WO2022036862A1 - 一种氨解液及氨解方法 - Google Patents

一种氨解液及氨解方法 Download PDF

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WO2022036862A1
WO2022036862A1 PCT/CN2020/125164 CN2020125164W WO2022036862A1 WO 2022036862 A1 WO2022036862 A1 WO 2022036862A1 CN 2020125164 W CN2020125164 W CN 2020125164W WO 2022036862 A1 WO2022036862 A1 WO 2022036862A1
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solid
ammonolysis
aminolysis
solution
phase synthesis
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French (fr)
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曹春艳
赵双
靳开宝
纪磊
韩承昊
朱知浩
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Genewiz Suzhou Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical

Definitions

  • the present application belongs to the technical field of nucleic acid synthesis, and relates to an ammonia solution and an ammonia solution method.
  • Short-chain DNA also known as oligonucleotide (Oligo)
  • Oligo oligonucleotide
  • Solid-phase synthesis uses controlled pore glass spheres (CPG) as the solid-phase carrier and phosphoramidite as the monomer. According to the 3'-5' direction, through four cycles of deprotection, condensation, oxidation and capping, the target sequence is synthesized. gradually attached to the solid support.
  • CPG controlled pore glass spheres
  • the advantage of the solid-phase synthesis method is that each step of the reaction is carried out on a solid-phase carrier, the unreacted raw materials can be removed by suction filtration after the reaction, and the post-processing steps are simple. Therefore, solid-phase synthesis is widely used in Oligo and peptide synthesis.
  • the 5'-OH of the phosphoramidite monomer is protected by a dimethoxytriphenyl (DMT) group, which can be dissolved in 2 ⁇ 3% CCl 3 COOH/CH 2 Cl 2 solution
  • DMT dimethoxytriphenyl
  • the exposed free hydroxyl group undergoes a nucleophilic substitution reaction with phosphoramidite, that is, a condensation reaction; after the condensation reaction, in order to prevent the unreacted hydroxyl group from entering the next round of condensation, it is necessary to Acetylation of unreacted free hydroxyl groups, commonly known as "capping"; finally, because phosphoramidites are very unstable, phosphoramidites need to be oxidized to +5-valent phosphorus with an oxidizing agent such as iodine solution.
  • an oxidizing agent such as iodine solution.
  • the aminolysis reaction mainly includes: (1) cleaving Oligo from the solid support; (2) exposing the 3'-OH of Oligo; (3) removing the protective groups Ac and Bz on A, C, and G bases , dmf excision; (4) excision of the cyanoethyl group on the phosphoric acid.
  • the ammonia hydrolysis reaction was initially carried out with ammonia water.
  • the main problems include long reaction time, 16 hours at 55°C and 8 hours at 65°C; during the reaction, the synthesis column needs to be soaked in ammonia water, which is easy to cause cross-contamination; due to ammonia water Oligo can be dissolved, and the post-processing steps are cumbersome; impurities in ammonia water will accumulate, causing Oligo pollution.
  • the gas-phase ammonolysis adopts ammonia gas as the active medium, and uses a high-pressure-resistant ammonolysis pot as the reaction vessel, and conducts the ammonolysis reaction under the reaction conditions of high temperature and high pressure.
  • the advantages of gas-phase ammonolysis are that the reaction is fast and efficient, and the operation is simple.
  • Microwave ammonolysis is a widely used process in recent years, mainly using microwave as a heating method, but in the actual operation process, microwave heating can easily cause uneven heating, and continuous heating will cause liquid to boil, resulting in losses and cross-contamination.
  • CN109956987A discloses a method for aminolysis after solid-phase synthesis of DNA, comprising the following steps: 1) adding an aminolysis composition to a synthesis column comprising a solid-phase carrier for synthesizing DNA fragments; 2) adding the The synthesis column is placed in a sealable container containing ammonolysis buffer, and the synthesis column is kept above the liquid level of the aminolysis buffer; and 3) the sealable container is microwaved so that the The solid support is in the vapor atmosphere of the aminolysis buffer; 4) the synthesis column is taken out, the solid support is treated with the eluent and the effluent is collected.
  • this method still has the problems of uneven heating by microwave heating, easy damage or cross-contamination.
  • the application provides an ammonolysis solution and an ammonolysis method.
  • the formula of the ammonolysis solution is mild, and the ammonolysis buffer solution not only provides an atmosphere for ammonolysis, but also assists the ammonolysis reaction.
  • the ammonolysis process does not use toxic gases, has high safety, It is environmentally friendly, does not damage the primer structure, and at the same time, the post-processing steps are simple to avoid cross-contamination.
  • the first application provides an aminolysis solution comprising amine, butanol, aminoalcohol and an alkaline aqueous solution.
  • the amine cooperates with two alcohols to cut the synthesized oligonucleotide from the solid support, and remove the protective groups Ac, Bz and dmf on the A, C, and G bases. It acts as the active ingredient of the ammonia solution, butanol acts as a reaction solvent, and amino alcohol has two functions.
  • a small amount of alkaline aqueous solution is added to the ammonolysis solution at the same time to play the role of a catalyst, and cooperate with amine, butanol and amino alcohol to help speed up the reaction rate of ammonolysis. Yield and quality of nucleotides.
  • the amine includes propylamine and/or butylamine.
  • the propylamine includes n-propylamine and/or isopropylamine.
  • the butylamine includes any one or a combination of at least two of n-butylamine, isobutylamine, sec-butylamine or tert-butylamine, preferably n-butylamine.
  • propylamine and/or butylamine are used as the active ingredients of the ammonolysis solution, and cooperate with butanol, amino alcohol and alkaline aqueous solution to exert high-efficiency ammonolysis activity, and realize efficient ammonolysis reaction under a specific ratio .
  • the butanol includes any one or a combination of at least two of n-butanol, isobutanol, sec-butanol or tert-butanol, preferably n-butanol.
  • the amino alcohol includes aminobutanol and/or aminopentanol.
  • the aminopentanol includes 5-amino-1-pentanol and/or 4-amino-1-pentanol, preferably 5-amino-1-pentanol.
  • the alkaline aqueous solution includes any one or a combination of at least two of LiOH aqueous solution, NaOH aqueous solution, KOH aqueous solution, Be(OH) 2 aqueous solution, Mg(OH) 2 aqueous solution or Ca(OH) 2 aqueous solution.
  • the concentration of the alkaline aqueous solution is 1-10M, such as 1M, 2M, 3M, 4M, 5M, 6M, 7M, 8M, 9M or 10M, preferably 5M.
  • the concentration of the alkaline aqueous solution is limited within the range of 1-10M, which is helpful for the efficient ammonolysis reaction.
  • the concentration of the alkaline aqueous solution should not be too low or too high, and if the concentration is too low, it cannot play a role in promoting the ammonolysis reaction. If the concentration is too high, the yield and purity of oligonucleotides will be affected. The eluted oligonucleotides are prone to salt peaks during mass spectrometry detection, and the oligonucleotides may appear whitish after drying.
