CN116024192A - A TbAgo-based nucleic acid cutting system and detection method and kit for target nucleic acid molecules - Google Patents
A TbAgo-based nucleic acid cutting system and detection method and kit for target nucleic acid molecules Download PDFInfo
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- CN116024192A CN116024192A CN202111255982.5A CN202111255982A CN116024192A CN 116024192 A CN116024192 A CN 116024192A CN 202111255982 A CN202111255982 A CN 202111255982A CN 116024192 A CN116024192 A CN 116024192A
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- nucleic acid
- tbago
- leu
- dna
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
技术领域Technical Field
本发明属于生物技术领域,具体涉及一种基于新型中高温Argonaute蛋白 TbAgo的核酸切割体系及由此开发的靶标核酸分子的检测方法和试剂盒。The present invention belongs to the field of biotechnology, and specifically relates to a nucleic acid cleavage system based on a novel medium- and high-temperature Argonaute protein TbAgo, and a detection method and kit for target nucleic acid molecules developed thereby.
背景技术Background Art
核酸检测技术已被广泛的应用于各种领域,如生物分子医学诊断、食品安全检测、环境监测、病原微生物检测、基因分型等。Nucleic acid detection technology has been widely used in various fields, such as biomolecular medical diagnosis, food safety testing, environmental monitoring, pathogenic microorganism detection, genotyping, etc.
尽管传统核酸检测技术,如定量聚合酶链式反应(qPCR)、高通量测序、DNA 印迹(southern blot)等仍被广泛应用,但这些技术均有缺陷,如费用昂贵、耗时较久且需专业人员操作等。Although traditional nucleic acid detection technologies, such as quantitative polymerase chain reaction (qPCR), high-throughput sequencing, and southern blot, are still widely used, these technologies have their own drawbacks, such as being expensive, time-consuming, and requiring professional personnel to operate.
近年来,CRISPR/Cas体系,即规律成簇的间隔短回文重复序列及其相关基因(Clustered regularly interspaced short palindromic repeats/CRISPR associatedgene,CRISPR/Cas)成为研究的热点,基于该体系的核酸检测技术也不断被开发,如SHERLOCK、DETECTOR、HOLMES等,虽然相较传统核酸检测技术更经济、便捷,但仍具有一定缺陷。如Cas蛋白对靶标核酸分子的识别与切割需依赖PAM位点,使得其可作用的位点受限;Cas蛋白需在向导RNA (gRNA)介导下才能发挥功能,而RNA不仅生产过程复杂,价格昂贵,而且易被环境中的核酸酶降解,使得整个体系稳定性较差;Cas蛋白在识别、结合特定核酸后被激发的反式切割活性为非特异性核酸酶活性,因此基于此开发的核酸检测技术无法达到单一体系检测多个靶标核酸分子的要求。In recent years, the CRISPR/Cas system, i.e., Clustered regularly interspaced short palindromic repeats/CRISPR associated gene (CRISPR/Cas), has become a hot topic of research, and nucleic acid detection technologies based on this system have also been continuously developed, such as SHERLOCK, DETECTOR, HOLMES, etc. Although more economical and convenient than traditional nucleic acid detection technologies, they still have certain defects. For example, the recognition and cutting of target nucleic acid molecules by Cas proteins rely on PAM sites, which limits the sites they can act on; Cas proteins can only function under the mediation of guide RNA (gRNA), and RNA is not only complicated in production process and expensive, but also easily degraded by nucleases in the environment, making the stability of the entire system poor; the trans-cutting activity stimulated by Cas proteins after recognizing and binding to specific nucleic acids is non-specific nuclease activity, so the nucleic acid detection technology developed based on this cannot meet the requirements of detecting multiple target nucleic acid molecules with a single system.
综上,仍须开发一种检测位点不受限、体系稳定并且可同时检测多个靶标核酸分子的核酸检测新技术。In summary, it is still necessary to develop a new nucleic acid detection technology that has no restrictions on detection sites, a stable system, and can detect multiple target nucleic acid molecules at the same time.
