WO2023240494A1 - Sequencing buffer and method for improving stability of dntp modified with reversible blocking group - Google Patents
Sequencing buffer and method for improving stability of dntp modified with reversible blocking group Download PDFInfo
- Publication number
- WO2023240494A1 WO2023240494A1 PCT/CN2022/098977 CN2022098977W WO2023240494A1 WO 2023240494 A1 WO2023240494 A1 WO 2023240494A1 CN 2022098977 W CN2022098977 W CN 2022098977W WO 2023240494 A1 WO2023240494 A1 WO 2023240494A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- sequencing
- reversible blocking
- blocking group
- dntp
- modified
- Prior art date
Links
- 238000012163 sequencing technique Methods 0.000 title claims abstract description 98
- 230000000903 blocking effect Effects 0.000 title claims abstract description 53
- 230000002441 reversible effect Effects 0.000 title claims abstract description 46
- 239000000872 buffer Substances 0.000 title claims abstract description 38
- 238000000034 method Methods 0.000 title claims abstract description 17
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 47
- 239000000126 substance Substances 0.000 claims abstract description 11
- 230000002860 competitive effect Effects 0.000 claims description 29
- KYNFOMQIXZUKRK-UHFFFAOYSA-N 2,2'-dithiodiethanol Chemical compound OCCSSCCO KYNFOMQIXZUKRK-UHFFFAOYSA-N 0.000 claims description 20
- 239000000243 solution Substances 0.000 claims description 18
- LQLQRFGHAALLLE-UHFFFAOYSA-N 5-bromouracil Chemical compound BrC1=CNC(=O)NC1=O LQLQRFGHAALLLE-UHFFFAOYSA-N 0.000 claims description 9
- 239000011259 mixed solution Substances 0.000 claims description 6
- 238000010944 pre-mature reactiony Methods 0.000 abstract description 5
- 230000008859 change Effects 0.000 abstract description 2
- 238000012360 testing method Methods 0.000 description 16
- 238000002360 preparation method Methods 0.000 description 15
- 239000000203 mixture Substances 0.000 description 14
- 238000006243 chemical reaction Methods 0.000 description 12
- 238000011156 evaluation Methods 0.000 description 6
- 238000006460 hydrolysis reaction Methods 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 230000002776 aggregation Effects 0.000 description 4
- 238000004220 aggregation Methods 0.000 description 4
- 230000007062 hydrolysis Effects 0.000 description 4
- 230000009467 reduction Effects 0.000 description 4
- 238000006722 reduction reaction Methods 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000011176 pooling Methods 0.000 description 3
- 239000002096 quantum dot Substances 0.000 description 3
- 239000000654 additive Substances 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 238000013401 experimental design Methods 0.000 description 2
- 238000009396 hybridization Methods 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 238000006116 polymerization reaction Methods 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000002271 resection Methods 0.000 description 2
- 239000004255 Butylated hydroxyanisole Substances 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- VLCDUOXHFNUCKK-UHFFFAOYSA-N N,N'-Dimethylthiourea Chemical compound CNC(=S)NC VLCDUOXHFNUCKK-UHFFFAOYSA-N 0.000 description 1
- VSWDORGPIHIGNW-UHFFFAOYSA-N Pyrrolidine dithiocarbamic acid Chemical compound SC(=S)N1CCCC1 VSWDORGPIHIGNW-UHFFFAOYSA-N 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- MDDIUTVUBYEEEM-UHFFFAOYSA-N azane;pyrrolidine-1-carbodithioic acid Chemical compound N.SC(=S)N1CCCC1 MDDIUTVUBYEEEM-UHFFFAOYSA-N 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 238000012165 high-throughput sequencing Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 238000006479 redox reaction Methods 0.000 description 1
- 238000005096 rolling process Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
Classifications
-
- 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
Definitions
- the present invention relates to the biological field. Specifically, the present invention relates to sequencing buffers and methods for improving the stability of dNTPs modified with reversible blocking groups.
- Sequencing by Synthesis requires the 3'-hydroxyl end of dNTPs to be connected to a removable blocking group (also known as a "reversible blocking group”). "), and this removable blocking group needs to meet two conditions: 1) It does not affect the efficient and accurate polymerization of modified dNTPs to the 3'-hydroxyl end of the sequencing strand by DNA polymerase; 2) It is stable enough: in its In the reaction buffer, the cleavable blocking group is stably connected to the 3'-hydroxyl end of dNTPs without breaking; 3) Easy to cleave: After one cycle of measurement, the cleavable blocking group can be efficiently cleaved. Remove and restore the 3'-hydroxyl structure of the sequencing strand.
- a removable blocking group also known as a "reversible blocking group”
- the reversible blocking groups of dNTPs are often not stable enough due to the structure of the sequencing buffer or modified dNTPs themselves.
- the disulfide bond of the reversible blocking group is easily reduced to a sulfhydryl group, and the reversible blocking group
- the ester bond is easily hydrolyzed, causing the blocking group of dNTPs with reversible blocking groups to fall off easily and lose its blocking function, which in turn causes the sequencing reaction to proceed ahead of schedule (the removable blocking group is lost, causing the polymerization reaction to stop. Stop, one cycle aggregates two or more modified or unmodified dNTPs); this is reflected in the sequencing data, showing a decrease in sequencing quality and an increase in error rate. Therefore, improving the stability of dNTPs modified with reversible blocking groups is one of the key factors for successful sequencing and remains to be studied.
- 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 the application of sequencing buffers and sequencing solutions in sequencing, methods for improving the stability of dNTPs modified with reversible blocking groups, and sequencing kits.
- the sequencing buffer includes competitive reagents that can be combined with The dNTP modified with the reversible blocking group competitively reacts to reduce the changes of the reversible blocking group (such as being reduced, hydrolyzed, etc.), thus ensuring its stability, better application in sequencing, and helping to improve sequencing Quality, greatly reducing the occurrence of Runon (premature reaction) and reducing the error rate.
- the invention provides a sequencing buffer.
- the sequencing buffer includes: a competitive reagent that can compete with a dNTP modified with a reversible blocking group to react with a specific substance.
- the dNTP modified with a reversible blocking group competes with the specific substance to react, thereby reducing changes in the reversible blocking group (such as being modified by the blocking group). reduction, hydrolysis, etc.) to ensure its stability, which further helps to improve the quality of sequencing, greatly reduces the occurrence of runon (advanced reactions), and reduces the error rate.
- the "specific substance” described in the present invention refers to a substance that can react with a reversible blocking group to change the group.
- it can be an oxygen-containing compound, an OH - containing compound or an H + -containing compound.
- the present invention does not strictly limit the source of the "specific substance".
- it can originate from other components in the sequencing buffer (such as oxygen dissolved in the solution) or the environment in which the sequencing buffer is located (such as OH contained in acidic or alkaline environments). - or H + ); the type of "reaction” is not strictly limited, it can be oxidation reaction, reduction reaction or hydrolysis reaction, etc.
- the sequencing buffer may also have the following additional technical features:
- the reversible blocking group includes an ester bond and/or a disulfide bond.
- a reversible reaction can occur and be reversibly ablated.
- Any dNTP with ester bonds and/or disulfide bonds can be used in the present invention, and the present invention is not strictly limited to this.
