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WO2018129644A1 - 一种提高核酸聚合测序质量的方法、反应体系和试剂盒 - Google Patents

一种提高核酸聚合测序质量的方法、反应体系和试剂盒 Download PDF

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WO2018129644A1
WO2018129644A1 PCT/CN2017/070711 CN2017070711W WO2018129644A1 WO 2018129644 A1 WO2018129644 A1 WO 2018129644A1 CN 2017070711 W CN2017070711 W CN 2017070711W WO 2018129644 A1 WO2018129644 A1 WO 2018129644A1
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nucleotide
sequencing
nucleic acid
modification
type
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French (fr)
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李计广
马可心
邱敏
陈奥
徐崇钧
章文蔚
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MGI Tech Co Ltd
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MGI Tech Co Ltd
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Priority to CN202210627105.4A priority Critical patent/CN114958998A/zh
Priority to CN202210628687.8A priority patent/CN115141880A/zh
Priority to PCT/CN2017/070711 priority patent/WO2018129644A1/zh
Priority to CN201780068192.1A priority patent/CN109937259B/zh
Publication of WO2018129644A1 publication Critical patent/WO2018129644A1/zh
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    • 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
    • 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 application relates to the field of nucleic acid sequencing, and in particular to a method, a reaction system and a kit for improving the quality of nucleic acid polymerization sequencing.
  • Nucleic acid polymerization sequencing ie, side-synthesis sequencing (abbreviated SBS) refers to the ability to add fluorescently labeled nucleotides during the SBS process (Metzker et. al., Genome Res 15(12): 1767-1776 (2005)). Helps identify template DNA bases (Prober et. al., Science 238:336-341 (1987)) to give DNA sequence information. In recent years, compared with other sequencing methods, polymerization sequencing has been favored for its high throughput and low price.
  • SBS side-synthesis sequencing
  • a reversible termination and a base with a fluorescent signal are synthesized by a DNA polymerase; specifically, a nucleotide having a modification at the 3' sugar hydroxyl group is used, thereby blocking the addition of other nucleotides, Fluorescent signals to distinguish between 4 different bases. After removal of the blocking and fluorophores, the naturally free 3' hydroxyl group is restored for addition to the next nucleotide and the fluorescent signal is removed to facilitate synthesis and detection of the next base.
  • the synthesis efficiency is very high, and in the case of incomplete synthesis in multiple copies, signal confusion will occur, affecting the read length and accuracy of sequencing.
  • Nucleotides with reversible blocking and fluorophores are required to be added in sequencing while synthesis. Due to the increased modification, the molecules of the nucleotides are larger, the molecular structure is special, and the nucleotides are not modified. In contrast, DNA polymerases are less efficient at identifying and synthesizing such modified dNTPs, limiting the read length and accuracy of sequencing.
  • the present application discloses a method for improving the quality of nucleic acid polymerization sequencing comprising the addition of a second type of nucleotide to the reaction sequence of the polymerization sequence, the 3' sugar hydroxyl group of the second type of nucleotide having a blocking modification but no fluorescent modification.
  • dNTPs having only blocking modification that is, a second type of nucleotide
  • the effect of blocking modified dNTPs on DNA polymerase recognition and synthesis is relatively small. Therefore, the addition of the second type of nucleotides can complement the incomplete reaction, increase the efficiency of the synthesis reaction, and reduce the risk of insufficient reaction; And because of the second category Nucleotides are not fluorescently modified, which reduces signal interference caused by unclear fluorescence refraction or unclean fluorescence of the excised cells, which improves the efficiency of excision and reduces the sequencing error rate; thereby improving the quality of sequencing.
  • the second type of nucleotide is used in the same amount as the first type of nucleotide in the reaction solution, and the first type of nucleotide is a 3' sugar hydroxyl group having both a blocking modification and a fluorescent modification.
  • the first nucleotide in the present application is a nucleotide which is normally added in the polymerization sequencing.
  • the second nucleotide and the first nucleotide are added in equal amounts.
  • the amount of the second type of nucleotide can be adjusted according to requirements, but as long as the second type of nucleotide is added, the synthesis reaction efficiency can be improved to a certain extent. The effect of sequencing error rate.