Argonaute(Ago)蛋白最早在一项描述拟南芥突变体的研究中被提及。目前已报道的Ago蛋白主要分为真核Ago(eAgo)和原核Ago(pAgo)两类。eAgo在真核生物中是RNA干扰(RNAi)途径的关键参与者,可以在转录后调节基因表达,从而防御入侵的RNA病毒并保护基因组的完整性。而pAgo可以结合单链的向导 DNA(gDNA)或向导RNA(gRNA),对与向导核酸序列互补的DNA或RNA 在特定位点进行切割,即在从向导核酸5’端起的第10和第11位之间产生切割,被认为可能也是原核生物的一种免疫机制。Argonaute (Ago) protein was first mentioned in a study describing Arabidopsis mutants. Currently reported Ago proteins are mainly divided into two categories: eukaryotic Ago (eAgo) and prokaryotic Ago (pAgo). eAgo is a key player in the RNA interference (RNAi) pathway in eukaryotes, which can regulate gene expression after transcription, thereby defending against invading RNA viruses and protecting the integrity of the genome. pAgo can bind to single-stranded guide DNA (gDNA) or guide RNA (gRNA) and cut DNA or RNA complementary to the guide nucleic acid sequence at specific sites, that is, cutting between the 10th and 11th positions from the 5' end of the guide nucleic acid, which is believed to be an immune mechanism of prokaryotes.
目前仅有极少数的pAgo被表征,这些Ago蛋白多为高温Ago,可在高温下结合单链gDNA或gRNA,切割单链DNA或单链RNA。相较CRISPR/Cas体系,pAgo对靶标核酸分子链进行切割时不依赖PAM位点,因此可通过对gDNA 的设计,达到对任意序列的核酸进行切割的目的;而gDNA则比gRNA具有更好的稳定性;同时由于切割行为需依赖gDNA的特定序列,因此在单一反应体系内的多个切割反应互不干扰,可达到单一体系同时检测多个靶标核酸分子的目的。Currently, only a very small number of pAgos have been characterized. Most of these Ago proteins are high-temperature Agos, which can bind to single-stranded gDNA or gRNA at high temperatures and cut single-stranded DNA or single-stranded RNA. Compared with the CRISPR/Cas system, pAgo does not rely on the PAM site when cutting the target nucleic acid molecule chain. Therefore, the purpose of cutting nucleic acids of any sequence can be achieved through the design of gDNA; and gDNA has better stability than gRNA; at the same time, because the cutting behavior depends on the specific sequence of gDNA, multiple cutting reactions in a single reaction system do not interfere with each other, and the purpose of simultaneously detecting multiple target nucleic acid molecules in a single system can be achieved.
基于现有核酸检测技术的缺点和pAgo体系的特点,开发新型基于pAgo蛋白的核酸检测技术具有重要的现实意义和广阔的应用前景。Based on the shortcomings of existing nucleic acid detection technologies and the characteristics of the pAgo system, the development of new nucleic acid detection technologies based on pAgo proteins has important practical significance and broad application prospects.
发明内容Summary of the invention
在本发明的第一方面,提供了一种核酸切割体系。In a first aspect of the present invention, a nucleic acid cleavage system is provided.
所述核酸切割体系包括:The nucleic acid cleavage system comprises:
(a)可编程核酸内切酶Argonaute(Ago);所述的可编程核酸内切酶 Argonaute来源于嗜热菌(Thermus brockianus),所述的可编程核酸内切酶 Argonaute是可编程核酸内切酶TbAgo。(a) Programmable endonuclease Argonaute (Ago); the programmable endonuclease Argonaute is derived from Thermus brockianus, and the programmable endonuclease Argonaute is the programmable endonuclease TbAgo.
(b)向导DNA(gDNA);和(b) guide DNA (gDNA); and
(c)核酸探针,其中若所述核酸探针被切割,所述的切割是可以被检测出的。(c) a nucleic acid probe, wherein if the nucleic acid probe is cleaved, the cleavage is detectable.
在另一优选例中,所述核酸切割体系的反应温度为50℃-95℃,较佳地为 50℃-85℃,更佳地为50℃-75℃。In another preferred embodiment, the reaction temperature of the nucleic acid cleavage system is 50°C-95°C, preferably 50°C-85°C, and more preferably 50°C-75°C.
在另一优选例中,所述的TbAgo包括野生型和突变型的TbAgo。所述TbAgo 与如SEQID NO:1所示氨基酸序列具有至少80%同一性。In another preferred embodiment, the TbAgo includes wild-type and mutant TbAgo, and the TbAgo has at least 80% identity with the amino acid sequence shown in SEQ ID NO:1.