- the reversible blocking group includes a disulfide bond;
- the competitive reagent includes 2-hydroxyethyl disulfide.
- the competitive reagent can be reduced competitively with the disulfide bond-modified dNTP, making the disulfide bond less susceptible to reduction, thereby improving the stability of the protected disulfide bond.
- the reversible blocking group includes an ester bond;
- the competitive reagent includes 5-bromouracil.
- the competitive reagent can be competitively hydrolyzed with the ester bond-modified dNTP, making the ester bond less susceptible to hydrolysis, thus improving the stability of the protected ester bond.
- the invention provides a sequencing solution.
- the sequencing solution includes: the aforementioned sequencing buffer and dNTPs modified with reversible blocking groups. Therefore, in the sequencing solution according to the embodiment of the present invention, the dNTP modified with a reversible blocking group has strong stability and is not prone to hydrolysis, reduction, etc., thus improving the sequencing quality and greatly reducing Runon (premature reaction) occurs, reducing the error rate.
- the concentration of the dNTP is 0.1 ⁇ M ⁇ 50mM, preferably 0.1 ⁇ M ⁇ 10mM.
- the concentration of the competitive reagent is 10 ⁇ M to 100 mM, preferably 1 to 10 mM. The inventor obtained the above concentration through extensive experiments, thereby better improving the stability of the reversible blocking group.
- the present invention proposes a method for improving the stability of dNTPs modified with reversible blocking groups.
- the method includes: contacting a competitive reagent with the dNTP modified with a reversible blocking group; wherein the competitive reagent can compete with the reversible blocking group to interact with a specific Substances react.
- the competitive reagent can compete with the reversible blocking group to interact with a specific Substances react.
- the dNTPs modified with reversible blocking groups can react competitively with specific substances, thereby reducing changes in the reversible blocking groups (such as being reduced, hydrolyzed, etc.) and ensuring their stability. It further helps to maintain the accuracy of sequencing, improve sequencing quality, greatly reduce the occurrence of Runon (premature reaction), and reduce the error rate.
- the reversible blocking group includes a disulfide bond;
- the competitive reagent includes 2-hydroxyethyl disulfide.
- the competitive reagent can be reduced competitively with the disulfide bond-modified dNTP, making the disulfide bond less susceptible to reduction, thereby improving the stability of the protected disulfide bond.
- the reversible blocking group includes an ester bond;
- the competitive reagent includes 5-bromouracil.
- the competitive reagent can be competitively hydrolyzed with the ester bond-modified dNTP, making the ester bond less susceptible to hydrolysis, thus improving the stability of the protected ester bond.
- the concentration of the dNTP in the mixed solution obtained by the contact, is 0.1 ⁇ M ⁇ 50mM, preferably 0.1 ⁇ M ⁇ 10mM.
- the concentration of the competitive reagent in the mixed solution obtained by the contact, is 10 ⁇ M ⁇ 100mM, preferably 1 ⁇ 10mM. The inventor obtained the above concentration through extensive experiments, thereby better improving the stability of the reversible blocking group.
- the present invention provides a sequencing kit.
- the sequencing kit includes: the aforementioned sequencing buffer or sequencing solution. Therefore, the dNTP with reversible blocking group modification in the sequencing kit according to the embodiment of the present invention has good stability, helps to improve the sequencing quality, greatly reduces the occurrence of Runon (premature reaction), and reduces the error rate.
- the present invention proposes the application of the aforementioned sequencing buffer, sequencing solution or sequencing kit in sequencing.
- the dNTPs modified with reversible blocking groups in the sequencing solution or sequencing kit described above have good stability, help to improve sequencing quality, reduce error rates, and are suitable for wide application.
- the present invention provides a sequencing method.
- the method includes: using the aforementioned sequencing buffer, sequencing solution or sequencing kit. Therefore, the sequencing method according to the embodiment of the present invention has high sequencing quality and low error rate, and is suitable for wide application.
- Sample name Standard library reagent V3 (26ng/tube, E320, Barcode 97-104, MGI 1000005033).
- HotMPS dNTPs Mix and HotMPS dNTPs Mix II were added to the LTE buffer containing 2-hydroxyethyl disulfide (HED) at a final concentration of 2mM as the test group and to the LTE buffer as the control group, respectively.
- HED 2-hydroxyethyl disulfide
- the reaction system was vortexed and mixed evenly, centrifuged for 5 seconds, and immediately placed in a PCR machine.
- the reaction conditions were as follows:
- HotMPS dNTPs Mix conc. 0.6 ⁇ M Enzyme DNA polymerase Enzyme conc. 10 ⁇ g/mL
- control group test freshly prepared reagent, runon (%) 0.54%; after being placed at 4°C for 24 hours, runon (%) increased to 0.73%;
- HED sequencing group The runon (%) of the freshly prepared reagent was reduced to 0.41%, and after being left for 24 hours, the runon (%) increased to 0.58%, which was much lower than the runon (%) of the control group after being left for 24 hours.
- the Runon (%) of 5-20mM HED is basically the same, both are lower than the Runon (%) of 2mM; the difference in MappingRate (%) may come from the differences between different chips; Q30 (%) with HED All were better than the control group; there was little difference between different concentrations.
- HED is helpful to improve the stability of HotMPS dNTPs Mix and HotMPS dNTPs Mix II in sequencing buffer; 5mM, 10mM and 20mM HED have basically the same effect on Runon, with 5-10mM being preferred.
- Tables 15-1 and 15-2 prepare two sets of reagents from the same batch, one of which is used as a control, and the other set is added with 5mM 5-bromouracil as a test group. After preparation, the control and test are tested on the machine within half an hour. The group performed SE50 sequencing.
- DNB preparation, DNB pooling, and loading are the same as in Example 1.
- Tables 8-1 and 8-2 prepare 6 sets of reagents from the same batch, of which 2 sets are used as controls, and 2 sets are added with a final concentration of 2mM DMTU (N,N'-dimethylthiourea, brand: Merck, product number : D188700) as test group 1, 2 sets were added with a final concentration of 2mM APDC (ammonium pyrrolidine dithiocarbamate, brand: Merck, product number: P8765) as test group 2, and the control and control were tested within half an hour after preparation.
- 2mM DMTU N,N'-dimethylthiourea, brand: Merck, product number : D188700
- 2mM APDC ammonium pyrrolidine dithiocarbamate, brand: Merck, product number: P8765
- 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.
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
A sequencing buffer and a method for improving the stability of dNTP modified with a reversible blocking group. The sequencing buffer comprises: a competing reagent, which can compete with dNTP modified with a reversible blocking group in reacting with a specific substance. The competing reagent in the sequencing buffer can compete with dNTP modified with a reversible blocking group in interacting with a specific substance, which reduces a change in the reversible blocking group (for example, being reduced and hydrolyzed, etc.), thereby ensuring the stability of the dNTP, allowing same to be better used in sequencing, helping to improve the quality of sequencing, greatly reducing the occurrence of premature reactions, and reducing the error rate.
Description
本发明涉及生物领域。具体地,本发明涉及测序缓冲液及提高具有可逆阻断基团修饰的dNTP的稳定性的方法。The present invention relates to the biological field. Specifically, the present invention relates to sequencing buffers and methods for improving the stability of dNTPs modified with reversible blocking groups.
