WO2018107350A1 - Method for purifying reversibly blocked deoxyribonucleotide triphosphate and sequencing method - Google Patents

Abstract

Disclosed in the present invention are a method for purifying a reversibly blocked deoxyribonucleotide triphosphate and a sequencing method, the method for purifying a reversibly blocked deoxyribonucleotide triphosphate comprising: using an adenylate cyclase to treat a 3' reversibly blocked dATP, and/or using guanylate cyclase to treat a 3' reversibly blocked dGTP. When used in sequencing, a reversibly blocked dNTP which has been treated may lead to lower phase shifting, especially for a pre-determined phase, so as to improve sequencing quality.

Description

Method and sequencing method for purifying reversible blocking of deoxyribonucleoside triphosphate Technical field

The invention relates to the field of sequencing technology, in particular to a method and a sequencing method for purifying reversible blocking deoxyribonucleoside triphosphate.

Background technique

In recent years, in the field of gene sequencing technology, compared with other sequencing methods, sequencing by synthesis (SBS) sequencing method has been favored for its high throughput and low price. In this sequencing method, 3' reversible blocking of dNTPs is the center of sequencing and is the most important and most important factor in sequencing quality. Upon addition of each 3' reversibly blocked dNTP, 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. However, if a very small amount of 3' blocking group is detached due to steps such as synthesis or storage, the phase phasing (Phasing) and the predetermined phase (Prephasing) are increased. The so-called "Phasing" refers to the SBS. Phenomenon, this phenomenon is caused by incomplete removal of the 3' blocking group and the fluorophore, and a portion of the DNA strand cannot be fully incorporated into the synthetic strand by the polymerase in a given sequencing cycle. "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.

In order to increase the stability of the 3' blocking group, 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. In general, 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.

Summary of the invention

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. .

According to a first aspect of the present invention, there is provided a method of purifying reversibly blocking deoxyribonucleoside triphosphate, the method comprising: treating a 3' reversibly blocked dATP with adenylate cyclase, and/or using Guanylate cyclase treats 3' reversibly blocked dGTP.

Further, the above treatment was carried out at 37 °C.

Further, the above processing time is 10 minutes or more.

Further, the above treatment was carried out in 200 mM Tris buffer containing 10 mM Mg ²⁺ and 100 mM NaCl.

Further, the above method further comprises: filtering the treated product to remove at least a portion of the protein.

Further, the above protein includes a protein having a molecular weight of more than 10,000.

According to a second aspect of the present invention, 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.

Further, 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.

Further, the above treatment was carried out at 37 °C.

Further, 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. In addition, 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.

DRAWINGS

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.

detailed description

The present invention will be further described in detail below with reference to the accompanying drawings.

In the 3' reversibly blocked dNTPs, most of the dNTPs carry a 3' blocking group, however, there are still a small number of dNTPs without a 3' blocking group. These unblocked dNTPs will result in different sequencing results. The effect of the degree, the higher the unblocked concentration, the faster the phase shift, and the worse the sequencing result.

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.

As shown in Figure 1, dATP (deoxyadenosine triphosphate) with a 3' blocking group cannot undergo cyclization under the action of adenylate cyclase; without 3' blocking The dATP of the group undergoes cyclization to form a circularized deoxyadenosine (cdAMP) under the action of adenylate cyclase.

As shown in Figure 2, dGTP (deoxyguanosine triphosphate) with a 3' blocking group cannot undergo cyclization under the action of guanylate cyclase; without 3' blocking The dGTP of the group undergoes cyclization to form cyclized deoxyguanosine monophosphate (cdGMP) under the action of guanylate cyclase.

In an embodiment of the invention, 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.

In the embodiment 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.

In the embodiment of the present invention, 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. An enzyme for acid cyclase or guanylate cyclase activity, and any enzyme having adenylate cyclase or guanylate cyclase activity and having other enzyme activities. That is, in the present invention, adenylate cyclase or guanylate cyclase adopts a broad concept.

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.