在另一优选例中,所述野生型TbAgo的氨基酸序列如NCBI序列号 WP_071678161.1所示。In another preferred example, the amino acid sequence of the wild-type TbAgo is shown in NCBI sequence number WP_071678161.1.
在另一优选例中,所述的gDNA是5’端磷酸化或5’端羟基化的单链DNA分子。In another preferred embodiment, the gDNA is a single-stranded DNA molecule with 5'-end phosphorylation or 5'-end hydroxylation.
在另一优选例中,所述的gDNA是5’端磷酸化的单链DNA分子。In another preferred embodiment, the gDNA is a single-stranded DNA molecule with 5' end phosphorylated.
在另一优选例中,所述的gDNA与所述核酸探针之间具有反向互补的片段,可引导TbAgo切割核酸探针,产生可被检出的信号值。In another preferred embodiment, the gDNA and the nucleic acid probe have reverse complementary fragments, which can guide TbAgo to cut the nucleic acid probe and generate a detectable signal value.
在另一优选例中,所述的gDNA的长度为10nt-35nt,更佳地14nt-35nt。In another preferred embodiment, the length of the gDNA is 10nt-35nt, more preferably 14nt-35nt.
在另一优选例中,所述的核酸探针是单链DNA(ssDNA)。In another preferred embodiment, the nucleic acid probe is single-stranded DNA (ssDNA).
在另一优选例中,所述的核酸探针是未经修饰的单链DNA。In another preferred embodiment, the nucleic acid probe is unmodified single-stranded DNA.
在另一优选例中,当所述核酸探针被切割,所述的切割能够通过电泳法被检测出。In another preferred embodiment, when the nucleic acid probe is cleaved, the cleavage can be detected by electrophoresis.
在另一优选例中,所述的电泳法是用14%的核酸Urea-PAG电泳检测法。In another preferred embodiment, the electrophoresis method is a 14% nucleic acid Urea-PAG electrophoresis detection method.
在另一优选例中,所述的核酸探针是仅5’端修饰荧光基团的单链DNA分子,所述的荧光基团是FAM。In another preferred embodiment, the nucleic acid probe is a single-stranded DNA molecule with a fluorescent group modified only at the 5' end, and the fluorescent group is FAM.
在另一优选例中,所述核酸探针是同时带有荧光基团和淬灭基团的核酸探针。In another preferred embodiment, the nucleic acid probe is a nucleic acid probe having both a fluorescent group and a quenching group.
在另一优选例中,所述荧光基团包括:FAM、HEX、CY5、CY3、VIC、JOE、 TET、5-TAMRA、ROX、Texas Red-X,或其组合。In another preferred embodiment, the fluorescent group includes: FAM, HEX, CY5, CY3, VIC, JOE, TET, 5-TAMRA, ROX, Texas Red-X, or a combination thereof.
在另一优选例中,所述猝灭基团包括:BHQ、TAMRA、DABCYL、DDQ,或其组合。In another preferred embodiment, the quenching group includes: BHQ, TAMRA, DABCYL, DDQ, or a combination thereof.
在另一优选例中,所述的荧光基团和淬灭基团各自独立地位于所述核酸探针的5’端与3’端。In another preferred embodiment, the fluorescent group and the quencher group are independently located at the 5' end and the 3' end of the nucleic acid probe.
在另一优选例中,当所述核酸探针被切割,所述的切割能够通过酶标仪被检测出。In another preferred embodiment, when the nucleic acid probe is cleaved, the cleavage can be detected by an enzyme reader.
在另一优选例中,所述核酸探针是同时带有荧光基团和生物素基团的核酸探针,所述的荧光基团是FAM。In another preferred embodiment, the nucleic acid probe is a nucleic acid probe carrying both a fluorescent group and a biotin group, and the fluorescent group is FAM.
在另一优选例中,所述的荧光基团和生物素基团各自独立地位于所述核酸探针的5’端与3’端。In another preferred embodiment, the fluorescent group and the biotin group are independently located at the 5' end and the 3' end of the nucleic acid probe.
在另一优选例中,当所述核酸探针被切割,所述的切割能够通过胶体金试纸条被检测出。In another preferred embodiment, when the nucleic acid probe is cleaved, the cleavage can be detected by a colloidal gold test strip.
在另一优选例中,所述的核酸切割体系还包含:(d)二价金属离子。In another preferred embodiment, the nucleic acid cleavage system further comprises: (d) divalent metal ions.
