WO2023240500A1 - 保存dNTP的方法 - Google Patents

保存dNTP的方法 Download PDF

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WO2023240500A1
WO2023240500A1 PCT/CN2022/098997 CN2022098997W WO2023240500A1 WO 2023240500 A1 WO2023240500 A1 WO 2023240500A1 CN 2022098997 W CN2022098997 W CN 2022098997W WO 2023240500 A1 WO2023240500 A1 WO 2023240500A1
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dntp
dntps
reagent
mix
preservation
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PCT/CN2022/098997
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French (fr)
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王静静
许颖颖
李长英
王雅蒙
林结桃
张桢
孟逸欣
龚梅花
李计广
贾曼
徐崇钧
蒋慧
刘健
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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/CN2022/098997 priority Critical patent/WO2023240500A1/zh
Publication of WO2023240500A1 publication Critical patent/WO2023240500A1/zh

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • 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

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  • the present invention relates to the biological field.
  • the present invention relates to methods of preserving dNTPs.
  • SBS Sequencing by Synthesis
  • the group needs to meet three conditions: 1) It does not affect the efficient and accurate polymerization of modified dNTPs to the 3'-hydroxyl end of the sequencing chain by DNA polymerase; 2) It is stable enough: it can be resected during storage and sequencing reactions.
  • the group is stably connected to the 3'-hydroxyl end of dNTPs without breaking; 3) Easy to remove: after a cycle of measurement, the removable blocking group can be efficiently cut off and restore the 3'-hydroxyl group of the sequencing chain. structure.
  • the blocking group of dNTPs When the removable blocking group of dNTPs is not stable enough, the blocking group detaches from the dNTPs to restore the 3'-hydroxyl group, causing the sequencing reaction to advance (the removable blocking group is lost, causing the polymerization reaction to be There is no stopping when stopping, and one cycle aggregates two or more modified or unmodified dNTPs); reflected in the sequencing data, the sequencing quality decreases and the error rate increases.
  • the ester bond can be used as a reversible blocking group in sequencing.
  • Its application has advantages and disadvantages: the advantage is that esters are in aqueous solutions, especially alkaline aqueous solutions. It is very easy to be cut or broken, and the speed is very fast, which is conducive to improving the sequencing speed; the disadvantage is that it is not stable enough in aqueous solutions, which is not conducive to long-term storage.
  • the present invention aims to solve at least one of the technical problems existing in the prior art to at least a certain extent.
  • the present invention proposes a dNTP preservation reagent, the application of the reagent in preserving dNTP and a method for preserving dNTP.
  • the dNTP preservation reagent of the present invention can dissolve dNTP (especially dNTP modified with a reversible blocking group), And improve the stability of dNTP, so that dNTP can be stored for a long time, which is beneficial to ensure the accuracy and efficiency of amplification and sequencing.
  • the present invention provides a dNTP preservation reagent.
  • the reagents include: dimethyl sulfoxide, sulfolane, tetrahydrofuran, dimethylformamide, dimethylacetamide, N,N-dimethylformamide, N-methylformamide and one or more of N-methylpyrrolidone.
  • the inventor found that using the above reagents can dissolve dNTPs (especially dNTPs modified with reversible blocking groups) and improve the stability of dNTPs, so that dNTPs can be stored for a long time, which is beneficial to ensuring the accuracy and efficiency of amplification and sequencing. .
  • the above-mentioned dNTP preservation reagent may also have the following additional technical features:
  • the reagent is used to improve the storage stability of dNTPs.
  • the dNTP has a reversible blocking group modification. Storing dNTPs modified with reversible blocking groups in the above reagents can effectively avoid hydrolysis of the reversible blocking groups and improve their stability.
  • the reversible blocking group is selected from at least one of the following: ester bond, amide, imide, ether bond, acid anhydride, acid chloride, carbonate and carbamate.
  • dNTPs with the above-mentioned reversible blocking groups have good stability in reagents and are not prone to hydrolysis, which helps to extend their shelf life and can be stored at room temperature for at least one year.
  • the invention proposes the use of reagents in preserving dNTPs.
  • the reagent is selected from the aforementioned dNTP preservation reagents.
