WO2019052322A1 - 一种基于高通量测序分析寡核苷酸序列杂质的方法及应用 - Google Patents

一种基于高通量测序分析寡核苷酸序列杂质的方法及应用 Download PDF

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WO2019052322A1
WO2019052322A1 PCT/CN2018/101878 CN2018101878W WO2019052322A1 WO 2019052322 A1 WO2019052322 A1 WO 2019052322A1 CN 2018101878 W CN2018101878 W CN 2018101878W WO 2019052322 A1 WO2019052322 A1 WO 2019052322A1
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oligonucleotide
sequence
throughput sequencing
oligonucleotide sequence
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郭良让
张佩琢
于雷
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苏州吉赛基因测序科技有限公司
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Priority to US16/648,529 priority Critical patent/US11597922B2/en
Priority to EP18857038.6A priority patent/EP3680346A4/en
Priority to JP2020537274A priority patent/JP7034299B2/ja
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms

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  • the invention belongs to the field of biotechnology, and particularly relates to a method and application for analyzing impurities of an oligonucleotide sequence based on high-throughput sequencing.
  • Antisense oligodeoxynucleotide technology is a therapeutic method for blocking and inhibiting the expression of genes involved in disease development by using synthetic or biosynthetic DNA or RNA complementary to RNA, which can be used to treat mutations caused by genes. Tumor or genetic disease.
  • Antisense technology is a new drug development method.
  • the drug developed by this technology is called antisense drug and involves antisense DNA, antisense RNA and ribozyme.
  • antisense drugs can hybridize with specific genes, interfere with the production of pathogenic proteins at the gene level, that is, interfere with the transmission of genetic information from nucleic acids to proteins.
  • Traditional drugs mainly act directly on the causative protein itself, and antisense drugs act on genes that produce proteins.
  • Antisense drugs have higher selectivity and efficiency than traditional drugs, and can be widely used in the treatment of various diseases such as infectious diseases, inflammation, cardiovascular diseases and tumors.
  • Gene therapy refers to the introduction of a foreign normal gene into a target cell to correct or compensate for diseases caused by genetic defects and abnormalities for therapeutic purposes. Broadly speaking, gene therapy can also include measures and new techniques for treating certain diseases taken at the DNA level.
  • Antisense DNA mainly refers to antisense oligodeoxynucleotide (AS-ODN), which is a short sequence complementary to one or more sites (complementary regions) of target gene mRNA.
  • AS-ODN antisense oligodeoxynucleotide
  • AS-ODN can act on different targets: binding to double-stranded DNA to regulate transcription; binding to mRNA precursors or splicing sites inhibits mRNA precursor splicing and affects splicing of mRNA from nucleus to cytoplasm; binding to mRNA in cytoplasm Translation; binding to specific proteins regulates gene expression.
  • antisense DNA is a synthetic sequence
  • there are cases of base insertion and lack during synthesis resulting in the synthesis of DNA containing a large amount of impurities, and the synthesized sequence is purified for use in pharmaceuticals, thus requiring purification of each sequence group.
  • Analysis and confirmation However, there is currently no good solution for the analysis of single-stranded oligonucleic acid sequences.
  • the technical problem to be solved by the present invention is how to quickly, accurately and comprehensively analyze the composition and purity and/or content of each component sequence in a synthetic oligonucleotide sequence.
  • the present invention first provides a method for constructing a high-throughput sequencing library for impurity analysis of oligonucleotide sequences.
  • the method for constructing a high-throughput sequencing library for oligonucleotide sequence impurity analysis comprises the following steps:
  • the primer used for the reverse extension and amplification consists of the oligonucleotide to be detected and the extension primer;
  • the extension primer sequence is sequentially composed of a DNA molecule represented by position 1-22 of the sequence 2 and N bases A or bases T or bases C or bases G; the N is an integer greater than or equal to 6;
  • the oligonucleotide may have a length of 8 to 120 bp.
  • the oligonucleotide may be single stranded DNA or double stranded DNA.
  • the oligonucleotide is a single-stranded DNA having a nucleotide sequence of sequence 5 and a size of 21 bp.
  • the poly tail in step 1), may be a poly A tail or a poly G tail or a poly C tail or a poly T tail; in a specific embodiment of the invention, the poly tail is a poly A tail;
  • the method of adding a poly tail to the 3' end of the oligonucleotide sequence to be detected is specifically as follows: 1.5 ⁇ L of the terminal transferase, 1 ⁇ L of the oligonucleotide to be detected, dATP (or dTTP or dCTP or dGTP) (25 ⁇ M) 0.5 ⁇ L, 5 ⁇ TdT Buffer 4 ⁇ L and nuclease-free water were mixed to obtain a reaction system (total volume of 20 ⁇ L). The final concentration of the oligonucleotide to be detected in the reaction system was 5 ⁇ M.
  • the N may be any integer greater than or equal to 6. In a specific embodiment of the invention, the N is specifically 20.
  • the extension primer sequence is the DNA molecule shown in SEQ ID NO: 1 or the DNA molecule shown in SEQ ID NO: 2 or the DNA molecule shown in SEQ ID NO: 3 or the DNA molecule shown in SEQ ID NO: 4. In a specific embodiment of the invention, the extension primer sequence is the DNA molecule shown in SEQ ID NO: 1.
  • the system for reverse extension and amplification reaction (total volume 50 ⁇ L) consisted of 2 ⁇ Phata Max Buffer 25 ⁇ L, dNTPS (10 mM) 2 ⁇ L, extension primer 2 ⁇ L, 1 ⁇ L of oligonucleotide to be detected, DNA polymerase I 1 ⁇ L and no Nuclease-containing water composition.
