 mM Tris-HCl, 0.05 to 0.2 M NaCl, 2 to 20 mM MgSO 4 , 0.2 to 10 mM DTT, 0.05 to 5% by mass.
  • the surfactant has a pH of 7-8.
  • the concentration of each component is based on the working concentration, and those skilled in the art can understand that the endonuclease buffer system can also be used as a storage solution.
  • the concentration of the various components in the form of the stock solution may be several times the working concentration, such as 10 times or 5 times. Therefore, the concentration of each component in the above endonuclease buffer system which is multiplied by an equal ratio is also understood to be included in the scope of protection of the present invention.
  • the concentration of each component in the endonuclease buffer system of the embodiment of the present invention is a range, that is, a concentration of Tris-HCl of 20 to 200 mM, a concentration of NaCl of 0.05 to 0.2 M, a concentration of MgSO 4 of 2 to 20 mM, and a concentration of DTT of 0.2 to 10 mM.
  • the surfactant concentration is 0.05 to 5% by mass, and the pH is 7 to 8.
  • the endonuclease buffer system in the examples of the present invention was optimized for the Endo IV enzyme. However, the inventors also verified the effect of the endonuclease buffer system on the excision efficiency of other endonucleases, for example, APN1, Tth Endo IV and APE 1 endonuclease, etc., indicating that the endonuclease buffer system can be widely used. It is used in a variety of different endonucleases.
  • the surfactant may be selected within a relatively wide range, such as a cationic surfactant, an anionic surfactant, a nonionic surfactant, and a zwitterionic surfactant.
  • a cationic surfactant include: ditetradecyldimethylammonium bromide, cetyltrimethylammonium bromide; typical as an anionic surfactant but Non-limiting examples include: polyoxyethylene lauryl ether carboxylic acid, glycolic acid ethoxy oleate, glycolic acid 4-tert-butyl phenyl ether, sodium lignin sulfonate, N-lauric acid amide Sodium sarcosinate, poly(2-propenylamine-2-methyl-1-propanesulfonic acid) solution; typical but non-limiting examples of nonionic surfactants include: Tween-20, Tween-80, Triton X-100, poly(isobutyl)
  • endonuclease buffer system comprising of 50mM Tris-HCl, 0.1M of NaCl, MgSO 2mM of DTT 4, 2mM, and 1 mass% of a surfactant, pH 7.5.
  • the endonuclease buffer system comprises 50 mM Tris-HCl, 0.1 M NaCl, 2 mM MgSO 4 , 2 mM DTT, 1% by mass Tween-80, pH 7.5.
  • the endonuclease buffer system further comprises 0.1 to 5% by mass of short-chain alcohols having a carbon number of less than 5, such as ethanol, propanol, isopropanol, n-butanol and uncle Butanol.
  • concentration of the short-chain alcohol is preferably 0.5% by mass.
  • the short chain alcohol is 0.5% by mass of tert-butanol.
  • the endonuclease buffer system comprises 50 mM Tris-HCl, 0.1 M NaCl, 2 mM MgSO 4 , 2 mM DTT, 1% by mass Tween-80, 0.5% by mass of uncle Butanol, pH 7.5.
  • Example 1 Excision efficiency of Endo IV enzyme in the range of buffer pH 4-9
  • a buffer of different pH (4-9) was prepared using 50 mM Tris-HCl, and 0.1 M NaCl, 2 mM MgSO 4 , 2 mM DTT was added to prepare an Endo IV enzyme working solution.
  • a 42 bp primer was used as a substrate for the enzyme, and the 3' end of the primer was a nucleotide having a phosphate group as a blocking group. Normal nucleotide primers were selected for the control experiment, and the default was that the control group had a removal efficiency of 100% under its own excision reagent.
  • the two channels of the chip ( Figure 1) are loaded with the same DNA Nano Ball (DNB), and the common nucleotide primers (L01) are loaded in the two channels (L01/L02). And a nucleotide primer (L02) having a phosphate group as a blocking group at the 3' end. Then, the chip was heated to 37 ° C, and the reaction buffer containing Endo IV and Endo IV was separately pumped and reacted for 5 min.
