WO2018099141A1 - Method and kit for detecting recombinase strand displacement activity - Google Patents

Method and kit for detecting recombinase strand displacement activity Download PDF

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WO2018099141A1
WO2018099141A1 PCT/CN2017/098210 CN2017098210W WO2018099141A1 WO 2018099141 A1 WO2018099141 A1 WO 2018099141A1 CN 2017098210 W CN2017098210 W CN 2017098210W WO 2018099141 A1 WO2018099141 A1 WO 2018099141A1
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stranded dna
strand displacement
recombinase
detecting
double
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盛司潼
孙文龙
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广州康昕瑞基因健康科技有限公司
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general
    • G01N2333/91245—Nucleotidyltransferases (2.7.7)

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  • the present invention relates to the field of molecular biology, and more particularly to a method and kit for detecting recombinase chain displacement activity.
  • Recombinase is a kind of proteinase that binds to a single-stranded DNA molecule and invades its homologous double-stranded DNA fragment, allowing a single-stranded DNA molecule to bind to a double-stranded DNA fragment in a homologous region to form a stable complex structure. Recombinases are involved in DNA repair and UV-induced mutations and are indispensable in organisms.
  • the detection methods for recombinase chain displacement activity are common: for example, isotopically labeled single-stranded M13 phage or Phi x174 phage, although the method of isotope labeling is simple and sensitive, the reagent cost of the method is high, and the general scientific research institutions and enterprises No unit can be carried out.
  • the literature reports that the chain displacement activity of recombinases is detected by agarose gel electrophoresis. When homologous recombination occurs, single-stranded DNA completely replaces one strand of homologous double-stranded DNA to produce a gap-free loop.
  • the DNA product can be found on the single-stranded DNA agarose electrophoresis map, but the length of the phage DNA using the method is substantially above 5000 bp, and the product which produces complete strand displacement has higher requirements on the system. Sufficient product can be detected, which requires repeated adjustments to the system.
  • the object of the present invention is to provide a method and a kit for detecting recombinase chain displacement activity, which aims to solve the problem that the detection of recombinase chain displacement activity in the prior art requires high reaction system and high cost.
  • the present invention provides a method for detecting recombinase strand displacement activity, comprising the steps of:
  • the 3' end of the single-stranded DNA molecule is immobilized on a magnetic bead.
  • the isolated product is detected to contain a double-stranded DNA structure, and it is determined that the recombinase has strand displacement activity.
  • the recombinant enzyme to be tested is RecA or uvsX.
  • the first sequence has a length of 16 bp to 100 bp.
  • the method further comprises the step of: heat treating the chain displacement complex, wherein the step of heat treatment is to incubate the chain displacement complex at 65 to 80 ° C for 5 to 20 minutes.
  • the method for detecting the isolated product in the step C is: any one of an electrophoresis method, a fluorescence detection method, an ultraviolet absorption value detection method, and a fluorescent dye method.
  • the strand displacement reaction system further comprises a single-stranded binding protein.
  • the invention also provides a kit for detecting a recombinase strand displacement activity, comprising a single-stranded DNA molecule, a double-stranded DNA fragment; the single-stranded DNA molecule comprising a first sequence, the first sequence and the double-stranded DNA fragment Partial sequences are homologously complementary.
  • the sequence of the single-stranded DNA molecule homologously complementary to the double-stranded DNA fragment is 16 bp to 100 bp in length.
  • a single-stranded DNA molecule can invade a double-stranded DNA fragment having a homologous complementary sequence and obtain a strand displacement complex under the action of a recombinase having a strand displacement activity, and after the recombinant enzyme is inactivated or denatured, the single-stranded DNA molecule is detached. Thereby, the double-stranded DNA fragment is re-obtained, and therefore, the present invention can indicate that the recombinant enzyme to be tested has strand displacement activity by detecting the double-stranded DNA fragment.
  • the method for detecting recombinase chain displacement activity of the invention has simple operation, low requirements on instruments, and no pollution to the environment.
  • FIG. 1 is a schematic view showing the detection of recombinase chain displacement activity in the first embodiment of the present invention.
  • Fig. 2 is a diagram showing the gel electrophoresis detection of the separated product of the third embodiment of the present invention.
  • the recombinase has a single-stranded DNA-binding activity, and when a double-stranded DNA fragment having a target sequence complementary to a single-stranded DNA molecule exists in the system, recombination having a strand displacement activity in the case where the ATP or ATP analog provides energy
  • the enzyme can form a stable complex with the single-stranded DNA molecule, and the complex searches for and invades the double-stranded DNA region which is homologously complementary to the single-stranded DNA molecule sequence, and undergoes a strand displacement reaction under the action of the recombinase.
  • the single-stranded DNA molecule contains a sequence homologously complementary to the partially double-stranded DNA fragment, a partial strand displacement reaction occurs on the double-stranded DNA fragment.
  • a single-stranded DNA molecule 2 invades a part of the double-stranded DNA fragment 3 to undergo a strand displacement reaction to form a strand displacement complex 4; and a purified strand replacement complex 4, And treating it to denature or inactivate the recombinant enzyme, the single-stranded DNA molecule 2 can be detached, and the isolated product can be obtained by separating the single-stranded DNA molecule 2 from the system; and detecting the isolated product, which contains the double-stranded DNA structure, It is determined that the recombinase to be tested has strand displacement activity; if the isolated product does not contain a double-stranded DNA structure, it is determined that the recombinase to be tested does not have strand displacement activity.