  • the final concentration of the extension primer in the reverse system was 4 ⁇ M; the final concentration of the oligonucleotide to be detected in the reverse system was 2 ⁇ M.
  • the method of the precipitation is sodium acetate precipitation; the method of reversely stretching and amplifying the product for sodium acetate precipitation is as follows:
  • step 3a 1/10 volume of sodium acetate, 2.5 volumes of absolute ethanol and 1 ⁇ L of glycoside are added to the reverse extension and the amplification product; the pH of the sodium acetate is 5.2; The concentration of the element is 20 mg/mL;
  • the centrifugation condition is 12000 rpm, and centrifugation at 4 ° C for 30 minutes;
  • the centrifugation condition is 12000 rpm, centrifugation at 4 ° C for 5 minutes; the ethanol is 80% by volume of ethanol;
  • step 3a) a step of leaving at -80 ° C for 30 minutes is further included; the step 3 c) is repeated once.
  • the method of sequentially performing the terminal repair, the addition of the A tail, the connector and the PCR amplification of the precipitated product is as follows:
  • the method for performing the terminal repair and the A tail of the precipitated product is as follows: 15 ⁇ L of the precipitated product, 3 ⁇ L of 10 ⁇ end repair buffer, 2 ⁇ L of T4 DNA polymerase, and T4 Polynucleotide Kinase (T4 PNK). 2 ⁇ L, Klenow DNA polymerase I 0.5 ⁇ L, Bst DNA Pol I large Fragment 0.5 ⁇ L, and 7 ⁇ L of nuclease-free water were mixed and reacted.
  • the method for adding the repaired product to the linker is as follows: 30 ⁇ L of the repaired product, 15 ⁇ L of 10 ⁇ T4 DNA Ligase Buffer, 2 ⁇ L of T4 DNA Ligase, 2 ⁇ L of the Y-type linker, and nuclease-free water. Mix 1 ⁇ L and react.
  • the method for PCR amplification of the addition product is as follows: 5 ⁇ L of the linker product, 2 ⁇ L of each primer, 1 ⁇ L of dNTP mix (10 mM), 25 ⁇ L of 2 ⁇ Phanta Max Buffer, Phanta Max Super Fide 1 ⁇ L of DNA Polymerase and 14 ⁇ L of nuclease-free water were mixed and reacted.
  • the primer had a final concentration of 1 ⁇ M in the reaction system.
  • step 4b) Also included between the step 4b) and the step 4c) is a step of purifying, which may be carried out using magnetic beads.
  • the step 4c) further includes a step of purifying, which can be carried out using a silica gel column.
  • the present invention further provides a product.
  • the product of the present invention is any of the following a1)-a3):
  • A2) a PCR reagent comprising the extension primer of a1);
  • A3 A kit comprising the extension primer of a1) or the PCR reagent of a2).
  • the final concentration of the extension primer in the PCR reagent is from 0.1 to 100 ⁇ M.
  • the extension primer has a final concentration in the PCR reagent of 100 [mu]M.
  • the present invention also provides a method for analyzing impurities of an oligonucleotide sequence.
  • the method for analyzing an impurity of an oligonucleotide sequence comprises the following steps:
  • the high-throughput sequencing library is subjected to high-throughput sequencing, and the nucleotide sequence components are analyzed based on the sequencing results.
  • the present invention finally provides a new use of the above method.
  • the present invention provides the use of the above method for the analysis of synthetic antisense oligonucleotide sequence components for gene therapy.
  • the invention also provides the use of the above method for the analysis of the purity and/or content of each component in an artificially synthesized antisense oligonucleotide sequence for gene therapy.
  • Figure 1 is a flow diagram of a method for analyzing oligonucleotide sequence impurities based on high throughput sequencing.
  • Figure 2 shows the results of electrophoresis detection of sodium acetate precipitation product.
  • Figure 3 shows the results of electrophoresis detection of PCR amplification products.
  • Figure 4 shows the results of electrophoresis detection of the purified PCR amplification product.
  • FIG. 5 shows the results of high-throughput sequencing data analysis.
  • T4 DNA polymerase in the following examples is a product of NEB (Beijing) Co., Ltd., catalog number M0203L.
  • DNA Polymerase I and Large (Klenow) Fragment are products of NEB Corporation, catalog number M0210S.
  • DNA Polymerase I, Large (Klenow) Fragment is called DNA polymerase I.
  • T4 Polynucleotide Kinase is a product of NEB with catalog number M0201. Klenow DNA polymerase I and Bst DNA Pol I large Fragment are products of NEB Corporation, catalog number M0275.
  • T4 DNA Ligase is a product of NEB Corporation with catalog number M0202L.
  • the terminal transferase is a product of Thermo Fisher Scientific (China) Co., Ltd., catalog number EP0162.
  • Deoxyadenosine triphosphate is a product of Thermo Fisher Scientific (China) Co., Ltd., catalog number 10216018.
  • Phanta Max Super Fide DNA Polymerase is a product of Nanjing Nuoweizan Biotechnology Co., Ltd., catalog number is P505.
  • 10 ⁇ end repair buffer formulation in the following examples: solute and its concentration of 900 mM MgCl 2 , 30 mM DTT, 10 mM ATP, 1 ⁇ g/ ⁇ L BSA and 4 mM dNTPs; solvent 8.3, 500 mM Tris-HCl buffer .