In the examples of the present invention, adenylate cyclase or guanylate cyclase is reacted at respective suitable enzyme reaction temperatures, preferably at respective optimum reaction temperatures. In one embodiment of the present invention, 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.

In the embodiment of the present invention, the enzyme reaction buffer may be any buffer suitable for adenylate cyclase or guanylate cyclase reaction. In one embodiment of the present invention, 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.

In an embodiment of the invention, preferably, 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.

In one embodiment of the invention, 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.

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.

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. In addition, 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 technical solutions and effects of the present invention are described in detail below by way of examples. It should be understood that the embodiments are not intended to limit the scope of the invention.

Example 1 Expression and purification of guanylate cyclase

Guanylate cyclase use of DNA 2.0 Electra ^TM Cloning Reagents Kit Kit Construction of expression vector for the His-tag using Ni-column affinity purification.

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.

Example 2 Activity detection of guanylate cyclase

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.

Table 1

Component volume 10× reaction buffer 2.5μl 10mM dGTP 2.5μl

Properly diluted enzyme solution 1μl H ₂ O Up to 25μl

After incubation at 37 ° C for 10 min, the reaction was stopped by 10 mM EDTA, and the mixture was assayed for the yield of dGTP converted to cyclic dGTP (guanine monophosphate nucleotide) by HPLC.

The results showed that the guanylate cyclase was cyclized by the above detection method at 37 ° C for 10 min to circulate the amount of dGTP more than 10 nmol. It indicates that guanylate cyclase activity is normal.

Example 3 Treatment of 3' reversibly blocked dGTP by guanylate cyclase and 3' reversible blocking of dGTP after sequencing

The 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.

Table 2

platform Unprocessed predetermined phase value Processed predetermined phase value BGISEQ-500 0.11 0.07 HISEQ-2500 0.15 0.10

Conclusion: 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.

The above is a further detailed description of the present invention in connection with the specific embodiments, and the specific embodiments of the present invention are not limited to the description. It will be apparent to those skilled in the art that the present invention may be made without departing from the spirit and scope of the invention.

Claims (10)

A method for purifying reversibly blocking deoxyribonucleoside triphosphate, the method comprising: treating a 3' reversibly blocked dATP with adenylate cyclase, and/or using guanylate cyclase The 3' reversibly blocked dGTP was processed. The method of purifying reversibly blocking deoxyribonucleoside triphosphate according to claim 1, wherein the treatment is carried out at 37 °C. The method of purifying reversibly blocking deoxyribonucleoside triphosphate according to claim 2, wherein the treatment time is 10 minutes or more. The method for purifying reversibly blocking deoxyribonucleoside triphosphate according to any one of claims 1 to 3, wherein the treatment is carried out in 200 mM Tris buffer containing 10 mM Mg ²⁺ and 100 mM NaCl. The method of purifying reversibly blocking deoxyribonucleoside triphosphate according to any one of claims 1 to 3, wherein the method further comprises: filtering the treated product to remove at least a portion of the protein. The method of purifying reversibly blocking deoxyribonucleoside triphosphate according to claim 5, wherein the protein comprises a protein having a molecular weight of more than 10,000. A sequencing method, characterized in that the method is a side synthesis sequencing method comprising adding a reversible blocking deoxyribonucleoside triphosphate to a synthetic chain, wherein the reversible blocking of deoxyribonucleoside triphosphates is included 3' reversibly blocked dATP treated with adenylate cyclase, and/or 3' reversibly blocked dGTP treated with guanylate cyclase. The sequencing method according to claim 7, wherein the method further comprises treating the 3' reversible blocking with adenylate cyclase prior to adding the reversible blocking deoxyribonucleoside triphosphate to the synthetic strand. dATP, and/or treatment of 3' reversibly blocked dGTP using guanylate cyclase. The sequencing method according to claim 8, wherein the treatment is performed at 37 °C. The sequencing method according to claim 8 or 9, further comprising: after processing The product is filtered to remove at least a portion of the protein.

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