  • the use of this reagent can dissolve dNTPs and improve the stability of dNTPs, so that dNTPs can be stored for a long time, which is beneficial to ensuring the accuracy and efficiency of amplification and sequencing.
  • the present invention proposes a method of preserving dNTPs.
  • the method includes: mixing the aforementioned dNTP preservation reagent with dNTP. Using the above reagents can dissolve dNTPs and improve the stability of dNTPs, so that dNTPs can be stored for a long time, which is beneficial to ensuring the accuracy and efficiency of amplification and sequencing.
  • the reagent content is 50% to 99.9%.
  • the inventor obtained the above ratio through extensive experiments, thereby effectively improving the stability of dNTP.
  • Figure 1 shows a schematic diagram of comparative analysis of Runon (advance reaction) curves according to one embodiment of the present invention.
  • DMSO dimethyl sulfoxide
  • HotMPS dNTPs Mix and HotMPS dNTPs Mix II in anhydrous DMSO or LTE buffer to prepare Hot mix and Cold mix respectively.
  • Divide each Hot mix and Cold mix into 4 parts (actually test 3 time points , make an extra copy in case the experiment fails and needs to be repeated), seal it with a sealing film, and store it at room temperature (25°C), 0 days (fresh reagent), 11 days (equivalent to a validity period of 6 months), 22 days ( Equivalent to a validity period of 12 months), take 1 portion and test SE50 on the computer.
  • the LTE group needs to add 1% DMSO to the sequencing reagent before running on the computer.
  • the DMSO group needs to add 1% LTE to the forward sequencing reagent before running on the computer to ensure the sequencing reaction buffer.
  • the components in the liquid are the same; compare the Q30%, Runon (%), and AvgErrorRate (%) of the data tested at different time points.
  • reaction system was shaken and mixed with a vortex oscillator, centrifuged in a mini centrifuge for 5 seconds, and immediately placed in a PCR machine.
  • the reaction conditions are as follows:
  • Runon (%) only increased by 23%; and after 22 days of storage, Runon only increased by 17% ( This time it is lower than that of Runon stored for 11 days (which may be due to experimental error); and after 22 days of storage in DMSO at room temperature, the Q30% of SE50 remains consistent with that of freshly prepared reagents (94.83% vs 94.01%), and the MappingRate decreased by 0.23% , the AvgErrorRate only increased by 2%, which fully proves that the dNTPs in HotMPS dNTPs Mix and HotMPS dNTPs Mix II can be stably stored in anhydrous DMSO.
  • HotMPS dNTPs Mix and HotMPS dNTPs Mix II were stored at room temperature in LTE for 22 days. Q30% dropped from 94.65% to 68.87%. Runon% was too high to be accurately calculated (it reached a plateau after 20 cycles), and the data was basically impossible to calculate. Aligned to the reference genome (MappingRate is only 9%), and the error rate of the 9% aligned data is as high as 3.7%;
  • HotMPS dNTPs Mix and HotMPS dNTPs Mix II were stored in DMSO for 22 days from fresh preparation to room temperature. Q30% did not decrease, Runon% increased by 17%, MappingRate decreased by 0.23%, and AvgErrorRate increased by 2%;
  • HotMPS dNTPs Mix and HotMPS dNTPs Mix II can be stored in anhydrous DMSO for a long time, which is consistent with theoretical reasoning.
  • the conclusions of this experiment are in line with expectations.
  • Example 1 The materials and consumables used (except for the organic solvent tested), and the experimental procedures are the same as in Example 1. Here, only the results of 0 days and 11 days at room temperature are compared; since it is expected to be more stable than in LTE, sequencing was performed for comparison.
  • dNTPs were stored in DMAC (dimethylacetamide) and DMF (N,N-dimethylformamide) at room temperature, and the Runon% on days 0 and 11 was consistent;
  • dNTPs were stored in NMP (N-methylpyrrolidone) at room temperature for 11 days. Although Runon% increased by 31%, the Q30% of SE100 was still 88.84%, which was still more stable than in LTE;
  • references to the terms “one embodiment,” “some embodiments,” “an example,” “specific examples,” or “some examples” or the like means that specific features are described in connection with the embodiment or example. , structures, materials or features are included in at least one embodiment or example of the invention. In this specification, the schematic expressions of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the specific features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, those skilled in the art may combine and combine different embodiments or examples and features of different embodiments or examples described in this specification unless they are inconsistent with each other.