  • DNB DNA Nano Ball
  • the buffer was washed away with an eluent, and the dNTPs reagent labeled with fluorescence was added to extend the 3' end of the primer, and the unreacted dNTPs were washed away, and then a laser was used to photograph and detect the fluorescence signal, thereby obtaining a control group.
  • the signal value of L01 and the signal value of experimental group L02. The scanned signal picture is shown in Figure 2.
  • the excision efficiency of the phosphate group was determined using L01 as a control. Table 1 shows the signal measured using NEB's Endo IV buffer and its excision efficiency.
  • Endo IV basic working solution was prepared: 50 mM Tris-HCl (pH 7.5), 0.1 M NaCl, 2 mM MgSO 4 , 2 mM DTT. Based on this, different surfactants were added to prepare Endo IV as the reaction working solution.
  • the two channels of the chip ( Figure 1) are loaded with the same DNA Nano Ball (DNB), and the common nucleotide primers (L01) are loaded in the two channels (L01/L02). And a nucleotide primer (L02) having a phosphate group as a blocking group at the 3' end. Then, the chip was heated to 37 ° C, and the reaction working solution containing Endo IV and Endo IV was separately pumped and reacted for 2 min.
  • DNB DNA Nano Ball
  • the concentration of each surfactant was 0.05% by mass, 0.1% by mass, 0.5% by mass, 1% by mass, 2% by mass, and 5% by mass, respectively; Both are ranges, indicating that the excision efficiency is within the corresponding range at the above several concentrations.
  • the Endo IV basic working solution was prepared: 50 mM Tris-HCl (pH 7.5), 0.1 M NaCl, 2 mM MgSO 4 , 2 mM DTT, 1% by mass Tween-80. On this basis, different short-chain alcohols were added to prepare the Endo IV reaction working solution.
  • the two channels of the chip ( Figure 1) are loaded with the same DNA Nano Ball (DNB), and the common nucleotide primers (L01) are loaded in the two channels (L01/L02). And a nucleotide primer (L02) having a phosphate group as a blocking group at the 3' end. Then, the chip was heated to 37 ° C, and the reaction working solution containing Endo IV and Endo IV was separately pumped and reacted for 2 min.
  • DNB DNA Nano Ball
  • the buffer was washed away with an eluent, and the dNTPs reagent labeled with fluorescence was added to extend the 3' end of the primer, and the unreacted dNTPs were washed away, and then a laser was used to photograph and detect the fluorescence signal, thereby obtaining a control group.
  • the experimental results of adding different short-chain alcohols are shown in Table 4.
  • the concentration of each short-chain alcohol was 0.1% by mass, 0.5% by mass, 1% by mass, 2% by mass, and 5% by mass, respectively; It is the range, indicating that the excision efficiency is within the corresponding range at the above several concentrations.
  • Short-chain alcohols have a positive effect on improving Endo IV resection; and by optimizing the above conditions, Endo IV can achieve 100% resection efficiency within 2 min.
  • a reaction working solution was prepared: 50 mM Tris-HCl (pH 7.5), 0.1 M NaCl, 2 mM MgSO 4 , 2 mM DTT, 1% by mass of Tween-80, and 0.5% by mass of t-butanol.
  • the efficiency of the reaction in the two buffer systems was determined by adding endonucleases Endo IV, APN1, Tth Endo IV and APE 1 from different sources in NEB buffer and the reaction working solution, respectively.
  • both channels of the chip were loaded with the same DNA nanosphere (DNB) and loaded with a phosphate group at the 3' end as a blocking group in both channels (L01/L02). Nucleotide primers. Then, the chip was heated to 37 ° C, and the reaction working solution containing different endonucleases was separately pumped for 2 min. Subsequently, the buffer was washed away with an eluent, and the dNTPs reagent labeled with fluorescence was added to extend the 3' end of the primer, and the unreacted dNTPs were washed away.