目前的高通量测序中使用最广泛的边合成边测序(Sequencing by Synthesis,SBS)技术要求dNTPs的3’-羟基端连接可切除的阻断基团(亦可称为“可逆阻断基团”),且这个可切除的阻断基团需要满足两个条件: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:在其反应缓冲液中,可切除的阻断基团稳定地连接在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。Sequencing by Synthesis (SBS), the most widely used technology in current high-throughput sequencing, requires the 3'-hydroxyl end of dNTPs to be connected to a removable blocking group (also known as a "reversible blocking group"). "), and this removable blocking group needs to meet two conditions: 1) It does not affect the efficient and accurate polymerization of modified dNTPs to the 3'-hydroxyl end of the sequencing strand by DNA polymerase; 2) It is stable enough: in its In the reaction buffer, the cleavable blocking group is stably connected to the 3'-hydroxyl end of dNTPs without breaking; 3) Easy to cleave: After one cycle of measurement, the cleavable blocking group can be efficiently cleaved. Remove and restore the 3'-hydroxyl structure of the sequencing strand.
在测序反应中,往往由于测序缓冲液或修饰的dNTPs本身结构的原因,导致dNTPs的可逆阻断基团不够稳定,例如可逆阻断基团二硫键易被还原成巯基,可逆阻断基团酯键易水解,导致可逆阻断基团的dNTPs的阻断基团容易脱落,失去阻断功能,进而使得测序反应超前进行(可切除的阻断基团掉了,导致聚合反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。因此,提高具有可逆阻断基团修饰的dNTP的稳定性,是测序成功的关键因素之一,仍有待研究。In sequencing reactions, the reversible blocking groups of dNTPs are often not stable enough due to the structure of the sequencing buffer or modified dNTPs themselves. For example, the disulfide bond of the reversible blocking group is easily reduced to a sulfhydryl group, and the reversible blocking group The ester bond is easily hydrolyzed, causing the blocking group of dNTPs with reversible blocking groups to fall off easily and lose its blocking function, which in turn causes the sequencing reaction to proceed ahead of schedule (the removable blocking group is lost, causing the polymerization reaction to stop. Stop, one cycle aggregates two or more modified or unmodified dNTPs); this is reflected in the sequencing data, showing a decrease in sequencing quality and an increase in error rate. Therefore, improving the stability of dNTPs modified with reversible blocking groups is one of the key factors for successful sequencing and remains to be studied.
发明内容Contents of the invention
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,本发明提出了测序缓冲液、测序溶液在测序中的应用、提高具有可逆阻断基团修饰的dNTP稳定性的方法和测序试剂盒,该测序缓冲液包括竞争试剂,其可与具有可逆阻断基团修饰的dNTP竞争性地发生反应,减少可逆阻断基团发生变化(如被还原、水解等),从而保证其稳定性,更好地应用于测序中,有助于提高测序质量,极大地减少了Runon(超前反应)发生,降低错误率。The present invention aims to solve at least one of the technical problems existing in the prior art to at least a certain extent. To this end, the present invention proposes the application of sequencing buffers and sequencing solutions in sequencing, methods for improving the stability of dNTPs modified with reversible blocking groups, and sequencing kits. The sequencing buffer includes competitive reagents that can be combined with The dNTP modified with the reversible blocking group competitively reacts to reduce the changes of the reversible blocking group (such as being reduced, hydrolyzed, etc.), thus ensuring its stability, better application in sequencing, and helping to improve sequencing Quality, greatly reducing the occurrence of Runon (premature reaction) and reducing the error rate.
在本发明的一个方面,本发明提出了一种测序缓冲液。根据本发明的实施例,所述测序缓冲液包括:竞争试剂,所述竞争试剂可与具有可逆阻断基团修饰的dNTP竞争性地与特定物质发生反应。In one aspect of the invention, the invention provides a sequencing buffer. According to an embodiment of the present invention, the sequencing buffer includes: a competitive reagent that can compete with a dNTP modified with a reversible blocking group to react with a specific substance.
根据本发明实施例的测序缓冲液中,通过添加竞争试剂,使其与具有可逆阻断基团修饰的dNTP竞争性地与特定物质发生反应,从而减少具有可逆阻断基团发生变化(如被还原、水解等),保证其稳定性,进一步有助于提高测序质量,极大地减少了Runon(超前反应)发生,降低错误率。In the sequencing buffer according to the embodiment of the present invention, by adding a competitive reagent, the dNTP modified with a reversible blocking group competes with the specific substance to react, thereby reducing changes in the reversible blocking group (such as being modified by the blocking group). reduction, hydrolysis, etc.) to ensure its stability, which further helps to improve the quality of sequencing, greatly reduces the occurrence of runon (advanced reactions), and reduces the error rate.
需要说明的是,本发明所描述的“特定物质”是指能够与可逆阻断基团反应而使基团发生变化的物质,例如可以为含氧化合物、含OH
-化合物或含H
+化合物。本发明对于“特定物质”的来源不作严格限定,例如可以来源于测序缓冲液中其他组成(如溶解在溶液中的氧气)或者测序缓冲液所处环境(如酸性、碱性环境中含有的OH
-或H
+);“发生反应”的类型也不做严格限定,可以为氧化反应、还原反应或水解反应等。
It should be noted that the "specific substance" described in the present invention refers to a substance that can react with a reversible blocking group to change the group. For example, it can be an oxygen-containing compound, an OH - containing compound or an H + -containing compound. The present invention does not strictly limit the source of the "specific substance". For example, it can originate from other components in the sequencing buffer (such as oxygen dissolved in the solution) or the environment in which the sequencing buffer is located (such as OH contained in acidic or alkaline environments). - or H + ); the type of "reaction" is not strictly limited, it can be oxidation reaction, reduction reaction or hydrolysis reaction, etc.
根据本发明的实施例,所述测序缓冲液还可以具有下列附加技术特征:According to embodiments of the present invention, the sequencing buffer may also have the following additional technical features:
根据本发明的实施例,所述可逆阻断基团包括酯键和/或二硫键。由此,可以进行可逆反应,被可逆的切除。任一具有酯键和/或二硫键的dNTP均可应用于本发明,对此,本发明不作严格限定。According to an embodiment of the present invention, the reversible blocking group includes an ester bond and/or a disulfide bond. As a result, a reversible reaction can occur and be reversibly ablated. Any dNTP with ester bonds and/or disulfide bonds can be used in the present invention, and the present invention is not strictly limited to this.
根据本发明的实施例,所述可逆阻断基团包括二硫键;所述竞争试剂包括2-羟乙基二硫化物。该竞争试剂可与二硫键修饰的dNTP竞争性地被还原,使得二硫键更不易被还原,从而提高了被保护的二硫键的稳定性。According to an embodiment of the present invention, the reversible blocking group includes a disulfide bond; the competitive reagent includes 2-hydroxyethyl disulfide. The competitive reagent can be reduced competitively with the disulfide bond-modified dNTP, making the disulfide bond less susceptible to reduction, thereby improving the stability of the protected disulfide bond.
根据本发明的实施例,所述可逆阻断基团包括酯键;所述竞争试剂包括5-溴尿嘧啶。该竞争试剂可与酯键修饰的dNTP竞争性地被水解,使得酯键更不易被水解,从而提高了被保护的酯键的稳定性。According to an embodiment of the present invention, the reversible blocking group includes an ester bond; the competitive reagent includes 5-bromouracil. The competitive reagent can be competitively hydrolyzed with the ester bond-modified dNTP, making the ester bond less susceptible to hydrolysis, thus improving the stability of the protected ester bond.