  • Example 1 A method for analyzing impurities of an oligonucleotide sequence based on high-throughput sequencing
  • oligonucleotide sequence was as follows: 5'-CAGAGCAGCTTGTCTTTCTTC-3' (SEQ ID NO: 5).
  • the oligonucleotide sequence was synthesized by Shanghai Jierui Bioengineering Co., Ltd.
  • Table 1 is the tailing reaction system
  • Table 2 is a reverse extension amplification system
  • the extension primer extpT sequence is as follows:
  • the reverse extension amplification system prepared in the step 1 was placed on a PCR machine to carry out the reaction procedure shown in Table 3.
  • Table 3 is the reverse extension amplification program
  • Table 4 is the end repair reaction system
  • step 2 The end-repair reaction system in step 1 is subjected to the procedure shown in Table 5 on the PCR machine.
  • Table 5 shows the PCR reaction procedure
  • the joint connection reaction system was prepared according to each reagent and the amount added in Table 6. After mixing, mix and centrifuge instantaneously.
  • Table 6 is the joint connection reaction system
  • AI5 underlined base for phosphorylation: 5- G ATCGGAAGAGCACACGTCTGAACTCCAGTCACACAGTGATCTCGTATGCCGTCTTCTGCTTG.
  • the above Y-type linker sequences were synthesized by Shanghai Jierui Bioengineering Co., Ltd.
  • the concentration of the linker in the linker reaction system was 1.6 ⁇ M.
  • the linker reaction reaction system was placed on a PCR machine at 16 ° C for 2 hours to obtain a linker product.
  • Table 7 shows the PCR amplification system
  • the primer mPF sequence is as follows (synthesized by Shanghai Jierui Bioengineering Co., Ltd.): AATGATACGGCACCCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT;
  • the primer mRPI5 sequence is as follows (synthesized by Shanghai Jierui Bioengineering Co., Ltd.): CAAGCAGAAGACGGCATACGAGATCACTGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC T .
  • underlined bases are subjected to thio modification.
  • the PCR amplification system was placed in a PCR machine and amplified according to the PCR reaction procedure shown in Table 8, to obtain a PCR amplification product.
  • the PCR amplification product was subjected to gel electrophoresis, and the results are shown in FIG.
  • Table 8 shows the PCR reaction procedure
  • the library constructed in step 8 was sequenced using the Hiseq 3000 platform in a single-ended 150 bp sequencing mode.
  • ExtractValid.pl extracts the forward-sequenced reads containing the linker using the cutadapt1.2.1 software (the URL of the cutadapt1.2.1 software is as follows: https://github.com/marcelm/cutadapt/releases/tag/v1.2.1)
  • FilterTN.pl to remove the read segment containing the N and PolyT connectors
  • trim_polytail.pl to remove the terminal containing the wrong base due to impure dNTP.
  • Poly tail use the self-written perl script FastQ_ReadFilterByLength.pl to filter long or short reads, using FASTX Toolkit 0.0.13 software (FASTX Toolkit 0.0.13 software URL is as follows: https://hannonlab.cshl.edu/fastx_toolkit/ The fastx_collapser module in the merge merges the repeats, removing only one number of reads.
  • Table 9 shows the results of data analysis.
  • the oligonucleotide sequence is CAGAGCAGCTTGTCTTTCTTC.
  • ncbi-blast-2.2.28+ software is available at: https://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/2.2.28/)
  • the oligonucleotide reference sequence is compared; the results are parsed by a self-written script comparison, and the parsed results are normalized, and the standardized results are classified, and finally the classification results are counted.
  • the analysis results of the components and contents in which the content is more than 0.1% are shown in Table 10.
  • the analysis results of the number of readings of N-1 and the number of readings of N+1 are shown in Table 11 and Table 12.
  • Table 11 and Table 12 As can be seen from the table, the oligonucleotide sequence of each component having a content greater than 0.1% and its content, the number of reads of N-1 and the content and ratio of each component in the number of reads of N+1.
  • Table 10 shows the components and content of more than 0.1%
  • Table 11 shows the results of the analysis of the number of readings of N-1.
  • Numbering Classification (compared to oligonucleic acid sequences) Number of readings ratio(%) 1 Number of reads with a portion of base missing at the 5' or 3' end 290439 68.22% 2 Number of reads with incorrect bases 2447 0.57% 3 Number of reads with missing base in the middle 76214 4 Number of reads with partial insertion or deletion at the 5' or 3' end 57425 17.9% 5 Number of reads with inserted bases 137 13.49% 6 Total number of N-1 reads 425718 0.03%
  • the oligonucleotide sequence is CAGAGCAGCTTGTCTTTCTTC.
  • Table 12 shows the results of the analysis of the number of reads of N+1.
  • the oligonucleotide sequence is CAGAGCAGCTTGTCTTTCTTC.
  • the present invention provides a method for analyzing oligonucleotide sequence impurities based on high throughput sequencing.
  • the method of the present invention comprises the steps of: constructing a high-throughput sequencing library for oligonucleotide sequence impurity analysis; performing high-throughput sequencing of the high-throughput sequencing library, and sequencing the oligonucleotide sequence according to the sequencing result
  • the analysis is performed in the high-throughput sequencing library; the extended primer sequence used in the construction of the high-throughput sequencing library is sequentially represented by the DNA molecule shown in position 1-22 of the sequence 2 and the N base A or the base T or the base C or the base.
  • G composition; the N is an integer greater than or equal to 6. It has been experimentally proved that the method for analyzing oligonucleotide sequence impurities based on high-throughput sequencing of the present invention can quickly, accurately and comprehensively analyze the purity and content of each component in the oligonucleotide sequence.