  • the volume ratio of the amine, butanol, amino alcohol and alkaline aqueous solution is (5-10):(1-5):1:(0.01-0.1), for example, it can be 5:1:1:0.01, 5:2:1:0.05, 5:5:1:0.1, 6:1:1:0.01, 6:2:1:0.05, 6:5:1:0.1, 7:1:1:0.01, 7: 2:1:0.05, 7:5:1:0.1, 8:1:1:0.01, 8:2:1:0.05, 8:5:1:0.1, 9:1:1:0.01, 9:2: 1:0.05, 9:5:1:0.1, 10:1:1:0.01, 10:2:1:0.05 or 10:5:1:0.1, preferably (6 ⁇ 8):(2 ⁇ 3): 1: (0.04 ⁇ 0.06).
  • amine, butanol, aminoalcohol and alkaline aqueous solution cooperate with each other in a specific ratio, and amine, butanol and aminoalcohol are used as the main functional components of the aminolysis solution, and the synthesized oligonucleotide is cut from the solid phase carrier. down, and remove the protective groups Ac, Bz, dmf on the A, C, G bases, and a trace amount of alkaline aqueous solution plays a catalytic role in aminolysis, which not only improves the yield of oligonucleotides, but also avoids the appearance of oligonucleotides. Whitening phenomenon, the obtained oligonucleotides are of high purity.
  • the volume ratio of n-butylamine, n-butanol, 5-amino-1-pentanol and NaOH/KOH aqueous solution is (5 ⁇ 10):(1 ⁇ 5):1:(0.01 ⁇ 0.1), preferably It is (6 ⁇ 8):(2 ⁇ 3):1:(0.04 ⁇ 0.06).
  • the ratio of the alkaline aqueous solution in the aminolysis solution should not be too low or too high. If the ratio is too low, it cannot play the role of promoting the aminolysis reaction. If the ratio is too high, the yield and purity of the oligonucleotide will be affected.
  • the present application provides an ammonolysis buffer comprising ammonia water and/or organic amines.
  • the ammonolysis buffer is vaporized into steam under heating conditions, providing a suitable ammonolysis environment and helping to improve the efficiency of ammonolysis; when ammonia water is used as the ammonolysis buffer, the vaporized ammonia can be A suitable environment for ammonolysis is provided, and the liquid-phase ammonolysis can be assisted to carry out the gas-phase ammonolysis reaction, thereby significantly improving the efficiency of ammonolysis and the yield of oligonucleotides, and shortening the reaction time.
  • the organic amine includes any one or a combination of at least two of diethylamine, ethylenediamine or pyridine.
  • the vaporized organic amine can not only provide a suitable ammonolysis environment, but also assist the liquid-phase ammonolysis to carry out the gas-phase ammonolysis reaction, which significantly improves the ammonolysis efficiency and the Oligonucleotide yield, shortened reaction time.
  • the application provides a method for aminolysis, the method comprising the following steps:
  • the solid phase carrier is immersed in the ammonia hydrolysis solution described in the first aspect, and the ammonolysis reaction can occur under mild heating conditions, and the ammonolysis buffer solution in the sealed container is gasified, which promotes the occurrence of the ammonolysis reaction and the Continuously, the synthesized oligonucleotide is cleaved from the solid-phase carrier, and the amino protecting group on the base is removed, which is beneficial to obtain an oligonucleotide product with high purity and high yield.
  • the solid phase carrier in step (1) comprises glass spheres with controlled pore size.
  • CPG Controlled Pore Glass
  • the solid phase carrier in step (1) is connected with a synthetic oligonucleotide, and the oligonucleotide is an artificially synthesized DNA short chain, usually composed of 20-60 bases, which can be used as primers, Sequencing adapters, probes, etc.
  • the ammonia solution of the present application can cut the artificially synthesized DNA short chain from the solid phase carrier, and excise the amino protecting groups Ac, Bz, and dmf on the A, C, and G bases.
  • the ammonia hydrolysis solution in step (1) is immersed in the solid-phase carrier.
  • the sealed container in step (2) comprises a metal sealed box.
  • a metal sealed box is used as the ammonia solution box to provide an atmosphere slightly higher than atmospheric pressure, and metal is a good conductor of heat, which is conducive to quickly transferring the heating temperature provided by the heating device to the inside of the ammonia solution box, and efficiently triggers the ammonolysis box.
  • the heating temperature in step (3) enables the aminolysis buffer to reach a vaporization temperature, and a vapor atmosphere of the aminolysis buffer is formed in the sealed container, so that the solid-phase synthesis column is in the atmosphere of the aminolysis buffer.
  • the aminolysis solution is promoted to play the role of aminolysis, and the synthesized oligonucleotides are cut off from the solid support.
  • the time of the described heating in step (3) is related to the synthesis specification, the formula of the aminolysis solution, the heating temperature, the volume of the sealed container and the completion degree of the desired aminolysis reaction, and is adjusted according to the specific implementation situation, and the time of the heating is preferably 30-60min, for example, can be 30min, 35min, 40min, 45min, 50min, 55min or 60min.
  • the cooling in step (3) is an ice bath for 5-15 minutes, preferably an ice bath for 10 minutes.
  • the eluent in step (4) includes water and/or Tris-HCl.
  • the concentration of the Tris-HCl is 10-30 mM, such as 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26mM, 27mM, 28mM, 29mM or 30mM, preferably 25mM.
  • the pH of the Tris-HCl is 7-8, such as 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8, preferably 7.2.
  • the step of drying and washing the solid phase synthesis column is further included.
  • the drying is to treat at 50-60° C. for 1-5 minutes to remove the ammonolysis buffer droplets condensed on the surface of the solid-phase synthesis column
  • the drying temperature can be, for example, 50° C., 51° C., 52° C., 53° C. °C, 54 °C, 55 °C, 56 °C, 57 °C, 58 °C, 59 °C or 60 °C, preferably 55 °C
  • the drying time can be, for example, 1 min, 2 min, 3 min, 4 min or 5 min, preferably 3 min.
  • the washing solution used in the washing includes 100% acetonitrile and/or 80-95% acetonitrile, for removing unreacted reagents and reaction by-products.
  • the washing includes: washing the dried solid-phase synthesis column with 100% acetonitrile and 80-95% acetonitrile in sequence.
  • the step of centrifugation is further included after washing the solid phase synthesis column.
  • the application provides a method for aminolysis, the method comprising the following steps:
  • the formula of the aminolysis solution of the present application is concise, and butylamine, butanol and aminopentanol cooperate with each other to cut the synthesized oligonucleotide from the solid phase carrier, and remove the protection on the A, C, and G bases Based on Ac, Bz, dmf, the ammonolysis solution is further added with an alkaline aqueous solution to speed up the reaction rate of ammonolysis;
  • the ammonolysis reaction of the present application is carried out in a sealed metal ammonolysis box, and the internal ammonolysis buffer is vaporized under heating conditions to provide a suitable ammonolysis environment for the ammonolysis reaction, and ammonia water is used as the ammonolysis buffer It not only provides a suitable ammonolysis environment, but also has the effect of gas-phase ammonolysis, which can assist the liquid-phase ammonolysis reaction;
  • Fig. 1 is the HPLC spectrogram of the oligonucleotide with the length of 23 bases;
  • Fig. 2 is the ESI mass spectrogram of the oligonucleotide with the length of 23 bases;
  • Fig. 3 is the HPLC spectrogram of the oligonucleotide of 60 bases in length
  • Fig. 4 is the ESI mass spectrogram of the oligonucleotide with the length of 60 bases;
  • Fig. 5 is the HPLC spectrogram of the oligonucleotide of 86 base length
  • Figure 6 is an ESI mass spectrum of an oligonucleotide of 86 bases in length.
