WO2023240563A1 - 一种包含巯基封闭试剂的测序试剂盒及其应用 - Google Patents

一种包含巯基封闭试剂的测序试剂盒及其应用 Download PDF

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WO2023240563A1
WO2023240563A1 PCT/CN2022/099258 CN2022099258W WO2023240563A1 WO 2023240563 A1 WO2023240563 A1 WO 2023240563A1 CN 2022099258 W CN2022099258 W CN 2022099258W WO 2023240563 A1 WO2023240563 A1 WO 2023240563A1
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sequencing
blocking
blocking reagent
nucleic acid
disulfide bond
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PCT/CN2022/099258
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贾曼
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深圳华大智造科技股份有限公司
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Priority to PCT/CN2022/099258 priority Critical patent/WO2023240563A1/zh
Priority to PCT/CN2023/100777 priority patent/WO2023241701A1/zh
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    • 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

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  • the invention belongs to the field of nucleic acid sequencing, and specifically relates to a kit containing a thiol blocking reagent, a method for reducing sequencing background signals based on the thiol blocking reagent, and its application.
  • DNA sequencing includes first-strand sequencing and second-strand sequencing. After the first-strand sequencing is completed, the first strand is used as a template to grow the second strand through multiple displacement amplification (MDA), and then the second strand of DNA is sequenced. During the second-strand growth process, multiple strand displacement amplification enzyme (phi29 DNA polymerase) is used. phi29 DNA polymerase is easily adsorbed non-specifically on the sequencing chip and is difficult to remove, causing residues. The residual phi29 DNA polymerase will be entangled with dNTPs (chemically modified deoxyribonucleotides) at the beginning of second-strand sequencing.
  • dNTPs chemically modified deoxyribonucleotides
  • modified dNTPs contains fluorescent dyes, non-specific binding of enzymes and dNTPs
  • the result of the entanglement is that the background signal of sequencing increases, causing the signal-to-noise ratio to decrease, thus affecting the quality of sequencing.
  • the present invention provides a thiol blocking reagent and a method for reducing sequencing background signals based on the thiol blocking reagent and its application.
  • the mechanism of the present invention first, the disulfide bond in the enzyme protein structure is opened, so that the disulfide bond becomes an exposed sulfhydryl group.
  • the other is an addition reaction, and N-ethylmaleimide is commonly used as shown in reaction (2) in Figure 1.
  • Michael Addition is a conjugate addition reaction between a compound that can provide a nucleophilic carbanion and an electrophilic conjugated system under base catalysis.
  • This invention is the first to use dithiothreitol (DTT) to open the disulfide bonds in the enzyme protein structure during the sequencing process, and uses a blocking reagent to irreversibly block all disulfide bonds, release the fluorescent dye, and completely remove it, thereby Reduce background signals and improve sequencing quality.
  • the sulfhydryl blocking reagents of the present invention include N-ethylmaleimide and/or iodoacetamide, which are often used in protein structures as protein markers and are important means for protein detection.
  • This invention applies sulfhydryl blocking reagents to sequencing technology for the first time. As a means to reduce second-strand background, it can well block the polymerase in the MDA process, which is more conducive to thorough cleaning and avoids its impact on second-strand sequencing. , which is beneficial to improving sequencing quality.
  • the first aspect of the present invention provides a sequencing kit.
  • the sequencing kit includes a thiol blocking reagent.
  • the thiol blocking reagent includes iodoacetamide and/or N-ethylmaleimide. .
  • the sulfhydryl blocking reagent is a 50 mM ammonium bicarbonate solution containing 50-100 mM N-ethylmaleimide.
  • the sulfhydryl blocking reagent is a 50mM ammonium bicarbonate solution containing 50mM N-ethylmaleimide.
  • the sulfhydryl blocking reagent is a 50mM ammonium bicarbonate solution containing 50-100mM iodoacetamide.
  • the sulfhydryl blocking reagent is a 50mM ammonium bicarbonate solution containing 100mM iodoacetamide.
  • the sequencing kit further includes a disulfide bond reducing agent.
  • the disulfide bond reducing agent is trishydroxymethylaminomethanephosphine (TCEP) or thiol such as ⁇ -mercaptoethanol ( ⁇ -ME) or dithiothreitol (DTT).
  • the sequencing kit further includes one or more of DNA polymerase, MDA polymerase, sequencing slide, and nucleotide mixture with fluorescent modification and reversible blocking.
  • MDA polymerase is phi29 DNA polymerase.
  • a second aspect of the present invention provides a method for reducing nucleic acid sequencing background signals.
