WO2023240564A1 - 含甘氨酸-NaOH缓冲液的切除试剂及其在核酸测序中的应用 - Google Patents

含甘氨酸-NaOH缓冲液的切除试剂及其在核酸测序中的应用 Download PDF

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WO2023240564A1
WO2023240564A1 PCT/CN2022/099260 CN2022099260W WO2023240564A1 WO 2023240564 A1 WO2023240564 A1 WO 2023240564A1 CN 2022099260 W CN2022099260 W CN 2022099260W WO 2023240564 A1 WO2023240564 A1 WO 2023240564A1
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sequencing
excision
reagent
excision reagent
blocking group
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PCT/CN2022/099260
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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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    • 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
    • C12Q1/6869—Methods for sequencing

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  • the invention belongs to the field of sequencing, and specifically relates to an excision reagent containing glycine-NaOH buffer, a kit containing the same and its application in polynucleotide sequencing.
  • First-generation sequencing is a sequencing technology based on the Sanger method.
  • second-generation sequencing is based on massively parallel sequencing technology, which can simultaneously complete the synthesis of complementary strands of the sequencing template and the acquisition of sequence data.
  • Third-generation sequencing is based on single-molecule sequencing and massively parallel sequencing (MPS) technology.
  • Next-generation sequencing is the current mainstream sequencing technology.
  • second-generation sequencing technology includes three consecutive steps: 1. Add dNTPs to the sequencing reaction system; 2. Detect the base type of dNTP added to the sequencing system; 3. Remove various enzymes and fluorescence in the reaction. Label or dNTP 3' end hindering group and other elution reactions. This series of consecutive steps achieves "sequencing while synthesising".
  • the current mainstream mcMPS (multi-copy base signal acquisition unit MPS) technology is based on the "discontinuous polymerization sequencing method" to realize the reading of nucleotide sequences.
  • MPS multi-copy base signal acquisition unit
  • the “base extension” of different nucleic acid molecules within the “unit” is not completely synchronized. There may be individual nucleic acid molecules that do not undergo the expected polymerization extension or that have more than one unexpected polymerization extension reaction.
  • MGISEQ-2000RS high-throughput rapid sequencing reagent set is fast and efficient, and can complete sequencing requirements in a short time. It is more suitable for samples with small data volume and urgent samples, especially for tumor detection.
  • MGISEQ-2000RS high-throughput sequencing kit uses combined probe anchored polymerization technology (cPAS) to polymerize DNA molecular anchors and fluorescent probes on DNA nanospheres (DNB), and uses a high-resolution imaging system to detect light The signal is collected, and the optical signal is digitally processed to obtain high-quality and high-accuracy sample sequence information.
  • cPAS combined probe anchored polymerization technology
  • DDB DNA nanospheres
  • the MGISEQ-2000RS high-throughput sequencing kit will also cause runon and lag phenomena during the sequencing process. Therefore, the rapid and effective removal of the 3' end blocking group is crucial to the speed and accuracy of sequencing. Importantly, the kit components need to be optimized to increase the speed and completeness of resection.
  • the present invention provides an excision reagent containing glycine-NaOH buffer and its application in nucleic acid sequencing.
  • an alkaline buffer solution is more conducive to the excision, that is, to speed up the excision and the completeness of the excision, thereby reducing the lead and lag phenomena in the sequencing process, especially hysteresis.
  • the Tris-HCl buffer system is used as the excision solution, which cannot meet the strong alkaline requirements, that is, the pH is greater than 9.0.
  • This invention applies the glycine-sodium hydroxide buffer system to the excision reagent for sequencing for the first time to speed up the excision efficiency and improve the sequencing quality.
  • the use of this kind of reagent in sequencing kits will improve the sequencing quality of the product and increase the product value and market share.
  • an excision reagent which includes a glycine-sodium hydroxide buffer.
  • the glycine-sodium hydroxide buffer further contains a 3' end blocking group cleaving agent.