  • the primers with lengths of 23 bases, 60 bases and 86 bases are respectively subjected to aminolysis reaction, and the sequences are:
  • SEQ ID NO:2 (60nt), target molecular weight is 18270.8:
  • SEQ ID NO:3 (86nt), target molecular weight is 26543.2:
  • a 50 nmol synthesis column was used, the activator was an acetonitrile solution containing 0.25M 5-ethylthiotetrazolium (ETT), the deprotection reagent was a dichloromethane solution containing 3% trichloroacetic acid, and the capping reagent was 10% acetic anhydride/ Acetonitrile and N-methylimidazole/pyridine/acetonitrile (ratio 14:10:76), oxidizing agent is tetrahydrofuran/pyridine/water (ratio 70:20:10) containing 0.05M iodine, synthesized in Dr.Oligo 192 on a synthesizer.
  • ETT 5-ethylthiotetrazolium
  • the deprotection reagent was a dichloromethane solution containing 3% trichloroacetic acid
  • the capping reagent was 10% acetic anhydride/ Acetonitrile and N-methylimidazole
  • Example 2 the aminolysis method of Example 1 was adopted, and 96 primers with a length of 40-60 bases were subjected to aminolysis, and the mass spectrometry qualification rate and yield were analyzed.
  • a 5 nmol synthesis column was used, the activator was an acetonitrile solution containing 0.25M 5-ethylthiotetrazolium (ETT), the deprotection reagent was a dichloromethane solution containing 3% trichloroacetic acid, and the capping reagent was 10% acetic anhydride/ Acetonitrile and N-methylimidazole/pyridine/acetonitrile (ratio 14:10:76), oxidizing agent is tetrahydrofuran/pyridine/water (ratio 70:20:10) containing 0.05M iodine, synthesized in Dr.Oligo 768 on a synthesizer.
  • ETT 5-ethylthiotetrazolium
  • the deprotection reagent was a dichloromethane solution containing 3% trichloroacetic acid
  • the capping reagent was 10% acetic anhydride/ Acetonitrile and N-methylimidazole
  • the primer yield and purity of liquid-phase ammonolysis and gas-phase ammonolysis are as shown in Table 1, and it can be seen that compared with gas-phase ammonolysis, the average yield of 96 primers obtained by the liquid-phase ammonolysis method of the present application is higher; Taking 75% mass spectrometry purity as the qualified standard, the qualified rate of mass spectrometry of liquid-phase ammonolysis is also slightly higher than that of gas-phase ammonolysis. It is shown that compared with the conventional gas-phase aminolysis, the liquid-phase aminolysis method of the present application has improved both the primers and the purity of the oligonucleotides.
  • Table 2 shows the comparison of NGS purity and cross-contamination between liquid-phase aminolysis and gas-phase aminolysis. It can be seen that the oligonucleotides prepared by liquid-phase aminolysis have high purity and low cross-contamination rates within and between plates.
  • a 50 nmol synthesis column is used to synthesize 96 primers with a length of 40-60 bases.
  • synthesis steps refer to Example 2.
  • ammonia hydrolysis is performed, the mass spectrometry qualification rate and yield are analyzed. The steps are as follows:
  • ammonia hydrolysis reaction was carried out by using the formula of different volume ratios of the ammonia hydrolysis solution, and the influence of the ammonolysis components on the effect of the ammonolysis was analyzed by comparing the mass spectrometry and the yield.
  • the specific experimental steps are as follows:
  • Example 2 Compared with Example 2, the formula of the ammonolysis solution is diethylenetriamine: 1M LiOH aqueous solution: the volume ratio of ethanol is 1:3:6, and other conditions are the same as in Example 2. Comparing mass spectrometry and yield, the influence of ammonolysis components on the effect of ammonolysis was analyzed. The specific experimental steps are those shown in Example 2.
  • the yield and purity of the primers of Examples 2 to 7 and Comparative Example 1 are shown in Table 3, illustrating that using this aminolysis formula, the yields and The purity can be guaranteed, and the scope of application is wide; the aminolysis activity of diethylenetriamine and ethanol used in the aminolysis solution used in Comparative Example 1 is weak, so it is easy to add a high proportion of alkaline water to ensure the aminolysis. The reaction occurs, but it will cause the problem of poor primer quality, serious whitening of the primer, many impurities, and complicated post-processing steps, which affect the structure and function of the primer.
  • the aminolysis solution of the present application has a simple formula, low cost, environmental friendliness, high safety, mild aminolysis process conditions, no damage to the oligonucleotide structure, and the aminolysis reaction occurs in a sealed container. , avoids the volatilization of the aminolysis solution and/or the aminolysis buffer, helps the continuous progress of the aminolysis reaction, has high aminolysis efficiency, and is easy to operate.
  • the aminolysis buffer not only provides a suitable aminolysis environment, but also has auxiliary ammonia The effect of aminolysis is further improved, and it has important application prospects in the field of solid-phase synthesis of nucleic acids.
  • the present application illustrates the detailed method of the present application through the above-mentioned embodiments, but the present application is not limited to the above-mentioned detailed method, which does not mean that the present application must rely on the above-mentioned detailed method for implementation.
  • Those skilled in the art should understand that any improvement to the application, the equivalent replacement of each raw material of the product of the application, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the application.

