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WO2018107350A1 - Procédé de purification de triphosphate de désoxyribonucléotide bloqué de manière réversible et procédé de séquençage - Google Patents

Procédé de purification de triphosphate de désoxyribonucléotide bloqué de manière réversible et procédé de séquençage Download PDF

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Publication number
WO2018107350A1
WO2018107350A1 PCT/CN2016/109597 CN2016109597W WO2018107350A1 WO 2018107350 A1 WO2018107350 A1 WO 2018107350A1 CN 2016109597 W CN2016109597 W CN 2016109597W WO 2018107350 A1 WO2018107350 A1 WO 2018107350A1
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Prior art keywords
sequencing
blocking
reversibly
purifying
reversibly blocked
Prior art date
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Ceased
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PCT/CN2016/109597
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English (en)
Chinese (zh)
Inventor
刘二凯
徐崇钧
章文蔚
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BGI Shenzhen Co Ltd
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BGI Shenzhen Co Ltd
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Application filed by BGI Shenzhen Co Ltd filed Critical BGI Shenzhen Co Ltd
Priority to CN201680090626.3A priority Critical patent/CN109983136B/zh
Priority to PCT/CN2016/109597 priority patent/WO2018107350A1/fr
Publication of WO2018107350A1 publication Critical patent/WO2018107350A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Definitions