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Abstract

一种基于高通量测序分析寡核苷酸序列杂质的方法及应用。方法包括如下步骤：构建用于寡核苷酸序列杂质分析的高通量测序文库；将所述高通量测序文库进行高通量测序，并根据测序结果对寡核苷酸序列组分进行分析；所述构建高通量测序文库中所使用的延伸引物序列依次由序列2第1-22位所示的DNA分子和N个碱基A或碱基T或碱基C或碱基G组成；所述N为大于等于6的整数。通过实验证明：基于高通量测序分析寡核苷酸序列杂质的方法可以快速、准确和全面的对寡聚核苷酸序列中各组分纯度和含量进行分析。

Description

一种基于高通量测序分析寡核苷酸序列杂质的方法及应用 技术领域
本发明属于生物技术领域，具体涉及一种基于高通量测序分析寡核苷酸序列杂质的方法及应用。
背景技术
近年来，国内外越来越多的制药企业纷纷涉足基因药物，投入巨资用于研制新型基因药物以对抗各种疾病。反义寡聚脱氧核苷酸技术是利用能与RNA互补的人工合成或生物合成的DNA或RNA，封闭和抑制与疾病发生相关基因表达的一种治疗手段，可用于治疗由于基因突变所致的肿瘤或遗传疾病。
反义技术是一种新的药物开发方法，利用这一技术研制的药物称为反义药物，涉及反义DNA、反义RNA及核酶(ribozyme)。根据核酸杂交原理，反义药物能与特定基因杂交，在基因水平上干扰致病蛋白的产生，即干扰遗传信息从核酸向蛋白质的传递。传统药物主要直接作用于致病蛋白本身，反义药物则作用于产生蛋白质的基因。与传统药物相比，反义药物具有更高的选择性和效率，可广泛用于多种疾病的治疗，如传染病、炎症、心血管疾病及肿瘤等。
基因治疗是指将外源正常基因导入靶细胞，以纠正或补偿因基因缺陷和异常引起的疾病，以达到治疗目的。从广义说，基因治疗还可包括从DNA水平采取的治疗某些疾病的措施和新技术。
反义DNA主要指反义寡聚脱氧核苷酸(antisense oligodeoxynucleotide，AS-ODN)，AS-ODN是一种与靶基因mRNA的一个或多个位点(互补区)互补、人工合成的短序列单链DNA片段，能抑制或减少靶基因的表达。AS-ODN可作用于不同目标：与双链DNA结合调节转录；与mRNA前体或剪接点结合抑制mRNA前体剪接，并影响剪接后的mRNA从核运至细胞质；与胞质中mRNA结合阻断翻译；与特异蛋白质结合调节基因表达。
由于反义DNA是人工合成的序列，在合成过程中存在碱基插入及缺少 的情况，导致合成的DNA含有大量的杂质，并且合成的序列纯化后用于制药，因此需要对纯化的各序列组分进行分析和确认。但对于单链寡核酸序列的分析，目前没有很好的解决办法。
发明公开
本发明要解决的技术问题是如何快速、准确和全面的分析人工合成的寡聚核苷酸序列中各组分序列的组成及其纯度和/或含量。
为了解决上述技术问题，本发明首先提供了一种用于寡核苷酸序列杂质分析的高通量测序文库的构建方法。
本发明提供的用于寡核苷酸序列杂质分析的高通量测序文库的构建方法包括如下步骤：
1)将待检测寡核苷酸序列的3’端加poly尾，得到加poly尾产物；
2)将所述加poly尾产物进行反向延伸并扩增，得到反向延伸并扩增产物；
所述反向延伸并扩增所使用的引物由所述待检测寡核苷酸和延伸引物组成；
所述延伸引物序列依次由序列2第1-22位所示的DNA分子和N个碱基A或碱基T或碱基C或碱基G组成；所述N为大于等于6的整数；
3)将所述反向延伸并扩增产物进行沉淀，得到沉淀后产物；
4)将所述沉淀后产物依次进行末端修复、加A尾、连接头和PCR扩增，得到高通量测序文库。
上述方法中，所述寡核苷酸的长度可为8-120bp。所述寡核苷酸可为单链DNA或双链DNA。在本发明的具体实施例中，所述寡核苷酸为单链DNA，其核苷酸序列为序列5，大小为21bp。
上述方法中，步骤1)中，所述poly尾可为poly A尾或poly G尾或poly C尾或poly T尾；在本发明的具体实施例中，所述poly尾为poly A尾；
所述将待检测寡核苷酸序列的3’端加poly尾的方法具体如下：将末端转移酶1.5μL、待检测寡核苷酸1μL、dATP(或dTTP或dCTP或dGTP)(25μM)0.5μL、5×TdT Buffer 4μL和不含核酸酶的水混匀，得到反应 体系(总体积为20μL)。所述待检测寡核苷酸在反应体系中的终浓度为5μM。
上述方法中，步骤2)中，所述N可为任一大于等于6的整数。在本发明的具体实施例中，所述N具体为20。当N为20时，所述延伸引物序列为序列1所示的DNA分子或序列2所示的DNA分子或序列3所示的DNA分子或序列4所示的DNA分子。在本发明的具体实施例中，所述延伸引物序列为序列1所示的DNA分子。
所述反向延伸并扩增反应的体系(总体积为50μL)由2×Phata Max Buffer 25μL、dNTPS(10mM)2μL、延伸引物2μL、待检测寡核苷酸1μL、DNA聚合酶I 1μL和不含核酸酶的水组成。所述延伸引物在反向体系中的终浓度为4μM；所述待检测寡核苷酸在反向体系中的终浓度为2μM。
上述方法中，步骤3)中，所述沉淀采用的方法为醋酸钠沉淀；将反向延伸并扩增产物进行醋酸钠沉淀的方法具体如下：
3a)向反向延伸并扩增产物中加入醋酸钠、无水乙醇和糖元；
3b)离心，弃上清液，收集沉淀；
3c)向所述沉淀中加入乙醇，离心，弃上清液，收集沉淀。