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Abstract

一种氨解液及氨解方法，所述氨解液包括胺、丁醇、氨基醇和碱性水溶液，所述氨解方法包括：(1)向装有固相载体的固相合成柱中加入氨解液；(2)将所述固相合成柱置于装有氨解缓冲液的密封容器内，并保持所述固相合成柱不接触氨解缓冲液；(3)加热所述密封容器，使固相载体处于氨解缓冲液的蒸汽气氛中，随后冷却；(4)从所述密封容器中取出固相合成柱，用洗脱液处理固相合成柱中的固相载体，并收集流出液。所述氨解液配方温和，氨解缓冲液不仅提供氨解氛围，而且辅助氨解反应，氨解工艺不使用有毒气体、安全性高、环境友好、不破坏引物结构，同时后处理步骤简单，避免交叉污染，可以实现大规模工业化应用。

Description

一种氨解液及氨解方法 技术领域
本申请属于核酸的合成技术领域，涉及一种氨解液及氨解方法。
背景技术
短链DNA又称为寡核苷酸(Oligo)，通常采用固相合成技术进行人工合成。固相合成以控制孔径玻璃球(CPG)为固相载体，以亚磷酰胺为单体，按照3’-5’方向，经过脱保护、缩合、氧化和盖帽四个反应的循环，将目标序列逐渐连接在固相载体上。固相合成方法的优势在于：每一步反应都在固相载体上进行，反应结束后可以将未反应的原料通过抽滤的方式除去，后处理步骤简单。因此，固相合成被广泛应用于Oligo和多肽合成。在现在大部分的Oligo合成工艺中，亚磷酰胺单体的5’-OH被二甲氧基三苯基(DMT)基团保护，可以在2～3％CCl 3COOH/CH 2Cl 2溶液中被切除，称为脱保护反应；脱保护后裸露出的自由羟基与亚磷酰胺发生亲核取代反应，即缩合反应；缩合反应结束后，为避免未反应的羟基进入下一轮缩合，需要将未反应的自由羟基乙酰化，俗称“盖帽”；最后，由于亚磷酰胺非常不稳定，需要用氧化剂如碘液将亚磷酰胺氧化为+5价磷。每连接一个碱基都需要经历四个反应，将目标碱基全部合成后，将Oligo从CPG上剪切下来。同时，由于A、C、G碱基含有活性氨基，会干扰缩合反应，所以一般预先采用乙酰基(Ac)、苯甲酰基(Bz)和二氮甲基甲脒基(dmf)将氨基保护，当反应结束后，再将Ac、Bz和dmf基团切除。在这里，将Oligo从CPG上剪切下来和脱除保护基的反应称为氨解反应。氨解反应的主要包括：(1)将Oligo从固相载体上切割下来；(2)将Oligo的3’-OH暴露；(3)将A、C、G碱基上的保护基Ac、Bz、dmf切除；(4)将磷酸上的氰乙基切除。
氨解反应最初采用氨水进行，其问题主要包括反应时间长，55℃反应需要16小时，65℃反应需要8小时；在反应过程中需要将合成柱浸泡在氨水中，容易造成交叉污染；由于氨水可以溶解Oligo，后处理步骤繁琐；氨水中的杂质会积累，造成Oligo污染。后来研发的甲胺:氨水＝1:1(AMA)的氨解反应体系大大降低了反应时间(M.P.Reddy,N.B.Hanna,and F.Farooqui,Nucleos.Nucleot.,1997,16,1589-1598.)，但是仍然存在交叉污染和后处理步骤繁琐等问题。气相氨解采用氨气为活性介质，以耐高压的氨解锅为反应容器，在高温高压的反应条件下进行氨解反应。气相氨解的优势在于反应快速高效、操作简单，但是存在对设备要求高、需要使用有毒气体、存在安全隐患、污染环境、对引物的结构破坏性较大的缺点。微波氨解是近年来广泛应用的工艺，主要以微波作为加热手段，但是在实际操作过程中，微波加热很容易造成受热不均匀，持续加热会引起液体爆沸，造成损失和引起交叉污染。
CN109956987A公开了一种用于在DNA固相合成后进行氨解的方法，包括以下步骤：1)向包括用于合成DNA片段的固相载体的合成柱内加入氨解组合物；2)将所述合成柱置于装有氨解缓冲液的可密封容器内，并保持所述合成柱位于所述氨解缓冲液的液面上方；以及3) 以微波处理所述可密封容器，使得所述固相载体处于所述氨解缓冲液的蒸汽气氛中；4)取出所述合成柱，以洗脱液处理所述固相载体并收集流出液。但是该方法仍然存在微波加热受热不均匀、容易造成损伤或引起交叉污染的问题。
因此，需要研发一种新的氨解方法，反应条件温和，不会破坏引物结构，解决现有技术存在的问题。
发明内容
本申请提供了一种氨解液及氨解方法，所述氨解液配方温和，氨解缓冲液不仅提供氨解氛围，而且辅助氨解反应，氨解工艺不使用有毒气体、安全性高、环境友好、不破坏引物结构，同时后处理步骤简单，避免交叉污染。
第一申请，本申请提供了一种氨解液，所述氨解液包括胺、丁醇、氨基醇和碱性水溶液。
本申请中，胺与两种醇相互配合，将合成的寡核苷酸从固相载体上切割下来，并去除A、C、G碱基上的保护基Ac、Bz、dmf，其中，胺的作用为氨解液的活性成分，丁醇的作用为反应溶剂，氨基醇兼具有两种作用。
本申请同时在氨解液中加入微量碱性水溶液，发挥催化剂的作用，与胺、丁醇和氨基醇相互配合，有助于加快氨解反应速率，既起到氨解催化作用，又不影响寡核苷酸的产率和品质。
优选地，所述胺包括丙胺和/或丁胺。
优选地，所述丙胺包括正丙胺和/或异丙胺。
优选地，所述丁胺包括正丁胺、异丁胺、仲丁胺或叔丁胺中的任意一种或至少两种的组合，优选为正丁胺。
本申请中，采用的丙胺和/或丁胺作为氨解液的活性成分，与丁醇、氨基醇和碱性水溶液相互配合，发挥高效的氨解活性，在特定配比下实现高效的氨解反应。
优选地，所述丁醇包括正丁醇、异丁醇、仲丁醇或叔丁醇中的任意一种或至少两种的组合，优选为正丁醇。
优选地，所述氨基醇包括氨基丁醇和/或氨基戊醇。
优选地，所述氨基戊醇包括5-氨基-1-戊醇和/或4-氨基-1-戊醇，优选为5-氨基-1-戊醇。
优选地，所述碱性水溶液包括LiOH水溶液、NaOH水溶液、KOH水溶液、Be(OH) 2水溶液、Mg(OH) 2水溶液或Ca(OH) 2水溶液中的任意一种或至少两种的组合。
优选地，所述碱性水溶液的浓度为1～10M，例如可以是1M、2M、3M、4M、5M、6M、7M、8M、9M或10M，优选为5M。
本申请中，将碱性水溶液的浓度限定在1～10M的范围内，有助于高效进行氨解反应，碱性水溶液的浓度不能过低或过高，浓度过低不能发挥促进氨解反应的作用，浓度过高会影响寡核苷酸的产率和纯度，洗脱得到的寡核苷酸在质谱检测时容易产生盐峰，干燥后寡核苷酸可能外观发白。