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Abstract

本发明提出了dNTP的保存试剂，所述dNTP的保存试剂包括：二甲基亚砜、环丁砜、四氢呋喃、二甲基甲酰胺、二甲基乙酰胺、N，N-二甲基甲酰胺、N-甲基甲酰胺和N-甲基吡咯烷酮中的一种或多种。采用本发明dNTP的保存试剂可溶解dNTP，尤其是具有可逆阻断基团修饰的dNTP，并提高dNTP的稳定性，使得dNTP可以长期保存，有利于保证扩增和测序的准确性和高效性。

Description

保存dNTP的方法 技术领域
本发明涉及生物领域。具体地，本发明涉及保存dNTP的方法。
背景技术
目前的高通量测序中使用最广泛的是边合成边测序(Sequencing by Synthesis,SBS)技术，要求dNTPs的3’-羟基端连接可切除的阻断基团，且这个可切除的阻断基团需要满足三个条件：1)不影响修饰的dNTPs被DNA聚合酶高效准确的聚合到测序链的3’-羟基端；2)足够稳定：在其储存和测序反应时，可切除的阻断基团稳定地连在dNTPs的3’-羟基端，不断裂；3)易切除：测完一个循环后，可切除的阻断基团可以被高效的切掉并恢复测序链的3’-羟基结构。当dNTPs的可切除的阻断基团不够稳定时，阻断基团脱离dNTPs使其恢复3’-羟基基团，导致测序反应超前进行(可切除的阻断基团掉了，导致聚合反应应该停止时没有停止，一个循环聚合上了两个或两个以上的修饰或未修饰的dNTPs)；反映在测序数据上，表现为测序质量下降，错误率升高。
酯键作为可断裂或可切除并形成羟基的基团，可以作为可逆阻断基团被应用在测序中，其应用有利有弊：优势是酯在水溶液中，尤其是偏碱性的水溶液中，非常容易被切除或断裂，且速度非常快，有利于提高测序速度；劣势是它在水相溶液中稳定性不够好，不利于长期保存。
因此，目前针对具有可逆阻断基团修饰的dNTP的保存仍有待研究。
发明内容
本发明旨在至少在一定程度上解决现有技术中存在的技术问题至少之一。为此，本发明提出了dNTP的保存试剂、试剂在保存dNTP中的应用和保存dNTP的方法，采用本发明的dNTP的保存试剂可以溶解dNTP(尤其是具有可逆阻断基团修饰的dNTP)，并提高dNTP的稳定性，使得dNTP可以长期保存，有利于保证扩增和测序的准确性和高效性。
在本发明的一个方面，本发明提出了一种dNTP的保存试剂。根据本发明的实施例，所述试剂包括：二甲基亚砜、环丁砜、四氢呋喃、二甲基甲酰胺、二甲基乙酰胺、N,N-二甲基甲酰胺、N-甲基甲酰胺和N-甲基吡咯烷酮中的一种或多种。发明人发现，采用上述试剂可以溶解dNTP(尤其是具有可逆阻断基团修饰的dNTP)，并提高dNTP的稳定性，使得dNTP可以长期保存，有利于保证扩增和测序的准确性和高效性。
根据本发明的实施例，上述dNTP的保存试剂还可以具有下列附加技术特征：
根据本发明的实施例，所述试剂用于提高dNTP的保存稳定性。
根据本发明的实施例，所述dNTP具有可逆阻断基团修饰。将具有可逆阻断基团修饰的dNTP保存于上述试剂中，可以有效地避免可逆阻断基团的水解，提高其稳定性。
根据本发明的实施例，所述可逆阻断基团选自下列的至少一种：酯键、酰胺、酰亚胺、醚键、酸酐、酰氯、碳酸酯和氨基甲酸酯。具有上述可逆阻断基团的dNTP在试剂中稳定性好，不易发生水解，从而有助于延长其保存期，可在室温下保存至少一年。
在本发明的另一方面，本发明提出了试剂在保存dNTP中的应用。根据本发明的实施例，所述试剂选自前面所述dNTP的保存试剂。采用该试剂可以溶解dNTP，并提高dNTP的稳定性，使得dNTP可以长期保存，有利于保证扩增和测序的准确性和高效性。
在本发明的又一方面，本发明提出了一种保存dNTP的方法。根据本发明的实施例，所述方法包括：将前面所述dNTP的保存试剂与dNTP混合。采用上述试剂可以溶解dNTP，并提高dNTP的稳定性，使得dNTP可以长期保存，有利于保证扩增和测序的准确性和高效性。
根据本发明的实施例，基于所述dNTP和试剂的总体积，所述试剂含量为50～99.9％。发明人经过大量实验得到上述配比，由此，可以有效地提高dNTP的稳定性。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1显示了根据本发明一个实施例的Runon(超前反应)曲线比较分析示意图。
具体实施方式
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1