在本发明的另一方面,本发明提出了一种测序溶液。根据本发明的实施例,所述测序溶液包括:前面所述测序缓冲液和具有可逆阻断基团修饰的dNTP。由此,根据本发明实施例的测序溶液中,具有可逆阻断基团修饰的dNTP的稳定性强,不易发生水解、被还原等,从而提高了测序质量,极大地减少了Runon(超前反应)发生,降低错误率。In another aspect of the invention, the invention provides a sequencing solution. According to an embodiment of the present invention, the sequencing solution includes: the aforementioned sequencing buffer and dNTPs modified with reversible blocking groups. Therefore, in the sequencing solution according to the embodiment of the present invention, the dNTP modified with a reversible blocking group has strong stability and is not prone to hydrolysis, reduction, etc., thus improving the sequencing quality and greatly reducing Runon (premature reaction) occurs, reducing the error rate.
根据本发明的实施例,所述dNTP的浓度为0.1μM~50mM,优选为0.1μM~10mM。根据本发明的另一实施例,所述竞争试剂的浓度为10μM~100mM,优选为1~10mM。发明人经过大量实验得到上述浓度,由此,可以更好地提高可逆阻断基团的稳定性。According to an embodiment of the present invention, the concentration of the dNTP is 0.1 μM ~ 50mM, preferably 0.1 μM ~ 10mM. According to another embodiment of the present invention, the concentration of the competitive reagent is 10 μM to 100 mM, preferably 1 to 10 mM. The inventor obtained the above concentration through extensive experiments, thereby better improving the stability of the reversible blocking group.
在本发明的又一方面,本发明提出了一种提高具有可逆阻断基团修饰的dNTP稳定性的方法。根据本发明的实施例,所述方法包括:使竞争试剂与所述具有可逆阻断基团修饰的dNTP进行接触;其中,所述竞争试剂可与所述可逆阻断基团竞争性地与特定物质发生反应。通过添加竞争试剂,使其与具有可逆阻断基团修饰的dNTP竞争性地与特定物质发生反应,从而减少具有可逆阻断基团发生变化(如被还原、水解等),保证其稳定性,进 一步有助于维持测序的准确性,提高测序质量,极大地减少了Runon(超前反应)发生,降低错误率。In yet another aspect of the present invention, the present invention proposes a method for improving the stability of dNTPs modified with reversible blocking groups. According to an embodiment of the present invention, the method includes: contacting a competitive reagent with the dNTP modified with a reversible blocking group; wherein the competitive reagent can compete with the reversible blocking group to interact with a specific Substances react. By adding competitive reagents, the dNTPs modified with reversible blocking groups can react competitively with specific substances, thereby reducing changes in the reversible blocking groups (such as being reduced, hydrolyzed, etc.) and ensuring their stability. It further helps to maintain the accuracy of sequencing, improve sequencing quality, greatly reduce the occurrence of Runon (premature reaction), and reduce the error rate.
根据本发明的实施例,所述可逆阻断基团包括二硫键;所述竞争试剂包括2-羟乙基二硫化物。该竞争试剂可与二硫键修饰的dNTP竞争性地被还原,使得二硫键更不易被还原,从而提高了被保护的二硫键的稳定性。According to an embodiment of the present invention, the reversible blocking group includes a disulfide bond; the competitive reagent includes 2-hydroxyethyl disulfide. The competitive reagent can be reduced competitively with the disulfide bond-modified dNTP, making the disulfide bond less susceptible to reduction, thereby improving the stability of the protected disulfide bond.
根据本发明的实施例,所述可逆阻断基团包括酯键;所述竞争试剂包括5-溴尿嘧啶。该竞争试剂可与酯键修饰的dNTP竞争性地被水解,使得酯键更不易被水解,从而提高了被保护的酯键的稳定性。According to an embodiment of the present invention, the reversible blocking group includes an ester bond; the competitive reagent includes 5-bromouracil. The competitive reagent can be competitively hydrolyzed with the ester bond-modified dNTP, making the ester bond less susceptible to hydrolysis, thus improving the stability of the protected ester bond.
根据本发明的实施例,在所述接触所得混合溶液中,所述dNTP的浓度为0.1μM~50mM,优选为0.1μM~10mM。根据本发明的另一实施例,在所述接触所得混合溶液中,所述竞争试剂的浓度为10μM~100mM,优选为1~10mM。发明人经过大量实验得到上述浓度,由此,可以更好地提高可逆阻断基团的稳定性。According to an embodiment of the present invention, in the mixed solution obtained by the contact, the concentration of the dNTP is 0.1 μM ~ 50mM, preferably 0.1 μM ~ 10mM. According to another embodiment of the present invention, in the mixed solution obtained by the contact, the concentration of the competitive reagent is 10 μM ~ 100mM, preferably 1 ~ 10mM. The inventor obtained the above concentration through extensive experiments, thereby better improving the stability of the reversible blocking group.
在本发明的又一方面,本发明提出了一种测序试剂盒。根据本发明的实施例,所述测序试剂盒包括:前面所述测序缓冲液或测序溶液。由此,根据本发明实施例的测序试剂盒中具有可逆阻断基团修饰的dNTP稳定性好,有助于提高测序质量,极大地减少了Runon(超前反应)发生,降低错误率。In yet another aspect of the present invention, the present invention provides a sequencing kit. According to an embodiment of the present invention, the sequencing kit includes: the aforementioned sequencing buffer or sequencing solution. Therefore, the dNTP with reversible blocking group modification in the sequencing kit according to the embodiment of the present invention has good stability, helps to improve the sequencing quality, greatly reduces the occurrence of Runon (premature reaction), and reduces the error rate.
在本发明的又一方面,本发明提出了前面所述测序缓冲液、测序溶液或测序试剂盒在测序中的应用。前面所述测序溶液或测序试剂盒中具有可逆阻断基团修饰的dNTP稳定性好,有助于提高测序质量,降低错误率,适于广泛应用。In yet another aspect of the present invention, the present invention proposes the application of the aforementioned sequencing buffer, sequencing solution or sequencing kit in sequencing. The dNTPs modified with reversible blocking groups in the sequencing solution or sequencing kit described above have good stability, help to improve sequencing quality, reduce error rates, and are suitable for wide application.
在本发明的又一方面,本发明提出了一种测序方法。根据本发明的实施例,所述方法包括:使用前面所述测序缓冲液、测序溶液或测序试剂盒。由此,根据本发明实施例的测序方法的测序质量高,错误率低,适于广泛应用。In yet another aspect of the present invention, the present invention provides a sequencing method. According to an embodiment of the present invention, the method includes: using the aforementioned sequencing buffer, sequencing solution or sequencing kit. Therefore, the sequencing method according to the embodiment of the present invention has high sequencing quality and low error rate, and is suitable for wide application.
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。Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
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。The solutions of the present invention will be explained below with reference to examples. Those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. If specific techniques or conditions are not specified in the examples, the techniques or conditions described in literature in the field or product instructions will be followed. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be purchased commercially.