优选地，所述胺、丁醇、氨基醇和碱性水溶液的体积比为(5～10):(1～5):1:(0.01～0.1)，例如可以是5:1:1:0.01、5:2:1:0.05、5:5:1:0.1、6:1:1:0.01、6:2:1:0.05、6:5:1:0.1、7:1:1:0.01、7:2:1:0.05、7:5:1:0.1、8:1:1:0.01、8:2:1:0.05、8:5:1:0.1、9:1:1:0.01、9:2:1:0.05、9:5:1:0.1、10:1:1:0.01、10:2:1:0.05或10:5:1:0.1，优选为(6～8):(2～3):1:(0.04～0.06)。
本申请中，胺、丁醇、氨基醇和碱性水溶液在特定配比下相互配合，胺、丁醇和氨基醇作为氨解液的主要功效成分，将合成的寡核苷酸从固相载体上切割下来，并去除A、C、G碱基上的保护基Ac、Bz、dmf，微量的碱性水溶液发挥氨解催化作用，不仅提高了寡核苷酸产率，而且避免了寡核苷酸外观发白现象，获得的寡核苷酸纯度高。
优选地，所述正丁胺、正丁醇、5-氨基-1-戊醇和NaOH/KOH水溶液的体积比为(5～10):(1～5):1:(0.01～0.1)，优选为(6～8):(2～3):1:(0.04～0.06)。
本申请中，碱性水溶液在氨解液中的比例同样不能过低或过高，比例过低不能发挥促进氨解反应的作用，比例过高会影响寡核苷酸的产率和纯度。
第二方面，本申请提供了一种氨解缓冲液，所述氨解缓冲液包括氨水和/或有机胺。
本申请中，氨解缓冲液在加热条件下气化为蒸汽，提供适宜的氨解环境，有助于提高氨解效率；当采用氨水作为氨解缓冲液时，气化后的氨气既可以提供适宜的氨解环境，又可以辅助液相氨解进行气相氨解反应，显著提高了氨解效率和寡核苷酸产率，缩短了反应时间。
优选地，所述有机胺包括二乙胺、乙二胺或吡啶中的任意一种或至少两种的组合。
本申请中，当采用有机胺作为氨解缓冲液时，气化后的有机胺既可以提供适宜的氨解环境，又可以辅助液相氨解进行气相氨解反应，显著提高了氨解效率和寡核苷酸产率，缩短了反应时间。
第三方面，本申请提供了一种氨解方法，所述方法包括以下步骤：
(1)向装有固相载体的固相合成柱中加入第一方面所述的氨解液；
(2)将所述固相合成柱置于装有第二方面所述的氨解缓冲液的密封容器内，并保持所述固相合成柱不接触氨解缓冲液；
(3)加热所述密封容器，使固相载体处于氨解缓冲液的蒸汽气氛中，随后冷却；
(4)从所述密封容器中取出固相合成柱，用洗脱液处理固相合成柱中的固相载体，并收集流出液。
本申请中，采用第一方面所述的氨解液浸没固相载体，在温和的加热条件下即可发生氨解反应，密封容器中的氨解缓冲液发生气化，促进氨解反应发生和持续进行，将合成的寡核苷酸从固相载体上切割下来，将碱基上的氨基保护基团去除，有利于获得纯度高、产率高的寡核苷酸产品。
优选地，步骤(1)所述固相载体包括控制孔径玻璃球。
根据本申请，控制孔径玻璃球(Controlled Pore Glass，CPG)的球体之间存在空隙，氨解液流动于其中进行氨解反应，将寡核苷酸从CPG上切割下来。
优选地，步骤(1)所述固相载体上连接有合成的寡核苷酸，所述寡核苷酸是人工合成的 DNA短链，通常由20～60个碱基组成，可以作为引物、测序接头、探针等，本申请的氨解液可以将人工合成的DNA短链从固相载体上切割下来，将A、C、G碱基上的氨基保护基团Ac、Bz、dmf切除。
优选地，步骤(1)所述氨解液浸没固相载体。
优选地，步骤(2)所述密封容器包括金属密封盒。
本申请中，采用金属密封盒作为氨解盒，提供一个稍高于大气压的氛围，金属是热的良导体，有利于将加热装置提供的加热温度快速传递到氨解盒内部，高效引发氨解缓冲液气化和氨解反应的发生；同时，密封的氨解盒有效避免了氨解液和氨解缓冲液的挥发损失，有助于高效完成氨解反应。
优选地，步骤(3)所述加热的温度一方面使氨解缓冲液达到气化的温度，在密封容器中形成氨解缓冲液的蒸汽气氛，从而使固相合成柱处于氨解缓冲液的蒸汽气氛中，促进氨解液发挥氨解作用，将合成的寡核苷酸从固相载体上剪切下来，所述加热的温度优选为45～100℃，例如可以是45℃、50℃、55℃、60℃、65℃、70℃、75℃、80℃、81℃、82℃、83℃、84℃、85℃、86℃、87℃、88℃、89℃、90℃、91℃、92℃、93℃、94℃、95℃、96℃、97℃、98℃、99℃或100℃，进一步优选为90～95℃。
优选地，步骤(3)所述加热的时间与合成规格、氨解液配方、加热温度、密封容器体积和期望氨解反应的完成程度有关，根据具体实施情况调整，所述加热的时间优选为30～60min，例如可以是30min、35min、40min、45min、50min、55min或60min。
优选地，步骤(3)所述冷却为冰浴5～15min，优选为冰浴冷却10min。
优选地，步骤(4)所述洗脱液包括水和/或Tris-HCl。
优选地，所述Tris-HCl的浓度为10～30mM，例如可以是10mM、11mM、12mM、13mM、14mM、15mM、16mM、17mM、18mM、19mM、20mM、21mM、22mM、23mM、24mM、25mM、26mM、27mM、28mM、29mM或30mM，优选为25mM。
优选地，所述Tris-HCl的pH为7～8，例如可以是7、7.1、7.2、7.3、7.4、7.5、7.6、7.7、7.8、7.9或8，优选为7.2。
优选地，在步骤(4)所述用洗脱液处理固相合成柱中的固相载体之前，还包括干燥和洗涤所述固相合成柱的步骤。
优选地，所述干燥为在50～60℃下处理1～5min，去除固相合成柱表面凝结的氨解缓冲液滴，所述干燥的温度例如可以是50℃、51℃、52℃、53℃、54℃、55℃、56℃、57℃、58℃、59℃或60℃，优选为55℃，所述干燥的时间例如可以是1min、2min、3min、4min或5min，优选为3min。
优选地，所述洗涤采用的洗涤液包括100％乙腈和/或80～95％乙腈，用于去除未反应的试剂和反应副产物。
优选地，所述洗涤包括：将干燥后的固相合成柱依次采用100％乙腈和80～95％乙腈洗涤。
优选地，在洗涤所述固相合成柱之后还包括离心的步骤。
作为优选技术方案，本申请提供了一种氨解方法，所述方法包括以下步骤：
(1)向装有连接了合成的寡核苷酸的控制孔径玻璃球的固相合成柱中加入第一方面所述的氨解液，浸没控制孔径玻璃球；
(2)将所述固相合成柱置于装有氨解缓冲液的金属密封容器内，并保持所述固相合成柱不接触氨解缓冲液，所述氨解缓冲液包括氨水和/或有机胺；
(3)将所述金属密封容器在45～100℃下加热30～60min，使连接了合成的寡核苷酸的控制孔径玻璃球处于氨解缓冲液的蒸汽气氛中，随后冰浴冷却5～15min；
(4)从所述密封容器中取出固相合成柱，50～60℃下干燥1～5min，并依次用100％乙腈和80～95％乙腈洗涤，离心后用水和/或10～25mM pH 7～8的Tris-HCl处理固相合成柱中的固相载体，并收集流出液。