所述步骤3a)中，向反向延伸并扩增产物中加入1/10体积的醋酸钠、2.5倍体积的无水乙醇和1μL的糖元；所述醋酸钠的pH为5.2；所述糖元浓度为20mg/mL；
所述步骤3b)中，所述离心的条件为12000rpm，4℃离心30分钟；
所述步骤3c)中，所述离心的条件为12000rpm，4℃离心5分钟；所述乙醇为体积分数为80％的乙醇；
所述步骤3a)和所述步骤3b)之间还包括-80℃放置30分钟的步骤；所述步骤3c)重复一次。
上述方法中，步骤4)中，将所述沉淀后产物依次进行末端修复、加A尾、连接头和PCR扩增的方法具体如下：
4a)将所述沉淀后产物进行末端修复加A尾，得到修复后产物；
4b)将所述修复后产物加接头，得到加接头产物；
4c)将所述加接头产物进行PCR扩增，得到扩增产物，即为用于寡核苷酸序列杂质分析的高通量测序文库。
所述步骤4a)中，将所述沉淀后产物进行末端修复加A尾的方法具体 如下：将沉淀后产物15μL、10×末端修复缓冲液3μL、T4 DNA聚合酶2μL、T4 Polynucleotide Kinase(T4 PNK) 2μL、Klenow DNA polymerase I 0.5μL、Bst DNA Pol I large Fragment 0.5μL和不含核酸酶的水7μL混匀，反应。
所述步骤4b)中，将所述修复后产物加接头的方法具体如下：将修复后产物30μL、10×T4 DNA Ligase Buffer 15μL、T4 DNA Ligase 2μL、Y型接头2μL和不含核酸酶的水1μL混匀，反应。
所述步骤4c)中，将所述加接头产物进行PCR扩增的方法具体如下：将加接头产物5μL、引物各2μL、dNTP mix(10mM)1μL、2×Phanta Max Buffer 25μL、Phanta Max Super Fide DNA Polymerase 1μL和不含核酸酶的水14μL混匀，反应。所述引物在反应体系中的终浓度为1μM。
所述步骤4b)和所述步骤4c)之间还包括纯化的步骤，所述纯化可采用磁珠进行。
所述步骤4c)后还包括纯化的步骤，所述纯化可采用硅胶柱进行。
为了解决上述技术问题，本发明又提供了一种产品。
本发明的产品为如下a1)-a3)中的任一种：
a1)上述延伸引物；
a2)含有a1)所述的延伸引物的PCR试剂；
a3)含有a1)所述的延伸引物或a2)所述的PCR试剂的试剂盒。
上述产品中，所述延伸引物在所述PCR试剂中的终浓度为0.1-100μM。在本发明的具体实施例中，所述延伸引物在所述PCR试剂中的终浓度为100μM。
上述产品在构建用于寡核苷酸序列杂质分析的高通量测序文库中的应用也属于本发明的保护范围。
上述产品在寡核苷酸序列杂质分析中的应用也属于本发明的保护范围。
为了解决上述技术问题，本发明还提供了一种寡核苷酸序列杂质的分析方法。
本发明提供的寡核苷酸序列杂质的分析方法包括如下步骤：
(1)按照上述方法构建用于寡核苷酸序列杂质分析的高通量测序文库；
(2)将所述高通量测序文库进行高通量测序，并根据测序结果对核苷酸序列组分进行分析。
为了解决上述技术问题，本发明最后提供了上述方法的新用途。
本发明提供了上述方法在分析人工合成的用于基因治疗的反义寡聚核苷酸序列组分中的应用。
本发明还提供了上述方法在分析人工合成的用于基因治疗的反义寡聚核苷酸序列中各组分纯度和/或含量中的应用。
通过实验证明：本发明的基于高通量测序分析寡核苷酸序列杂质的方法可以快速、准确和全面的对寡聚核苷酸序列中各组分纯度和含量进行分析。
附图说明
图1为基于高通量测序分析寡核苷酸序列杂质的方法流程图。
图2为醋酸钠沉淀产物的电泳检测结果。
图3为PCR扩增产物的电泳检测结果。
图4为纯化后的PCR扩增产物的电泳检测结果。
图5为高通量测序数据分析结果。
实施发明的最佳方式
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
下述实施例中的T4 DNA聚合酶为NEB(北京)有限公司的产品，产品目录号为M0203L。DNA Polymerase I和Large(Klenow)Fragment均为NEB公司的产品，产品目录号为M0210S，在下文中，DNA Polymerase I、Large(Klenow)Fragment称为DNA聚合酶I。T4 Polynucleotide Kinase为NEB公司的产品，产品目录号为M0201。Klenow DNA polymerase I和Bst DNA Pol I large Fragment为NEB公司的产品，产品目录号为M0275。T4 DNA Ligase为NEB公司的产品，产品目录号为M0202L。末端转移酶为赛默飞世尔科技(中国)有限公司的产品，产品目录号为EP0162。脱氧腺苷三磷酸为赛默飞世尔科技(中国)有限公司的产品，产品目录号为 10216018。Phanta Max Super Fide DNA Polymerase为南京诺唯赞生物科技有限公司的产品，产品目录号为P505。
下述实施例中的10×末端修复缓冲液配方：溶质及其浓度为900mM MgCl 2、30mM DTT、10mM ATP、1μg/μL BSA和4mM dNTPs；溶剂为pH8.3、500mM的Tris-HCl缓冲液。
实施例1、一种基于高通量测序分析寡核苷酸序列杂质的方法
一、寡核苷酸加poly尾