  • the method includes the following steps:
  • step (1) the three-dimensional spatial structure is opened by destroying the disulfide bond, and the exposed active group is a thiol group; and/or,
  • step (2) the blocking uses a thiol blocking reagent alone or in the sequencing kit as described in the first aspect; and/or,
  • the fluorescent substance may be a nucleotide individually labeled with a label, such as Cy5 fluorescence, ROX fluorescence, Cy3 fluorescence or EF700 fluorescence.
  • independent means that the reagent does not come from the sequencing kit described in the first aspect, but can be prepared separately from the same reagent.
  • the fluorescent substance can be a label combined with labeled nucleotides, so that different luciferases can be connected to the nucleotides.
  • the molecular tag used to label a nucleotide and the tag that specifically binds thereto may be any pairing of molecules capable of specifically binding to each other. Specific binding between pairing members enables the linkage of nucleotides to luciferase.
  • Exemplary pair members include, but are not limited to: (a) haptens or antigenic compounds in combination with corresponding antibodies or binding portions or fragments thereof, such as digoxin-digoxin antibodies, N3G-N3G antibodies, FITC-FITC antibodies; ; (b) Nucleic acid aptamers and proteins; (c) Non-immune binding pairs (such as biotin-avidin, biotin-streptavidin, biotin-neutral avidin); (d) hormones - Hormone binding proteins; (e) receptors - receptor agonists or antagonists; (f) lectins - carbohydrates; (g) enzymes - enzyme cofactors; (h) enzymes - enzyme inhibitors; and (i) Complementary pairs of oligonucleotides or polynucleotides capable of forming nucleic acid duplexes.
  • haptens or antigenic compounds in combination with corresponding antibodies or binding portions or fragments thereof, such as digoxin-digoxin
  • step (4) the cleaning is performed using an elution reagent.
  • step (1) through the disulfide bond reducing agent alone or in the sequencing kit described in the first aspect, such as trishydroxymethylaminomethanephosphine (TCEP) or thiol such as ⁇ -mercaptoethanol ( ⁇ -ME) or dithiothreitol (DTT) is used to reduce the disulfide bond and destroy the disulfide bond to open the three-dimensional space.
  • TCEP trishydroxymethylaminomethanephosphine
  • thiol such as ⁇ -mercaptoethanol ( ⁇ -ME) or dithiothreitol (DTT)
  • the thiol blocking reagent is 50mM hydrogen carbonate containing 50-100mM N-ethylmaleimide alone or in the sequencing kit described in the first aspect. ammonium solution.
  • the sulfhydryl blocking reagent is a 50mM ammonium bicarbonate solution containing 50mM N-ethylmaleimide.
  • the thiol blocking reagent is a 50 mM ammonium bicarbonate solution containing 50-100 mM iodoacetamide alone or in the sequencing kit described in the first aspect.
  • the sulfhydryl blocking reagent is a 50mM ammonium bicarbonate solution containing 100mM iodoacetamide.
  • step (1) after the disulfide bond reducing agent is mixed with the enzyme protein, the disulfide bond reducing agent is eluted; and/or in step (2), the disulfide bond reducing agent is added After the thiol blocking reagent reacts, a second elution is performed.
  • the elution reagents used for elution and secondary elution are elution reagents in the sequencing reagent tank of the MGISEQ-2000RS high-throughput sequencing kit; and/or, the enzyme protein For MDA polymerase, such as phi29 DNA polymerase.
  • a third aspect of the present invention provides a nucleic acid sequencing method, which method includes the following steps:
  • sequencing reagents such as dNTP molecules and DNA polymerase
  • nucleic acid sequencing includes the following steps:
  • the nucleic acid sequencing is performed in a gene sequencer MGISEQ-2000RS; the cycle is performed 50-400 times, such as 50, 100, 150, 200 or 400 times; and/or the polynucleotide is DNA or RNA.
  • a fourth aspect of the present invention provides the use of the sequencing kit described in the first aspect in nucleic acid sequencing or preparation of reagents for nucleic acid sequencing.
  • This invention uses a sequencing kit containing a sulfhydryl blocking reagent for the first time. It is based on the strategy of opening the disulfide bond of the enzyme protein and then irreversibly blocking it to better release the entangled dye substances, thereby reducing the background signal and improving the signal during sequencing. Noise ratio and sequencing quality.
  • the use of such solutions and reagent sets in sequencing scripts and sequencing kits will improve the sequencing quality of the product and increase the value and market share of the product.
  • Figure 1 shows the mechanism of irreversibly blocking the sulfhydryl group of DNA polymerase in the present invention.