  • Another aspect of the present application discloses the use of the method of the present application in nucleic acid sequencing.
  • the method of the present application is applicable to the sequencing of nucleic acid sequences of the human genome and other animal, plant and microbial species; the main applications include sequencing of WES, WGS, RNA, DNA, etc.; in one embodiment of the present application, the method of the present application is applied to the large intestine DNA sequencing of Bacillus.
  • a further aspect of the present application discloses a nucleic acid sequencing method, which comprises simultaneously adding two types of nucleotides to a reaction solution in a process of sequencing while synthesizing, the first nucleotide being a 3' sugar hydroxyl group and having a blocking effect.
  • Modified and fluorescently modified nucleotides the second type of nucleotide is a 3' sugar hydroxyl group having only a blocking modification but no fluorescent modification.
  • the method for improving the quality of nucleic acid polymerization sequencing of the present application can be applied to various sequencing platforms based on edge synthesis sequencing, and the application only needs to be added to the reaction solution without changing the sequencing conditions and parameters.
  • the second type of nucleotide can be.
  • Another aspect of the present application discloses a reaction system for improving the quality of nucleic acid sequencing, comprising a reaction solution for sequencing while synthesizing, wherein a second type of nucleotide is added to the reaction solution, and a third nucleotide of the second type is added.
  • the saccharide hydroxyl group has a blocking modification but no fluorescent modification.
  • the reaction solution comprises a reaction buffer, a DNA polymerase and a first type of nucleotide, and the first type of nucleotide is a 3' sugar hydroxyl group having both a blocking modification and a fluorescent modification.
  • a further aspect of the present application discloses a nucleic acid sequencing kit comprising the reaction system of the present application.
  • the key inventive idea of the present application is to add dNTPs having only blocking modification, that is, a second type of nucleotide, in the reaction solution which is synthesized while synthesizing, so as to improve the synthesis efficiency;
  • the present application further proposes a reaction system for improving the quality of nucleic acid sequencing, that is, a reaction solution prepared by sequencing while adding a second type of nucleotide.
  • a reaction system for improving the quality of nucleic acid sequencing that is, a reaction solution prepared by sequencing while adding a second type of nucleotide.
  • the reagent system of the present application can also be made into a nucleic acid sequencing kit.
  • the method for improving the quality of nucleic acid polymerization sequencing of the present application adds dNTPs having only blocking modification to the reaction liquid, and since such dNTPs have no fluorescent modification, it is more conducive to DNA polymerase recognition and synthesis, and can supplement the incomplete reaction portion. Increasing the efficiency of the synthesis reaction and reducing the risk of insufficient reaction; at the same time, because there is no fluorescence modification, the signal interference caused by the unclear fluorescence removal or the uncleaned fluorescence of the resection is reduced, and the resection efficiency is improved and the reduction is improved. Sequencing error rate.
  • FIG. 1 is a schematic structural view of a second type of nucleotide in the embodiment of the present application.
  • FIG. 2 is a schematic structural view of a first type of nucleotide in the embodiment of the present application.
  • Figure 5 is a graph showing the variation in sequencing error rate for each cycle in the examples of the present application.
  • the present application proposes to add dNTPs having only blocking modification on the basis of the existing polymerization sequencing reaction solution;
  • the second type of nucleotide can be used to supplement the incomplete part, which guarantees the synthesis efficiency and reduces the risk of insufficient reaction;
  • the fluorescence excision is also reduced. Unclean, or signal interference caused by unclear fluorescent elution, improves the efficiency of resection and reduces the sequencing error rate.
  • the nucleic acid sequencing method of this example was performed on a BGISEQ-500 sequencing platform, and the sequenced sample was an E. coli standard library.
  • the specific plan is as follows:
  • the dNTP 1 mixture is prepared, that is, the first nucleotide; the first nucleotide of this example, as shown in FIG. 2, has both a fluorescent dye and a blocking group on the nucleotide.
  • A, T, G, and C represent adenine nucleotides, thymidine nucleotides, guanine nucleotides, and cytosine nucleotides, respectively.