实施例1 2-羟乙基二硫化物作为竞争试剂Example 1 2-hydroxyethyl disulfide as competitive reagent
1、实验材料1. Experimental materials
表1试剂、耗材信息Table 1 Reagents and consumables information
2、仪器2. Instruments
表2仪器信息Table 2 Instrument information
仪器instrument | 型号model |
基因扩增仪Gene cycler | 63376337 |
测序仪sequencer | MGISEQ-2000RSMGISEQ-2000RS |
Qubit仪Qubit instrument | Qubit4.0Qubit4.0 |
电子天平Electronic balance | MS204TSMS204TS |
3、实验样本3. Experimental samples
样品名称:标准文库试剂V3(26ng/支,E320,Barcode 97-104,华大智造1000005033)。Sample name: Standard library reagent V3 (26ng/tube, E320, Barcode 97-104, MGI 1000005033).
4、实验设计4. Experimental design
分别将HotMPS dNTPs Mix和HotMPS dNTPs Mix II进行如下实验:Conduct the following experiments on HotMPS dNTPs Mix and HotMPS dNTPs Mix II respectively:
将HotMPS dNTPs Mix和HotMPS dNTPs Mix II分别加入到含有终浓度为2mM的2- 羟乙基二硫化物(HED)的LTE缓冲液中作为测试组以及加入到LTE缓冲液中作为对照组。分别在加入后的半小时内上机,和在4℃放置24小时后上机测SE50,测序酶上机前加入。比较新鲜配制的试剂(半小时内)和4℃放置24小时两个时间点的数据,尤其是Runon(%)、Q30(%)、AvgErrorRate(%)、TotalReads(M)等指标。HotMPS dNTPs Mix and HotMPS dNTPs Mix II were added to the LTE buffer containing 2-hydroxyethyl disulfide (HED) at a final concentration of 2mM as the test group and to the LTE buffer as the control group, respectively. Put the sequencing enzyme on the machine within half an hour after adding it, and put it on the machine after placing it at 4°C for 24 hours to measure SE50. Add the sequencing enzyme before putting it on the machine. Compare the data at two time points of freshly prepared reagents (within half an hour) and 24 hours at 4°C, especially Runon (%), Q30 (%), AvgErrorRate (%), TotalReads (M) and other indicators.
继续进行浓度梯度测试:设置HED终浓度为2、5、10、20mM几个梯度,将HotMPS dNTPs Mix和HotMPS dNTPs Mix II加入到含有HED的LTE缓冲液或者不含HED的LTE缓冲液中,进行SE50测序。比较测序数据,尤其是Runon(%)、Q30(%)、AvgErrorRate(%)、TotalReads(M)等指标,选择出最合适的浓度。Continue the concentration gradient test: set the final concentration of HED to 2, 5, 10, and 20mM gradients, add HotMPS dNTPs Mix and HotMPS dNTPs Mix II to the LTE buffer containing HED or the LTE buffer without HED, and proceed. SE50 sequencing. Compare the sequencing data, especially Runon (%), Q30 (%), AvgErrorRate (%), TotalReads (M) and other indicators, and select the most appropriate concentration.
5、评价指标及标准5. Evaluation indicators and standards
表3评价标准Table 3 Evaluation Criteria
评价指标Evaluation index | 合格标准Eligibility criteria |
Runon(%)Runon(%) | 越低越好The lower the better |
Q30(%)Q30(%) | 越高越好The higher the better |
AvgErrorRate(%)AvgErrorRate(%) | 越低越好The lower the better |
TotalReads(M)TotalReads(M) | 越高越好The higher the better |
6、实验步骤6. Experimental steps
6.1 DNB制备6.1 DNB preparation
按下表体系加入试剂及文库Add reagents and libraries according to the system in the table below
表4 DNB制备反应体系Table 4 DNB preparation reaction system
组份Component | 加入量(μL)Adding volume (μL) |
文库ssDNALibrary ssDNA | 33 |
LTE缓冲液LTE buffer | 1717 |
DNB制备缓冲液DNB preparation buffer | 2020 |
总体积total capacity | 4040 |
将上述反应体系用漩涡振荡器震荡混匀,离心机离心5s,置于PCR仪中进行引物杂交,反应条件见下表:Vortex and mix the above reaction system with a vortex shaker, centrifuge for 5 seconds, and place it in a PCR machine for primer hybridization. The reaction conditions are shown in the table below:
表5 DNB制备反应引物杂交条件Table 5 DNB preparation reaction primer hybridization conditions
温度temperature | 时间time |
热盖(105℃)Hot lid (105℃) | OnOn |
95℃95℃ | 1min1min |
65℃65℃ | 1min1min |
40℃40℃ | 1min1min |
4℃4℃ | HoldHold |
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。当PCR仪达到4℃后取出PCR管,离心机离心5s后,在冰上往此PCR管中加入如下组分:Take out the DNB polymerase mixture II (LC) and place it on an ice box, centrifuge briefly for 5 seconds, and place it on an ice box for later use. When the PCR machine reaches 4°C, take out the PCR tube, centrifuge for 5 seconds, and add the following components to the PCR tube on ice:
表6 DNB制备反应组分2Table 6 DNB preparation reaction components 2
组分Components | 加入量(μL)Adding volume (μL) |
DNB聚合酶混合液IDNB polymerase mix I | 4040 |
DNB聚合酶混合液II(LC)DNB polymerase mix II (LC) | 44 |
反应体系用漩涡振荡器震荡混匀,离心机离心5s,即刻置于PCR仪中,反应条件如下:The reaction system was vortexed and mixed evenly, centrifuged for 5 seconds, and immediately placed in a PCR machine. The reaction conditions were as follows:
表7 DNB制备滚环扩增条件Table 7 DNB preparation rolling circle amplification conditions
温度temperature | 时间time |
热盖(35℃)Hot lid (35℃) | OnOn |
30℃30℃ | 25min25min |
4℃4℃ | HoldHold |
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。When the temperature of the PCR machine reaches 4°C, immediately add 20 μL of DNB stop buffer and pipet and mix slowly with a wide-mouth pipette 5-8 times.
用Qubit测DNB浓度。Use Qubit to measure DNB concentration.
6.2 DNB pooling及加载6.2 DNB pooling and loading
6.2.1取DNB 100μL,再加入32μL DLBⅡ,然后用扩口枪头混匀。6.2.1 Take 100μL of DNB, then add 32μL of DLBⅡ, and then mix evenly with an expanded pipette tip.
6.2.2 DNB加载6.2.2 DNB loading
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。Use MGIDL-200H to load DNB onto the slide. After the slide is loaded, remove the slide and wait for sequencing.
6.3上机测序6.3 On-machine sequencing
按照表8-1和8-2,分别配制4套同批次试剂,其中2套作为对照,另外2套加入终浓度为2mM 2-羟乙基二硫化物作为测试组,配好后半小时内上机测对照和测试组的其中的1套;4℃放置24小时后测另外1套;选用相同的脚本进行SE50测序。According to Tables 8-1 and 8-2, prepare 4 sets of the same batch of reagents, 2 sets of which are used as controls, and the other 2 sets are added with a final concentration of 2mM 2-hydroxyethyl disulfide as the test group. Half an hour after preparation Test one set of the control and test groups on the computer; test the other set after placing it at 4°C for 24 hours; use the same script for SE50 sequencing.