  • Figure 2 shows the results of different blocking reagents on test cycle number and 0.1% error rate
  • control group 50mM ammonium bicarbonate solution without any blocking reagent
  • iodoacetamide 50mM bicarbonate containing 100mM iodoacetamide.
  • Ammonium solution; N-ethylmaleimide Add 50mM ammonium bicarbonate solution containing 50mM N-ethylmaleimide.
  • Figure 3 shows the results of different blocking reagents in reducing the background signal of sequencing; control group: 50mM ammonium bicarbonate solution without adding any blocking reagent; iodoacetamide: adding 50mM ammonium bicarbonate solution containing 100mM iodoacetamide; N - Ethylmaleimide: Add a solution of 50mM ammonium bicarbonate containing 50mM N-ethylmaleimide.
  • Figure 4 shows the results of different blocking reagents improving the signal-to-noise ratio of sequencing
  • control group 50mM ammonium bicarbonate solution without any blocking reagent
  • iodoacetamide 50mM ammonium bicarbonate solution containing 100mM iodoacetamide
  • N-ethylmaleimide Add 50mM ammonium bicarbonate solution containing 50mM N-ethylmaleimide.
  • MGISEQ-2000RS sequencer MGIDL-200H loader, and MGISEQ-2000RS sequencing slide.
  • the excitation wavelengths of the instruments are 532 and 650 nm respectively.
  • the nucleotide mixture with fluorescence modification and reversible blocking mentioned in this example includes: dATP-1, which refers to adenine nucleotide with both reversible blocking group modification and Cy5 fluorescence modification; dTTP- 1.
  • dGTP-1 which refers to guanine nucleotides with both reversible blocking group modification and Cy3 fluorescence modification
  • dCTP-1 which refers to a cytosine nucleotide with both reversible blocking group modification and EF700 fluorescence modification.
  • the above-mentioned dATP-1, dTTP-1, dGTP-1, and dCTP-1 are all from MGI’s MGISEQ-2000RS high-throughput sequencing kit.
  • the nucleotide mixture with fluorescent modification and reversible blocking can be diverse.
  • the nucleotide mixture can be mixed with a simple reversible blocking group nucleotide or other modification types.
  • Step 1 Load the above-mentioned DNA nanospheres onto the prepared above-mentioned chip (i.e., sequencing slide);
  • Step 2 Pump the prepared dNTP molecule mixture (from the above-mentioned MGISEQ-2000RS high-throughput sequencing reagent set) into the chip, and use DNA polymerase to add the dNTP molecules to the parent strand of DNA for first-strand sequencing;
  • Step 3 After the first-strand sequencing is completed, enter the MDA process to make the second-strand grow. After the second-strand growth is completed, use the excess MDA enzyme (from the above-mentioned MGISEQ-2000RS high-throughput sequencing reagent set) with the elution reagent ( from MGISEQ-2000RS high-throughput sequencing reagent set) eluted cleanly;
  • Step 4 Pump DTT to open the disulfide bond in the enzyme protein, and then elute the DTT; pump in the sulfhydryl blocking reagent, that is, an ammonium bicarbonate solution containing N-ethylmaleimide, and react for 30 minutes. , use the above elution reagent again to elute the solution cleanly for second-strand sequencing.
  • the sulfhydryl blocking reagent that is, an ammonium bicarbonate solution containing N-ethylmaleimide
  • the #13 hole in the kit contains 50mM ammonium bicarbonate solution containing 100mM iodoacetamide.
  • the fluorescent modification and reversible blocking are sequentially polymerized on the MGISEQ-2000RS sequencing platform.
  • Nucleotide mixture and then use the above elution reagent to elute the free nucleotides, so that polymerization and leveling can be performed while signal acquisition. At this time, the polymerized nucleotide mixture is reversibly blocked and modified. Nucleotides.
  • the nucleotide mixture with fluorescent modification and reversible blocking is sequentially polymerized on the MGISEQ-2000RS sequencing platform, and then the free nucleotides are eluted using elution reagents.
  • the steps include elution, signal collection under photographic solution, use of excision reagent to remove the protecting group, and use of elution reagent for washing. Then the Q30 decrease of each cycle and the sequencing error rate curve of each cycle are calculated to evaluate the sequencing quality.
  • the #13 hole in the kit contains a 50mM ammonium bicarbonate solution containing 50mM N-ethylmaleimide.
  • the fluorescence is sequentially polymerized on the MGISEQ-2000RS sequencing platform.