与现有技术相比，本申请具有如下有益效果：
(1)本申请的氨解液配方简洁，丁胺、丁醇和氨基戊醇相互配合，将合成的寡核苷酸从固相载体上切割下来，并去除A、C、G碱基上的保护基Ac、Bz、dmf，氨解液进一步添加有碱性水溶液，加快了氨解反应速率；
(2)本申请的氨解反应在密封的金属氨解盒中进行，内部的氨解缓冲液在加热的条件下气化，为氨解反应提供适宜的氨解环境，氨水作为氨解缓冲液不仅提供了适宜的氨解环境，而且具有气相氨解的效果，可以辅助液相氨解反应；
(3)本申请的氨解工艺条件温和、成本低廉、环境友好、安全性高，具有重要的应用价值。
附图说明
图1为23个碱基长度的寡核苷酸的HPLC谱图；
图2为23个碱基长度的寡核苷酸的ESI质谱图；
图3为60个碱基长度的寡核苷酸的HPLC谱图；
图4为60个碱基长度的寡核苷酸的ESI质谱图；
图5为86个碱基长度的寡核苷酸的HPLC谱图；
图6为86个碱基长度的寡核苷酸的ESI质谱图。
具体实施方式
为进一步阐述本申请所采取的技术手段及其效果，以下结合实施例和附图对本申请作进一步地说明。可以理解的是，此处所描述的具体实施方式仅仅用于解释本申请，而非对本申请的限定。
实施例中未注明具体技术或条件者，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，均为可通过正规渠道商购获得的常规产品。
实施例1
本实施例对长度分别为23个碱基、60个碱基和86个碱基的引物进行氨解反应，序列分别为：
SEQ ID NO:1(23nt)，目标分子量为7030.6：
5’-CATTCGTAGCTCGGATCGTGTAC-3’；
SEQ ID NO:2(60nt)，目标分子量为18270.8：
5’-GCATGGCGACCTTATCAGTCTACGTCCTTTGTCTTCCTAAGACCGCTTGGCCTCCGACTT-3’；
SEQ ID NO:3(86nt)，目标分子量为26543.2：
5’-GCCTGCTTGAGCAGGCTGAAGTTGGTGGCACGCGTTTAATTAACCATATCATCTAGTGTTGCATATGAGACAAGGACTTTTCCCAT-3’。
步骤如下：
引物合成
使用50nmol合成柱，活化剂为含有0.25M 5-乙硫基四氮唑(ETT)的乙腈溶液，脱保护试剂为含有3％三氯乙酸的二氯甲烷溶液，盖帽试剂为10％乙酸酐/乙腈和N-甲基咪唑/吡啶/乙腈(比例为14:10:76)，氧化剂为含有0.05M碘的四氢呋喃/吡啶/水(比例为70:20:10)，合成反应在Dr.Oligo 192合成仪上进行。
液相氨解
(1)向固相合成柱中加入20μL氨解液，浸没连接有合成的引物的CPG，氨解液的配方为正丁胺:正丁醇:5-氨基-1-戊醇:NaOH水溶液(5M)的体积比为6:3:1:0.05；
(2)将20mL氨水倒入规格为170mm×130mm×95mm的金属氨解盒中；
(3)小心将包含多个固相合成柱的合成板放入氨解盒中，静置1min，将金属盒盖紧，用螺丝固定密封，保证固相合成柱不接触氨解缓冲液；
(4)小心将金属氨解盒放入烘箱，90℃加热60min，反应结束后，将金属氨解盒取出，放入冰水浴中冷却10min；
(5)取出金属氨解盒，松开螺丝，取出合成板，放入烘箱中55℃干燥3min，取出放凉至室温；
(6)用100％乙腈洗涤两次，每次加入100μL，随后用100μL 90％乙腈/水洗涤一次；
(7)取出合成板，3000rpm下离心1min；
(8)加入100μL 25mM、pH7.2的Tris-HCl洗脱液洗脱，收集流出液进行质谱分析。
长度分别为23个碱基、60个碱基和86个碱基的引物的HPLC谱图和ESI质谱图分别如图1、图2、图3、图4、图5和图6所示，可以看出，液相氨解处理的不同长度的引物的HPLC纯度、质谱均能够达到行业标准，证明本申请的氨解方法对不同长度的寡核苷酸均具有较好的氨解效果。
实施例2
本实施例采用实施例1的氨解方法，对96条长度为40～60碱基的引物进行氨解，分析 质谱合格率和产量。
步骤如下：
引物合成
使用5nmol合成柱，活化剂为含有0.25M 5-乙硫基四氮唑(ETT)的乙腈溶液，脱保护试剂为含有3％三氯乙酸的二氯甲烷溶液，盖帽试剂为10％乙酸酐/乙腈和N-甲基咪唑/吡啶/乙腈(比例为14:10:76)，氧化剂为含有0.05M碘的四氢呋喃/吡啶/水(比例为70:20:10)，合成反应在Dr.Oligo 768合成仪上进行。
液相氨解
(1)向固相合成柱中加入20μL氨解液，浸没连接有合成的引物的CPG，氨解液的配方为正丁胺:正丁醇:5-氨基-1-戊醇:NaOH水溶液(5M)的体积比为7:2:1:0.05；
(2)将20mL氨水倒入规格为170mm×130mm×95mm的金属氨解盒中；
(3)小心将包含多个固相合成柱的合成板放入氨解盒中，静置1min，将金属盒盖紧，用螺丝固定密封，保证固相合成柱不接触氨解缓冲液；
(4)小心将金属氨解盒放入烘箱，90℃加热60min，反应结束后，将金属氨解盒取出，放入冰水浴中冷却10min；
(5)取出金属氨解盒，松开螺丝，取出合成板，放入烘箱中55℃干燥3min，取出放凉至室温；
(6)用100％乙腈洗涤两次，每次加入200μL，随后用200μL 90％乙腈/水洗涤一次；
(7)取出合成板，3000rpm离心1min；
(8)加入200μL 25mM、pH7.2的Tris-HCl洗脱液洗脱，收集流出液进行质谱分析和酶标仪定量。
气相氨解
(1)将下机后的合成板直接放入气相氨解锅中，90℃、80～100MPsi条件下反应40min；
(2)小心将合成板从氨解锅中取出，放入烘箱中55℃干燥3min，取出放凉至室温；
(3)用100％乙腈洗涤两次，每次加入200μL，随后用200μL 90％乙腈/水洗涤一次；
(4)取出合成板，3000rpm离心1min；
(5)加入200μL 25mM、pH7.2的Tris-HCl洗脱液洗脱，收集流出液进行质谱分析和酶标仪定量。
液相氨解和气相氨解的引物产量和纯度如表1所示，可以看出，与气相氨解相比，采用本申请的液相氨解方法获得的96条引物的平均产量更高；以75％的质谱纯度为合格标准，液相氨解的质谱合格率也比气相氨解的合格率稍高。说明本申请的液相氨解方法与常规的气相氨解相比，寡核苷酸的引物和纯度均得到提升。
表1 96条引物的平均产量和纯度
氨解方法 产量(nmol) 纯度(％)
气相氨解 3.26 96.9
液相氨解 3.59 98.9