  • the 3' end of the single-stranded DNA molecule comprises a biotin label and is immobilized on a magnetic bead comprising streptavidin by a biotin label.
  • the solution makes it possible to conveniently purify the chain displacement complex immobilized on the magnetic beads by separating the magnetic beads during the operation of the step B; at the same time, it is also ensured that the single chain is separated during the operation of the step C.
  • the steps of DNA molecules are simple.
  • the first sequence on the single-stranded DNA molecule is from 16 bp to 100 bp in length.
  • the single-stranded DNA molecule immobilized on the solid phase carrier can be firmly bound to the double-stranded DNA fragment to form a stable strand displacement complex under the action of the recombinase.
  • the first sequence may be a partial sequence of a single-stranded DNA molecule, or may be a complete sequence of a single-stranded DNA molecule.
  • the first sequence is the entire sequence of a single-stranded DNA molecule.
  • the present scheme reduces the difficulty in synthesizing single-stranded DNA molecules by ensuring that the single-stranded DNA molecules are intruded after the double-stranded DNA fragments are intruded.
  • the double-stranded DNA fragment is 50 to 200 bp in length.
  • the double-stranded DNA fragment of the present scheme is simple in design and simple in synthesis.
  • the double-stranded DNA fragment has a length of not less than 200 bp.
  • the uninvaded double strands can be stably coupled by hydrogen bonding, thereby ensuring the stability of the strand-replacement complex structure.
  • the step further comprises the step of inactivating or denaturation of the recombinant enzyme to be tested, and the step of inactivating or denaturation of the recombinant enzyme to be tested may be the step of adding a proteolytic enzyme to the strand displacement complex to decompose the recombinant enzyme, or The heat treatment is performed on the chain displacement composite.
  • the step of inactivating or denaturation of the recombinant enzyme to be tested is: heat treating the strand displacement complex, and the step of heat treatment is to incubate the strand displacement complex at 65 to 80 ° C for 5 to 20 minutes.
  • the recombinant enzyme is denatured by heat treatment, and the operation is simple, no other impurities are introduced, and the temperature range of the solution can denature the recombinant enzyme, thereby separating and detaching the single-stranded DNA molecule immobilized on the solid phase carrier and reforming the double-stranded DNA. But does not cause double-stranded DNA to form a single strand.
  • the buffer comprises the following components: 20 to 100 mM Tris-HCl, 5 to 14 mM MgCl2, 0.1 to 1.0 mM DTT, and a pH of 7.5 to 8.0.
  • the buffer of this protocol is suitable for a variety of recombinant enzymes to function.
  • the method for detecting the isolated product in the step C is: an electrophoresis method, a fluorescence detection method, an ultraviolet absorption value detection method, and a fluorescent dye method.
  • the method for detecting separated products used in the invention is simple in operation, low in requirements on instruments, and does not cause environmental pollution.
  • the recombinant enzyme to be tested is RecA or uvsX.
  • the strand displacement reaction system further comprises a single-stranded binding protein.
  • the displaced single-stranded portion binds to the single-stranded binding protein, and further replacement of the replaced portion of the strand-replacement complex can be prevented.
  • a second embodiment of the present invention provides a recombinase strand displacement activity detecting kit, the kit comprising a single-stranded DNA molecule, a double-stranded DNA fragment; the single-stranded DNA molecule comprising a first sequence, the first The sequence is homologously complementary to a partial sequence on the double-stranded DNA fragment.
  • the kit further comprises a buffer suitable for the activity of the recombinase, the buffer comprising the following components: 20 to 100 mM Tris-HCl, 5 to 14 mM MgCl2, 0.1 to 1.0 mM DTT, pH 7.5 to 8.0.
  • a buffer suitable for the activity of the recombinase comprising the following components: 20 to 100 mM Tris-HCl, 5 to 14 mM MgCl2, 0.1 to 1.0 mM DTT, pH 7.5 to 8.0.
  • the kit further comprises an ATP or ATP analog.
  • the ATP or analog thereof provides energy for the strand displacement reaction.
  • the single-stranded DNA molecule comprises a biotin label on the 3' end and is immobilized on streptavidin-containing magnetic beads by biotin labeling.
  • the kit of the present invention can rapidly and simply separate and purify the chain displacement complex in the subsequent reaction process.
  • the first sequence on the single-stranded DNA molecule is from 16 bp to 100 bp in length.
  • the single-stranded DNA molecule immobilized on the solid phase carrier can be firmly bound to the double-stranded DNA fragment to form a stable strand displacement complex under the action of the recombinase.
  • the first sequence may be a partial sequence of a single-stranded DNA molecule, or may be a complete sequence of a single-stranded DNA molecule.
  • the first sequence is the entire sequence of a single-stranded DNA molecule.
  • the present scheme reduces the difficulty in synthesizing single-stranded DNA molecules by ensuring that the single-stranded DNA molecules are intruded after the double-stranded DNA fragments are intruded.
  • the double-stranded DNA fragment is 50 to 200 bp in length.
  • the double-stranded DNA fragment of the present scheme is simple in design and It's simple.
  • the double-stranded DNA fragment has a length of not less than 200 bp.
  • the uninvaded double strands can be stably coupled by hydrogen bonding, thereby ensuring the stability of the strand-replacement complex structure.
  • a method for detecting recombinase strand displacement activity is proposed.