  • the invention relates to the field of sequencing technology, in particular to a method and a sequencing method for purifying reversible blocking deoxyribonucleoside triphosphate.
  • sequencing by synthesis (SBS) sequencing method has been favored for its high throughput and low price.
  • SBS sequencing by synthesis
  • 3' reversible blocking of dNTPs is the center of sequencing and is the most important and most important factor in sequencing quality.
  • the presence of the 3' blocking group prevents additional nucleotides from being added to the synthetic strand. After removal of the blocking group, the naturally free 3' hydroxyl group is restored for the addition of the next nucleotide.
  • phase phasing Phase phasing
  • Prephasing the phase phasing
  • SBS SBS
  • Prephasing was caused by nucleotide incorporation in the absence of a potent 3' blocking group, and the incorporation event was performed in advance for 1 cycle. This makes the phase shift of the sequencing process more serious and faster, thereby reducing read length, increasing error rate, and reducing sequencing quality. This is reflected in different sequencing platforms.
  • Illumina has made a number of modifications to the blocking group in the patent application WO2014139596A1, using the phasing and predetermined phases of its sequencing platform to judge the advantages and disadvantages of the blocking group.
  • reversibly blocked dNTPs are purified by high performance liquid chromatography (HPLC) after synthesis, and their purity is characterized by nuclear magnetic resonance, mass spectrometry or the like.
  • HPLC purification of reversibly blocked dNTPs has the following disadvantages: dNTPs blocked by polar separation and unblocked dNTPs, the difference is too small, and separation is difficult; Even if it can be separated, it has low efficiency and cannot be effectively removed when it contains traces of unblocked dNTPs.
  • Unblocked dNTP sources include reversible blocking of dNTP degradation and the introduction of two possibilities in the synthesis step. Regardless of which one does not result in complete separation during the purification step, the sequencing results will be affected to varying degrees. The higher the unblocked concentration, the faster the phase shift and the worse the sequencing results.
  • the invention provides a method and a sequencing method for purifying reversible blocking deoxyribonucleoside triphosphate, and the processed reversible blocking dNTP is used for sequencing, which can bring lower phase shift, especially a predetermined phase, and improve sequencing quality. .
  • a method of purifying reversibly blocking deoxyribonucleoside triphosphate comprising: treating a 3' reversibly blocked dATP with adenylate cyclase, and/or using Guanylate cyclase treats 3' reversibly blocked dGTP.
  • the above processing time is 10 minutes or more.
  • the above method further comprises: filtering the treated product to remove at least a portion of the protein.
  • the above protein includes a protein having a molecular weight of more than 10,000.
  • the present invention provides a sequencing method which is a synthetic side sequencing method comprising adding a reversible blocking deoxyribonucleoside triphosphate to a synthetic chain, wherein the reversible blocking of a deoxyribose nucleus Among the glycosides is a 3' reversibly blocked dATP treated with adenylate cyclase, and/or a 3' reversibly blocked dGTP treated with guanylate cyclase.
  • the above method further comprises adding a reversible blocking deoxyribonucleoside triphosphate to the synthetic Prior to the chain, 3' reversibly blocked dATP was treated with adenylate cyclase, and/or 3' reversibly blocked dGTP was treated with guanylate cyclase.
  • the method further comprises: filtering the treated product to remove at least a portion of the protein.
  • the invention applies cyclase (adenylate cyclase and guanylate cyclase) to the field of sequencing technology for purifying reversible blocking of deoxyribonucleoside triphosphate. Since cyclase can only use unblocked dNTPs, it has a very high specificity, which enables the ratio of unblocked dNTPs to be lower in total dNTPs, even by conventional means.
  • the present invention can be used for sequencing after the cyclase treatment, only the filter protein is required, and the cyclized deoxyribonucleoside (cdNMP) does not have any influence on sequencing.
  • Figure 1 is the result of the reaction of blocked and non-blocked dATP after adenylyl cyclase treatment, wherein Block represents a 3' blocking group.
  • Figure 2 is the result of the reaction of blocked and non-blocked dGTP after guanylate cyclase treatment, wherein Block represents a 3' blocking group.
  • a method for purifying a reversible blocking deoxyribonucleoside triphosphate according to an embodiment of the present invention, wherein the basic scheme is: treating a 3' reversibly blocked dATP with adenylate cyclase, and/or treating with guanylate cyclase 3' reversible blocking of dGTP.
  • dATP deoxyadenosine triphosphate
  • cdAMP circularized deoxyadenosine
  • dGTP deoxyguanosine triphosphate
  • cdGMP cyclized deoxyguanosine monophosphate
  • the blocking group can be any group in the art for blocking the 3' hydroxyl group of a dNTP, typically a non-limiting one comprising an azide methylene group.
  • Other useful blocking groups also include those blocking groups such as those disclosed in international application WO 2014139596 A1.
  • the type of the blocking group in the present invention is not particularly limited, and any group in the art for blocking the 3' hydroxyl group of the dNTP can be used as a blocking group in the present invention according to the principle of the present invention.
  • the 3′ reversibly blocked dATP or the 3′ reversibly blocked dGTP to be purified may be purified by HPLC, or may be purified by other methods, or even after purification.
  • the present invention is not particularly limited thereto.