不同浓度的添加剂测试:按照表8-1和8-2,分别配制5套同批次试剂,其中1套作为对照,其他4套分别加入终浓度为2mM、5mM、10mM、20mM的HED,选用相同的脚本进行SE50测序。Testing of additives at different concentrations: According to Tables 8-1 and 8-2, prepare 5 sets of reagents from the same batch, of which 1 set is used as a control, and the other 4 sets are added with final concentrations of 2mM, 5mM, 10mM, and 20mM HED. Select The same script performed SE50 sequencing.
实验条件:Experimental conditions:
表8-1 Hot dNTP试剂槽配制组分Table 8-1 Hot dNTP reagent tank preparation components
HotMPS dNTPs Mix conc.HotMPS dNTPs Mix conc. | 0.6μM0.6μM |
EnzymeEnzyme | DNA聚合酶DNA polymerase |
Enzyme conc.Enzyme conc. | 10μg/mL10μg/mL |
表8-2 Cold dNTP试剂槽配制组分Table 8-2 Cold dNTP reagent tank preparation components
HotMPS dNTPs Mix II conc.HotMPS dNTPs Mix II conc. | 2μM2μM |
EnzymeEnzyme | DNA聚合酶DNA polymerase |
Enzyme conc.Enzyme conc. | 10μg/mL10μg/mL |
脚本条件:Script conditions:
表9脚本条件Table 9 script conditions
步骤step | Tm(℃)Tm(℃) | Time(s)Time(s) |
Hot聚合Hot aggregation | 6060 | 120120 |
Cold聚合Cold aggregation | 6060 | 120120 |
切除resection | 6565 | 120120 |
7、实验结果与分析7. Experimental results and analysis
如表10所示,对照组测试:新鲜配制的试剂,runon(%)0.54%;4℃放置24小时后,runon(%)增加至0.73%;As shown in Table 10, the control group test: freshly prepared reagent, runon (%) 0.54%; after being placed at 4°C for 24 hours, runon (%) increased to 0.73%;
HED测序组:新鲜配制的试剂runon(%)降低至0.41%,放置24小时,runon(%)增加至0.58%,远低于对照组放24小时的Runon(%)。HED sequencing group: The runon (%) of the freshly prepared reagent was reduced to 0.41%, and after being left for 24 hours, the runon (%) increased to 0.58%, which was much lower than the runon (%) of the control group after being left for 24 hours.
由此表明,2-羟乙基二硫化物的添加对提高HotMPS dNTPs Mix和HotMPS dNTPs Mix II在测序缓冲液中的稳定性有帮助。This shows that the addition of 2-hydroxyethyl disulfide is helpful to improve the stability of HotMPS dNTPs Mix and HotMPS dNTPs Mix II in sequencing buffer.
表10不同添加剂的比较Table 10 Comparison of different additives
如表11所示,5-20mM HED的Runon(%)基本一致,均比2mM的Runon(%)低;MappingRate(%)的差异可能来自于不同的芯片间差异;加HED的Q30(%)均比对照组好;不同浓度之间差异不大。As shown in Table 11, the Runon (%) of 5-20mM HED is basically the same, both are lower than the Runon (%) of 2mM; the difference in MappingRate (%) may come from the differences between different chips; Q30 (%) with HED All were better than the control group; there was little difference between different concentrations.
表11 HED浓度测试比较Table 11 HED concentration test comparison
8、实验结论8. Experimental conclusion
HED对提高HotMPS dNTPs Mix和HotMPS dNTPs Mix II在测序缓冲液中的稳定性有帮助;5mM、10mM和20mM的HED对Runon的影响基本一致,优选5-10mM。HED is helpful to improve the stability of HotMPS dNTPs Mix and HotMPS dNTPs Mix II in sequencing buffer; 5mM, 10mM and 20mM HED have basically the same effect on Runon, with 5-10mM being preferred.
实施例2 5-溴尿嘧啶作为竞争试剂Example 2 5-bromouracil as a competitive reagent
1、实验材料1. Experimental materials
表12试剂、耗材信息Table 12 Reagents and consumables information
2、仪器2. Instruments
表13仪器信息Table 13 Instrument information
仪器instrument | 型号model |
基因扩增仪Gene cycler | 63376337 |
测序仪sequencer | MGISEQ-2000RSMGISEQ-2000RS |
Qubit仪Qubit instrument | Qubit4.0Qubit4.0 |
电子天平Electronic balance | MS204TSMS204TS |
3、实验样本3. Experimental samples
同实施例1。Same as Example 1.
4、实验设计4. Experimental design
配置两个SE50测序试剂盒,一个试剂盒作为对照,一个试剂盒的测序缓冲液中加入终浓度为5mM 5-溴尿嘧啶,室温加速6小时后,在MGISEQ-2000RS测序仪上进行SE50测序,比较Runon(%)、Q30(%)、AvgErrorRate(%)和TotalReads(M)。Configure two SE50 sequencing kits, one kit is used as a control, and a final concentration of 5mM 5-bromouracil is added to the sequencing buffer of one kit. After accelerating at room temperature for 6 hours, perform SE50 sequencing on the MGISEQ-2000RS sequencer. Compare Runon(%), Q30(%), AvgErrorRate(%) and TotalReads(M).
5、评价指标及标准5. Evaluation indicators and standards
表14评价标准Table 14 Evaluation Criteria
评价指标Evaluation index | 合格标准Eligibility criteria |
Runon(%)Runon(%) | 越低越好The lower the better |
Q30(%)Q30(%) | 越高越好The higher the better |
AvgErrorRate(%)AvgErrorRate(%) | 越低越好The lower the better |
TotalReads(M)TotalReads(M) | 越高越好The higher the better |
6、实验步骤6. Experimental steps
6.1 DNB制备6.1 DNB preparation
同实施例1。Same as Example 1.
6.2:DNB pooling及加载6.2: DNB pooling and loading
同实施例1。Same as Example 1.
6.3上机测序6.3 On-machine sequencing
按照表15-1和15-2,分别配制2套同批次试剂,其中1套作为对照,另外1套加入5mM5-溴尿嘧啶作为测试组,配好后半小时内上机测对照和测试组进行SE50测序。According to Tables 15-1 and 15-2, prepare two sets of reagents from the same batch, one of which is used as a control, and the other set is added with 5mM 5-bromouracil as a test group. After preparation, the control and test are tested on the machine within half an hour. The group performed SE50 sequencing.