  • DNB DNA nanosphere
  • the signal value of the control group L01 and the signal value of the experimental group L02 can be obtained, respectively.
  • the excision efficiency of the phosphate group was determined using L01 as a control.
  • the experimental results of adding different endonucleases are shown in Table 5:
  • the optimized buffer system not only has a significant increase in the reaction rate of Endo IV, but also has an effect of increasing the reaction rate for endonucleases from other species.

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Abstract

一种能提高Endo IV酶活性的内切酶缓冲液，包括20～200mM的Tris-HCl，0.05～0.2M的NaCl，2～20mM的MgSO 4，0.2～10mM的DTT，0.05～5质量％的表面活性剂，pH为7～8。所述缓冲液还可进一步包括0.1～5质量％的碳原子数小于5的短链醇类。

Description

一种高效的内切酶缓冲液体系 技术领域
本发明涉及酶技术领域，具体涉及一种高效的内切酶缓冲液体系。
背景技术
Endo IV(Endonuclease IV)是一种核酸内切酶，可作用于多种DNA氧化损伤，能够水解完整的脱嘌呤/脱嘧啶(AP)位点。能够切除与AP位点相连的第一个磷酸二酯键，在3’末端留下一个-OH，而在5’末端留下一个脱氧核糖5’磷酸。Endo IV酶也具有3’-二酯酶活性，可以从DNA的3’端释放出磷酸甘油醛、完整的脱氧核糖5’磷酸以及磷酸盐(David J.Hosfield1999)。Endo IV酶可应用于DNA损伤和修复研究(Demple&Harrison 1994)、DNA结构研究(Hosfield et al.1999)、抗肿瘤药物研究(Levin&Demple 1996)和SNP分析(Kutyavin et al.2006)。
Endo IV酶的许多实际的应用依赖于其快速高效的切除磷酸二酯键的效率，目前常用的Endo IV酶的缓冲液是NEB公司的缓冲液3，包括100mM NaCl、50mM Tris-HCl、10mM MgCl2、1mM DTT(二硫苏糖醇)、pH 7.9(25℃)。但是，在此缓冲液体系中，Endo IV酶的切除效率比较低，切除时间在60分钟以上(Wouter Meuleman 2013)，不能满足DNA损伤的及时修复以及一些阻断基团的高效切除(Backman et al.1996)，影响了Endo IV酶在实际中的应用。
发明内容
本发明提供一种高效的内切酶缓冲液体系，通过改变酶的反应缓冲液体系，解决了Endo IV酶在现有的缓冲液中反应时间长，切除效率低的问题。
一种高效的内切酶缓冲液体系，包括20～200mM的Tris-HCl，0.05～0.2M的NaCl，2～20mM的MgSO4，0.2～10mM的DTT，0.05～5质量％的表面活性剂，pH为7～8。
优选地，包括50mM的Tris-HCl，0.1M的NaCl，2mM的MgSO4，2mM的DTT，1质量％的表面活性剂，pH为7.5。