1、在寡核苷酸序列的3’端加poly A尾，按照表1中的各试剂及加入量配制加尾反应体系。寡核苷酸序列如下：5’-CAGAGCAGCTTGTCTTTCTTC-3’(序列5)。寡核苷酸序列由上海捷瑞生物工程有限公司合成。
表1为加尾反应体系
试剂 加入量(μL)
不含核酸酶的水 13
寡核苷酸(100μM) 1
dATP(25μM) 0.5
5×TdT Buffer 4
末端转移酶 1.5
总体积 20μL
2、37℃反应25min。
3、70℃反应10min，失活末端转移酶。
二、反向延伸并扩增
1、向上述步骤一得到的反应产物中加入表2中所示的各试剂，配制反向延伸扩增体系。
表2为反向延伸扩增体系
试剂 加入量(μL)
不含核酸酶的水 19
2×Phanta Max Buffer 25
dNTPS(10mM) 2
延伸引物extpT(100μM) 2
寡核苷酸(100μM) 1
DNA聚合酶I 1
总体积 50
延伸引物extpT序列如下：
5’-GAGACACGAATAGACGGCACGATTTTTTTTTTTTTTTTTTTT-3’(序列1)。
2、将步骤1中配制的反向延伸扩增体系置于PCR仪上进行表3所示的反应程序。
表3为反向延伸扩增程序
Figure PCTCN2018101878-appb-000001
三、醋酸钠沉淀
1、取步骤二得到的产物，加入1/10体积的醋酸钠(pH5.2)、2.5倍体积的无水乙醇和1μL浓度为20mg/mL的糖元。
2、-80℃放置30分钟。
3、12000rpm，4℃离心30分钟，弃上清，收集沉淀。
4、向沉淀中加入1mL 80％的乙醇，然后12000rpm，4℃离心5分钟，弃上清，收集沉淀。
5、重复步骤4一次。
6、室温晾干，加入30μL TE溶解。
7、沉淀产物质检。具体步骤如下：配制12％PAGE凝胶，200V电泳40分钟，于暗盒中染色10分钟，于凝胶成像系统中成像拍照。电泳检测结果如图2所示。
四、末端修复
1、按照表4中的各试剂及加入量配制末端修复反应体系。配制后混匀，瞬时离心。
表4为末端修复反应体系
试剂 加入量(μL)
不含核酸酶的水 7
10×末端修复缓冲液 3
T4 DNA聚合酶 2
T4 Polynucleotide Kinase(T4 PNK) 2
Klenow DNA polymerase I 0.5
Bst DNA Pol I large Fragment 0.5
步骤三产物 15
总体积 30
2、将步骤1中的末端修复反应体系在PCR仪上执行表5所示的程序。
表5为PCR反应程序
segment1 20℃30分钟
segment2 65℃30分钟
segment3 10℃保存
五、接头连接
1、按照表6中的各试剂及加入量配制接头连接反应体系。配制后混匀，瞬时离心。
表6为接头连接反应体系
试剂 加入量(μL)
末端修复产物 30
不含核酸酶的水 11
10×T4 DNA Ligase Buffer 5
Y型接头(40μM) 2
T4 DNA Ligase 2
总体积 50
上述Y型接头由UAF和AI5组成，其序列分别如下：
UAF(下划线标记的碱基进行硫代修饰)：5-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATC T；
AI5(下划线标记的碱基进行磷酸修饰)：5- GATCGGAAGAGCACACGTCTGAACTCCAGTCACACAGTGATCTCGTATGCCGTCTTCTGCTTG。
上述Y型接头序列均由上海捷瑞生物工程有限公司合成。接头在接头连接反应体系中的浓度均为1.6μM。
2、将接头连接反应体系置于PCR仪上16℃反应2小时，得到接头连接产物。
六、接头连接产物纯化
1、振荡Ampure XP磁珠(贝克曼库尔特有限公司生产的Agencourt AMPure XP Kit，产品目录号为A63880)，充分混匀；
2、向上述步骤五中获得的连接产物中加入1×的Ampure XP磁珠，用移液器混匀10次，室温放置1分钟；
3、置于磁力架上吸附5分钟，弃上清；
4、向磁珠中加入200μL新鲜配制的80％的乙醇，室温放置30s，弃上清；
5、重复一次上述步骤4；
6、加入50μL浓度为10mM Tris-Hcl(pH8.0)洗脱，转移上清至新的离心管中；
7、加入1倍体积的磁珠，用移液器混匀10次，室温放置1分钟；
8、向磁珠中加入200μL新鲜配制的80％的乙醇，室温放置30s，弃上清；
9、重复一次上述步骤8；
10、开盖室温放置10分钟；
11、加入50μL浓度为10mM Tris-Hcl(pH8.0)，用移液器混匀，室温放置1分钟；
12、置于磁力架上5分钟，转移上清至新的离心管中，得到纯化后的接头连接产物。
七、接头连接产物PCR扩增
1、向纯化后的接头连接产物中加入表7中的各试剂，加入量也按照表7所示，配制PCR扩增体系。
表7为PCR扩增体系
试剂 加入量(μL)
不含核酸酶的水 14
mpF(25μM) 2
mpRI5(25μM) 2
dNTP mix(10mM) 1
2×Phanta Max Buffer 25
Phanta Max Super Fide DNA Polymerase 1
引物mPF序列如下(由上海捷瑞生物工程有限公司合成)：AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT；
引物mRPI5序列如下(由上海捷瑞生物工程有限公司合成)：CAAGCAGAAGACGGCATACGAGATCACTGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC T。上述引物序列中，下划线标记的碱基进行硫代修饰。