实验条件:Experimental conditions:
表15-1 Hot dNTP试剂槽配制组分Table 15-1 Hot dNTP reagent tank preparation components
表15-2 Cold dNTP试剂槽配制组分Table 15-2 Cold dNTP reagent tank preparation components
脚本条件:Script conditions:
表16脚本条件Table 16 Script Conditions
步骤step | Tm(C)Tm(C) | Time(s)Time(s) |
Hot聚合Hot aggregation | 6060 | 120120 |
Cold聚合Cold aggregation | 6060 | 120120 |
切除resection | 6565 | 120120 |
7、实验结果与分析7. Experimental results and analysis
表17实验结果Table 17 Experimental results
| 5-Br(5mM)5-Br(5mM) | 对照control |
Q30(%)Q30(%) | 93.3293.32 | 92.8592.85 |
ESR(%)ESR(%) | 85.1185.11 | 84.7884.78 |
MappingRate(%)MappingRate(%) | 99.3799.37 | 99.3399.33 |
AvgErrorRate(%)AvgErrorRate(%) | 0.380.38 | 0.430.43 |
Lag(%)Lag(%) | 0.150.15 | 0.170.17 |
Runon(%)Runon(%) | 0.520.52 | 0.580.58 |
在室温加速6小时后,加5-溴尿嘧啶的实验组Runon(%)降低了10%,AvgErrorRate(%)降低了11.6%。由此,表明5-溴尿嘧啶的添加有助于提高HotMPS dNTPs Mix和HotMPS dNTPs Mix II的稳定性。After 6 hours of acceleration at room temperature, the Runon (%) of the experimental group with 5-bromouracil was reduced by 10%, and the AvgErrorRate (%) was reduced by 11.6%. This shows that the addition of 5-bromouracil helps improve the stability of HotMPS dNTPs Mix and HotMPS dNTPs Mix II.
对比例Comparative ratio
实验材料(除2-羟乙基二硫化物)、仪器、实验样本同实施例1。The experimental materials (except for 2-hydroxyethyl disulfide), instruments, and experimental samples were the same as in Example 1.
实验步骤:Experimental steps:
1、DNB制备、DNB pooling、加载同实施例1。1. DNB preparation, DNB pooling, and loading are the same as in Example 1.
2、上机测序2. On-machine sequencing
按照表8-1和8-2,分别配制6套同批次试剂,其中2套作为对照,2套加入终浓度为2mM DMTU(N,N′-二甲基硫脲,品牌:Merck,货号:D188700)作为测试组1,2套加入终浓度为2mM APDC(吡咯烷二硫代甲酸铵盐,品牌:Merck,货号:P8765)作为测试组2,配好后半小时内上机测对照和测试组1、测试组2的其中的1套;4℃放置24小时后测另外1套;选用相同的脚本(同实施例1)进行SE50测序。According to Tables 8-1 and 8-2, prepare 6 sets of reagents from the same batch, of which 2 sets are used as controls, and 2 sets are added with a final concentration of 2mM DMTU (N,N'-dimethylthiourea, brand: Merck, product number : D188700) as test group 1, 2 sets were added with a final concentration of 2mM APDC (ammonium pyrrolidine dithiocarbamate, brand: Merck, product number: P8765) as test group 2, and the control and control were tested within half an hour after preparation. One set of test group 1 and test group 2; test the other set after placing it at 4°C for 24 hours; use the same script (same as Example 1) for SE50 sequencing.
结果如表18所示,可以看出,测序缓冲液中加入DMTU和APDTC,在4℃放置24小时后,比对照组还差。由此表明DMTU和APDTC不能提高HotMPS dNTPs Mix和HotMPS dNTPs Mix II在测序缓冲液中的稳定性。The results are shown in Table 18. It can be seen that adding DMTU and APDTC to the sequencing buffer and leaving it at 4°C for 24 hours was worse than the control group. This shows that DMTU and APDTC cannot improve the stability of HotMPS dNTPs Mix and HotMPS dNTPs Mix II in sequencing buffer.
表18 DMTU和APDTC的测序结果分析Table 18 Analysis of sequencing results of DMTU and APDTC
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。In the description of this specification, reference 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.
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。Although the embodiments of the present invention have been shown and described above, it can be understood that the above-mentioned embodiments are illustrative and should not be construed as limitations of the present invention. Those of ordinary skill in the art can make modifications to the above-mentioned embodiments within the scope of the present invention. The embodiments are subject to changes, modifications, substitutions and variations.
Claims (19)
- 一种测序缓冲液,其特征在于,包括:A sequencing buffer, characterized in that it includes:竞争试剂,所述竞争试剂可与具有可逆阻断基团修饰的dNTP竞争性地与特定物质发生反应。Competitive reagents, which can compete with dNTPs modified with reversible blocking groups to react with specific substances.
- 根据权利要求1所述的测序缓冲液,其特征在于,所述可逆阻断基团包括酯键和/或二硫键。The sequencing buffer according to claim 1, wherein the reversible blocking group includes an ester bond and/or a disulfide bond.
- 根据权利要求1或2所述的测序缓冲液,其特征在于,所述可逆阻断基团包括二硫键,所述竞争试剂包括2-羟乙基二硫化物。The sequencing buffer according to claim 1 or 2, wherein the reversible blocking group includes a disulfide bond, and the competitive reagent includes 2-hydroxyethyl disulfide.
- 根据权利要求1或2所述的测序缓冲液,其特征在于,所述可逆阻断基团包括酯键,所述竞争试剂包括5-溴尿嘧啶。The sequencing buffer according to claim 1 or 2, wherein the reversible blocking group includes an ester bond, and the competitive reagent includes 5-bromouracil.
- 一种测序溶液,其特征在于,包括:权利要求1~4任一项所述测序缓冲液和具有可逆阻断基团修饰的dNTP。A sequencing solution, characterized by comprising: the sequencing buffer according to any one of claims 1 to 4 and a dNTP modified with a reversible blocking group.
- 根据权利要求5所述的测序溶液,其特征在于,所述dNTP的浓度为0.1μM~50mM。The sequencing solution according to claim 5, wherein the concentration of the dNTP is 0.1 μM ~ 50 mM.
- 根据权利要求5或6所述的测序溶液,其特征在于,所述dNTP的浓度为0.1μM~10mM。The sequencing solution according to claim 5 or 6, wherein the concentration of the dNTP is 0.1 μM to 10 mM.
- 根据权利要求5所述的测序溶液,其特征在于,所述竞争试剂的浓度为10μM~100mM。The sequencing solution according to claim 5, wherein the concentration of the competitive reagent is 10 μM to 100 mM.
- 根据权利要求5-8任一项所述的测序溶液,其特征在于,所述竞争试剂的浓度为1~10mM。The sequencing solution according to any one of claims 5 to 8, wherein the concentration of the competitive reagent is 1 to 10 mM.
- 一种提高具有可逆阻断基团修饰的dNTP稳定性的方法,其特征在于,包括:A method for improving the stability of dNTPs modified with reversible blocking groups, which is characterized by including:使竞争试剂与所述具有可逆阻断基团修饰的dNTP进行接触;Contact the competitive reagent with the dNTP modified with a reversible blocking group;其中,所述竞争试剂可与所述可逆阻断基团竞争性地与特定物质发生反应。Wherein, the competitive reagent can compete with the reversible blocking group to react with a specific substance.
- 根据权利要求10所述的方法,其特征在于,所述可逆阻断基团包括二硫键,所述竞争试剂包括2-羟乙基二硫化物。The method of claim 10, wherein the reversible blocking group includes a disulfide bond and the competitive reagent includes 2-hydroxyethyl disulfide.
- 根据权利要求10或11所述的方法,其特征在于,所述可逆阻断基团包括酯键,所述竞争试剂包括5-溴尿嘧啶。The method according to claim 10 or 11, wherein the reversible blocking group includes an ester bond, and the competitive reagent includes 5-bromouracil.
- 根据权利要求10所述的方法,其特征在于,在所述接触所得混合溶液中,所述竞争试剂的浓度为10μM~100mM。The method according to claim 10, characterized in that, in the mixed solution obtained by the contact, the concentration of the competitive reagent is 10 μM to 100 mM.