优选地，上述表面活性剂选自阳离子型表面活性剂、阴离子型表面活性剂、非离子型表面活性剂和两性离子型表面活性剂。
优选地，上述表面活性剂是Tween-80。
优选地，包括50mM的Tris-HCl，0.1M的NaCl，2mM的MgSO4，2mM的DTT，1质量％的Tween-80，pH为7.5。
进一步地，还包括0.1～5质量％的碳原子数小于5的短链醇类。
优选地，上述短链醇类的浓度是0.5质量％。
优选地，上述短链醇类选自乙醇、丙醇、异丙醇、正丁醇和叔丁醇。
优选地，上述短链醇类是0.5质量％的叔丁醇。
优选地，包括50mM的Tris-HCl，0.1M的NaCl，2mM的MgSO4，2mM的DTT，1质量％的Tween-80，0.5质量％的叔丁醇，pH为7.5。
原有的Endo IV酶的缓冲液经过优化后，能够极大提高Endo IV酶对DNA双链中3’端磷酸及磷酸单酯核苷酸阻断基团的切除效率，缩短切除时间，拓宽Endo IV酶在实际中的应用。
附图说明
图1为本发明实施例中芯片及流程示意图；
图2为本发明实施例中L01和L02在芯片上的信号图片。
具体实施方式
下面通过具体实施方式结合附图对本发明作进一步详细说明。在以下的实施方式中，很多细节描述是为了使得本发明能被更好的理解。然而，本领域技术人员可以毫不费力的认识到，其中部分特征在不同情况下是可以省略的，或者可以由其他元件、材料、方法所替代。在某些情况下，本发明相关的一些操作并没有在说明书中显示或者描述，这是为了避免本发明的核心部分被过多的描述所淹没，而对于本领域技术人员而言，详细描述这些相关操作并不是必要的，他们根据说明书中的描述以及本领域的一般技术知识即可完整了解相关操作。
本发明实施例提供一种使Endo IV酶能够高效完成对多核苷酸链中3’磷酸及磷酸单酯核苷酸阻断基团的切除的内切酶缓冲液体系。Endo IV酶能够切断磷酸二酯键的位置，在3’末端留下一个-OH。但对于在3’端使用磷酸或者单磷酸酯基团阻断的核苷酸来说，Endo IV酶在NEB的缓冲液体系中切除3’端磷酸基团的效率并不高。本发明实施例更换新的Endo IV酶的 工作缓冲液，主要包括：不同pH及浓度的Tris-HCl缓冲液、氯化钠、硫酸镁、二硫苏糖醇(DTT)，以及优选添加的多种表面活性剂和短链醇类，极大提高了Endo IV酶对多核苷酸链中3’磷酸核苷酸阻断基团的切除效率。
在本发明实施例中，高效的内切酶缓冲液体系，包括20～200mM的Tris-HCl，0.05～0.2M的NaCl，2～20mM的MgSO4，0.2～10mM的DTT，0.05～5质量％的表面活性剂，pH为7～8。
需要说明的是，在本发明实施例的内切酶缓冲液体系中各种成分的浓度是按照工作浓度计的，本领域技术人员能够理解，内切酶缓冲液体系也可以以储存液(备用液)的形式提供，这种储存液形式中各种成分的浓度可能是工作浓度的若干倍，如10倍或5倍等。因此，上述内切酶缓冲液体系中各种成分的浓度按照等比例成倍增加的浓度，也应当理解为是包括在本发明的保护范围之内。
在本发明实施例的内切酶缓冲液体系中各种成分的浓度均是范围，即Tris-HCl浓度20～200mM、NaCl浓度0.05～0.2M、MgSO4浓度2～20mM、DTT浓度0.2～10mM、表面活性剂浓度0.05～5质量％，以及pH为7～8。发明人在上述各浓度的范围内进行了大量实验验证，表明在上述浓度范围内变化时，Endo IV酶的切除效率不会受到明显的影响。
在本发明实施例的内切酶缓冲液体系是针对Endo IV酶进行优化得到的。但是，发明人也验证了该内切酶缓冲液体系对其它内切酶的切除效率的影响，例如，APN1、Tth Endo IV和APE 1内切酶等，表明该内切酶缓冲液体系能够广泛地用于各种不同的内切酶。