  • the 3' end blocking group excluding agent is, for example, THPP, TCEP or DTT.
  • the excision reagent is located in hole No. 9 of the sequencing reagent tank.
  • the pH range of the glycine-sodium hydroxide buffer is greater than 9, specifically 9-12, more specifically 9.6-10.6, such as 9.6, 10 or 10.6.
  • the pH of the glycine-sodium hydroxide buffer is 10.6.
  • the concentration of the 3' end blocking group cleaving agent, such as THPP is 5-30mM, such as 5mM, 10mM, 20mM or 30mM.
  • the concentration of the 3'-end blocking group excluding agent is 20mM.
  • the second aspect of the present invention provides a sequencing kit, which includes the excision reagent according to the first aspect of the present invention.
  • the sequencing kit further includes one or more of DNA nanospheres, sequencing slides, MDA polymerase, dNTPs and scanning reagents.
  • the third aspect of the present invention provides a method for excluding the 3' end blocking group of a polynucleotide.
  • the method uses the excision reagent described in the first aspect of the present invention to excise the 3' end blocking group. Cut off the group, exposing the 3' terminal hydroxyl group.
  • the conditions for resection are as follows; the temperature is 30-60°C, and/or the time is 1-5 s.
  • the temperature is 30-40°C, 40-50°C or 50-60°C, such as 30°C, 40°C, 50°C or 60°C.
  • the temperature is 50°C and the time is 2 seconds.
  • the fourth solution of the present invention provides a sequencing method, which method includes the following steps:
  • a sequencing reagent such as a dNTP molecule and DNA polymerase
  • the method includes the following steps:
  • the cycle is performed 50-400 times, such as 50, 100, 150, 200 or 400 times.
  • the scanning reagent is located in hole No. 10 of the sequencing reagent tank.
  • the fifth aspect of the present invention provides an excision reagent as described in the first aspect of the present invention or a sequencing kit as described in the second aspect of the present invention for use in polynucleotide sequencing or preparation of polynucleosides.
  • Application of acid sequencing reagents are described in the first aspect of the present invention or a sequencing kit as described in the second aspect of the present invention for use in polynucleotide sequencing or preparation of polynucleosides.
  • the reagents and raw materials used in the present invention are all commercially available.
  • This invention applies the glycine-sodium hydroxide buffer system to the excision reagent for sequencing for the first time to speed up the excision efficiency and improve the sequencing quality.
  • the use of this kind of reagent in sequencing kits will improve the sequencing quality of the product and increase the value and market share of the product.
  • Figure 1 shows the effect of glycine-sodium hydroxide buffer system on Lag (lag) in sequencing.
  • Figure 2 shows the effect of the glycine-sodium hydroxide buffer system on Runon (lead) in sequencing.
  • Ammonium bicarbonate (analytical grade) N-ethylmaleimide, ultrapure water, Escherichia coli single-stranded circular DNA as template, standard library reagent V3.0, primer sequence: CAACTCCTTGGCTCACAGAACATGGCTACGATCCGACTT (SEQ ID NO: 1).
  • DNA polymerase and DNA nanospheres were both from MGI (MGISEQ-2000RS high-throughput sequencing kit PE150). The following experiments all use E. coli single-stranded circular DNA as a template, and use the MGISEQ-2000RS high-throughput sequencing kit to prepare DNA nanospheres and load them onto a sequencing slide for subsequent sequencing.
  • 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. It refers to thymine nucleotides with both reversible blocking group modification and ROX fluorescence modification; dGTP-1, which refers to guanine nucleotides with both reversible blocking group modification and Cy3 fluorescence modification; and dCTP-1, which refers to a cytosine nucleotide with both reversible blocking group modification and EF700 fluorescence modification.