- 根据权利要求10或13所述的方法,其特征在于,在所述接触所得混合溶液中,所述竞争试剂的浓度为1~10mM。The method according to claim 10 or 13, characterized in that, in the mixed solution obtained by the contact, the concentration of the competitive reagent is 1 to 10 mM.
- 根据权利要求10所述的方法,其特征在于,在所述接触所得混合溶液中,所述dNTP的浓度为0.1μM~50mM。The method according to claim 10, characterized in that, in the mixed solution obtained by the contact, the concentration of the dNTP is 0.1 μM to 50 mM.
- 根据权利要求10或15所述的方法,其特征在于,在所述接触所得混合溶液中,所述dNTP的浓度为0.1μM~10mM。The method according to claim 10 or 15, characterized in that, in the mixed solution obtained by the contact, the concentration of the dNTP is 0.1 μM to 10 mM.
- 一种测序试剂盒,其特征在于,包括:权利要求1~4任一项所述测序缓冲液或权利要求5~9任一项所述测序溶液。A sequencing kit, characterized by comprising: the sequencing buffer according to any one of claims 1 to 4 or the sequencing solution according to any one of claims 5 to 9.
- 权利要求1~4任一项所述测序缓冲液、权利要求5~9任一项所述测序溶液或权利要求17所述测序试剂盒在测序中的应用。Application of the sequencing buffer according to any one of claims 1 to 4, the sequencing solution according to any one of claims 5 to 9, or the sequencing kit according to claim 17 in sequencing.
- 一种测序方法,其特征在于,包括:使用权利要求1~4任一项所述测序缓冲液、权利要求5~9任一项所述测序溶液或权利要求17所述测序试剂盒。A sequencing method, characterized by comprising: using the sequencing buffer according to any one of claims 1 to 4, the sequencing solution according to any one of claims 5 to 9, or the sequencing kit according to claim 17.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
PCT/CN2022/098977 WO2023240494A1 (en) | 2022-06-15 | 2022-06-15 | Sequencing buffer and method for improving stability of dntp modified with reversible blocking group |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
PCT/CN2022/098977 WO2023240494A1 (en) | 2022-06-15 | 2022-06-15 | Sequencing buffer and method for improving stability of dntp modified with reversible blocking group |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2023240494A1 true WO2023240494A1 (en) | 2023-12-21 |
Family
ID=89193023
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/CN2022/098977 WO2023240494A1 (en) | 2022-06-15 | 2022-06-15 | Sequencing buffer and method for improving stability of dntp modified with reversible blocking group |
Country Status (1)
Country | Link |
---|---|
WO (1) | WO2023240494A1 (en) |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1553953A (en) * | 2000-07-07 | 2004-12-08 | ά�������\����˾ | Real-time sequence determination |
US20070003938A1 (en) * | 2005-06-30 | 2007-01-04 | Perlegen Sciences, Inc. | Hybridization of genomic nucleic acid without complexity reduction |
US20100190168A1 (en) * | 2008-12-02 | 2010-07-29 | Helicos Biosciences Corporation | Competitor molecules useful for lowering nonspecific adsorption of dye labeled nucleotides |
CN108239669A (en) * | 2016-12-23 | 2018-07-03 | 深圳华大智造科技有限公司 | Detect the method and kit of non-blacked impurity content in reversible dNTP |
CN110691854A (en) * | 2017-10-25 | 2020-01-14 | 深圳华大生命科学研究院 | Nucleic acid sequencing method and nucleic acid sequencing kit |
CN113272448A (en) * | 2018-11-09 | 2021-08-17 | 深圳华大智造科技股份有限公司 | Massively parallel sequencing Using unlabeled nucleotides |
-
2022
- 2022-06-15 WO PCT/CN2022/098977 patent/WO2023240494A1/en unknown
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1553953A (en) * | 2000-07-07 | 2004-12-08 | ά�������\����˾ | Real-time sequence determination |
US20070003938A1 (en) * | 2005-06-30 | 2007-01-04 | Perlegen Sciences, Inc. | Hybridization of genomic nucleic acid without complexity reduction |
US20100190168A1 (en) * | 2008-12-02 | 2010-07-29 | Helicos Biosciences Corporation | Competitor molecules useful for lowering nonspecific adsorption of dye labeled nucleotides |
CN108239669A (en) * | 2016-12-23 | 2018-07-03 | 深圳华大智造科技有限公司 | Detect the method and kit of non-blacked impurity content in reversible dNTP |
CN110691854A (en) * | 2017-10-25 | 2020-01-14 | 深圳华大生命科学研究院 | Nucleic acid sequencing method and nucleic acid sequencing kit |
CN113272448A (en) * | 2018-11-09 | 2021-08-17 | 深圳华大智造科技股份有限公司 | Massively parallel sequencing Using unlabeled nucleotides |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JPS5825980B2 (en) | Cyclic nucleotide quantification method | |
AU2004292673A1 (en) | Method for adjusting the quantification range of individual analytes in a multiplexed assay | |
CN109116040B (en) | Method for detecting ***e based on dimercapto aptamer | |
CN106645050B (en) | It is a kind of for detecting the aptamers and its application of lactoferrin content | |
JP2009536023A (en) | Analyte-linked enzyme-immunochemical detection method using an endogenous calibrator | |
JPS62253399A (en) | Determination of lipid-bonding sialic acid | |
CN108169145A (en) | A kind of kit for measuring serum complement C1q and its preparation application method | |
CN113493932B (en) | Method and kit for constructing capture library with high detection performance | |
CN110596405A (en) | Kit for detecting content of heart-type fatty acid binding protein by latex enhanced immunoturbidimetry | |
CN111118116A (en) | Multiple miRNA detection method based on gold nanoparticle aggregation and dispersion | |
WO2023240494A1 (en) | Sequencing buffer and method for improving stability of dntp modified with reversible blocking group | |
US20090130696A1 (en) | HDL Cholesterol Sensor Using Selective Surfactant | |
US3649198A (en) | Diagnostic method for the determination of uric acid in blood | |
EP2460888B1 (en) | Method and kit for measurement of dehydrogenase or substrate for the dehydrogenase | |
CN111830254B (en) | Matrix metalloproteinase-3 determination kit and preparation method thereof | |
Liu et al. | Human serum biomarker detection based on a cascade signal amplification strategy by a DNA molecule machine | |
WO2023240500A1 (en) | Method for preserving dntp | |
JP2023519365A (en) | Method for detecting analytes with different abundances | |
Wang et al. | Amplification-free quantitative detection of genomic DNA using lateral flow strips for milk authentication | |
CN112326613A (en) | Method for detecting thrombin content | |
CN115925704B (en) | Method for detecting thrombin based on perylene derivative probe without marking | |
CN115976221B (en) | Internally-doped reference substance for quantitative detection of BCR or TCR rearrangement as well as preparation method and application thereof | |
CN115792231B (en) | DNase I biosensor based on thrombin aptamer-regulated enzyme cascade reaction | |
CN117089593A (en) | Method for measuring activity of DNA ligase and enzyme activity detection system of DNA ligase | |
JP4616516B2 (en) | Immunological assay |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 22946188 Country of ref document: EP Kind code of ref document: A1 |