在另一优选例中,所述的二价金属离子为Mn2+、Mg2+、Ca2+、Co2+、Mn2+、 Zn2+、Fe2+或Cu2+,较佳的为Mn2+、Mg2+或Co2+,最佳的为Mn2+。In another preferred embodiment, the divalent metal ion is Mn 2+ , Mg 2+ , Ca 2+ , Co 2+ , Mn 2+ , Zn 2+ , Fe 2+ or Cu 2+ , preferably Mn 2+ , Mg 2+ or Co 2+ , and most preferably Mn 2+ .
在另一优选例中,所述的核酸切割体系中,Mn2+的浓度为0μM-1000μM,较佳地100μM-1000μM,更佳地250μM-1000μM。In another preferred embodiment, in the nucleic acid cleavage system, the concentration of Mn 2+ is 0 μM-1000 μM, preferably 100 μM-1000 μM, and more preferably 250 μM-1000 μM.
在另一优选例中,所述的核酸切割体系还包括:(e)缓冲液。In another preferred embodiment, the nucleic acid cleavage system further comprises: (e) a buffer.
在另一优选例中,所述缓冲液中,NaCl的浓度为0mM-1000mM,较佳地为0mM-500mM,最佳的为100mM-250mM。In another preferred embodiment, the concentration of NaCl in the buffer is 0mM-1000mM, preferably 0mM-500mM, and most preferably 100mM-250mM.
在另一优选例中,当在所述gDNA与核酸探针的反向互补区域中,从5’端起第7位至第14位以及第1、3、5位中的任一个碱基存在与所述核酸探针的错配时,会显著降低所述可编程核酸内切酶TbAgo的切割率。In another preferred example, when in the reverse complementary region between the gDNA and the nucleic acid probe, there is a mismatch with the nucleic acid probe at any base from the 7th to the 14th base from the 5' end and at the 1st, 3rd, and 5th bases, the cleavage rate of the programmable nuclease TbAgo will be significantly reduced.
在另一优选例中,所述的显著降低可编程核酸内切酶TbAgo切割率是指:相同反应条件下,所述可编程核酸内切酶TbAgo的切割率降低≥70%,较佳地降低≥80%,更佳地降低≥90%。In another preferred example, the significantly reduced cleavage rate of the programmable endonuclease TbAgo means that under the same reaction conditions, the cleavage rate of the programmable endonuclease TbAgo is reduced by ≥70%, preferably by ≥80%, and more preferably by ≥90%.
在另一优选例中,所述的核酸切割体系中,所述荧光核酸探针的浓度为0.1 μM-2μM,较佳地0.5μM-2μM,更佳地0.5μM-1μM,最佳地为0.5μM。In another preferred embodiment, in the nucleic acid cleavage system, the concentration of the fluorescent nucleic acid probe is 0.1 μM-2 μM, preferably 0.5 μM-2 μM, more preferably 0.5 μM-1 μM, and most preferably 0.5 μM.
在另一优选例中,所述的核酸切割体系中,所述可编程核酸内切酶TbAgo 的浓度为10nM-10μM,较佳地500nM-5μM,最佳地为2.5μM。In another preferred embodiment, in the nucleic acid cleavage system, the concentration of the programmable endonuclease TbAgo is 10 nM-10 μM, preferably 500 nM-5 μM, and most preferably 2.5 μM.
在另一优选例中,所述的核酸切割体系中,所述gDNA的浓度为10nM-10 μM,较佳地100nM-1μM,最佳地为500nM。In another preferred embodiment, in the nucleic acid cleavage system, the concentration of the gDNA is 10 nM-10 μM, preferably 100 nM-1 μM, and most preferably 500 nM.
在本发明的第二方面,提供了一种靶标核酸分子的检测体系。In a second aspect of the present invention, a detection system for a target nucleic acid molecule is provided.
所述的检测体系包含如本发明第一方面所述的核酸切割体系。The detection system comprises the nucleic acid cleavage system as described in the first aspect of the present invention.
所述靶标核酸分子为DNA分子或RNA分子。The target nucleic acid molecule is a DNA molecule or an RNA molecule.
在另一优选例中,所述的检测体系还包含检测的对象,即靶标核酸分子。In another preferred embodiment, the detection system further comprises a detection object, namely a target nucleic acid molecule.