  • the adenylate cyclase or the guanylate cyclase may be a commercially available enzyme, or an enzyme obtained by an enzyme engineering method or the like, and also includes any modified adenosine.
  • the cyclized deoxyadenylate and deoxyguanosine produced by the method of the present invention cannot be incorporated into the synthetic strand in SBS sequencing, thus effectively reducing the predetermined phase phenomenon during sequencing. At the same time, the cyclized deoxyadenylate and deoxyguanosine do not adversely affect the sequencing process.
  • adenylate cyclase or guanylate cyclase is reacted at respective suitable enzyme reaction temperatures, preferably at respective optimum reaction temperatures.
  • the reaction is carried out at 37 ° C, and the reaction time may be 1 minute or longer, preferably 10 minutes or longer. Such as 20 minutes, 30 minutes, 60 minutes, and so on.
  • the enzyme reaction buffer may be any buffer suitable for adenylate cyclase or guanylate cyclase reaction.
  • the reaction was carried out in a 200 mM Tris buffer containing 10 mM Mg 2+ and 100 mM NaCl, and a remarkable excellent effect was obtained.
  • the treated product is filtered to remove at least a portion of the protein, particularly a protein having a molecular weight greater than 10,000.
  • a suitable filter can be used for the filtration treatment. Removal of macromolecular proteins by filtration can effectively avoid the influence of macromolecular proteins on subsequent sequencing reactions.
  • the product after filtration can be directly used in the sequencing reaction, and of course, it can be appropriately diluted or concentrated as needed, or even other purification treatments.
  • adenylate cyclase (ADCY2) is synthesized and purified in E. coli and then added to a 3' to be purified in 200 mM Tris buffer containing 10 mM Mg 2+ and 100 mM NaCl. Reversible blocking of dATP, addition of adenylate cyclase, incubation at 37 ° C for a certain period of time, filtering protein with molecular weight greater than 10,000, verified by sequencing platform that adenylate cyclase treatment of 3' reversible blocking dATP is lower Scheduled phase. Adjusting the concentration of Mg 2+ and the concentration of adenylate cyclase to change the K m value of dATP can achieve a better purification effect.
  • ADCY2 adenylate cyclase
  • the embodiment of the invention further provides a sequencing method, which is a synthetic side sequencing method, comprising: reversibly blocking deoxyribonucleoside triphosphate into a synthetic chain, wherein reversible blocking of deoxyribonucleoside triphosphate includes use Adenylate cyclase treated 3' reversibly blocked dATP, and/or 3' reversibly blocked dGTP treated with guanylate cyclase.
  • a sequencing method which is a synthetic side sequencing method, comprising: reversibly blocking deoxyribonucleoside triphosphate into a synthetic chain, wherein reversible blocking of deoxyribonucleoside triphosphate includes use Adenylate cyclase treated 3' reversibly blocked dATP, and/or 3' reversibly blocked dGTP treated with guanylate cyclase.
  • the side-synthesis sequencing method is a sequencing method known in the field of sequencing, and the present invention is not described herein.
  • the key difference between the present invention and the existing side-synthesis sequencing method is that the present invention uses adenylate cyclase-treated 3' reversibly blocked dATP, and/or guanylate cyclase-treated 3 Reversible blocking of dGTP, replacing or at least partially replacing existing 3' reversibly blocked dATP and/or 3' reversibly blocked dGTP, especially 3' reversibly blocked dATP and/or HPLC-purified only 3' reversible blocking of dGTP.
  • 3' reversibly blocked dATP and/or 3' reversible blocking used in the sequencing method of the present invention The dGTP may be pre-treated or prepared temporarily prior to sequencing. In the case of temporary preparation, the sequencing method of the embodiment of the present invention may be considered to further include: treating the 3' reversible resistance with adenylate cyclase before adding the reversible blocking deoxyribonucleoside triphosphate to the synthetic strand. Broken dATP, and/or treatment of 3' reversibly blocked dGTP using guanylate cyclase.
  • the invention applies cyclase (adenylate cyclase and guanylate cyclase) to the field of sequencing technology for purifying reversible blocking of deoxyribonucleoside triphosphate. Since cyclase can only use unblocked dNTPs, it has a very high specificity, which enables the ratio of unblocked dNTPs to be lower in total dNTPs, even by conventional means.
  • the present invention can be used for sequencing after the cyclase treatment, only the filter protein is required, and the cyclized deoxyribonucleoside (cdNMP) does not have any influence on sequencing.
  • the purified guanylate cyclase was subjected to relevant purity detection, and electrophoresis was carried out by SDS-PAGE using a concentrated gel of 5% and a separation gel of 12%.
  • the purity of the purified guanylate cyclase can reach 95% or more.
  • the detection of guanylate cyclase activity was carried out by HPLC detection of the detection product.
  • the enzyme and substrate were reacted at 37 ° C for 10 min to cyclize 10 nmol dGTP into an enzyme unit, and the polymerization activity was up to standard.
  • the reaction solution was added in the following manner in Table 1 in a 96-well plate.
  • guanylate cyclase treatment was performed on the 3' reversibly blocked dGTP, and guanylate cyclase buffer (200 mM Tris containing 10 mM Mg 2+ and 100 mM NaCl) was added to a certain amount of 3' reversibly blocked dGTP. Buffer) and guanylate cyclase, incubated at 37 ° C for 10 minutes, respectively, the treated and untreated 3' reversibly blocked dGTP were configured as BGISEQ-500 or Illumina sequencing platform sequencing reagent, sequencing 20 The length of the base, compared to the predetermined phase value of the guanine. As shown in table 2.
  • Guanylate cyclase can reduce the quality of sequencing by reducing the predetermined phase caused by the incomplete blocking of 3' reversible blocking of dGTP in sequencing reagents.

