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WO2018107350A1 - 提纯可逆阻断脱氧核糖核苷三磷酸的方法和测序方法 - Google Patents

提纯可逆阻断脱氧核糖核苷三磷酸的方法和测序方法 Download PDF

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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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sequencing
blocking
reversibly
purifying
reversibly blocked
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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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Priority to CN201680090626.3A priority Critical patent/CN109983136B/zh
Priority to PCT/CN2016/109597 priority patent/WO2018107350A1/zh
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    • 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 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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Abstract

本发明公开了一种提纯可逆阻断脱氧核糖核苷三磷酸的方法和测序方法，其中提纯可逆阻断脱氧核糖核苷三磷酸的方法包括：使用腺苷酸环化酶处理3'可逆阻断的dATP，和/或使用鸟苷酸环化酶处理3'可逆阻断的dGTP。经过处理的可逆阻断dNTP用于测序，能够带来更低的移相，尤其是预定相，提高了测序质量。

Description

提纯可逆阻断脱氧核糖核苷三磷酸的方法和测序方法 技术领域
本发明涉及测序技术领域，尤其涉及一种提纯可逆阻断脱氧核糖核苷三磷酸的方法和测序方法。
背景技术
近年来，在基因测序技术领域，与其它测序方法相比，边合成边测序(sequencing by synthesis,SBS)的测序方法以其通量高、价格低而获得青睐。在这一测序方法中，3′可逆阻断的dNTP是测序的中心也是测序成本最主要和最影响测序质量的一环。在加入每个3′可逆阻断的dNTP后，3’阻断基团的存在阻止其它核苷酸加入合成的链中。在去除阻断基团后，恢复天然游离的3’羟基基团用于加入下一个核苷酸。但是如果由于合成或者存储等步骤导致非常少量的3′阻断基团脱落，就会增加测序的定相(Phasing)和预定相(Prephasing)，所谓“定相(Phasing)”是指SBS中的现象，该现象由3’阻断基团和荧光基团的不完全除去引起，不能通过聚合酶在给定的测序循环中将DNA链的一部分完全并入到合成链中。“预定相(Prephasing)”是由在不存在有效的3’阻断基团的情况下，核苷酸并入所引起的，并且该并入事件预先进行了1个循环。这使测序过程移相发生的更严重、更快，从而降低读长，增加错误率，降低测序质量。这在不同的测序平台上都有体现。
为了提高3’阻断基团的稳定性，Illumina公司在专利申请WO2014139596A1中对阻断基团做了很多改性，利用其测序平台的定相和预定相来评判阻断基团的优劣。一般，可逆阻断的dNTP在合成之后，用高效液相色谱(HPLC)进行纯化，以及用核磁共振、质谱等表征其纯度。HPLC纯化可逆阻断的dNTP存在以下不足：依靠极性分离阻断的dNTP和未阻断的dNTP，差异太小，分离困难； 即使能分离开，在含有痕量未阻断的dNTP时，效率也较低而不能有效去除。
未阻断的dNTP来源包括可逆阻断的dNTP降解和合成步骤中引入两种可能性。不管哪一种在纯化步骤中如果未能得到完全的分离，测序的结果将会受到不同程度的影响，未阻断的浓度越高移相越快，测序结果越差。
发明内容
本发明提供一种提纯可逆阻断脱氧核糖核苷三磷酸的方法和测序方法，经过处理的可逆阻断dNTP用于测序，能够带来更低的移相，尤其是预定相，提高了测序质量。