表2为液相氨解和气相氨解的NGS纯度与交叉污染对比，可以看出，液相氨解制备的寡核苷酸纯度高、板内和板间交叉污染率低。
表2
Figure PCTCN2020125164-appb-000001
实施例3
本实施例采用50nmol合成柱合成96条长度为40～60碱基的引物，合成步骤参见实施例2，进行氨解后分析质谱合格率和产量，步骤如下：
(1)向固相合成柱中加入20μL氨解液，浸没连接有合成的引物的CPG，氨解液的配方为异丁胺:正丁醇:5-氨基-1-戊醇:KOH水溶液(6M)的体积比为6:2:1:0.04；
(2)将20mL氨水倒入规格为170mm×130mm×95mm的金属氨解盒中；
(3)小心将包含多个固相合成柱的合成板放入氨解盒中，静置1min，将金属盒盖紧，用螺丝固定密封，保证固相合成柱不接触氨解缓冲液；
(4)小心将金属氨解盒放入烘箱，95℃加热40min，反应结束后，将金属氨解盒取出，放入冰水浴中冷却10min；
(5)取出金属氨解盒，松开螺丝，取出合成板，放入烘箱中55℃干燥3min，取出放凉至室温；
(6)用100％乙腈洗涤两次，每次加入200μL，随后用200μL 90％乙腈/水洗涤一次；
(7)取出合成板，3000rpm离心1min；
(8)加入200μL 25mM、pH7.2的Tris-HCl洗脱液洗脱，收集流出液进行质谱分析和酶标仪定量。
实施例4
本实施例采用50nmol合成柱合成的96条长度为40～60碱基的引物，合成步骤参见实施例2，进行氨解后分析质谱合格率和产量，步骤如下：
(1)向固相合成柱中加入20μL氨解液，浸没连接有合成的引物的CPG，氨解液的配方为正丙胺:异丁醇:4-氨基-1-戊醇:LiOH(4M)的体积比为6:2:1:0.06；
(2)将20mL二乙胺倒入规格为170mm×130mm×95mm的金属氨解盒中；
(3)小心将包含多个固相合成柱的合成板放入氨解盒中，静置1min，将金属盒盖紧，用螺丝固定密封，保证固相合成柱不接触氨解缓冲液；
(4)小心将金属氨解盒放入烘箱，85℃加热50min，反应结束后，将金属氨解盒取出，放入冰水浴中冷却10min；
(5)取出金属氨解盒，松开螺丝，取出合成板，放入烘箱中55℃干燥3min，取出放凉至室温；
(6)用100％乙腈洗涤两次，每次加入200μL，随后用200μL 90％乙腈/水洗涤一次；
(7)取出合成板，3000rpm离心1min；
(8)加入200μL 20mM、pH7.2的Tris-HCl洗脱液洗脱，收集流出液进行质谱分析和酶标仪定量。
实施例5
本实施例采用50nmol合成柱合成的96条长度为40～60碱基的引物，合成步骤参见实施例2，进行氨解后分析质谱合格率和产量，步骤如下：
(1)向固相合成柱中加入20μL氨解液，浸没连接有合成的引物的CPG，氨解液的配方为仲丁胺:异丁醇:4-氨基-1-戊醇:Be(OH) 2水溶液(2M)的体积比为8:3:1:0.05；
(2)将20mL二乙胺倒入规格为170mm×130mm×95mm的金属氨解盒中；
(3)小心将包含多个固相合成柱的合成板放入氨解盒中，静置1min，将金属盒盖紧，用螺丝固定密封，保证固相合成柱不接触氨解缓冲液；
(4)小心将金属氨解盒放入烘箱，100℃加热30min，反应结束后，将金属氨解盒取出，放入冰水浴中冷却10min；
(5)取出金属氨解盒，松开螺丝，取出合成板，放入烘箱中55℃干燥3min，取出放凉至室温；
(6)用100％乙腈洗涤两次，每次加入200μL，随后用200μL 80％乙腈/水洗涤一次；
(7)取出合成板，3000rpm离心1min；
(8)加入200μL纯水洗脱液洗脱，收集流出液进行质谱分析和酶标仪定量。
实施例6
本实施例采用50nmol合成柱合成的96条长度为40～60碱基的引物，合成步骤参见实施例2，进行氨解后分析质谱合格率和产量，步骤如下：
(1)向固相合成柱中加入20μL氨解液，浸没连接有合成的引物的CPG，氨解液的配方为叔丁胺:仲丁醇:氨基丁醇:Mg(OH) 2水溶液(1M)的体积比为5:1:1:0.1；
(2)将20mL乙二胺倒入规格为170mm×130mm×95mm的金属氨解盒中；
(3)小心将包含多个固相合成柱的合成板放入氨解盒中，静置1min，将金属盒盖紧， 用螺丝固定密封，保证固相合成柱不接触氨解缓冲液；
(4)小心将金属氨解盒放入烘箱，60℃加热35min，反应结束后，将金属氨解盒取出，放入冰水浴中冷却10min；
(5)取出金属氨解盒，松开螺丝，取出合成板，放入烘箱中55℃干燥3min，取出放凉至室温；
(6)用100％乙腈洗涤两次，每次加入200μL，随后用200μL 95％乙腈/水洗涤一次；
(7)取出合成板，3000rpm离心1min；
(8)加入200μL纯水洗脱液洗脱，收集流出液进行质谱分析和酶标仪定量。
实施例7
本实施例利用不同体积比的氨解液配方进行氨解反应，对比质谱和产量，分析氨解组分对氨解效果的影响。具体实验步骤如下：
(1)向固相合成柱中加入20μL氨解液，浸没连接有合成的引物的CPG，氨解液的配方为异丙胺:叔丁醇:氨基丁醇:Ca(OH) 2(10M)的体积比为10:5:1:0.01；
(2)将20mL吡啶倒入规格为170mm×130mm×95mm的金属氨解盒中；
(3)小心将包含多个固相合成柱的合成板放入氨解盒中，静置1min，将金属盒盖紧，用螺丝固定密封，保证固相合成柱不接触氨解缓冲液；
(4)小心将金属氨解盒放入烘箱，45℃加热35min，反应结束后，将金属氨解盒取出，放入冰水浴中冷却10min；
(5)取出金属氨解盒，松开螺丝，取出合成板，放入烘箱中55℃干燥3min，取出放凉至室温；
(6)用100％乙腈洗涤两次，每次加入200μL，随后用200μL 95％乙腈/水洗涤一次；
(7)取出合成板，3000rpm离心1min；
(8)加入200μL纯水洗脱液洗脱，收集流出液进行质谱分析和酶标仪定量。
对比例1
与实施例2相比，氨解液的配方为二亚乙基三胺:1M LiOH水溶液:乙醇的体积比为1:3:6，其他条件与实施例2相同。对比质谱及产量，分析氨解组分对氨解效果的影响。具体实验步骤为实施例2所示步骤。
实施例2～7和对比例1的引物产量和纯度如表3所示，说明使用本氨解配方，对低产量引物(采用5nmol合成)和高产量引物(采用50nmol合成)，其产率和纯度都能有所保证，适用范围广；对比例1使用的氨解液功效成分二亚乙基三胺和乙醇的氨解活性较弱，因此需要添加高配比的碱性水容易以保证氨解反应发生，但是会造成引物品质差的问题，引物发白严重，杂质较多，且后处理步骤繁琐，影响引物的结构和功能。
表3不同氨解条件下96条引物的平均产量和纯度