在本发明实施例中，表面活性剂可以在比较宽泛的范围内选择，例如阳离子型表面活性剂、阴离子型表面活性剂、非离子型表面活性剂和两性离子型表面活性剂。其中，作为阳离子型表面活性剂的典型但非限定性的实例包括：溴化双十四烷基二甲基铵、十六烷基三甲基溴化铵；作为阴离子型表面活性剂的典型但非限定性的实例包括：聚氧乙烯月桂醚羧酸、乙醇酸乙氧基油醚、二醇酸乙氧酸4-叔丁基苯基醚、木素磺酸钠盐、N-月桂酸酰肌氨酸钠盐、聚(2-丙烯胺-2甲基-1-丙磺酸)溶液；作为非离子型表面活性剂的典型但非限定性的实例包括：Tween-20、Tween-80、Triton X-100、聚(异丁烯-alt-马来酸酐)、聚(马来酸酐-alt-1-十八碳烯)、聚乙二醇单油酸酯、聚氧代乙烯山梨醇六油酸酯、聚(乙烯二醇)山嵛醚甲丙烯酰酸、PEG 4000、PEG 6000、PEG 8000；作为两性离子型表面活性剂的典型但非限定性的实例包括：十二烷基二甲基氧化胺。在一个最优的实施例中，表面活性剂是Tween-80。
在本发明的一个优选实施例中，内切酶缓冲液体系包括50mM的Tris-HCl，0.1M的NaCl，2mM的MgSO4，2mM的DTT，1质量％的表面活性剂，pH为7.5。在本发明的一个更优选 实施例中，内切酶缓冲液体系包括50mM的Tris-HCl，0.1M的NaCl，2mM的MgSO4，2mM的DTT，1质量％的Tween-80，pH为7.5。
在本发明的进一步改进的实施例中，内切酶缓冲液体系还包括0.1～5质量％的碳原子数小于5的短链醇类，例如乙醇、丙醇、异丙醇、正丁醇和叔丁醇。短链醇类的浓度优选是0.5质量％。在一个最优的实施例中，短链醇类是0.5质量％的叔丁醇。
在本发明的一个优选实施例中，内切酶缓冲液体系包括50mM的Tris-HCl，0.1M的NaCl，2mM的MgSO4，2mM的DTT，1质量％的Tween-80，0.5质量％的叔丁醇，pH为7.5。
以下通过实施例详细说明本发明的技术方案和效果，应当理解，实施例仅是示例性的，不能理解为对本发明保护范围的限制。
实施例1：在缓冲液pH 4～9的范围内Endo IV酶的切除效率
使用50mM的Tris-HCl配制不同pH(4～9)的缓冲液，并加入0.1M NaCl、2mM MgSO4、2mM DTT配制成Endo IV酶工作液。以一段42bp的引物作为酶作用的底物，这段引物的3’端是一个带有磷酸基团作为阻断基团的核苷酸。对照实验选取普通核苷酸引物，并默认为对照组在其自身的切除试剂下的切除效率为100％。
首先，将芯片两条通道(如图1)都装载上相同的DNA纳米球(DNA Nano Ball，DNB)，并分别在两条通道(L01/L02)中装载上普通核苷酸引物(L01)和3’端带有磷酸基团作为阻断基团的核苷酸引物(L02)。然后，将芯片加热到37℃，分别泵入不含Endo IV和含有Endo IV的反应缓冲液，反应5min。随后，使用洗脱液将缓冲液清洗掉，并加入标记有荧光的dNTPs试剂来延伸引物的3’端，将未反应的dNTPs洗掉后使用激光进行拍照，检测荧光信号，能够分别得到对照组L01的信号值以及实验组L02的信号值。扫描得到的信号图片如图2所示。以L01作为对照来确定磷酸基团的切除效率。表1是使用NEB的Endo IV缓冲液测的信号及其切除效率。
表1 NEB的Endo IV缓冲液切除5min后测的信号及其切除效率
Figure PCTCN2017095470-appb-000001
使用以上方法对不同pH(4～9)的缓冲液进行5min切除测试，并拍照检测信号值，实验结果如表2：
表2 使用不同pH的Endo IV缓冲液切除后信号值
Figure PCTCN2017095470-appb-000002
结论：同一条件下，pH 7～8范围内具有较好的切除效率，pH 7.5的缓冲液有最好的切除效率。
实施例2：在Endo IV工作液中加入表面活性剂对切除效率的影响