根据本发明的第一方面，本发明提供一种提纯可逆阻断脱氧核糖核苷三磷酸的方法，该方法包括：使用腺苷酸环化酶处理3′可逆阻断的dATP，和/或使用鸟苷酸环化酶处理3′可逆阻断的dGTP。
进一步地，在37℃下进行上述处理。
进一步地，上述处理时间是10分钟以上。
进一步地，在含有10mM Mg2+和100mM NaCl的200mM Tris缓冲液中进行上述处理。
进一步地，上述方法还包括：对处理后的产物进行过滤以去除至少部分蛋白。
进一步地，上述蛋白包括分子量大于1万的蛋白。
根据本发明的第二方面，本发明提供一种测序方法，该方法是边合成边测序方法，包括使可逆阻断脱氧核糖核苷三磷酸加入合成的链中，其中上述可逆阻断脱氧核糖核苷三磷酸中包括使用腺苷酸环化酶处理过的3′可逆阻断的dATP，和/或使用鸟苷酸环化酶处理过的3′可逆阻断的dGTP。
进一步地，上述方法还包括在使可逆阻断脱氧核糖核苷三磷酸加入合成的 链中之前，使用腺苷酸环化酶处理3′可逆阻断的dATP，和/或使用鸟苷酸环化酶处理3′可逆阻断的dGTP。
进一步地，在37℃下进行上述处理。
进一步地，还包括：对处理后的产物进行过滤以去除至少部分蛋白。
本发明将环化酶(腺苷酸环化酶和鸟苷酸环化酶)应用于测序技术领域，用于提纯可逆阻断脱氧核糖核苷三磷酸。由于环化酶只能使用未阻断的dNTP，有非常高的特异性，能够使未阻断的dNTP在总dNTP的比例更低，甚至常规手段无法检测。另外，本发明在环化酶处理之后，仅需要过滤蛋白即可将dNTP用于测序，环化的脱氧核糖核苷(cdNMP)并不对测序产生任何影响。
附图说明
图1是阻断和非阻断的dATP在腺苷酸环化酶处理后的反应结果，其中Block表示3′阻断基团。
图2是阻断和非阻断的dGTP在鸟苷酸环化酶处理后的反应结果，其中Block表示3′阻断基团。
具体实施方式
下面通过具体实施方式结合附图对本发明作进一步详细说明。
在3′可逆阻断的dNTP中，大部分dNTP都带有3′阻断基团，然而仍有少量的dNTP不带3′阻断基团，这些未阻断的dNTP会使测序结果受到不同程度的影响，未阻断的浓度越高移相越快，测序结果越差。
本发明实施例的提纯可逆阻断脱氧核糖核苷三磷酸的方法，其基本方案是：使用腺苷酸环化酶处理3′可逆阻断的dATP，和/或使用鸟苷酸环化酶处理3′可逆阻断的dGTP。
如图1所示，带有3′阻断基团(Block)的dATP(三磷酸脱氧腺苷)在腺苷酸环化酶的作用下，不能发生环化反应；而不带3′阻断基团的dATP在腺苷酸环化酶的作用下，发生环化生成环化的脱氧腺苷酸(cdAMP)。
如图2所示，带有3′阻断基团(Block)的dGTP(三磷酸脱氧鸟苷)在鸟苷酸环化酶的作用下，不能发生环化反应；而不带3′阻断基团的dGTP在鸟苷酸环化酶的作用下，发生环化生成环化的脱氧鸟苷酸(cdGMP)。
本发明实施例中，阻断基团可以是任何本领域中用于阻断dNTP的3′羟基的基团，典型单非限定性的包括叠氮亚甲基。其它的可用阻断基团还包括例如国际申请WO2014139596A1中公开的那些阻断基团。本发明对阻断基团的种类没有特别限定，根据本发明的原理，任何本领域中用于阻断dNTP的3′羟基的基团都可以作为本发明中的阻断基团。
本发明实施例中，待纯化的3′可逆阻断的dATP或3′可逆阻断的dGTP，可以是HPLC纯化过的，也可以是采用其它方法纯化过的，甚至是合成后尚未纯化的，本发明对此没有特别限定。
本发明实施例中，腺苷酸环化酶或鸟苷酸环化酶，可以是市售的酶，也可以是通过酶工程等方法获得的酶，并且也包括任何改造过的仍然具有腺苷酸环化酶或鸟苷酸环化酶活性的酶，还包括任何具有腺苷酸环化酶或鸟苷酸环化酶活性同时具有其它酶活性的酶。就是说，在本发明中，腺苷酸环化酶或鸟苷酸环化酶采用广义的概念。
本发明方法产生的环化的脱氧腺苷酸和脱氧鸟苷酸不能在SBS测序中并入合成的链中，因此有效地降低了测序过程中的预定相现象。同时，环化的脱氧腺苷酸和脱氧鸟苷酸也不会对测序过程造成负面影响。
本发明实施例中，腺苷酸环化酶或鸟苷酸环化酶在各自适宜的酶反应温度下进行反应，优选在各自的最适反应温度下进行反应。在本发明的一个实施例中，在37℃下进行反应，反应时间可以是1分钟以上，优选10分钟以上，例 如20分钟、30分钟、60分钟等。
本发明实施例中，酶反应缓冲液可以是任何适于腺苷酸环化酶或鸟苷酸环化酶反应的缓冲液。在本发明的一个实施例中，在含有10mM Mg2+和100mM NaCl的200mM Tris缓冲液中进行反应，能取得明显优异的效果。