  • dATP-1 which refers to adenine nucleotide with both reversible blocking group modification and Cy5 fluorescence modification
  • dTTP- 1 It refers to thymine nucleotides with both reversible blocking group modification and ROX fluorescence modification
  • dGTP-1 which refers to
  • Control group 1 Use the MGISEQ-2000RS high-throughput sequencing kit and perform SE50 sequencing on the MGISEQ-2000RS sequencing platform according to the experimental process.
  • the nucleosides with fluorescence modification and reversible blocking are sequentially polymerized on the MGISEQ-2000RS sequencing platform.
  • Acid mixture then use an elution reagent to elute the free nucleotides, collect signals under a photographing solution, use an excision reagent to remove the protecting group, and use an elution reagent for washing. Then the Lag and Runon decreases of each cycle and the sequencing error rate curve of each cycle are calculated to evaluate the sequencing quality.
  • Experimental Group 1 Use the MGISEQ-2000RS high-throughput sequencing kit, then remove the reagent in the No. 9 hole in the kit and replace it with the buffer solution containing the excision reagent of the present invention, and sequentially polymerize the fluorescence modification and The nucleotide mixture is reversibly blocked, and then the free nucleotides are eluted using elution reagents, so that polymerization and leveling can be performed while the signal is being collected. At this time, the polymerized nucleotide mixture is reversibly blocked.
  • the modified nucleotides were subjected to SE50 sequencing on the MGISEQ-2000RS sequencing platform according to the same experimental process as the control group, and then the Lag and Runon decreases in each cycle and the sequencing error rate curve in each cycle were calculated to evaluate the sequencing quality.
  • glycine and sodium hydroxide Accurately weigh a certain amount of glycine and sodium hydroxide, and configure them into glycine-sodium hydroxide buffer solutions with different pH values according to Table 1, X mL 0.2mol/L glycine + Y mL 0.2mol/L sodium hydroxide, and dilute with water to 200 mL, then weigh a certain amount of THPP (tris(3-hydroxypropyl)phosphine) and dissolve it in the above buffer solution, and prepare a 20 mM THPP solution for later use.
  • THPP tris(3-hydroxypropyl)phosphine
  • Step 1 Load the DNA nanospheres onto the prepared sequencing slide
  • Step 2 Pump the prepared dNTP molecule mixture into the sequencing slide, and use MDA polymerase to add the dNTP molecules to the parent strand of DNA;
  • Step 3 Determine the type of base by taking photos with scanning reagent
  • Step 4 Use the excision reagent to expose the 3’ hydroxyl group, the temperature is 50°C, and the excision time is 2 seconds;
  • DNA nanospheres, MDA polymerase, dNTPs and scanning reagents can be derived from the MGISEQ-2000RS high-throughput sequencing kit or other similar kits. Wherein, the scanning reagent is located in hole No. 10 of the sequencing reagent tank.
  • the alkaline solution of glycine-sodium hydroxide is beneficial to improving sequencing Q30 and reducing Lag and Runon.
  • the glycine-sodium hydroxide buffer system can also be used in combination with TCEP and DTT excision reagents to achieve good sequencing results.
  • DNA sequencing is a very complex process, and various reagents in each link include many ingredients.
  • the inventor also tried to use a boric acid-sodium borate buffer system, but compared with the glycine-sodium hydroxide buffer system at the same pH value Shows poor sequencing quality. This may be caused by the boric acid-sodium borate buffer not being suitable for the entire reagent environment. There may be the following reasons: 1. Poor biocompatibility, destroying DNA nanospheres; 2. The pH value is thermally unstable; 3. Mixed with the excision reagent. Chemical reactions occur with other ingredients; 4. Poor buffering capacity, etc.; but the specific reasons are unknown.
  • the inventor also tried a carbonic acid-sodium carbonate (pH 10.6) buffer system, but it was only effective in the strategy of using the azide series as the exposed hydroxyl group at the 3' end, and was ineffective in other cleavable blocking groups.
  • the carbonate-sodium carbonate system buffer solution has problems such as instability and easy decomposition when heated, and is not suitable for long-term storage as a commercial product. This also shows that when selecting the buffer composition for sequencing, it is not only the buffer efficiency that affects the sequencing effect, but also other comprehensive factors such as its composition and thermal stability.