  • Modified and reversibly blocked nucleotide mixture and then use elution reagents to elute the free nucleotides, so that polymerization and leveling can be performed while signal acquisition is performed. At this time, the polymerized nucleotide mixture is reversible.
  • Block the modified nucleotides perform SE50 sequencing on the MGISEQ-2000RS sequencing platform according to the same experimental process as Example 1, and then count the Q30 decrease of each cycle and the sequencing error rate curve of each cycle to evaluate the sequencing quality.
  • the #13 hole in the kit contains a 50mM ammonium bicarbonate solution.
  • the background signal value is the lowest when adding 50mM ammonium bicarbonate solution containing 50mM N-ethylmaleimide; followed by adding 100mM iodoacetamide 50mM ammonium bicarbonate solution; the 50mM ammonium bicarbonate solution without any blocking reagent has the highest background signal value.
  • the background signal value of adding 100mM iodoacetamide in 50mM ammonium bicarbonate solution is comparable to that of adding 50mM N-ethylmaleimide in 50mM ammonium bicarbonate solution, but Still lower than the background signal value of 50mM ammonium bicarbonate solution without any blocking reagent.

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Abstract

一种包含巯基封闭试剂的测序试剂盒及其应用。还公开了一种降低测序背景信号的方法，其包括以下步骤：(1)打开所述酶蛋白的三维空间结构，将活性基团裸露；(2)将裸露的活性基团进行封闭，所述封闭为不可逆封闭；(3)释放荧光物质；(4)清洗。首次基于将酶蛋白的二硫键打开再进行不可逆封闭，以更好地释放缠绕的染料类物质的策略，来降低测序中的背景信号、提高信噪比以及测序质量，有利于高效的高通量测序。

Description

一种包含巯基封闭试剂的测序试剂盒及其应用 技术领域
本发明属于核酸测序领域，具体涉及一种含巯基封闭试剂的试剂盒、基于巯基封闭试剂降低测序背景信号的方法及其应用。
背景技术
DNA测序包括一链测序和二链测序，在一链测序结束后，以一链为模板，通过多重置换扩增(multiple displacement amplification，MDA)长出二链，然后再对DNA二链进行测序。在二链生长过程中会使用到多重链置换扩增酶(phi29 DNA聚合酶)，phi29 DNA聚合酶很容易非特异性地吸附在测序的芯片上，较难清除，造成残留。残留的phi29 DNA聚合酶在二链测序的开始阶段会和dNTP(化学修饰过的脱氧核糖核苷酸)相互缠绕，由于修饰过的dNTP的分子结构带有荧光染料，非特异性结合的酶和dNTP的缠绕的结果是使得测序的背景信号升高，造成信噪比下降，从而影响测序质量。