配制Endo IV基本工作液：50mM Tris-HCl(pH7.5)、0.1M NaCl、2mM MgSO4、2mM DTT。并在此基础上加入不同的表面活性剂来配制成Endo IV作为反应工作液。
首先，将芯片两条通道(如图1)都装载上相同的DNA纳米球(DNA Nano Ball，DNB)，并分别在两条通道(L01/L02)中装载上普通核苷酸引物(L01)和3’端带有磷酸基团作为阻断基团的核苷酸引物(L02)。然后，将芯片加热到37℃，分别泵入不含Endo IV和含有Endo IV的反应工作液，反应2min。随后，使用洗脱液将缓冲液清洗掉，并加入标记有荧光的dNTPs试剂来延伸引物的3’端，将未反应的dNTPs洗掉后使用激光进行拍照，检测荧光信号，能够分别得到对照组L01的信号值以及实验组L02的信号值。以L01作为对照来确定磷酸基团的 切除效率。加入不同表面活性剂的实验结果如表3，具体实验中，每种表面活性剂浓度分别取0.05质量％、0.1质量％、0.5质量％、1质量％、2质量％和5质量％；切除效率均是范围，表示在上述几个浓度下，切除效率均在对应范围内。
表3 工作液中加入不同表面活性剂Endo IV的四种碱基信号值的平均切除效率
表面活性剂 浓度/质量％ 切除效率/％
Tween-20 0.05～5 90～95
Tween-80 0.05～5 92～99
聚氧乙烯月桂醚羧酸 0.05～5 88～95
Triton X-100 0.05～5 90～94
乙醇酸乙氧基油醚 0.05～5 88～95
乙醇酸乙氧酸4-壬基苯醚 0.05～5 85～90
二醇酸乙氧酸4-叔丁基苯基醚 0.05～5 86～90
双十四烷基二甲基溴化铵 0.05～5 90～95
聚(异丁烯-alt-马来酸酐) 0.05～5 88～97
聚乙二醇单油酸酯 0.05～5 86～93
聚氧代乙烯山梨醇六油酸酯 0.05～5 88～98
十二烷基二甲基氧化胺 0.05～5 92～99
十六烷基三甲基溴化铵 0.05～5 90～98
木素磺酸钠盐 0.05～5 88～95
聚(马来酸酐-alt-1-十八碳烯) 0.05～5 90～98
N-月桂酸酰肌氨酸钠盐 0.05～5 90～95
聚(2-丙烯胺-2甲基-1-丙磺酸)溶液 0.05～5 92～97
聚(乙烯二醇)山嵛醚甲丙烯酰酸 0.05～5 93～98
PEG 4000 0.05～5 92～97
PEG 6000 0.05～5 93～98
PEG 8000 0.05～5 93～99
结论：在合适浓度的表面活性剂的作用下，Endo IV的切除效率有大幅提升，2min内切除效率接近100％。
实施例3：短链醇类对Endo IV的切除效率的影响
配制Endo IV基本工作液：50mM Tris-HCl(pH7.5)、0.1M NaCl、2mM MgSO4、2mM DTT、1质量％Tween-80。并在此基础上加入不同的短链醇类来配制成Endo IV反应工作液。
首先，将芯片两条通道(如图1)都装载上相同的DNA纳米球(DNA Nano Ball，DNB)，并分别在两条通道(L01/L02)中装载上普通核苷酸引物(L01)和3’端带有磷酸基团作为阻断基团的核苷酸引物(L02)。然后，将芯片加热到37℃，分别泵入不含Endo IV和含有Endo IV的反应工作液，反应2min。随后，使用洗脱液将缓冲液清洗掉，并加入标记有荧光的dNTPs试剂来延伸引物的3’端，将未反应的dNTPs洗掉后使用激光进行拍照，检测荧光信号，能够分别得到对照组L01的信号值以及实验组L02的信号值。以L01作为对照来确定磷酸基团的切除效率。加入不同的短链醇类的实验结果如表4，具体实验中，每种短链醇类浓度分别取0.1质量％、0.5质量％、1质量％、2质量％和5质量％；切除效率均是范围，表示在上述几个浓度下，切除效率均在对应范围内。
表4 工作液中加入短链醇类Endo IV的四种碱基信号值的平均切除效率
短链醇类 浓度/质量％ 切除效率/％