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  • Chemical & Material Sciences (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne un procédé de purification d'un triphosphate de désoxyribonucléotide bloqué de manière réversible et un procédé de séquençage, le procédé de purification d'un triphosphate de désoxyribonucléotide bloqué de manière réversible consistant à : utiliser une adénylate cyclase pour traiter un dATP bloqué de manière réversible en 3' et/ou utiliser la guanylate cyclase pour traiter un dGTP bloqué de manière réversible en 3'. Lorsqu'il est utilisé dans le séquençage, un dNTP bloqué de manière réversible qui a été traité peut conduire à un décalage de phase inférieur, en particulier pendant une phase prédéterminée, de façon à améliorer la qualité de séquençage.
PCT/CN2016/109597 2016-12-13 2016-12-13 Procédé de purification de triphosphate de désoxyribonucléotide bloqué de manière réversible et procédé de séquençage Ceased WO2018107350A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN201680090626.3A CN109983136B (zh) 2016-12-13 2016-12-13 提纯可逆阻断脱氧核糖核苷三磷酸的方法和测序方法
PCT/CN2016/109597 WO2018107350A1 (fr) 2016-12-13 2016-12-13 Procédé de purification de triphosphate de désoxyribonucléotide bloqué de manière réversible et procédé de séquençage

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PCT/CN2016/109597 WO2018107350A1 (fr) 2016-12-13 2016-12-13 Procédé de purification de triphosphate de désoxyribonucléotide bloqué de manière réversible et procédé de séquençage

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022083686A1 (fr) * 2020-10-21 2022-04-28 深圳华大生命科学研究院 Nucléoside ou nucléotide modifié
US12031179B2 (en) 2020-10-30 2024-07-09 Singular Genomics Systems, Inc. Methods and compositions for reducing nucleotide impurities

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021104514A1 (fr) * 2019-11-29 2021-06-03 Bgi Shenzhen Co., Ltd. Synthèse enzymatique d'oligonucléotides

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101942000A (zh) * 2010-04-13 2011-01-12 深圳华因康基因科技有限公司 携带修饰物的核苷酸及其制备方法和用于基因测序的方法
WO2014139596A1 (fr) * 2013-03-15 2014-09-18 Illumina Cambridge Limited Nucléosides ou nucléotides modifiés

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Publication number Priority date Publication date Assignee Title
CN106202846B (zh) * 2015-04-30 2018-12-25 中国科学院青岛生物能源与过程研究所 口腔微生物群落检测模型的构建方法

Patent Citations (2)

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CN101942000A (zh) * 2010-04-13 2011-01-12 深圳华因康基因科技有限公司 携带修饰物的核苷酸及其制备方法和用于基因测序的方法
WO2014139596A1 (fr) * 2013-03-15 2014-09-18 Illumina Cambridge Limited Nucléosides ou nucléotides modifiés

Non-Patent Citations (1)

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Title
TANG, DAONIAN ET AL.: "Synthesis and Application of Four Fluorescence Labeled Nucleotides Through Disulfide as Reversible Terminators in DNA Sequencing by Synthesis", CHEMICAL JOURNAL OF CHINESE UNIVERSITIES, vol. 35, 30 November 2014 (2014-11-30), pages 2346 - 2352, XP055299457 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022083686A1 (fr) * 2020-10-21 2022-04-28 深圳华大生命科学研究院 Nucléoside ou nucléotide modifié
US12031179B2 (en) 2020-10-30 2024-07-09 Singular Genomics Systems, Inc. Methods and compositions for reducing nucleotide impurities
EP4200444A4 (fr) * 2020-10-30 2024-09-25 Singular Genomics Systems, Inc. Procédés et compositions pour réduire la teneur en impuretés nucléotidiques

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CN109983136A (zh) 2019-07-05

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