  • A, strand displacement reaction system 1 ⁇ L of ATP ⁇ S solution with a concentration of 6 mM; 2 ⁇ g of the recombinase to be tested; 2 ⁇ L of 10 ⁇ recombinase buffer: 700 mM Tris-HCl, 100 mM MgCl 2 , 5 mM DTT, pH 7.6; 7.0 ⁇ L pre-fixed at single-stranded DNA molecules on magnetic beads (per ⁇ L containing 10 6 beads); 4 L concentration of 10ng / ⁇ L of double-stranded DNA fragment; the reaction was mixed and incubated at 37 °C 20 minutes.
  • the recombinant enzymes to be tested in the two strand displacement reaction systems are RecA and uvsX, respectively; the single-stranded DNA molecule sequence is SEQ ID NO: 1; the double-stranded DNA fragment consists of SEQ ID NO: 2 and SEQ ID NO: 3;
  • a blank control group was established, and the blank control group contained no recombinant enzyme to be tested, and the remaining components were identical to the components of the chain displacement reaction system;
  • Lane 0 is a molecular size marker
  • 1 is a blank control group
  • 2 is an isolated product of a chain displacement reaction system containing RecA
  • 3 is a separation product of a chain displacement reaction system containing uvsX.
  • double-stranded DNA could not be detected in the isolated product of the strand displacement reaction system without the addition of the recombinase, and the electrophoresis bands were observed at 464 bp in the isolated products of the chain displacement reaction system containing RecA and uvsX. This indicates that both RecA and uvsX have strand displacement activity.

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Abstract

Disclosed is a method for detecting a recombinase strand displacement activity, comprising: preparing a strand displacement reaction system and performing a strand displacement reaction to generate a strand displacement complex, wherein the reaction system comprises recombinase to be tested, single-stranded DNA molecules, double-stranded DNA fragments, ATP or ATP analogues, and a buffer suitable for the recombinase to be tested to achieve activity, the single-stranded DNA molecules comprise a first sequence, and the first sequence is homologous to and complementary with a partial sequence of the double-stranded DNA fragments; terminating the strand displacement reaction, separating and purifying the strand displacement complex; separating and removing the single-stranded DNA molecules in the strand displacement complex to obtain an isolated product, and detecting the isolated product to determine the recombinase strand displacement activity. The present invention also relates to a kit for detecting the recombinase strand displacement activity. The method for detecting the recombinase strand displacement activity is simple to operate, has few requirements for instruments, and does not cause environmental pollution.

Description

重组酶链置换活性检测方法及试剂盒Recombinase chain displacement activity detection method and kit 技术领域Technical field
本发明涉及分子生物学领域，更具体地说，涉及一种重组酶链置换活性检测方法及试剂盒。The present invention relates to the field of molecular biology, and more particularly to a method and kit for detecting recombinase chain displacement activity.
背景技术Background technique
重组酶是一类蛋白质酶，它可以结合单链DNA分子并能入侵其同源的双链DNA片段，使单链DNA分子与双链DNA片段在同源区域互补结合，形成稳定复合物结构。重组酶参与DNA修复和紫外线诱导的突变，在生物体中是不可缺少的。Recombinase is a kind of proteinase that binds to a single-stranded DNA molecule and invades its homologous double-stranded DNA fragment, allowing a single-stranded DNA molecule to bind to a double-stranded DNA fragment in a homologous region to form a stable complex structure. Recombinases are involved in DNA repair and UV-induced mutations and are indispensable in organisms.
目前对重组酶链置换活性的检测方法常见的有：例如采用同位素标记单链M13噬菌体或Phi x174噬菌体，同位素标记的方法虽然简便灵敏度高，但该方法的试剂成本高，一般的科研机构和企业单位都无法开展。此外，文献报道采用琼脂糖凝胶电泳的方法检测重组酶的链置换活性，当发生同源重组时，单链DNA会将同源的双链DNA的一条链完全置换下来，产生具有缺口的环状DNA产物，可以在单链DNA琼脂糖电泳图上方发现该链置换的产物，但采用该方法的噬菌体DNA的长度基本都在5000bp以上，产生完全链置换的产物对体系的要求较高，产生足够的产物才能检测到，这就需要反复地调节体系。At present, the detection methods for recombinase chain displacement activity are common: for example, isotopically labeled single-stranded M13 phage or Phi x174 phage, although the method of isotope labeling is simple and sensitive, the reagent cost of the method is high, and the general scientific research institutions and enterprises No unit can be carried out. In addition, the literature reports that the chain displacement activity of recombinases is detected by agarose gel electrophoresis. When homologous recombination occurs, single-stranded DNA completely replaces one strand of homologous double-stranded DNA to produce a gap-free loop. The DNA product can be found on the single-stranded DNA agarose electrophoresis map, but the length of the phage DNA using the method is substantially above 5000 bp, and the product which produces complete strand displacement has higher requirements on the system. Sufficient product can be detected, which requires repeated adjustments to the system.
因此，需要一种对体系要求低，检测成本低的重组酶链置换活性检测方法及试剂盒。Therefore, there is a need for a method and kit for detecting recombinase strand displacement activity which requires low system requirements and low detection cost.
发明内容Summary of the invention
本发明的目的在于提供一种重组酶链置换活性检测方法及试剂盒、旨在解决现有技术中重组酶链置换活性检测对反应体系要求高，成本高的问题。The object of the present invention is to provide a method and a kit for detecting recombinase chain displacement activity, which aims to solve the problem that the detection of recombinase chain displacement activity in the prior art requires high reaction system and high cost.