本发明实施例中，优选地，对处理后的产物进行过滤以去除至少部分蛋白，尤其是分子量大于1万的蛋白。过滤处理可以使用合适的滤膜。通过过滤处理去除大分子蛋白，能够有效避免大分子蛋白对后续测序反应的影响。通过过滤处理后的产物可以直接用于测序反应中，当然也可以根据需要进行合适的稀释或浓缩，甚至是其它纯化处理等。
在本发明的一个实施例中，腺苷酸环化酶(ADCY2)在大肠杆菌中合成并提纯表征之后，在含有10mM Mg2+和100mM NaCl的200mM Tris缓冲液中，加入要纯化的3′可逆阻断的dATP，加入腺苷酸环化酶，37℃孵育一定时间，过滤分子量大于1万的蛋白，通过测序平台验证腺苷酸环化酶处理的3′可逆阻断的dATP有更低的预定相。调节Mg2+的浓度和腺苷酸环化酶的浓度以改变dATP的Km值能够达到更好的纯化效果。
本发明实施例还提供一种测序方法，该方法是边合成边测序方法，包括使可逆阻断脱氧核糖核苷三磷酸加入合成的链中，其中可逆阻断脱氧核糖核苷三磷酸中包括使用腺苷酸环化酶处理过的3′可逆阻断的dATP，和/或使用鸟苷酸环化酶处理过的3′可逆阻断的dGTP。
边合成边测序方法，是测序领域中已知的测序方法，本发明不在赘述。本发明与现有边合成边测序方法的关键不同就在于，本发明使用腺苷酸环化酶处理过的3′可逆阻断的dATP，和/或使用鸟苷酸环化酶处理过的3′可逆阻断的dGTP，代替或者至少部分代替现有的3′可逆阻断的dATP和/或3′可逆阻断的dGTP，尤其是仅经过HPLC纯化的3′可逆阻断的dATP和/或3′可逆阻断的dGTP。
本发明实施例的测序方法中使用的3′可逆阻断的dATP和/或3′可逆阻断的 dGTP可以是预先处理备用的，也可以是在测序之前临时制备的。在临时制备的情况下，本发明实施例的测序方法，可以认为还包括：在使可逆阻断脱氧核糖核苷三磷酸加入合成的链中之前，使用腺苷酸环化酶处理3′可逆阻断的dATP，和/或使用鸟苷酸环化酶处理3′可逆阻断的dGTP。
本发明将环化酶(腺苷酸环化酶和鸟苷酸环化酶)应用于测序技术领域，用于提纯可逆阻断脱氧核糖核苷三磷酸。由于环化酶只能使用未阻断的dNTP，有非常高的特异性，能够使未阻断的dNTP在总dNTP的比例更低，甚至常规手段无法检测。另外，本发明在环化酶处理之后，仅需要过滤蛋白即可将dNTP用于测序，环化的脱氧核糖核苷(cdNMP)并不对测序产生任何影响。
以下通过实施例详细说明本发明的技术方案和效果，应当理解，实施例意在用于示例性的说明本发明，并不构成对本发明保护范围的限制。
实施例1鸟苷酸环化酶的表达纯化
鸟苷酸环化酶采用DNA 2.0的ElectraTMCloning Reagents Kit试剂盒进行表达载体的构建，利用His标签进行Ni柱亲和纯化。
纯化后的鸟苷酸环化酶进行相关的纯度检测，利用浓缩胶为5％、分离胶为12％的SDS-PAGE进行电泳检测。纯化后的鸟苷酸环化酶纯度能达到95％以上。
实施例2鸟苷酸环化酶的活性检测
鸟苷酸环化酶活性检测采用检测产物HPLC检测，定义酶与底物于37℃反应10min环化10nmol dGTP为一个酶活单位，且聚合活性达标。
于96孔板中按下表1加入反应液。
表1
组分 体积
10×反应缓冲液 2.5μl
10mM dGTP 2.5μl
适当稀释的酶液 1μl
H2O 至25μl
37℃孵育10min，10mM EDTA终止反应后，混匀，利用HPLC测定其中dGTP转化为环状dGTP(鸟嘌呤单磷酸核苷酸)的产率。
结果显示：鸟苷酸环化酶利用上述检测方法其在37℃反应10min环化dGTP的量大于10nmol。表明鸟苷酸环化酶活性正常。
实施例3鸟苷酸环化酶处理3′可逆阻断的dGTP以及处理后3′可逆阻断的dGTP在测序中的表现
对3′可逆阻断的dGTP进行鸟苷酸环化酶处理，在一定量的3′可逆阻断的dGTP中加入鸟苷酸环化酶缓冲液(含有10mM Mg2+和100mM NaCl的200mM Tris缓冲液)和鸟苷酸环化酶，37℃孵育10分钟，分别将处理过的和未处理过的3′可逆阻断的dGTP配置成BGISEQ-500或者Illumina测序平台的测序试剂，测序20个碱基的长度，对比其中鸟嘌呤的预定相值。如表2所示。
表2
平台 未处理的预定相值 处理过的预定相值
BGISEQ-500 0.11 0.07
HISEQ-2500 0.15 0.10
结论：鸟苷酸环化酶能降低测序试剂中由3′可逆阻断的dGTP未完全阻断所带来的预定相，从而提高测序质量。
以上内容是结合具体的实施方式对本发明所作的进一步详细说明，不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干简单推演或替换，都应当视为属于本发明的保护范围。