现有的技术中对于降低非特异性荧光物质的吸附常采用的办法为用洗脱溶剂进行洗脱，要想将非特异性结合的荧光物质有效清除，需加大洗脱液的浓度以及延长洗脱时间，且不能达到有效去除非特异性吸附的效果，上述两者都是对于高效测序是不利的。
发明内容
为了解决现有技术中荧光染料清洗不彻底造成测序质量差的技术问题，本发明提供了一种巯基封闭试剂以及基于巯基封闭试剂降低测序背景信号的方法及其应用。
本发明的机理：首先将酶蛋白结构中的二硫键打开，使得二硫键成为裸露的巯基。封闭酶蛋白中裸露的巯基主要有两种方法：一种是卤素的取代反 应，常用碘代乙酰胺如图1反应(1)所示。另外一种是加成反应，常用N-乙基马来酰亚胺如图1反应(2)所示。迈克尔加成反应(Michael Addition)在碱催化下能提供亲核负碳离子的化合物和一个亲电共轭体系发生的共轭加成反应。
本发明首创在测序过程中用二硫苏糖醇(DTT)将酶蛋白结构中二硫键打开，并采用封闭试剂将二硫键全部不可逆地封闭，释放荧光染料，并将其彻底清除，进而降低背景信号提高测序质量。本发明所述巯基封闭试剂包括N-乙基马来酰亚胺和/或碘代乙酰胺，其常常被用于蛋白结构作为蛋白标记，是蛋白检测的重要手段。本发明首次将巯基封闭试剂应用在测序技术中，作为降低二链背景的手段，可以对MDA过程中的聚合酶进行很好地封闭，更有利于实现彻底清洗，避免其对二链测序的影响，有利于提高测序质量。
为了解决上述技术问题，本发明第一方面提供一种测序试剂盒，所述测序试剂盒包括巯基封闭试剂，所述巯基封闭试剂包括碘代乙酰胺和/或N-乙基马来酰亚胺。
在某些技术方案中，所述巯基封闭试剂为包含50-100mM的N-乙基马来酰亚胺的50mM碳酸氢铵溶液。优选地，所述巯基封闭试剂为包含50mM的N-乙基马来酰亚胺的50mM碳酸氢铵溶液。
在某些技术方案中，所述巯基封闭试剂为包含50-100mM的碘代乙酰胺的50mM碳酸氢铵溶液。优选地，所述巯基封闭试剂为包含100mM的碘代乙酰胺的50mM碳酸氢铵溶液。
在一些技术方案中，所述测序试剂盒还包括二硫键还原剂。优选地，所述二硫键还原剂为三羟甲基氨基甲烷磷化氢液(TCEP)或硫醇如β-巯基乙醇(β-ME)或二硫苏糖醇(DTT)。
在一些技术方案中，所述测序试剂盒还包括DNA聚合酶、MDA聚合酶、测序载片以及带荧光修饰和可逆阻断的核苷酸混合液中的一种或多种。优选地，所述MDA聚合酶为phi29 DNA聚合酶。
本发明第二方面提供一种降低核酸测序背景信号的方法，所述方法包括以下步骤：
(1)打开核酸测序过程中的酶蛋白的三维空间结构，将活性基团裸露；
(2)用本发明第一方面所述的测序试剂盒中的巯基封闭试剂将所述裸露的活性基团进行封闭，所述封闭为不可逆封闭；
(3)释放荧光物质；
(4)任选地，清洗。
在某些技术方案中，步骤(1)中，通过破坏二硫键来打开所述三维空间结构，且所述裸露的活性基团为巯基；和/或，
步骤(2)中，所述封闭使用单独的或者如第一方面所述的测序试剂盒中的巯基封闭试剂；和/或，
步骤(3)中，所述荧光物质可以为标记物单独标记的核苷酸，所述标记物例如为Cy5荧光、ROX荧光、Cy3荧光或EF700荧光。
本发明中，“单独的”指该试剂非来自于第一方面所述的测序试剂盒，而可以是另外配制相同的试剂。
步骤(3)中，所述荧光物质可以为标记物组合标记的核苷酸，从而使得可以在核苷酸上连接不同的荧光素酶。如本文所用，用于标记核苷酸的所述分子标记和与其特异性结合的标记可以是任何能够彼此特异性结合的分子配对。配对成员之间的特异性结合实现核苷酸与荧光素酶的连接。示例性的配对成员包括但不限于：(a)与相应抗体或其结合部分或片段组合的半抗原或抗原性化合物，例如地高辛-地高辛抗体，N3G-N3G抗体，FITC-FITC抗体；(b)核酸适配体和蛋白质；(c)非免疫结合对(例如生物素-抗生物素蛋白、生物素-链霉亲和素、生物素-中性抗生蛋白)；(d)激素-激素结合蛋白；(e)受体-受体激动剂或拮抗剂；(f)凝集素-碳水化合物；(g)酶-酶辅因子；(h)酶-酶抑制剂；和(i)能够形成核酸双链体的互补的寡核苷酸或多核苷酸对。
步骤(4)中，所述清洗使用洗脱试剂来进行。