为了实现发明目的，本发明提供了一种重组酶链置换活性检测方法，包括以下步骤：In order to achieve the object of the invention, the present invention provides a method for detecting recombinase strand displacement activity, comprising the steps of:
A、制备链置换反应体系并进行链置换反应以生成链置换复合体，所述反应体系包括待测重组酶，单链DNA分子，双链DNA片段，ATP或ATP类似物，以及适宜所述待测重组酶发挥活性的缓冲液，所述单链DNA分子上包含第一序列，所述第一序列与双链DNA片段上部分序列同源互补；A. preparing a strand displacement reaction system and performing a strand displacement reaction to form a strand displacement complex, the reaction system comprising a recombinant enzyme to be tested, a single-stranded DNA molecule, a double-stranded DNA fragment, an ATP or an ATP analog, and the like Detecting a buffer in which a recombinant enzyme functions, the single-stranded DNA molecule comprising a first sequence, the first sequence being homologously complementary to a partial sequence on the double-stranded DNA fragment;
B、终止链置换反应，分离提纯所述链置换复合体；B, terminating the chain displacement reaction, separating and purifying the chain displacement complex;
C、分离去除链置换复合体中的单链DNA分子以获得分离产物，检测分离产物，确定重 组酶的链置换活性。C. separating and removing the single-stranded DNA molecule in the strand displacement complex to obtain an isolated product, detecting the isolated product, and determining the weight The strand displacement activity of the enzyme group.
优选的，所述单链DNA分子的3’末端固定在磁珠上。Preferably, the 3' end of the single-stranded DNA molecule is immobilized on a magnetic bead.
优选的，检测所述分离产物中含有双链DNA结构，确定重组酶具有链置换活性。Preferably, the isolated product is detected to contain a double-stranded DNA structure, and it is determined that the recombinase has strand displacement activity.
优选的，所述待测重组酶为RecA或uvsX。Preferably, the recombinant enzyme to be tested is RecA or uvsX.
优选的，所述第一序列的长度为16bp至100bp。Preferably, the first sequence has a length of 16 bp to 100 bp.
优选的，步骤B结束后，还包括以下步骤：热处理所述链置换复合体，所述热处理的步骤为将链置换复合体置于65至80℃条件下孵育5至20分钟。Preferably, after the end of step B, the method further comprises the step of: heat treating the chain displacement complex, wherein the step of heat treatment is to incubate the chain displacement complex at 65 to 80 ° C for 5 to 20 minutes.
优选的，步骤C中用于检测分离产物的方法为：电泳法，荧光检测法，紫外吸光值检测方法以及荧光染料法中的任意一种方法。Preferably, the method for detecting the isolated product in the step C is: any one of an electrophoresis method, a fluorescence detection method, an ultraviolet absorption value detection method, and a fluorescent dye method.
优选的，所述链置换反应体系中还包括单链结合蛋白。Preferably, the strand displacement reaction system further comprises a single-stranded binding protein.
本发明还提供了一种重组酶链置换活性检测试剂盒，包括单链DNA分子，双链DNA片段；所述单链DNA分子上包含第一序列，所述第一序列与双链DNA片段上部分序列同源互补。The invention also provides a kit for detecting a recombinase strand displacement activity, comprising a single-stranded DNA molecule, a double-stranded DNA fragment; the single-stranded DNA molecule comprising a first sequence, the first sequence and the double-stranded DNA fragment Partial sequences are homologously complementary.
优选的，所述单链DNA分子上与双链DNA片段同源互补的序列长度为16bp至100bp。Preferably, the sequence of the single-stranded DNA molecule homologously complementary to the double-stranded DNA fragment is 16 bp to 100 bp in length.
单链DNA分子能够在具有链置换活性的重组酶的作用下，入侵与其具有同源互补序列的双链DNA片段并获得链置换复合体，重组酶失活或变性后，单链DNA分子脱落后从而重新获得双链DNA片段，因此，本发明通过检测到双链DNA片段，即可表明待测重组酶具有链置换活性。本发明的重组酶链置换活性检测方法操作简单、对仪器要求低，不会对环境造成污染。A single-stranded DNA molecule can invade a double-stranded DNA fragment having a homologous complementary sequence and obtain a strand displacement complex under the action of a recombinase having a strand displacement activity, and after the recombinant enzyme is inactivated or denatured, the single-stranded DNA molecule is detached. Thereby, the double-stranded DNA fragment is re-obtained, and therefore, the present invention can indicate that the recombinant enzyme to be tested has strand displacement activity by detecting the double-stranded DNA fragment. The method for detecting recombinase chain displacement activity of the invention has simple operation, low requirements on instruments, and no pollution to the environment.
附图说明DRAWINGS
图1是本发明第一实施例重组酶链置换活性检测示意图。BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a schematic view showing the detection of recombinase chain displacement activity in the first embodiment of the present invention.
图2是本发明第三实施例分离产物Page胶电泳检测图。Fig. 2 is a diagram showing the gel electrophoresis detection of the separated product of the third embodiment of the present invention.
具体实施方式detailed description
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.