2、以45μL/管分装至PCR管中，然后加入5μL连接产物。
3、将PCR扩增体系置于PCR仪中，按照表8所示的PCR反应程序进行扩增，得到PCR扩增产物。将PCR扩增产物进行凝胶电泳检测，结果如图3所示。
表8为PCR反应程序
Figure PCTCN2018101878-appb-000002
八、PCR扩增产物纯化
1、切取上述电泳中所需条带，用上海捷瑞生物工程有限公司的琼脂糖凝胶DNA回收试剂盒(离心柱型，GK2042-50)进行凝胶回收。
2、向凝胶中加入400μL Binding Solution，置于50℃水浴锅中，至胶块溶解。
3、期间每隔2分钟摇动一次。
4、将溶好的胶块转移至硅胶柱中，室温放置2分钟，6000转/分离心1分钟，弃废液。
5、向硅胶柱中加入500μL Washing Solution，室温放置3分钟。
6、12000转/分离心1分钟，弃废液。
7、重复一次步骤6。
8、12000转/分离心1分钟，转移硅胶柱至新的1.5mL离心管中。
9、向硅胶柱中加入30μL不含核酸酶的水，室温放置2分钟。
10、12000转/分离心1分钟，收集上清液，得到纯化后PCR扩增产物。将纯化后PCR扩增产物进行电泳检测，结果如图4所示，其中，M：20bp DNA Ladder；1：文库胶回收产物，从图中可以看出，成功得到高通量测序文库。
九、高通量测序及数据分析
1、高通量测序
取步骤八构建好的文库用Hiseq 3000平台用单端150bp测序模式进行测序。
2、高通量测序结果分析
使用trimmomatic-0.33软件(trimmomatic-0.33软件的网址如下：https://www.usadellab.org/cms/index.php？page＝trimmomatic)去除读段3’末端低质量的碱基，用自行编写的per脚本ExtractValid.pl提取含有接头的正向测序的读段，使用cutadapt1.2.1软件(cutadapt1.2.1软件的网址如下：https://github.com/marcelm/cutadapt/releases/tag/v1.2.1)去除读段中的PolyA接头，用自行编写的per脚本FilterTN.pl去除含有N及PolyT接头的读段，用自行编写的perl脚本trim_polytail.pl去除末端由于dNTP不纯而加入的含有错误碱基的Poly尾，用自行编写perl脚本FastQ_ReadFilterByLength.pl过滤过长或短的读段，用FASTX Toolkit  0.0.13软件(FASTX Toolkit 0.0.13软件的网址如下：https://hannonlab.cshl.edu/fastx_toolkit/)中的fastx_collapser模块合并重复序列，去除只有一个数目的读段。根据如下公式计算寡核苷酸各组分的纯度：各组分比率(％)＝该组分读段数目/各组分读段数目之和×100％。
结果如表9和图5所示。从结果可以看出：在获得的9664589条寡核苷酸序列中，与寡核苷酸序列(CAGAGCAGCTTGTCTTTCTTC)完全一致的寡核苷酸序列共有7830352条，其比率为81.02％。与寡核苷酸序列相比，5’端缺少部分序列的寡核苷酸序列共有1331990条，其比率为13.78％；与寡核苷酸序列相比，3’端缺少部分序列的寡核苷酸序列共有144439条，其比率为1.49％，与寡核苷酸序列相比，5’端和3’端均缺少部分序列的寡核苷酸序列共有18697条，其比率为0.19％，还有一些其他情况(如插入、缺失、掺入错误的碱基等)的寡核苷酸序列，共有339111条，其比率为3.51％。上述结果表明：本发明的方法可以准确、全面的分析寡核苷酸序列中各组分的含量及其所占比率。
表9为数据分析结果
类别(与寡核酸序列相比) 数目(条) 比率(％)
完全一致 7830352 81.02％
5’端缺少部分序列 1331990 13.78％
3’端缺少部分序列 144439 1.49％
5’端和3’端均缺少部分序列 18697 0.19％
其它情况 339111 3.51％
总数 9664589  
注：寡核苷酸序列为CAGAGCAGCTTGTCTTTCTTC。
3、数据分析
首先使用ncbi-blast-2.2.28+软件(ncbi-blast-2.2.28+软件的网址如下：https://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/2.2.28/)与寡核苷酸参考序列进行比对；用自行编写的脚本对比对结果进行解析， 并对解析的结果进行标准化，再对标准化的结果进行分类，最后对分类的结果进行统计。
含量大于0.1％的组分及含量的分析结果如表10所示。N-1的读段数和N+1的读段数的分析结果如表11和表12所示。从表中可以看到，含量大于0.1％的各组分的寡核苷酸序列及其含量，N-1的读段数和N+1的读段数中各组分的含量及比率。
表10为含量大于0.1％的组分及含量
组分编号 组分 比率(％)
1-7830352 CAGAGCAGCTTGTCTTTCTTC 81.02％
2-209704 GCTTGTCTTTCTTC 2.17％
3-204757 AGAGCAGCTTGTCTTTCTTC 2.12％