在某些技术方案中，步骤(1)中，通过单独的或第一方面所述测序试剂盒中的二硫键还原剂例如三羟甲基氨基甲烷磷化氢液(TCEP)或硫醇如β-巯基乙醇(β-ME)或二硫苏糖醇(DTT)来还原二硫键，以破坏二硫键来打开所述三维空间。
在某些技术方案中，步骤(2)中，所述巯基封闭试剂为单独的或第一方面所述测序试剂盒中的包含50-100mM的N-乙基马来酰亚胺的50mM碳酸氢铵溶液。优选地，所述巯基封闭试剂为包含50mM的N-乙基马来酰亚胺的50mM碳酸氢铵溶液。
在某些技术方案中，步骤(2)中，所述巯基封闭试剂为单独的或第一方面所述测序试剂盒中的包含50-100mM的碘代乙酰胺的50mM碳酸氢铵溶液。优选地，所述巯基封闭试剂为包含100mM的碘代乙酰胺的50mM碳酸氢铵溶液。
在某些技术方案中，步骤(1)中，将所述二硫键还原剂与所述酶蛋白混合后，洗脱二硫键还原剂；和/或，步骤(2)中，加入所述巯基封闭试剂反应后，进行二次洗脱。
在某些技术方案中，所述洗脱和二次洗脱使用的洗脱试剂均为MGISEQ-2000RS高通量测序试剂盒的测序试剂槽中的洗脱试剂；和/或，所述酶蛋白为MDA聚合酶，例如phi29 DNA聚合酶。
本发明第三方面提供一种核酸测序方法，所述方法包括以下步骤：
(1)将待测核酸加载到测序载片上；
(2)使待测核酸与测序试剂接触，例如dNTP分子和DNA聚合酶，进行一链测序；
(3)一链测序完成后，加入聚合酶扩增二链；
(4)可选地，洗脱；
(5)使用本发明第一方面所述的测序试剂盒或本发明第二方面所述的 方法对裸露的活性基团进行封闭；
(6)可选地，洗脱；
(7)进行二链测序。
具体地，所述核酸测序包括以下步骤：
(1)使用MGISEQ-2000RS高通量测序试剂盒，将DNA纳米球加载到测序载片上；
(2)将dNTP分子混合液泵入所述测序载片，用DNA聚合酶将dNTP分子加入到DNA的母链上，进行一链测序；
(3)一链测序完成后，加入MDA聚合酶扩增二链，并将多余的MDA聚合酶用洗脱试剂洗脱；
(4)使用本发明第二方面所述的方法打开测序体系中残余的MDA酶的三维结构，并将裸露的基团封闭，反应例如30min后使用所述洗脱试剂洗脱，并进行二链测序。
优选地，所述核酸测序在基因测序仪MGISEQ-2000RS中进行；所述循环进行50-400次，例如50、100、150、200或400次；和/或，所述多核苷酸为DNA或RNA。
本发明第四方面提供第一方面所述的测序试剂盒在核酸测序或制备用于核酸测序的试剂中的应用。
本发明的积极进步效果：
本发明首次使用包含巯基封闭试剂的测序试剂盒，基于将酶蛋白的二硫键打开再进行不可逆封闭，以更好地释放缠绕的染料类物质的策略，来降低测序中的背景信号、提高信噪比以及测序质量。此种方案和试剂组用在测序脚本中和测序试剂盒中将提高产品的测序质量，提高产品的价值和市场占有率。
附图说明
图1为本发明不可逆封闭DNA聚合酶巯基的机理。
图2为不同封闭试剂对测试循环数与0.1％错误率的结果；对照组：没有加任何封闭试剂的50mM碳酸氢铵溶液；碘代乙酰胺：添加含100mM的碘代乙酰胺的50mM碳酸氢铵溶液；N-乙基马来酰亚胺：添加含50mM的N-乙基马来酰亚胺的50mM碳酸氢铵溶液。
图3为不同封闭试剂降低测序的背景信号的结果；对照组：没有加任何封闭试剂的50mM碳酸氢铵溶液；碘代乙酰胺：添加含100mM的碘代乙酰胺的50mM碳酸氢铵溶液；N-乙基马来酰亚胺：添加含50mM的N-乙基马来酰亚胺的50mM碳酸氢铵溶液。
图4为不同封闭试剂提高测序的信噪比的结果；对照组：没有加任何封闭试剂的50mM碳酸氢铵溶液；碘代乙酰胺：添加含100mM的碘代乙酰胺的50mM碳酸氢铵溶液；N-乙基马来酰亚胺：添加含50mM的N-乙基马来酰亚胺的50mM碳酸氢铵溶液。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
本发明的不可逆封闭DNA聚合酶巯基的机理如图1所示。
实施例中使用的器材、试剂以及原料
实验器材:
MGISEQ-2000RS测序仪，MGIDL-200H加载仪，MGISEQ-2000RS测序载片，仪器的激发波长分别为532和650nm。
实验所用试剂及原料(见表1)：
表1 实验所用试剂及原料
Figure PCTCN2022099258-appb-000001