  • dNTP 1 mixture Final concentration (nmol/L) dCTP_1 100 dGTP_1 100 dATP_1 100 dTTP_1 100
  • dCTP_1 refers to cytosine nucleotides with both blocking and fluorescent modifications
  • dGTP_1 refers to guanine nucleotides with both blocking and fluorescent modifications
  • dATP_1 refers to both blocking and fluorescent modifications.
  • the adenine nucleotide, dTTP_1 refers to a thymidine nucleotide having both blocking and fluorescent modifications.
  • the dNTP 2 mixture is configured, that is, the second type of nucleotide; the second nucleotide of this example, as shown in Figure 1, has only a blocking group on the nucleotide, and no fluorescence.
  • a dye wherein A, T, G, and C represent adenine nucleotides, thymidine nucleotides, guanine nucleotides, and cytosine nucleotides, respectively.
  • dNTP 2 mixture Final concentration (nmol/L) dCTP_2 100 dGTP_2 100 dATP_2 100 dTTP_2 100
  • dCTP_2 refers to a cytosine nucleotide having only a blocking modification
  • dGTP_2 refers to a guanine nucleotide having only a blocking modification
  • dATP_2 refers to an adenine nucleotide having only a blocking modification
  • dTTP_2 is Refers to a thymidine nucleotide that only has a blocking modification.
  • SE50 sequencing was performed using the BGISEQ-500 platform, the experimental group with the synthetic reagent 1 was added as the control group, and the experimental group with the synthetic reagent 2 was added as the experimental group, and the fluorescence signal curves of each cycle in the two groups were statistically analyzed. Sequencing quality change curve for each cycle and sequencing error rate curve for each cycle.
  • the sequencing results are shown in Figures 3 to 5.
  • the fluorescence signal change curve of each cycle is shown in Fig. 3.
  • the abscissa is the sequencing read length
  • the ordinate is the sequencing signal
  • “ ⁇ ” is the curve of the experimental group
  • “ ⁇ ” is the curve of the control group