在对dNTP保存稳定性的研究过程中发现，将dNTP溶解于二甲基亚砜、环丁砜、四 氢呋喃、二甲基甲酰胺、二甲基乙酰胺、N-甲基吡咯烷酮中，均可以提高酯键修饰的dNTP的稳定性，酯键不易水解，可于室温下(22～28℃)保存至少一年，其中，二甲基亚砜(DMSO)效果最佳。下面以DMSO为例，详细描述研究过程及结果分析。
1、实验方案设计
分别将HotMPS dNTPs Mix和HotMPS dNTPs Mix II中的dNTP溶解在无水DMSO或LTE缓冲液分别配成Hot mix和Cold mix，将每个Hot mix和Cold mix各分成4份(实际测试3个时间点，多备份1份以防实验失败需要重复)，用封口膜封好后，保存在室温(25℃)，0天(新鲜试剂)、11天(相当于效期6个月)、22天(相当于效期12个月)分别取1份上机测试SE50，LTE组上机前需要向测序试剂加入1％DMSO，DMSO组上机前向测序试剂需加入1％LTE，以保证测序反应缓冲液中的成分相同；比较不同时间点测试的数据的Q30％、Runon(％)、AvgErrorRate(％)。
2、试剂、耗材、仪器和测试样本
2.1主要试剂、耗材
表1试剂、耗材信息
Figure PCTCN2022098997-appb-000001
Figure PCTCN2022098997-appb-000002
2.2仪器
表2仪器信息
仪器 型号
基因扩增仪 6337
测序仪 MGISEQ-2000RS
Qubit仪 Qubit4.0
电子天平 MS204TS
2.3实验样本
表3样本信息
Figure PCTCN2022098997-appb-000003
2.4评价指标及标准
表4评价标准
评价指标 合格标准 备注
Runon(％) 越低越好 /
Q30(％) 越高越好 /
MappingRate(％) 越高越好 /
AvgErrorRate(％) 越低越好 /
3、实验步骤
3.1 Hot mix(DMSO)、Hot mix(LTE)、Cold mix(DMSO)和Cold mix(LTE)配制
表5 Hot mix(DMSO)
Figure PCTCN2022098997-appb-000004
表6 Hot mix(LTE)
Figure PCTCN2022098997-appb-000005
表7 Cold mix(DMSO)
Figure PCTCN2022098997-appb-000006
表8 Cold mix(LTE)
Figure PCTCN2022098997-appb-000007
将以上四个mix分别分装成4份后，封口膜封好，放在室温保存。分别于配置当天、第10天后、第22天后拿其中一份进行SE50上机测序。
3.2DNB制备
1)按下表体系加入试剂及文库：
表9 DNB制备反应体系
Figure PCTCN2022098997-appb-000008
将上述反应体系用漩涡振荡器震荡混匀，迷你离心机离心5s，置于PCR仪中进行引物杂交，反应条件见下表：
表10 DNB制备反应引物杂交条件
温度 时间
热盖(105℃) On
95℃ 1min
65℃ 1min
40℃ 1min
4℃ Hold
2)取出DNB聚合酶混合液II(LC)置于冰盒上，短暂离心5s，置于冰盒上备用。
3)当PCR仪达到4℃后取出PCR管，迷你离心机离心5s后，在冰上往此PCR管中加入如下组分：
表11 DNB制备反应组分2
组分 加入量(μL)
DNB聚合酶混合液I 40
DNB聚合酶混合液II(LC) 4
4)反应体系用漩涡振荡器震荡混匀，迷你离心机离心5s，即刻置于PCR仪中，反应条件如下：
表12 DNB制备滚环扩增条件
温度 时间
热盖(35℃) On
30℃ 25min
4℃ Hold
5)当PCR仪温度达到4℃后立即加入20μL DNB终止缓冲液，用阔口吸头缓慢地吹打混匀5-8次。
6)用Qubit测DNB浓度。
3.3 DNB pooling及加载
1)取DNB 100μL，再加入32μL DLBⅡ，然后用扩口枪头混匀。
2)DNB加载
用MGIDL-200H将DNB加载到载片上，待载片加载完成，将载片取下，待上机测序。
3.4上机测序
分别配制2套同批次试剂，分别加入LTE中保存的Hot mix和Cold mix以及DMSO中保存的Hot mix和Cold mix，选用相同的脚本进行SE50测序。
实验条件：
表13-1 Hot mix试剂槽配制组分
Hot mix conc. 0.6μM
Enzyme DNA聚合酶
Enzyme conc. 10μg/mL