碳酸氢铵(分析纯)，N-乙基马来酰亚胺(分析纯)，超纯水，大肠杆菌单链环DNA为模板即标准文库试剂V3.0，引物序列：CAACTCCTTGGCTCACAGAACATGGCTACGATCCGACTT(SEQ ID NO:1)。DNA聚合酶和MDA酶来自BGI，DNA纳米球来源于BGI。以下实验均为以大肠杆菌单链环DNA为模板，并使用MGISEQ-2000RS高通量测序试剂套装(FCL PE150，货号1000012555，华大智造)完成DNA纳米球的制备及加载到芯片(华大智造，货号为1000008403)，以备后续测序。本实施例中提到的带荧光修饰及可逆阻断的核苷酸混合液包括：dATP-1，其是指同时具有可逆阻断基团修饰和Cy5荧光修饰的腺嘌呤核苷酸；dTTP-1，其是指同时具有可逆阻断基团修饰和ROX荧光修饰的胸腺嘧啶核苷酸；dGTP-1，其是指同时具有可逆阻断基团修饰和Cy3荧光修饰的鸟嘌呤核苷酸；以 及dCTP-1，其是指同时具有可逆阻断基团修饰和EF700荧光修饰的胞嘧啶核苷酸。上述dATP-1、dTTP-1、dGTP-1、dCTP-1均来自华大智造MGISEQ-2000RS高通量测序试剂盒。不同的平台，带荧光修饰及可逆阻断的核苷酸混合液可以多样，比如可以在此核苷酸混合液中参杂单纯的可逆阻断基团核苷酸或其他的修饰类型。
实验方法和步骤
(a)巯基封闭试剂的配制：
精确称量一定量的碳酸氢铵，溶于超纯水中并配成50mM碳酸氢铵水溶液待用。继续精确称量一定量的N-乙基马来酰亚胺和碘代乙酰胺，配置成含100mM的碘代乙酰胺的50mM碳酸氢铵溶液和含50mM的N-乙基马来酰亚胺的50mM碳酸氢铵溶液。
(b)核酸测序方法，参照说明书进行，简述如下：
第一步：将上述DNA纳米球加载到准备好的上述芯片(即测序载片)上；
第二步：将准备好的dNTP分子混合液(来自上述MGISEQ-2000RS高通量测序试剂套装)泵入芯片，用DNA聚合酶将dNTP分子加入到DNA的母链上进行一链测序；
第三步：待一链测序完成后，进入MDA过程，使得二链长出，完成二链生长后，将多余的MDA酶(来自上述MGISEQ-2000RS高通量测序试剂套装)用洗脱试剂(来自MGISEQ-2000RS高通量测序试剂套装)洗脱干净；
第四步：将DTT泵入打开酶蛋白中的二硫键，随后将DTT洗脱干净；泵入巯基封闭试剂即含N-乙基马来酰亚胺的碳酸氢铵溶液，进行反应30分钟，再次用上述洗脱试剂将溶液洗脱干净进行二链测序。
实施例1
使用MGISEQ-2000RS高通量测序试剂盒，试剂盒中#13号孔位为含有100mM的碘代乙酰胺的50mM的碳酸氢铵溶液，在MGISEQ-2000RS测序平台依次聚合带荧光修饰及可逆阻断的核苷酸混合液，然后使用上述洗脱试剂对游离的核苷酸进行洗脱，这样可以在信号采集的同时进行聚合补平，此时聚合的核苷酸混合液为可逆阻断修饰的核苷酸。
按照实验流程在MGISEQ-2000RS测序平台进行SE50测序，简言之，在MGISEQ-2000RS测序平台依次聚合带荧光修饰及可逆阻断的核苷酸混合液，然后使用洗脱试剂对游离的核苷酸进行洗脱、在拍照溶液下进行信号采集、使用切除试剂以切除保护基团、使用洗脱试剂进行洗涤的步骤。然后统计每个循环的Q30下降幅度，每个循环的测序错误率曲线来评估测序质量。
实施例2
使用MGISEQ-2000RS高通量测序试剂盒，试剂盒中#13号孔位为含有50mM的N-乙基马来酰亚胺的50mM的碳酸氢铵溶液，在MGISEQ-2000RS测序平台依次聚合带荧光修饰及可逆阻断的核苷酸混合液，然后使用洗脱试剂对游离的核苷酸进行洗脱，这样可以在信号采集的同时进行聚合补平，此时聚合的核苷酸混合液为可逆阻断修饰的核苷酸，按照与实施例1相同的实验流程在MGISEQ-2000RS测序平台进行SE50测序，然后统计每个循环的Q30下降幅度，每个循环的测序错误率曲线来评估测序质量。
对比例1
使用MGISEQ-2000RS高通量测序试剂盒，试剂盒中#13号孔位是50mM的碳酸氢铵的溶液。按照与实施例1相同的实验流程在MGISEQ-2000RS测序平台进行SE50测序，然后统计每个循环的Q30下降幅度，每个循环的测序错误率曲线来评估测序质量。
效果实施例1
结果分析：