本发明第一实施例，提出了一种重组酶链置换活性检测方法，包括以下步骤：In the first embodiment of the present invention, a method for detecting recombinase chain displacement activity is proposed, which comprises the following steps:
A、制备链置换反应体系并进行链置换反应以生成链置换复合体，所述反应体系包括待测重组酶，单链DNA分子，双链DNA片段，ATP或ATP类似物，以及适宜所述待测重组酶发挥活性的缓冲液，所述单链DNA分子上包含第一序列，所述第一序列与双链DNA片段上部分序列同源互补；A. preparing a strand displacement reaction system and performing a strand displacement reaction to form a strand displacement complex, the reaction system comprising a recombinant enzyme to be tested, a single-stranded DNA molecule, a double-stranded DNA fragment, an ATP or an ATP analog, and the like Detecting a buffer in which a recombinant enzyme functions, the single-stranded DNA molecule comprising a first sequence, the first sequence being homologously complementary to a partial sequence on the double-stranded DNA fragment;
B、终止链置换反应，分离提纯所述链置换复合体； B, terminating the chain displacement reaction, separating and purifying the chain displacement complex;
C、分离去除链置换复合体中的单链DNA分子以获得分离产物，检测分离产物，确定重组酶的链置换活性。C. Separating and removing the single-stranded DNA molecule in the strand displacement complex to obtain an isolated product, detecting the isolated product, and determining the strand displacement activity of the recombinase.
重组酶具有单链DNA结合活性，当体系中存在与单链DNA分子具有同源互补的靶序列的双链DNA片段时，在ATP或ATP类似物提供能量的情况下，具有链置换活性的重组酶可以与单链DNA分子结合形成稳定的复合体，复合体寻找并入侵与单链DNA分子序列同源互补的双链DNA区域，在重组酶的作用下，发生链置换反应。本发明中，由于单链DNA分子上包含与部分双链DNA片段同源互补的序列，因此，在双链DNA片段上，发生部分链置换反应。The recombinase has a single-stranded DNA-binding activity, and when a double-stranded DNA fragment having a target sequence complementary to a single-stranded DNA molecule exists in the system, recombination having a strand displacement activity in the case where the ATP or ATP analog provides energy The enzyme can form a stable complex with the single-stranded DNA molecule, and the complex searches for and invades the double-stranded DNA region which is homologously complementary to the single-stranded DNA molecule sequence, and undergoes a strand displacement reaction under the action of the recombinase. In the present invention, since the single-stranded DNA molecule contains a sequence homologously complementary to the partially double-stranded DNA fragment, a partial strand displacement reaction occurs on the double-stranded DNA fragment.
如图1所示，在具备链置换活性的重组酶1的作用下，单链DNA分子2入侵部分双链DNA片段3发生链置换反应，形成链置换复合体4；提纯链置换复合体4，并对其进行处理使重组酶变性或失活，可以使单链DNA分子2脱落，从体系中分离出单链DNA分子2后，得到分离产物；检测分离产物，其中含有双链DNA结构，则确定待测重组酶具有链置换活性；分离产物中不含双链DNA结构，则确定待测重组酶不具有链置换活性。As shown in Fig. 1, under the action of recombinase 1 having strand displacement activity, a single-stranded DNA molecule 2 invades a part of the double-stranded DNA fragment 3 to undergo a strand displacement reaction to form a strand displacement complex 4; and a purified strand replacement complex 4, And treating it to denature or inactivate the recombinant enzyme, the single-stranded DNA molecule 2 can be detached, and the isolated product can be obtained by separating the single-stranded DNA molecule 2 from the system; and detecting the isolated product, which contains the double-stranded DNA structure, It is determined that the recombinase to be tested has strand displacement activity; if the isolated product does not contain a double-stranded DNA structure, it is determined that the recombinase to be tested does not have strand displacement activity.
优选的，所述单链DNA分子3’末端包含生物素标记，并通过生物素标记固定在包含链霉亲和素的磁珠上。本方案使得在步骤B的操作过程中，可以通过分离磁珠的方法很方便的将固定在磁珠上的链置换复合体提纯出来；同时还可以保证在步骤C的操作过程中，分离单链DNA分子的步骤简单。Preferably, the 3' end of the single-stranded DNA molecule comprises a biotin label and is immobilized on a magnetic bead comprising streptavidin by a biotin label. The solution makes it possible to conveniently purify the chain displacement complex immobilized on the magnetic beads by separating the magnetic beads during the operation of the step B; at the same time, it is also ensured that the single chain is separated during the operation of the step C. The steps of DNA molecules are simple.
优选的，所述单链DNA分子上的第一序列的长度为16bp至100bp。本方案使得固定在固相载体上的单链DNA分子在重组酶的作用下，入侵双链DNA片段后能够牢固地结合在双链DNA片段上形成稳定的链置换复合体。Preferably, the first sequence on the single-stranded DNA molecule is from 16 bp to 100 bp in length. The single-stranded DNA molecule immobilized on the solid phase carrier can be firmly bound to the double-stranded DNA fragment to form a stable strand displacement complex under the action of the recombinase.
需要说明的是，所述第一序列可以是单链DNA分子的部分序列，也可以是单链DNA分子的全部序列。It should be noted that the first sequence may be a partial sequence of a single-stranded DNA molecule, or may be a complete sequence of a single-stranded DNA molecule.
优选的，所述第一序列为单链DNA分子的全部序列。本方案在保证了单链DNA分子入侵双链DNA片段后连接稳定的基础上，降低了单链DNA分子的合成难度。Preferably, the first sequence is the entire sequence of a single-stranded DNA molecule. The present scheme reduces the difficulty in synthesizing single-stranded DNA molecules by ensuring that the single-stranded DNA molecules are intruded after the double-stranded DNA fragments are intruded.