  • the sequencing quality variation curve of each cycle is shown in Fig. 4.
  • the abscissa is the sequencing read length
  • the ordinate is the sequencing quality value
  • “ ⁇ ” is the curve of the experimental group
  • “ ⁇ ” is the curve of the control group
  • the sequencing error rate curve for each cycle is shown in Figure 5.
  • the abscissa is the sequencing read length.
  • the ordinate is the sequencing error rate
  • “ ⁇ ” is the curve of the experimental group
  • “ ⁇ ” is the curve of the control group; the results show that with the increase of the sequencing cycle, the sequencing error rate begins to increase, and the error rate of the experimental group increases faster than the comparison.
  • the group should be slow, indicating that the experimental group has a lower error rate.

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Abstract

公开了一种提高核酸聚合测序质量的方法、反应体系和试剂盒。本申请的提高核酸聚合测序质量的方法，包括在聚合测序的反应液中加入第二类核苷酸，第二类核苷酸的3'糖羟基具有阻断修饰但没有荧光修饰。

Description

一种提高核酸聚合测序质量的方法、反应体系和试剂盒 技术领域
本申请涉及核酸测序领域，特别是涉及一种提高核酸聚合测序质量的方法、反应体系和试剂盒。
背景技术
核酸聚合测序即边合成边测序(缩写SBS)，是指在SBS过程中(Metzker et.al.,Genome Res 15(12):1767-1776(2005))，加入经过荧光标记的核苷酸能够帮助识别模板DNA碱基(Prober et.al.,Science 238:336-341(1987))，从而给出DNA序列信息。近年来，与其它测序方法相比，聚合测序以其通量高、价格低而获得青睐。在这一测序方法中，通过DNA聚合酶合成可逆终止和带有荧光信号的碱基；具体的，使用在3’糖羟基具有修饰的核苷酸，从而阻断其它核苷酸的加入，通过荧光信号来区分4种不同的碱基。在去除阻断和荧光基团后，恢复天然游离的3’羟基基团用于加入下一个核苷酸，并且去除荧光信号，以便于下一个碱基的合成和检测。在SBS中，对于合成效率要求很高，在多个拷贝中一旦出现合成不完全的情况，就会导致信号的混乱，影响测序的读长和准确性。而在边合成边测序中要求加入的带有可逆阻断和荧光基团的核苷酸，由于修饰的增加导致该核苷酸的分子较大，分子结构比较特殊，与没有修饰的核苷酸相比，DNA聚合酶识别和合成这种带修饰的dNTPs的效率较低，限制了聚合测序的读长和准确度。
发明内容
本申请的目的是提供一种改进的新的提高核酸聚合测序质量的方法、反应体系和试剂盒。
为了实现上述目的，本申请采用了以下技术方案：
本申请公开了一种提高核酸聚合测序质量的方法，包括在聚合测序的反应液中加入第二类核苷酸，第二类核苷酸的3’糖羟基具有阻断修饰但没有荧光修饰。
需要说明的是，本申请的关键在于，在聚合测序的反应液中加入了仅仅具有阻断修饰的dNTPs，即第二类核苷酸。阻断修饰的dNTPs对于DNA聚合酶识别和合成的影响相对较小，因此，第二类核苷酸的加入，能够补充反应不完全的部分，提高合成反应的效率，降低反应不充分的风险；并且，由于第二类 核苷酸没有荧光修饰，降低了因为荧光切除不干净，或者切除的荧光洗脱不干净导致的信号干扰，提高了切除的效率，降低了测序错误率；从而起到提高测序质量的作用。可以理解，本申请的方法，其关键在于，在聚合测序的反应液中加入了仅仅具有阻断修饰的第二类核苷酸，至于反应液其它组分，以及具体的反应条件，可以参考现有的聚合测序方法。