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Abstract

提供了一种含甘氨酸-NaOH缓冲液的切除试剂及其在核酸测序中的应用。还提供了一种包含所述切除试剂的测序试剂盒、切除多核苷酸3'端阻断基团的方法以及测序方法。在多核苷酸测序过程中，所述方案能加快3'端阻断基团切除效率，有效提升测序Q30和降低Lag和Runon，提高测序质量。

Description

含甘氨酸-NaOH缓冲液的切除试剂及其在核酸测序中的应用 技术领域
本发明属于测序领域，具体涉及一种含甘氨酸-NaOH缓冲液的切除试剂、含其的试剂盒及其在多核苷酸测序中的应用。
背景技术
一代测序是基于Sanger方法的测序技术，与之相比，二代测序基于大规模平行测序技术，它能同时完成测序模板互补链的合成和序列数据的获取。三代测序则基于单分子测序和大规模平行测序(MPS)技术。二代测序是目前的主流测序技术。一般来说，二代测序技术包括三个连续的步骤：1.往测序反应体系中加入dNTP；2.检测加入到测序体系中dNTP的碱基类型；3.去除反应中的各种酶、荧光标记物或dNTP 3’端的阻碍基团等洗脱反应。这样一系列连续的步骤从而实现“边合成边测序”。
目前主流的mcMPS(多拷贝碱基信号采集单元MPS)技术均基于“非连续聚合测序法”来实现核苷酸序列的读取，在每一轮的聚合反应中，同一个“碱基信号采集单元”内不同核酸分子的“碱基延伸”并非完全同步，可能会有个别的核酸分子未发生预期的聚合延伸或者发生了未预期的大于1次的聚合延伸反应。例如针对特定的核酸分子，测序仪在完成1次聚合延伸和信号采集后，标记在可逆末端终止子3’端的Block(阻断基团)不能正常去除还原为“-OH”，这将直接导致该核酸分子在下一个测序循环中无法正常进行聚合延伸，从而在后续的测序循环中持续产生“滞后信号”(Phasing或称Lag)。同理，如果作为聚合反应原料的可逆末端终止子的3'端阻断基团缺失或非有效阻断，则特定的核酸分子将完成大于1个碱基的聚合延伸反应，从而在后 续的测序循环中持续产生“超前信号”(Pre-phasing或称Runon)，即马拉松效应。随着测序反应的进行，同一个“碱基信号采集单元”中不同核酸分子的“马拉松效应”会越来越严重。
MGISEQ-2000RS高通量快速测序试剂套装具有快速、高效的特点，可以在短时间内完成测序需求，更适用于少数据量样本及紧迫样本，特别是肿瘤检测。MGISEQ-2000RS高通量测序试剂盒使用联合探针锚定聚合技术(cPAS)，通过将DNA分子锚和荧光探针在DNA纳米球(DNB)上进行聚合，并利用高分辨率成像***对光信号进行采集，光信号经过数字化处理后获得高质量高准确度的样本序列信息。
MGISEQ-2000RS高通量测序试剂盒在测序过程中同样会出现超前(Runon)和滞后(Lag)现象，因此，3’端阻断基团的快速有效的切除对测序的速度和准确度至关重要，需要优化试剂盒组分，以加快切除的速度和彻底性。
发明内容
为了解决现有技术中测序时3’端阻断基团的切除不够快速有效的问题，本发明提供了一种含甘氨酸-NaOH缓冲液的切除试剂及其在核酸测序中的应用。发明人发现，基于dNTP的3’端可逆阻断基团的切除，偏碱性的缓冲溶液更有利于切除即加快切除速度和切除彻底性，来降低测序过程中的超前和滞后现象，尤其是滞后现象。通常情况下使用Tris-HCl缓冲体系作为切除溶液，不能满足强碱性要求，即pH大于9.0的要求。经分析，叠氮与THPP的反应以及酯的水解形成3’端羟基的过程均需要在强碱性条件下进行。本发明首次将甘氨酸-氢氧化钠缓冲体系应用在测序的切除试剂中加快切除效率，提高测序质量。此种试剂用在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。
为解决上述问题，本发明的方案之一提供了一种切除试剂，所述切除试剂包含甘氨酸-氢氧化钠缓冲液。
在具体实施方案中，所述甘氨酸-氢氧化钠缓冲液还包含3’端阻断基团切除剂。所述3’端阻断基团切除剂例如为THPP、TCEP或DTT。使用时，所述切除试剂位于测序试剂槽9号孔位中。