优选的，所述双链DNA片段的长度为50至200bp。本方案的双链DNA片段设计简单，合成简单。Preferably, the double-stranded DNA fragment is 50 to 200 bp in length. The double-stranded DNA fragment of the present scheme is simple in design and simple in synthesis.
优选的，所述双链DNA片段的长度不小于200bp。本方案的双链DNA片段被单链DNA分子部分置换后，仍然能够保证未被入侵的双链之间通过氢键稳定地结合，从而保证链置换复合体结构的稳定性。Preferably, the double-stranded DNA fragment has a length of not less than 200 bp. After the double-stranded DNA fragment of the present invention is partially replaced by a single-stranded DNA molecule, the uninvaded double strands can be stably coupled by hydrogen bonding, thereby ensuring the stability of the strand-replacement complex structure.
步骤B结束后，还包括使待测重组酶失活或变性的步骤，所述使待测重组酶失活或变性的步骤可以是向链置换复合体中加入蛋白质分解酶分解重组酶，也可以是对链置换复合体进行热处理。 After the step B, the step further comprises the step of inactivating or denaturation of the recombinant enzyme to be tested, and the step of inactivating or denaturation of the recombinant enzyme to be tested may be the step of adding a proteolytic enzyme to the strand displacement complex to decompose the recombinant enzyme, or The heat treatment is performed on the chain displacement composite.
优选的，所述使待测重组酶失活或变性的步骤为：热处理所述链置换复合体，所述热处理的步骤为将链置换复合体置于65至80℃条件下孵育5至20分钟。采用热处理法使重组酶变性，操作简单，不会引入其他杂质，且本方案的温度范围可以使重组酶变性，从而使固定在固相载体上的单链DNA分子分离脱落并重新形成双链DNA，但不会导致双链DNA形成单链。Preferably, the step of inactivating or denaturation of the recombinant enzyme to be tested is: heat treating the strand displacement complex, and the step of heat treatment is to incubate the strand displacement complex at 65 to 80 ° C for 5 to 20 minutes. . The recombinant enzyme is denatured by heat treatment, and the operation is simple, no other impurities are introduced, and the temperature range of the solution can denature the recombinant enzyme, thereby separating and detaching the single-stranded DNA molecule immobilized on the solid phase carrier and reforming the double-stranded DNA. But does not cause double-stranded DNA to form a single strand.
优选的，所述缓冲液包括以下组分：20至100mM Tris-HCl，5至14mM MgCl2，0.1至1.0mM DTT，pH为7.5至8.0。本方案的缓冲液，适用于多种重组酶发挥作用。Preferably, the buffer comprises the following components: 20 to 100 mM Tris-HCl, 5 to 14 mM MgCl2, 0.1 to 1.0 mM DTT, and a pH of 7.5 to 8.0. The buffer of this protocol is suitable for a variety of recombinant enzymes to function.
优选的，步骤C中用于检测分离产物的方法为：电泳法，荧光检测法，紫外吸光值检测方法以及荧光染料法。本发明采用的检测分离产物的方法，操作简便，对仪器要求低，不会产生环境污染。Preferably, the method for detecting the isolated product in the step C is: an electrophoresis method, a fluorescence detection method, an ultraviolet absorption value detection method, and a fluorescent dye method. The method for detecting separated products used in the invention is simple in operation, low in requirements on instruments, and does not cause environmental pollution.
优选的，所述待测重组酶为RecA或uvsX。Preferably, the recombinant enzyme to be tested is RecA or uvsX.
优选的，所述链置换反应体系中还包括单链结合蛋白。当双链DNA片段上一部分被单链DNA分子置换后，被置换出的单链部分与单链结合蛋白结合，能够防止进一步对链置换复合体中被置换的部分重新进行替换。Preferably, the strand displacement reaction system further comprises a single-stranded binding protein. When a part of the double-stranded DNA fragment is replaced with a single-stranded DNA molecule, the displaced single-stranded portion binds to the single-stranded binding protein, and further replacement of the replaced portion of the strand-replacement complex can be prevented.
本发明第二实施例提出了一种重组酶链置换活性检测试剂盒，所述试剂盒包括单链DNA分子，双链DNA片段；所述单链DNA分子上包含第一序列，所述第一序列与双链DNA片段上部分序列同源互补。A second embodiment of the present invention provides a recombinase strand displacement activity detecting kit, the kit comprising a single-stranded DNA molecule, a double-stranded DNA fragment; the single-stranded DNA molecule comprising a first sequence, the first The sequence is homologously complementary to a partial sequence on the double-stranded DNA fragment.
优选的，所述试剂盒还包括适于重组酶发挥活性的缓冲液，所述缓冲液包括以下组分：20至100mM Tris-HCl，5至14mM MgCl2，0.1至1.0mM DTT，pH为7.5至8.0。Preferably, the kit further comprises a buffer suitable for the activity of the recombinase, the buffer comprising the following components: 20 to 100 mM Tris-HCl, 5 to 14 mM MgCl2, 0.1 to 1.0 mM DTT, pH 7.5 to 8.0.
优选的，所述试剂盒还包括ATP或ATP类似物。所述ATP或其类似物为链置换反应提供能量。Preferably, the kit further comprises an ATP or ATP analog. The ATP or analog thereof provides energy for the strand displacement reaction.