优选的，第二类核苷酸的用量，与反应液中第一类核苷酸的用量相同，第一类核苷酸为3’糖羟基同时具有阻断修饰和荧光修饰的核苷酸。
需要说明的是，本申请中第一类核苷酸就是聚合测序中正常添加的核苷酸，本申请优选的方案中，第二类核苷酸和第一类核苷酸是等量加入的。可以理解，在一些特殊的设计方案中，第二类核苷酸的用量可以根据需求进行调整，但是，只要加入了第二类核苷酸都可以在一定程度上起到提高合成反应效率、降低测序错误率的效果。
本申请的另一面公开了本申请的方法在核酸测序中的应用。本申请的方法适用于人类基因组和其它动物、植物和微生物物种的核酸序列测序；主要应用包括WES、WGS、RNA、DNA等测序；本申请的一个实施例中，将本申请的方法用于大肠杆菌的DNA测序。
本申请的再一面公开了一种核酸测序方法，包括在边合成边测序的过程中，于其反应液中同时添加两类核苷酸，第一核苷酸为3’糖羟基同时具有阻断修饰和荧光修饰的核苷酸，第二类核苷酸为3’糖羟基仅具有阻断修饰但没有荧光修饰的核苷酸。
可以理解，本申请的提高核酸聚合测序质量的方法，可以应用于各种基于边合成边测序的测序平台，在不改变其测序条件和参数的情况下，只需要在其反应液中添加本申请的第二类核苷酸即可。
本申请的另一面公开了一种提高核酸测序质量的反应体系，包括用于边合成边测序的反应液，该反应液中添加有第二类核苷酸，第二类核苷酸的3’糖羟基具有阻断修饰但没有荧光修饰。
优选的，反应液包括反应缓冲液、DNA聚合酶和第一类核苷酸，第一类核苷酸为3’糖羟基同时具有阻断修饰和荧光修饰的核苷酸。
本申请的再一面公开了一种含有本申请的反应体系的核酸测序试剂盒。
可以理解，本申请的关键发明思路在于，在边合成边测序的反应液中添加仅仅具有阻断修饰的dNTPs，即第二类核苷酸，以提高合成效率；基于该发明思路，为了使用方便，本申请进一步提出了一种提高核酸测序质量的反应体系，即添加有第二类核苷酸的边合成边测序的反应液。采用本申请的反应试剂体系， 可以有效的提高测序质量。同样的，为了使用方便，也完全可以将本申请的反应试剂体系制成核酸测序试剂盒。
由于采用以上技术方案，本申请的有益效果在于：
本申请的提高核酸聚合测序质量的方法，在其反应液中添加仅仅具有阻断修饰的dNTPs，由于这类dNTPs没有荧光修饰，更利于DNA聚合酶识别和合成，能够补充反应不完全的部分，提高合成反应的效率，降低反应不充分的风险；与此同时，由于没有荧光修饰，降低了因荧光切除不干净，或者切除的荧光洗脱不干净导致的信号干扰，提高了切除效率，降低了测序错误率。
附图说明
图1是本申请实施例中第二类核苷酸的结构示意图；
图2是本申请实施例中第一类核苷酸的结构示意图；
图3是本申请实施例中每个循环的荧光信号变化曲线；
图4是本申请实施例中每个循环的测序质量变化曲线；
图5是本申请实施例中每个循环的测序错误率变化曲线。
具体实施方式
边合成边测序的关键就在于，在反应液中添加同时具有阻断修饰和荧光修饰的dNTPs，但是，本申请的发明人经过大量的试验和研究发现，阻断修饰和荧光修饰的dNTPs会影响DNA聚合酶的识别和合成效率，从而限制聚合测序的读长和准确度。经过深入研究发现，其中荧光修饰是影响DNA聚合酶的识别和合成效率的主要原因；因此，本申请提出，在现有的聚合测序反应液的基础上，添加仅仅具有阻断修饰的dNTPs；这样，一方面，可以通过第二类核苷酸补充反应不完全的部分，保障合成效率，降低反应不充分的风险；另一方面，由于第二类核苷酸没有荧光修饰，也减低了荧光切除不干净，或者切除的荧光洗脱不干净导致的信号干扰，提高了切除的效率，降低了测序错误率。
下面通过具体实施例和附图对本申请作进一步详细说明。以下实施例仅对本申请进行进一步说明，不应理解为对本申请的限制。
实施例
本例的核酸测序方法在BGISEQ-500测序平台上进行，测序样品为大肠杆菌标准文库。具体方案如下：
(1)使用BGISEQ-500的测序平台，并按照说明书使用与该平台配套的测序试剂；
(2)按照表1配方配制dNTP 1混合液，即第一类核苷酸；本例的第一类核苷酸，如图2所示，核苷酸上同时具有荧光染料和阻断基团，其中，A,T,G,C分别表示腺嘌呤核苷酸、胸腺嘧啶核苷酸、鸟嘌呤核苷酸、胞嘧啶核苷酸。
表1 dNTP 1混合液配方表
dNTP 1混合液 终浓度(nmol/L)
dCTP_1 100
dGTP_1 100
dATP_1 100
dTTP_1 100
表中，dCTP_1是指同时具有阻断修饰和荧光修饰的胞嘧啶核苷酸，dGTP_1是指同时具有阻断修饰和荧光修饰的鸟嘌呤核苷酸，dATP_1是指同时具有阻断修饰和荧光修饰的腺嘌呤核苷酸，dTTP_1是指同时具有阻断修饰和荧光修饰的胸腺嘧啶核苷酸。