在具体实施方案中，所述甘氨酸-氢氧化钠缓冲液的pH范围大于9，具体地9-12，更具体地为9.6-10.6，例如9.6、10或10.6。
较佳地，所述甘氨酸-氢氧化钠缓冲液的pH为10.6。
在具体实施方案中，所述3’端阻断基团切除剂例如THPP的浓度为5-30mM，例如5mM、10mM、20mM或30mM。
较佳地，所述3’端阻断基团切除剂的浓度为20mM。
为解决上述问题，本发明的方案之二提供了一种测序试剂盒，所述测序试剂盒包括如本发明的方案之一所述的切除试剂。
在具体实施方案中，所述测序试剂盒还包括DNA纳米球、测序载片、MDA聚合酶、dNTP和扫描试剂中的一种或多种。
为解决上述问题，本发明的方案之三提供了一种切除多核苷酸3’端阻断基团的方法，所述方法使用本发明的方案之一所述的切除试剂来切除3’端阻断基团，裸露出3’端羟基。
在具体实施方案中，切除的条件如下；温度为30-60℃，和/或，时间为1-5s。优选地，温度为30-40℃、40-50℃或50-60℃，例如30℃、40℃、50℃或60℃。优选地，温度为50℃，时间为2s。
为解决上述问题，本发明的方案之四提供了一种测序方法，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，所述测序试剂例如为dNTP分子和DNA聚合酶；
(3)记录信号；
(4)使用如本发明的方案之一所述的切除试剂或如本发明的方案之二所述的测序试剂盒，切除3’端阻断基团，裸露出3’端羟基；以上步骤循环多次，直到测序结束。
具体地，所述方法包括以下步骤：
(1)使用本发明的方案之二所述的测序试剂盒，加载DNA纳米球；
(2)用MDA聚合酶将dNTP分子加入到DNA的母链上；
(3)记录信号；
(4)使用如本发明的方案之一所述的切除试剂或如本发明的方案之二所述的测序试剂盒，切除3’端阻断基团，裸露出3’端羟基；以上步骤循环多次，直到测序结束。
优选地，所述循环进行50-400次，例如50、100、150、200或400次。使用时，所述扫描试剂位于测序试剂槽10号孔位中。
为解决上述问题，本发明的方案之五提供了一种如本发明的方案之一所述的切除试剂或如本发明的方案之二所述的测序试剂盒在多核苷酸测序或制备多核苷酸测序的试剂中的应用。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果为：
本发明首次将甘氨酸-氢氧化钠缓冲体系应用在测序的切除试剂中加快 切除效率，提高测序质量。此种试剂用在测序试剂盒中将由于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为甘氨酸-氢氧化钠缓冲体系对于测序中Lag(滞后)的影响。
图2为甘氨酸-氢氧化钠缓冲体系对于测序中Runon(超前)的影响。
具体实施方式
实施例1试剂的制备和测序的进行
1.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532nm和650nm。
2.实验所用试剂及原料
碳酸氢铵(分析纯)N-乙基马来酰亚胺，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DNA纳米球均来自华大智造(MGISEQ-2000RS高通量测序试剂盒PE150)。以下实验均为以大肠杆菌单链环DNA为模板，并使用MGISEQ-2000RS高通量测序试剂盒完成DNA纳米球的制备及加载到测序载片，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。(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。)
对照组1：使用MGISEQ-2000RS高通量测序试剂盒，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Lag、Runon下降幅度，每个循环的测序错误率曲线来评估测序质量。
实验组1：使用MGISEQ-2000RS高通量测序试剂盒，然后去除试剂盒中9号孔位的试剂并更换成本发明的含切除试剂的缓冲溶液，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，按照与对照组相同的实验流程在MGISEQ-2000RS测序平台进行SE50测序，然后统计每个循环的Lag、Runon下降幅度，每个循环的测序错误率曲线来评估测序质量。