优选的，所述单链DNA分子的3’末端上包括生物素标记，且通过生物素标记固定在含链霉亲和素的磁珠上。本方案的试剂盒，在后续反应过程中能够快速、简单的对链置换复合体进行分离提纯。Preferably, the single-stranded DNA molecule comprises a biotin label on the 3' end and is immobilized on streptavidin-containing magnetic beads by biotin labeling. The kit of the present invention can rapidly and simply separate and purify the chain displacement complex in the subsequent reaction process.
优选的，所述单链DNA分子上的第一序列的长度为16bp至100bp。本方案使得固定在固相载体上的单链DNA分子在重组酶的作用下，入侵双链DNA片段后能够牢固地结合在双链DNA片段上形成稳定的链置换复合体。Preferably, the first sequence on the single-stranded DNA molecule is from 16 bp to 100 bp in length. The single-stranded DNA molecule immobilized on the solid phase carrier can be firmly bound to the double-stranded DNA fragment to form a stable strand displacement complex under the action of the recombinase.
需要说明的是，所述第一序列可以是单链DNA分子的部分序列，也可以是单链DNA分子的全部序列。It should be noted that the first sequence may be a partial sequence of a single-stranded DNA molecule, or may be a complete sequence of a single-stranded DNA molecule.
优选的，所述第一序列为单链DNA分子的全部序列。本方案在保证了单链DNA分子入侵双链DNA片段后连接稳定的基础上，降低了单链DNA分子的合成难度。Preferably, the first sequence is the entire sequence of a single-stranded DNA molecule. The present scheme reduces the difficulty in synthesizing single-stranded DNA molecules by ensuring that the single-stranded DNA molecules are intruded after the double-stranded DNA fragments are intruded.
优选的，所述双链DNA片段的长度为50至200bp。本方案的双链DNA片段设计简单，合 成简单。Preferably, the double-stranded DNA fragment is 50 to 200 bp in length. The double-stranded DNA fragment of the present scheme is simple in design and It's simple.
优选的，所述双链DNA片段的长度不小于200bp。本方案的双链DNA片段被单链DNA分子部分置换后，仍然能够保证未被入侵的双链之间通过氢键稳定地结合，从而保证链置换复合体结构的稳定性。Preferably, the double-stranded DNA fragment has a length of not less than 200 bp. After the double-stranded DNA fragment of the present invention is partially replaced by a single-stranded DNA molecule, the uninvaded double strands can be stably coupled by hydrogen bonding, thereby ensuring the stability of the strand-replacement complex structure.
本发明的第三实施例，提出了一种重组酶链置换活性检测方法。In a third embodiment of the present invention, a method for detecting recombinase strand displacement activity is proposed.
建立两个链置换反应体系分别检测RecA和uvsX的链置换活性，以及一个未添加重组酶的空白对照组：Two strand displacement reaction systems were established to detect the strand displacement activity of RecA and uvsX, respectively, and a blank control group without added recombinase:
A、链置换反应体系：1μL浓度为6mM的ATPγS溶液；2μg待测重组酶；2μL 10×重组酶缓冲液：700mM Tris-HCl，100mM MgCl2，5mM DTT，pH为7.6；7.0μL预先固定在磁珠上的单链DNA分子(每μL含106个磁珠)；4μL浓度为10ng/μL的双链DNA片段；将反应体系混匀，并于37℃孵育20分钟。其中两个链置换反应体系中的待测重组酶分别为RecA和uvsX；单链DNA分子序列为SEQ ID NO:1；双链DNA片段由SEQ ID NO:2和SEQ ID NO:3组成；A, strand displacement reaction system: 1 μL of ATPγS solution with a concentration of 6 mM; 2 μg of the recombinase to be tested; 2 μL of 10×recombinase buffer: 700 mM Tris-HCl, 100 mM MgCl 2 , 5 mM DTT, pH 7.6; 7.0 μL pre-fixed at single-stranded DNA molecules on magnetic beads (per μL containing 10 6 beads); 4 L concentration of 10ng / μL of double-stranded DNA fragment; the reaction was mixed and incubated at 37 ℃ 20 minutes. The recombinant enzymes to be tested in the two strand displacement reaction systems are RecA and uvsX, respectively; the single-stranded DNA molecule sequence is SEQ ID NO: 1; the double-stranded DNA fragment consists of SEQ ID NO: 2 and SEQ ID NO: 3;
建立一个空白对照组，空白对照组中不含待测重组酶，其余组分与链置换反应体系组分相同；A blank control group was established, and the blank control group contained no recombinant enzyme to be tested, and the remaining components were identical to the components of the chain displacement reaction system;
B、终止链置换反应，去除上清，使用1×重组酶缓冲液洗涤磁珠一次，得到固定在磁珠上的链置换复合体；B. Terminating the strand displacement reaction, removing the supernatant, and washing the magnetic beads once with 1×recombinase buffer to obtain a chain replacement complex immobilized on the magnetic beads;
C、在65℃条件下孵育15min后，吸取上清得到分离产物，对两个反应体系及对照组的分离产物分别使用12％的Page胶检测。C. After incubating for 15 min at 65 ° C, the supernatant was aspirated to obtain the isolated product, and the separated products of the two reaction systems and the control group were respectively detected by using 12% Page glue.