其中，根据图2可知，不同封闭试剂在进行测试循环时的碱基达到千分之一错误的比例是不同的，小错误率的碱基比例越高越好。具体地，添加含50mM的N-乙基马来酰亚胺的50mM碳酸氢铵溶液的千分之一错误的比例最高；其次是添加含100mM的碘代乙酰胺的50mM碳酸氢铵溶液；没有加任何封闭试剂的50mM碳酸氢铵溶液的千分之一错误的比例最低。由此可知含50mM的N-乙基马来酰亚胺的50mM碳酸氢铵溶液的效果最佳。
根据图3可知，在A、G、T的循环中，添加含50mM的N-乙基马来酰亚胺的50mM碳酸氢铵溶液的背景信号值最低；其次是添加含100mM的碘代乙酰胺的50mM碳酸氢铵溶液；没有加任何封闭试剂的50mM碳酸氢铵溶液的背景信号值最高。在C循环中，添加含100mM的碘代乙酰胺的50mM碳酸氢铵溶液的背景信号值与添加含50mM的N-乙基马来酰亚胺的50mM碳酸氢铵溶液的背景信号值相当，但是仍然都低于没有加任何封闭试剂的50mM碳酸氢铵溶液的背景信号值。
根据图4可知，在A、C、G、T的循环中，添加含50mM的N-乙基马来酰亚胺的50mM碳酸氢铵溶液的信噪比均是最高；其次是添加含100mM的碘代乙酰胺的50mM碳酸氢铵溶液；没有加任何封闭试剂的50mM碳酸氢铵溶液的信噪比均是最低。
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。

Claims (10)

  1. 一种测序试剂盒，所述试剂盒包括巯基封闭试剂。
  2. 根据权利要求1所述的测序试剂盒，其特征在于，所述巯基封闭试剂包括碘代乙酰胺和/或N-乙基马来酰亚胺，和/或所述巯基封闭试剂的浓度为50-100mM；
    较佳地，所述巯基封闭试剂为包括50-100mM N-乙基马来酰亚胺的50mM碳酸氢铵溶液，和/或所述巯基封闭试剂为包含50-100mM的碘代乙酰胺的50mM碳酸氢铵溶液。
  3. 根据权利要求1或2所述的测序试剂盒，其特征在于，所述测序试剂盒还包括二硫键还原剂；所述二硫键还原剂优选为三羟甲基氨基甲烷磷化氢液(TCEP)或硫醇如β-巯基乙醇(β-ME)或二硫苏糖醇(DTT)。
  4. 一种降低核酸测序背景信号的方法，其特征在于，所述方法包括以下步骤：
    (1)打开核酸测序过程中的酶蛋白的三维空间结构，将活性基团裸露；
    (2)用权利要求1-3任一项所述的试剂盒将裸露的活性基团进行封闭，所述封闭为不可逆封闭；
    (3)释放荧光物质；
    (4)任选地，清洗。
  5. 如权利要求4所述的方法，其特征在于，步骤(1)中，通过破坏二硫键来打开所述三维空间结构，且所述裸露的活性基团为巯基；和/或，
    步骤(2)中，所述封闭使用如权利要求1所述的测序试剂盒中的巯基封闭试剂；和/或，
    步骤(3)中，所述荧光物质可以为标记物单独标记的核苷酸，所述标记物例如为Cy5荧光、ROX荧光、Cy3荧光或EF700荧光；和/或所述荧光物质可以为标记物组合标记的核苷酸；和/或，
    步骤(4)中，所述清洗使用洗脱试剂来进行。
  6. 如权利要求5所述的方法，其特征在于，步骤(1)中，通过二硫键还原剂例如三羟甲基氨基甲烷磷化氢液(TCEP)或硫醇如β-巯基乙醇(β-ME)、二硫苏糖醇(DTT)来还原二硫键，以破坏二硫键来打开所述三维空间。
  7. 如权利要求6所述的方法，其特征在于，步骤(1)中，将所述二硫键还原剂与所述酶蛋白混合后，洗脱二硫键还原剂；和/或，步骤(2)中，加入所述巯基封闭试剂反应后，进行二次洗脱。
  8. 如权利要求4所述的方法，其特征在于，所述酶蛋白为MDA聚合酶，例如phi29 DNA聚合酶。
  9. 一种核酸测序方法，其特征在于，所述方法包括以下步骤：
    (1)将待测核酸加载到测序载片上；
    (2)使待测核酸与测序试剂接触，例如dNTP分子和DNA聚合酶，进行一链测序；
    (3)一链测序完成后，加入聚合酶扩增二链；
    (4)可选地，洗脱；
    (5)使用如权利要求1-3任一项所述的测序试剂盒或权利要求4-8任一项所述的方法对裸露的活性基团进行封闭；
    (6)可选地，洗脱；
    (7)进行二链测序。
  10. 一种如权利要求1-3任一项所述的测序试剂盒在核酸测序中或制备用于核酸测序的试剂的应用。
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