3.实验方法和步骤
(a)切除试剂的配置：
精确称量一定量的甘氨酸和氢氧化钠，按照表1分别配置成不同pH值的甘氨酸-氢氧化钠缓冲溶液，X mL 0.2mol/L甘氨酸+Y mL 0.2mol/L氢氧化钠，加水稀释至200mL，然后称量一定量的THPP(三(3-羟丙基)膦)溶于上述缓冲溶液中，并配制成20mM的THPP溶液待用。
表1甘氨酸-氢氧化钠缓冲液配置
pH 0.2mol/L甘氨酸(X mL) 0.2mol/L氢氧化钠(Y mL)
8.6 50 4.0
8.8 50 6.0
9.0 50 8.8
9.2 50 12.0
9.4 50 16.8
9.6 50 22.4
9.8 50 27.2
10.0 50 32.0
10.4 50 38.6
10.6 50 45.5
(b)DNA测序方法
第一步：将DNA纳米球加载到准备好的测序载片上；
第二步：将准备好的dNTP分子混合液泵入测序载片，用MDA聚合酶将dNTP分子加入到DNA的母链上；
第三步：通过扫描试剂拍照确定碱基的类型；
第四步：使用切除试剂将3’端羟基裸露出来，温度为50℃，切除时间为2s；
照前四步骤进行多次循环测序，直到测序结束。
DNA纳米球、MDA聚合酶、dNTP和扫描试剂可来源于MGISEQ-2000RS高通量测序试剂盒或其他同类试剂盒。其中，所述扫描试剂位于测序试剂槽的10号孔位。
实施例2各缓冲体系的效果
表2不同缓冲体系的pH值对测序质量的影响
Figure PCTCN2022099260-appb-000001
Figure PCTCN2022099260-appb-000002
从表2中可以看出，和Tris-HCl的缓冲体系相比，甘氨酸-氢氧化钠的偏碱性的溶液有利于提升测序Q30和降低Lag和Runon。
从图1中可以看出和Tris-HCl相比，pH值为10的甘氨酸-氢氧化钠缓冲体系能有效降低测序中Lag。
从图2中可以看出和Tris-HCl相比，pH值为10的甘氨酸-氢氧化钠缓冲体系能有效降低测序中Runon。
除HTPP外，甘氨酸-氢氧化钠缓冲体系同样可以与TCEP、DTT切除试剂联合使用，达到良好的测序效果。
DNA测序是一个非常复杂的过程，各个环节的各种试剂均包括很多种成分，发明人还尝试用硼酸-硼酸钠缓冲体系，但在相同的PH值下与甘氨酸-氢氧化钠缓冲体系相比显示测序质量差。其可能是硼酸-硼酸钠缓冲液不适合整套试剂环境所造成的，可能有以下原因：1.生物相容性差，破坏DNA纳米球；2.pH值热不稳定；3.和切除试剂里的其他成分发生化学反应；4.缓冲能力差等；但具体原因未明。
另外，发明人还尝试过碳酸-碳酸钠(pH10.6)缓冲体系，但其仅在以叠氮系列作为3’端裸露羟基的策略中有效，在其他可切断阻逆基团中无效。除此之外，碳酸-碳酸钠体系缓冲液存在不稳定、加热易分解等问题，不宜作为商品长时间存放。这也表明在选择测序的缓冲液组成时，影响测序效果的不只是缓冲效率，还有其成分组成、热稳定性等其他多方面综合因素。
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. 一种切除试剂，其特征在于，所述切除试剂包含甘氨酸-氢氧化钠缓冲液。
  2. 如权利要求1所述的切除试剂，其特征在于，所述切除试剂还包含3’端阻断基团切除剂，例如THPP、TCEP或DTT。
  3. 如权利要求1所述的切除试剂，其特征在于，所述甘氨酸-氢氧化钠缓冲液的pH范围为大于9，优选地9-12，更优选地9.6-10.6，例如9.6、10或10.6。
  4. 如权利要求2所述的切除试剂，其特征在于，所述3’端阻断基团切除剂的浓度为5-30mM，例如5mM、10mM、20mM或30mM。
  5. 一种测序试剂盒，其特征在于，所述测序试剂盒包括如权利要求1～4任一项所述的切除试剂。
  6. 如权利要求5所述的测序试剂盒，其特征在于，所述测序试剂盒还包括测序载片、聚合酶、dNTP和扫描试剂中的一种或多种。