检测结果如图2所示，泳道0为分子大小标记物，1为空白对照组，2为含RecA的链置换反应体系的分离产物，3为含uvsX的链置换反应体系的分离产物。从图中可以看出，未添加重组酶的链置换反应体系的分离产物中无法检测到双链DNA，在含RecA和uvsX的链置换反应体系的分离产物均在464bp处观察到电泳条带，这说明RecA和uvsX均具备链置换活性。The detection results are shown in Fig. 2. Lane 0 is a molecular size marker, 1 is a blank control group, 2 is an isolated product of a chain displacement reaction system containing RecA, and 3 is a separation product of a chain displacement reaction system containing uvsX. As can be seen from the figure, double-stranded DNA could not be detected in the isolated product of the strand displacement reaction system without the addition of the recombinase, and the electrophoresis bands were observed at 464 bp in the isolated products of the chain displacement reaction system containing RecA and uvsX. This indicates that both RecA and uvsX have strand displacement activity.
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。 The above is only the preferred embodiment of the present invention, and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection of the present invention. Within the scope.

Claims (10)

  1. 一种重组酶链置换活性检测方法，其特征在于，包括以下步骤：A method for detecting recombinase chain displacement activity, comprising the steps of:
    A、制备链置换反应体系并进行链置换反应以生成链置换复合体，所述反应体系包括待测重组酶，单链DNA分子，双链DNA片段，ATP或ATP类似物，以及适宜所述待测重组酶发挥活性的缓冲液，所述单链DNA分子上包含第一序列，所述第一序列与双链DNA片段上部分序列同源互补；A. preparing a strand displacement reaction system and performing a strand displacement reaction to form a strand displacement complex, the reaction system comprising a recombinant enzyme to be tested, a single-stranded DNA molecule, a double-stranded DNA fragment, an ATP or an ATP analog, and the like Detecting a buffer in which a recombinant enzyme functions, the single-stranded DNA molecule comprising a first sequence, the first sequence being homologously complementary to a partial sequence on the double-stranded DNA fragment;
    B、终止链置换反应，分离提纯所述链置换复合体；B, terminating the chain displacement reaction, separating and purifying the chain displacement complex;
    C、分离去除链置换复合体中的单链DNA分子以获得分离产物，检测分离产物，确定重组酶的链置换活性。C. Separating and removing the single-stranded DNA molecule in the strand displacement complex to obtain an isolated product, detecting the isolated product, and determining the strand displacement activity of the recombinase.
  2. 根据权利要求1所述的重组酶链置换活性检测方法，其特征在于，所述单链DNA分子的3’末端固定在磁珠上。The method for detecting a recombinase strand displacement activity according to claim 1, wherein the 3' end of the single-stranded DNA molecule is immobilized on a magnetic bead.
  3. 根据权利要求1所述的重组酶链置换活性检测方法，其特征在于，检测所述分离产物中含有双链DNA结构，确定待测重组酶具有链置换活性。The method for detecting a recombinase strand displacement activity according to claim 1, wherein the isolated product contains a double-stranded DNA structure, and the recombinase to be tested has a strand displacement activity.
  4. 根据权利要求1所述的重组酶链置换活性检测方法，其特征在于，所述待测重组酶为RecA或uvsX。The method for detecting recombinase strand displacement activity according to claim 1, wherein the recombinant enzyme to be tested is RecA or uvsX.
  5. 根据权利要求1所述的重组酶链置换活性检测方法，其特征在于，所述第一序列的长度为16bp至100bp。The method for detecting a recombinase strand displacement activity according to claim 1, wherein the first sequence has a length of 16 bp to 100 bp.
  6. 根据权利要求1所述的重组酶链置换活性检测方法，其特征在于，步骤B结束后，还包括以下步骤：热处理所述链置换复合体，所述热处理的步骤为将链置换复合体置于65至80℃条件下孵育5至20分钟。The method for detecting a recombinase strand displacement activity according to claim 1, wherein after the step B, the method further comprises the step of: heat treating the chain displacement complex, wherein the step of heat treating comprises placing the chain replacement complex Incubate for 5 to 20 minutes at 65 to 80 °C.
  7. 根据权利要求1所述的重组酶链置换活性检测方法，其特征在于，步骤C中用于检测分离产物的方法为：电泳法，荧光检测法，紫外吸光值检测方法以及荧光染料法中的任意一种方法。The method for detecting recombinase strand displacement activity according to claim 1, wherein the method for detecting the isolated product in the step C is: any one of an electrophoresis method, a fluorescence detection method, an ultraviolet absorption value detection method, and a fluorescent dye method. a way.
  8. 根据权利要求1所述的重组酶链置换活性检测方法，其特征在于，所述链置换反应体系中还包括单链结合蛋白。The method for detecting a recombinase strand displacement activity according to claim 1, wherein the strand displacement reaction system further comprises a single-stranded binding protein.
  9. 一种重组酶链置换活性检测试剂盒，其特征在于，包括单链DNA分子，双链DNA片段；所述单链DNA分子上包含第一序列，所述第一序列与双链DNA片段上部分序列同源互补。A kit for detecting a recombinase chain displacement activity, comprising: a single-stranded DNA molecule, a double-stranded DNA fragment; the single-stranded DNA molecule comprising a first sequence, and the first sequence and a portion of the double-stranded DNA fragment The sequences are homologously complementary.
  10. 根据权利要求9所述的重组酶链置换活性检测试剂盒，其特征在于，所述单链DNA分子上与双链DNA片段同源互补的序列长度为16bp至100bp。 The recombinase strand displacement activity detecting kit according to claim 9, wherein the sequence of the single-stranded DNA molecule homologously complementary to the double-stranded DNA fragment is 16 bp to 100 bp in length.
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