4-182418 CAGCTTGTCTTTCTTC 1.89％
5-165497 GAGCAGCTTGTCTTTCTTC 1.71％
6-157170 GCAGCTTGTCTTTCTTC 1.63％
7-154399 AGCAGCTTGTCTTTCTTC 1.60％
8-133435 CTTGTCTTTCTTC 1.38％
9-122557 AGCTTGTCTTTCTTC 1.27％
10-85682 CAGAGCAGCTTGTCTTTCTT 0.89％
11-34227 CAGAGCAGCTTGTCTTTCTC 0.35％
12-18571 CAGAGCAGCTTGTCTTTCT 0.19％
13-18146 CAGAGCAGCTTGTCTTCTTC 0.19％
14-15125 CAGAGAGCTTGTCTTTCTTC 0.16％
15-13947 CGGAGCAGCTTGTCTTTCTTC 0.14％
16-13400 CTGAGCAGCTTGTCTTTCTTC 0.14％
17-11075 CAGAGCAGCTTGTCTTTC 0.11％
18-10938 CAAGCAGCTTGTCTTTCTTC 0.11％
19-9391 CGAGCAGCTTGTCTTTCTTC 0.10％
  其它 2.83％
表11为N-1的读段数的分析结果
编号 分类(与寡核酸序列相比) 读段数 比率(％)
1 5’端或3’端缺失一部分碱基的读段数 290439 68.22％
2 含有错误碱基的读段数 2447 0.57％
3 中间含有缺失碱的读段数 76214  
4 5’端或3’端有部分插入或缺失的读段数 57425 17.9％
5 含有插入碱基的读段数 137 13.49％
6 N-1读段的总数 425718 0.03％
注：寡核苷酸序列为CAGAGCAGCTTGTCTTTCTTC。
表12为N+1的读段数的分析结果
编号 分类(与寡核酸序列相比) 读段数 比率(％)
1 含有错误碱基的读段数 73 0.56
2 含有插入碱基的读段数 8571 65.32
3 5’或3’端部分碱基无法匹配的读段数 4568 34.81
4 中间含有缺失碱基的读段数 0 0
5 N+1读段总数 13122  
注：寡核苷酸序列为CAGAGCAGCTTGTCTTTCTTC。
工业应用
本发明提供了一种基于高通量测序分析寡核苷酸序列杂质的方法。本发明的方法包括如下步骤：构建用于寡核苷酸序列杂质分析的高通量测序文库；将所述高通量测序文库进行高通量测序，并根据测序结果对寡核苷酸序列组分进行分析；所述构建高通量测序文库中所使用的延伸引物序列依次由序列2第1-22位所示的DNA分子和N个碱基A或碱基T或碱基C或碱基G组成；所述N为大于等于6的整数。通过实验证明：本发明的基于高通量测序分析寡核苷酸序列杂质的方法可以快速、准确和全面的对寡聚核苷酸序列中各组分纯度和含量进行分析。

Claims (13)

  1. 一种用于寡核苷酸序列杂质分析的高通量测序文库的构建方法，包括如下步骤：
    1)将待检测寡核苷酸序列的3’端加poly尾，得到加poly尾产物；
    2)将所述加poly尾产物进行反向延伸并扩增，得到反向延伸并扩增产物；
    所述反向延伸并扩增所使用的引物由所述待检测寡核苷酸和延伸引物组成；
    所述延伸引物序列依次由序列2第1-22位所示的DNA分子和N个碱基A或碱基T或碱基C或碱基G组成；所述N为大于等于6的整数；
    3)将所述反向延伸并扩增产物进行沉淀，得到沉淀后产物；
    4)将所述沉淀后产物依次进行末端修复、加A尾、连接头和PCR扩增，得到高通量测序文库。
  2. 根据权利要求1所述的方法，其特征在于：所述延伸引物序列为序列1或序列2或序列3或序列4所示的DNA分子。
  3. 根据权利要求2所述的方法，其特征在于：所述延伸引物序列为序列1所示的DNA分子。
  4. 根据权利要求1所述的方法，其特征在于：所述寡核苷酸为单链DNA或双链DNA。
  5. 根据权利要求4所述的方法，其特征在于：所述寡核苷酸为单链DNA。
  6. 根据权利要求1所述的方法，其特征在于：所述寡核苷酸的长度为8-120bp。
  7. 产品，为如下a1)-a3)中的任一种：
    a1)权利要求1中所述的延伸引物；
    a2)含有a1)所述的延伸引物的PCR试剂；
    a3)含有a1)所述的延伸引物或a2)所述的PCR试剂的试剂盒。
  8. 根据权利要求7所述的产品，其特征在于：所述延伸引物在所述PCR试剂中的终浓度为0.1-100μM。
  9. 权利要求7或8所述的产品在构建用于寡核苷酸序列杂质分析的高通量测序文库中的应用。
  10. 权利要求7或8所述的产品在寡核苷酸序列杂质分析中的应用。
  11. 一种寡核苷酸序列杂质的分析方法，包括如下步骤：
    (1)按照权利要求1-6任一所述的方法构建用于寡核苷酸序列杂质分析的高通量测序文库；
    (2)将所述高通量测序文库进行高通量测序，并根据测序结果对寡核苷酸序列组分进行分析。
  12. 权利要求11所述的方法在分析人工合成的用于基因治疗的反义寡聚脱氧核苷酸序列组分中的应用。
  13. 权利要求11所述的方法在分析人工合成的用于基因治疗的反义寡聚脱氧核苷酸序列中各组分纯度和/或含量中的应用。
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