乙醇 0.1～5 99～99.5
丙醇 0.1～5 99～99.8
异丙醇 0.1～5 99～100
正丁醇 0.1～5 99～99.9
叔丁醇 0.1～5 99～100
结论：短链醇类对提高Endo IV的切除有积极的影响；而且通过以上条件的优化，Endo IV能够在2min内达到100％的切除效率。
实施例4：不同种类的内切酶在此缓冲液体系中的比较
配制反应工作液：50mM Tris-HCl(pH7.5)、0.1M NaCl、2mM MgSO4、2mM DTT、1质量％Tween-80和0.5质量％叔丁醇。分别在NEB缓冲液和此反应工作液加入不同的来源的内切酶Endo IV、APN1、Tth Endo IV和APE 1，测定在这两种缓冲液体系中的反应效率。
首先，将芯片两条通道都装载上相同的DNA纳米球(DNA Nano Ball，DNB)，并分别在两条通道(L01/L02)中都装载3’端带有磷酸基团作为阻断基团的核苷酸引物。然后，将芯片加热到37℃，分别泵入含有不同内切酶的反应工作液，反应2min。随后，使用洗脱液将缓冲液清洗掉，并加入标记有荧光的dNTPs试剂来延伸引物的3’端，将未反应的dNTPs洗掉后 使用激光进行拍照，检测荧光信号，能够分别得到对照组L01的信号值以及实验组L02的信号值。以L01作为对照来确定磷酸基团的切除效率。加入不同的内切酶的实验结果如表5：
表5 NEB缓冲液和优化后的反应工作液中加入不同的内切酶的平均切除效率
Figure PCTCN2017095470-appb-000003
结论：优化后的缓冲液体系不仅仅对Endo IV的反应速率有明显的提高，而且对其它种属来源的内切酶同样有提高反应速率的作用。
以上应用了具体个例对本发明进行阐述，只是用于帮助理解本发明，并不用以限制本发明。对于本发明所属技术领域的技术人员，依据本发明的思想，还可以做出若干简单推演、变形或替换。

Claims (10)

  1. 一种高效的内切酶缓冲液体系，其特征在于，包括20～200mM的Tris-HCl，0.05～0.2M的NaCl，2～20mM的MgSO4，0.2～10mM的DTT，0.05～5质量％的表面活性剂，pH为7～8。
  2. 根据权利要求1所述的内切酶缓冲液体系，其特征在于，包括50mM的Tris-HCl，0.1M的NaCl，2mM的MgSO4，2mM的DTT，1质量％的表面活性剂，pH为7.5。
  3. 根据权利要求1或2所述的内切酶缓冲液体系，其特征在于，所述表面活性剂选自阳离子型表面活性剂、阴离子型表面活性剂、非离子型表面活性剂和两性离子型表面活性剂。
  4. 根据权利要求3所述的内切酶缓冲液体系，其特征在于，所述表面活性剂是Tween-80。
  5. 根据权利要求3所述的内切酶缓冲液体系，其特征在于，包括50mM的Tris-HCl，0.1M的NaCl，2mM的MgSO4，2mM的DTT，1质量％的Tween-80，pH为7.5。
  6. 根据权利要求1或2所述的内切酶缓冲液体系，其特征在于，还包括0.1～5质量％的碳原子数小于5的短链醇类。
  7. 根据权利要求6所述的内切酶缓冲液体系，其特征在于，所述短链醇类的浓度是0.5质量％。
  8. 根据权利要求6所述的内切酶缓冲液体系，其特征在于，所述短链醇类选自乙醇、丙醇、异丙醇、正丁醇和叔丁醇。
  9. 根据权利要求6所述的内切酶缓冲液体系，其特征在于，所述短链醇类是0.5质量％的叔丁醇。
  10. 根据权利要求6所述的内切酶缓冲液体系，其特征在于，包括50mM的Tris-HCl，0.1M的NaCl，2mM的MgSO4，2mM的DTT，1质量％的Tween-80，0.5质量％的叔丁醇，pH为7.5。
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