表13-2 Cold mix试剂槽配制组分
Cold mix conc. 4μM
Enzyme DNA聚合酶
Enzyme conc. 10μg/mL
脚本条件：
表14脚本条件
步骤 Tm(℃) Time(s)
Hot聚合 65 30
Cold聚合 65 60
Cleavage 50 45
4、实验结果与分析
结果如表15和图1所示。1)HotMPS dNTPs Mix和HotMPS dNTPs Mix II中的dNTP 在LTE缓冲液中室温保存11天，Runon(％)升高了118％；保存22天，Runon已经高至计算不准确(Runon的曲线在20cycle时已经到达平台期)；2)HotMPS dNTPs Mix和HotMPS dNTPs Mix II中的dNTP在DMSO中室温保存11天Runon(％)只升高了23％；而保存22天，Runon只增加了17％(此次比保存11天Runon低可能是来源于实验误差)；且在DMSO中室温保存22天后，SE50的Q30％仍与新鲜配制的试剂保持一致(94.83％vs 94.01％)，MappingRate下降了0.23％，AvgErrorRate只升高了2％，充分证明了HotMPS dNTPs Mix和HotMPS dNTPs Mix II中的dNTP在无水DMSO中可以稳定保存。
表15测序数据比较
Figure PCTCN2022098997-appb-000009
5、实验结论
HotMPS dNTPs Mix和HotMPS dNTPs Mix II中的dNTP在LTE中室温保存22天，Q30％从94.65％下降至68.87％，Runon％已经高到无法准确计算(20cycle后就到达平台期)，数据已经基本无法比对到参考基因组上(MappingRate只有9％)，而且这9％比对上的数据的错误率有高达3.7％；
HotMPS dNTPs Mix和HotMPS dNTPs Mix II中的dNTP保存在DMSO，从新鲜配制到室温保存22天，Q30％没有下降，Runon％增加了17％，MappingRate下降了0.23％，AvgErrorRate增加了2％；
所以HotMPS dNTPs Mix和HotMPS dNTPs Mix II中的dNTP在无水DMSO可以长期保存，符合理论推理，本次实验结论符合预期。
实施例2
使用的物料耗材(除测试的有机溶剂外)，实验步骤等同实施例1，在此处只比较0天和室温11天的结果；由于预计会比在LTE中稳定，因此进行测序进行比较。
表16其他有机化合物与DMSO的比较
Figure PCTCN2022098997-appb-000010
注：以上测试使用的dNTPs纯度比第一个表格中所用的dNTPs纯度更高；所以0天的Runon％比第一个表格中的低；
dNTPs在DMAC(二甲基乙酰胺)和DMF(N,N-二甲基甲酰胺)中室温保存，0天和11天的Runon％一致；
dNTPs在NMP(N-甲基吡咯烷酮)中室温保存11天，虽然Runon％增加了31％，但SE100的Q30％仍有88.84％，仍比在LTE中更稳定；
dNTPs在NMF(N-甲基甲酰胺)中保存，虽然Runon％增加了140％，但Q30％仍有86.71％，也比在LTE中更稳定。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (7)

  1. 一种dNTP的保存试剂，其特征在于，所述试剂包括：二甲基亚砜、环丁砜、四氢呋喃、二甲基甲酰胺、二甲基乙酰胺、N,N-二甲基甲酰胺、N-甲基甲酰胺和N-甲基吡咯烷酮中的一种或多种。
  2. 根据权利要求1所述的dNTP的保存试剂，其特征在于，所述试剂用于提高dNTP的保存稳定性。
  3. 根据权利要求1或2所述的dNTP的保存试剂，其特征在于，所述dNTP具有可逆阻断基团修饰。
  4. 根据权利要求3所述的dNTP的保存试剂，其特征在于，所述可逆阻断基团选自下列的至少一种：酯键、酰胺、酰亚胺、醚键、酸酐、酰氯、碳酸酯和氨基甲酸酯。
  5. 试剂在保存dNTP中的应用，其特征在于，所述试剂选自权利要求1-4任一项所述dNTP的保存试剂。
  6. 一种保存dNTP的方法，其特征在于，包括：将权利要求1-4任一项所述的dNTP的保存试剂与dNTP混合。
  7. 根据权利要求6所述的方法，其特征在于，基于所述dNTP和dNTP的保存试剂的总体积，所述试剂含量为50～99.9％。
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