  7. 一种切除核苷酸3’端阻断基团的方法，其特征在于，所述方法使用如权利要求1～4任一项所述的切除试剂来切除3’端阻断基团，裸露出3’端羟基。
  8. 如权利要求7所述的方法，其特征在于，切除的条件如下；温度为30-60℃，和/或，时间为1-5s；优选地，温度为30-40℃、40-50℃或50-60℃，例如30℃、40℃、50℃或60℃；优选地，温度为50℃，时间为2s。
  9. 一种测序方法，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，所述测序试剂例如为dNTP分子和 DNA聚合酶；
    (3)记录信号；
    (4)使用如权利要求1-4任一项所述的切除试剂或如权利要求5或6所述的测序试剂盒，切除3’端阻断基团，裸露出3’端羟基；
    (5)以上步骤循环多次，直到测序结束；
    优选地，所述循环进行50-400次，例如50、100、150、200或400次。
  10. 如权利要求1-4任一项所述的切除试剂或权利要求5或6所述的测序试剂盒在多核苷酸测序或制备多核苷酸测序的试剂中的应用。
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991017264A1 (en) * 1990-05-02 1991-11-14 Imperial Chemical Industries Plc Assay for specific nucleic acid sequences
CN107002067A (zh) * 2014-09-11 2017-08-01 宝生物工程株式会社 利用热稳定性错配内切核酸酶的方法
US20170268048A1 (en) * 2014-08-20 2017-09-21 Huawei Yang Test kit and method for testing target nucleic acid in sample
WO2019080725A1 (zh) * 2017-10-25 2019-05-02 深圳华大生命科学研究院 一种核酸测序方法以及一种核酸测序试剂盒
CN114317787A (zh) * 2021-12-29 2022-04-12 成都大学 吸附磁珠及其试剂盒和用途以及检测金黄色葡萄球菌的方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991017264A1 (en) * 1990-05-02 1991-11-14 Imperial Chemical Industries Plc Assay for specific nucleic acid sequences
US20170268048A1 (en) * 2014-08-20 2017-09-21 Huawei Yang Test kit and method for testing target nucleic acid in sample
CN107002067A (zh) * 2014-09-11 2017-08-01 宝生物工程株式会社 利用热稳定性错配内切核酸酶的方法
WO2019080725A1 (zh) * 2017-10-25 2019-05-02 深圳华大生命科学研究院 一种核酸测序方法以及一种核酸测序试剂盒
CN114317787A (zh) * 2021-12-29 2022-04-12 成都大学 吸附磁珠及其试剂盒和用途以及检测金黄色葡萄球菌的方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
GATES FREDERICK T, LINN STUART: "Endonuclease V of Escherichia coli", JBC - THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 252, no. 5, 10 March 1977 (1977-03-10), pages 1647 - 1653, XP093117709 *

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