Claims (10)

  1. 一种提纯可逆阻断脱氧核糖核苷三磷酸的方法，其特征在于，所述方法包括：使用腺苷酸环化酶处理3′可逆阻断的dATP，和/或使用鸟苷酸环化酶处理3′可逆阻断的dGTP。
  2. 根据权利要求1所述的提纯可逆阻断脱氧核糖核苷三磷酸的方法，其特征在于，在37℃下进行所述处理。
  3. 根据权利要求2所述的提纯可逆阻断脱氧核糖核苷三磷酸的方法，其特征在于，所述处理时间是10分钟以上。
  4. 根据权利要求1-3任一项所述的提纯可逆阻断脱氧核糖核苷三磷酸的方法，其特征在于，在含有10mM Mg2+和100mM NaCl的200mM Tris缓冲液中进行所述处理。
  5. 根据权利要求1-3任一项所述的提纯可逆阻断脱氧核糖核苷三磷酸的方法，其特征在于，所述方法还包括：对处理后的产物进行过滤以去除至少部分蛋白。
  6. 根据权利要求5所述的提纯可逆阻断脱氧核糖核苷三磷酸的方法，其特征在于，所述蛋白包括分子量大于1万的蛋白。
  7. 一种测序方法，其特征在于，所述方法是边合成边测序方法，包括使可逆阻断脱氧核糖核苷三磷酸加入合成的链中，其中所述可逆阻断脱氧核糖核苷三磷酸中包括使用腺苷酸环化酶处理过的3′可逆阻断的dATP，和/或使用鸟苷酸环化酶处理过的3′可逆阻断的dGTP。
  8. 根据权利要求7所述的测序方法，其特征在于，所述方法还包括在使可逆阻断脱氧核糖核苷三磷酸加入合成的链中之前，使用腺苷酸环化酶处理3′可逆阻断的dATP，和/或使用鸟苷酸环化酶处理3′可逆阻断的dGTP。
  9. 根据权利要求8所述的测序方法，其特征在于，在37℃下进行所述处理。
  10. 根据权利要求8或9所述的测序方法，其特征在于，还包括：对处理后 的产物进行过滤以去除至少部分蛋白。
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