(3)按照表2配方配置dNTP 2混合液，即第二类核苷酸；本例的第二类核苷酸，如图1所示，核苷酸上仅具有阻断基团，没有荧光染料，其中，A,T,G,C分别表示腺嘌呤核苷酸、胸腺嘧啶核苷酸、鸟嘌呤核苷酸、胞嘧啶核苷酸。
表2 dNTP 2混合液配方表
dNTP 2混合液 终浓度(nmol/L)
dCTP_2 100
dGTP_2 100
dATP_2 100
dTTP_2 100
表中，dCTP_2是指仅具有阻断修饰的胞嘧啶核苷酸，dGTP_2是指仅具有阻断修饰的鸟嘌呤核苷酸，dATP_2是指仅具有阻断修饰的腺嘌呤核苷酸，dTTP_2是指仅具有阻断修饰的胸腺嘧啶核苷酸。
(4)利用BGISEQ-500平台，比较增加dNTP 2混合液来实现补充反应的 效果，再保持其他试剂不变的情况下，取出试剂盒的#5和#6号孔位的试剂，更换成实验组需要用到的试剂；
(5)按照表3进行合成试剂1的配制，混合均匀后备用；
表3合成试剂1配方表
合成试剂1 用量(mL)
反应缓冲液 48
DNA聚合酶 1
dNTP 1混合液 1
(6)按照表4进行合成试剂2的配制，混合均匀后备用；
表4合成试剂2配方表
合成试剂2 用量(mL)
反应缓冲液 48
DNA聚合酶 1
dNTP 2混合液 1
(7)在对照组的试剂盒#5孔位和#6孔位分别加入等量的合成试剂1；在实验组的试剂盒#5孔位加入合成试剂1，#6孔位加入合成试剂2，两种试剂的用量比例为1:1。
(8)使用BGISEQ-500平台进行SE50的测序，添加合成试剂1的试验组作为对照组，添加合成试剂2的试验组作为实验组，分别统计分析两组试验中每个循环的荧光信号变化曲线、每个循环的测序质量变化曲线以及每个循环的测序错误率变化曲线。
测序结果如图3至图5所示。其中，每个循环的荧光信号变化曲线如图3所示，图中，横坐标是测序读长，纵坐标是测序信号，“○”为实验组的曲线，“▲”为对照组的曲线；结果显示，随着测序循环的增加，测序的信号慢慢降低，实验组的荧光信号降低速度比对照组要慢，说明测序合成和切除更充分。
每个循环的测序质量变化曲线如图4所示，图中，横坐标是测序读长，纵坐标是测序质量值，“○”为实验组的曲线，“▲”为对照组的曲线；结果显示，随着测序循环的增加，测序的质量降低，实验组的测序质量值降低速度比对照组要慢，说明实验组的测序质量更高。
每个循环的测序错误率变化曲线如图5所示，图中，横坐标是测序读长， 纵坐标是测序错误率，“○”为实验组的曲线，“▲”为对照组的曲线；结果显示，随着测序循环的增加，测序错误率开始增加，实验组的错误率增加速度比对照组要慢，说明实验组错误率更低。
可见，在聚合测序的反应液中添加本申请的仅仅具有阻断修饰的dNTPs，即第二类核苷酸，能够有效的提高测序质量，降低测序错误率。
以上内容是结合具体的实施方式对本申请所作的进一步详细说明，不能认定本申请的具体实施只局限于这些说明。对于本申请所属技术领域的普通技术人员来说，在不脱离本申请构思的前提下，还可以做出若干简单推演或替换，都应当视为属于本申请的保护范围。

Claims (9)

  1. 一种提高核酸聚合测序质量的方法，其特征在于：包括在聚合测序的反应液中加入第二类核苷酸，所述第二类核苷酸的3’糖羟基具有阻断修饰但没有荧光修饰。
  2. 根据权利要求1所述的方法，其特征在于：所述第二类核苷酸的用量，与反应液中第一类核苷酸的用量相同，所述第一类核苷酸为3’糖羟基同时具有阻断修饰和荧光修饰的核苷酸。
  3. 根据权利要求1或2所述的方法在核酸测序中的应用。
  4. 根据权利要求3所述的核酸包含DNA或RNA。
  5. 根据权利要求1或2所述的方法在人类基因组、动物、植物和/或微生物核酸测序中的应用。
  6. 一种核酸测序方法，其特征在于：包括在边合成边测序的过程中，于其反应液中同时添加两类核苷酸，第一核苷酸为3’糖羟基同时具有阻断修饰和荧光修饰的核苷酸，第二类核苷酸为3’糖羟基仅具有阻断修饰但没有荧光修饰的核苷酸。
  7. 一种提高核酸测序质量的反应体系，包括用于边合成边测序的反应液，其特征在于：所述反应液中添加有第二类核苷酸，所述第二类核苷酸的3’糖羟基具有阻断修饰但没有荧光修饰。
  8. 根据权利要求7所述的反应体系，其特征在于：所述反应液包括反应缓冲液、DNA聚合酶和第一类核苷酸，所述第一类核苷酸为3’糖羟基同时具有阻断修饰和荧光修饰的核苷酸。
  9. 一种含有权利要求7或8所述的反应体系的核酸测序试剂盒。
PCT/CN2017/070711 2017-01-10 2017-01-10 一种提高核酸聚合测序质量的方法、反应体系和试剂盒 Ceased WO2018129644A1 (zh)

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