编号 产量(nmol) 纯度(％) 外观
实施例2 3.59 98.9 未见发白现象
实施例3 35.26 98.5 未见发白现象
实施例4 36.60 99.4 未见发白现象
实施例5 33.49 97.6 未见发白现象
实施例6 35.45 98.8 未见发白现象
实施例7 34.03 98.0 未见发白现象
对比例1 33.22 97.8 发白
综上所述，本申请的氨解液配方简洁、成本低廉、环境友好、安全性高，氨解工艺条件温和，不会造成对寡核苷酸结构的破坏，氨解反应在密封容器中发生，避免了氨解液和/或氨解缓冲液的挥发，有助于氨解反应持续进行，氨解效率高，操作简便，氨解缓冲液不仅提供了适宜的氨解环境，而且具有辅助氨解的效果，进一步提高了氨解效率，在核酸的固相合成领域具有重要的应用前景。
申请人声明，本申请通过上述实施例来说明本申请的详细方法，但本申请并不局限于上述详细方法，即不意味着本申请必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本申请的任何改进，对本申请产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本申请的保护范围和公开范围之内。

Claims (31)

  1. 一种氨解液，包括：胺、丁醇、氨基醇和碱性水溶液；
    其中所述胺、丁醇、氨基醇和碱性水溶液的体积比为(5～10):(1～5):1:(0.01～0.1)。
  2. 根据权利要求1所述的氨解液，其中，所述胺包括丙胺和/或丁胺。
  3. 根据权利要求2所述的氨解液，其中，所述丙胺包括正丙胺和/或异丙胺。
  4. 根据权利要求2所述的氨解液，其中，所述丁胺包括正丁胺、异丁胺、仲丁胺或叔丁胺中的任意一种或至少两种的组合。
  5. 根据权利要求1所述的氨解液，其中，所述丁醇包括正丁醇、异丁醇、仲丁醇或叔丁醇中的任意一种或至少两种的组合。
  6. 根据权利要求1所述的氨解液，其中，所述氨基醇包括氨基丁醇和/或氨基戊醇。
  7. 根据权利要求6所述的氨解液，其中，氨基戊醇包括5-氨基-1-戊醇和/或4-氨基-1-戊醇。
  8. 根据权利要求1所述的氨解液，其中，所述碱性水溶液包括LiOH水溶液、NaOH水溶液、KOH水溶液、Be(OH) 2水溶液、Mg(OH) 2水溶液或Ca(OH) 2水溶液中的任意一种或至少两种的组合。
  9. 根据权利要求1所述的氨解液，其中，所述碱性水溶液的浓度为1～10M。
  10. 根据权利要求1所述的氨解液，其中，所述胺、丁醇、氨基醇和碱性水溶液的体积比为(6～8):(2～3):1:(0.04～0.06)。
  11. 根据权利要求8所述的氨解液，其中，所述胺是正丁胺，所述丁醇是正丁醇，所述氨基醇是5-氨基-1-戊醇，并且所述正丁胺、正丁醇、5-氨基-1-戊醇和碱性水溶液的体积比为(5～10):(1～5):1:(0.01～0.1)。
  12. 根据权利要求11所述的氨解液，其中，所述正丁胺、正丁醇、5-氨基-1-戊醇和碱性水溶液的体积比为(6～8):(2～3):1:(0.04～0.06)。
  13. 一种氨解方法，包括以下步骤：
    (1)向装有固相载体的固相合成柱中加入权利要求1-12任一项所述的氨解液；
    (2)将所述固相合成柱置于装有氨解缓冲液的密封容器内，并保持所述固相合成柱不接触氨解缓冲液；
    (3)加热所述密封容器，使固相载体处于氨解缓冲液的蒸汽气氛中，随后冷却；
    (4)从所述密封容器中取出固相合成柱，用洗脱液处理固相合成柱中的固相载体，并收集流出液。
  14. 根据权利要求13所述的方法，其中，步骤(1)所述固相载体包括控制孔径玻璃球。
  15. 根据权利要求13所述的方法，其中，步骤(1)所述固相载体上连接有合成的寡核苷酸。
  16. 根据权利要求13所述的方法，其中，步骤(1)所述氨解液浸没固相载体。
  17. 根据权利要求13所述的方法，其中，步骤(2)所述密封容器包括金属密封盒。
  18. 根据权利要求13所述的方法，其中，步骤(2)所述氨解缓冲液包括氨水和/或有机 胺。
  19. 根据权利要求18所述的方法，其中，所述有机胺包括二乙胺、乙二胺或吡啶中的任意一种或至少两种的组合。
  20. 根据权利要求13所述的方法，其中，步骤(3)所述加热的温度为45～100℃。
  21. 根据权利要求13所述的方法，其中，步骤(3)所述加热的时间为30～60min。
  22. 根据权利要求13所述的方法，其中，步骤(3)所述冷却为冰浴5～15min。
  23. 根据权利要求13所述的方法，其中，步骤(4)所述洗脱液包括水和/或Tris-HCl。
  24. 根据权利要求23所述的方法，其中，所述Tris-HCl的浓度为10～30mM。
  25. 根据权利要求24所述的方法，其中，所述Tris-HCl的pH为7～8。
  26. 根据权利要求13所述的方法，其中，在步骤(4)所述用洗脱液处理固相合成柱中的固相载体之前，还包括干燥和洗涤所述固相合成柱的步骤。
  27. 根据权利要求26所述的方法，其中，所述干燥为在50～60℃下处理1～5min。
  28. 根据权利要求26所述的方法，其中，所述洗涤采用的洗涤液包括100％乙腈和/或80～95％乙腈。
  29. 根据权利要求28所述的方法，其中，所述洗涤包括：将干燥后的固相合成柱依次采用100％乙腈和80～95％乙腈洗涤。
  30. 根据权利要求26所述的方法，其中，在洗涤所述固相合成柱之后还包括离心的步骤。
  31. 根据权利要求13所述的方法，其中，所述方法包括以下步骤：
    (1)向装有连接了合成的寡核苷酸的控制孔径玻璃球的固相合成柱中加入权利要求1-12任一项所述的氨解液，浸没控制孔径玻璃球；
    (2)将所述固相合成柱置于装有氨解缓冲液的金属密封容器内，并保持所述固相合成柱不接触氨解缓冲液，所述氨解缓冲液包括氨水和/或有机胺；
    (3)将所述金属密封容器在45～100℃下加热30～60min，使连接了合成的寡核苷酸的控制孔径玻璃球处于氨解缓冲液的蒸汽气氛中，随后冰浴冷却5～15min；
    (4)从所述密封容器中取出固相合成柱，50～60℃下干燥1～5min，并依次用100％乙腈和80～95％乙腈洗涤，离心后用水和/或10～25mM pH7～8的Tris-HCl处理固相合成柱中的固相载体，并收集流出液。
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