在另一优选例中,所述的检测体系中的靶标核酸分子来源于病原微生物、基因突变、植物、动物、病毒,以及以上的任意组合。In another preferred embodiment, the target nucleic acid molecule in the detection system is derived from pathogenic microorganisms, gene mutations, plants, animals, viruses, and any combination thereof.
在另一优选例中,所述的检测体系可检测的靶标核酸分子的种类为N,N取 1、2、3、4、5等自然数。In another preferred embodiment, the number of target nucleic acid molecules that can be detected by the detection system is N, and N is a natural number such as 1, 2, 3, 4, 5, etc.
在另一优选例中,所述的检测体系中的靶标核酸分子包括合成的或天然核酸分子。In another preferred embodiment, the target nucleic acid molecule in the detection system includes a synthetic or natural nucleic acid molecule.
在另一优选例中,所述的检测体系中的靶标核酸分子包括野生型或突变型核酸分子。In another preferred embodiment, the target nucleic acid molecule in the detection system includes a wild-type or mutant nucleic acid molecule.
在另一优选例中,所述的检测体系还包含扩增靶标核酸分子的试剂。In another preferred embodiment, the detection system further comprises a reagent for amplifying the target nucleic acid molecule.
在另一优选例中,所述的检测体系中的向导DNA包含一条或多条不同的 gDNA。In another preferred embodiment, the guide DNA in the detection system comprises one or more different gDNAs.
在另一优选例中,所述的检测体系中的核酸探针包含一条或多条不同的核酸探针。In another preferred embodiment, the nucleic acid probe in the detection system comprises one or more different nucleic acid probes.
在另一优选例中,所述的检测体系中的核酸探针与靶标核酸分子一一对应,具体的,指序列互补。In another preferred embodiment, the nucleic acid probes in the detection system correspond one-to-one to the target nucleic acid molecules, specifically, they are sequence complementary.
在另一优选例中,所述的检测体系中,所述的可编程核酸内切酶TbAgo会在gDNA的引导下识别并切割靶标核酸分子,从而产生新的gDNA,新的gDNA 与对应的核酸探针是序列互补的。In another preferred embodiment, in the detection system, the programmable endonuclease TbAgo will recognize and cut the target nucleic acid molecule under the guidance of gDNA, thereby generating new gDNA, and the new gDNA is sequence complementary to the corresponding nucleic acid probe.
在另一优选例中,所述的检测体系中,所述的可编程核酸内切酶TbAgo会在新的gDNA的引导下识别并切割对应核酸探针,从而产生相应的可检出的信号。In another preferred embodiment, in the detection system, the programmable endonuclease TbAgo will recognize and cut the corresponding nucleic acid probe under the guidance of the new gDNA, thereby generating a corresponding detectable signal.
在另一优选例中,所述的检测体系中,所述可编程核酸内切酶TbAgo的浓度为10nM-10μM,较佳地500nM-5μM,最佳地为5μM。In another preferred example, in the detection system, the concentration of the programmable nuclease TbAgo is 10nM-10μM, preferably 500nM-5μM, and optimally 5μM.
在另一优选例中,所述的检测体系中,所述靶标核酸分子的浓度为1fM-200 pM,较佳地1fM-1000fM,最佳地1fM-100fM。In another preferred embodiment, in the detection system, the concentration of the target nucleic acid molecule is 1fM-200 pM, preferably 1fM-1000fM, and most preferably 1fM-100fM.
在另一优选例中,所述的检测体系中,所述gDNA的浓度为10nM-10μM,较佳地100nM-1μM,最佳地为250nM。In another preferred embodiment, in the detection system, the concentration of the gDNA is 10nM-10μM, preferably 100nM-1μM, and most preferably 250nM.
在另一优选例中,所述的检测体系中,核酸探针浓度为100nM-1000nM, 较佳地为500nM-1000nM。In another preferred embodiment, in the detection system, the concentration of the nucleic acid probe is 100nM-1000nM, preferably 500nM-1000nM.
在另一优选例中,所述的检测体系还包括(d)二价金属离子,如本发明的第一方面所述。In another preferred embodiment, the detection system further comprises (d) divalent metal ions, as described in the first aspect of the present invention.
在另一优选例中,所述的检测体系还包括(e)缓冲液,如本发明的第一方面所述。In another preferred embodiment, the detection system further comprises (e) a buffer, as described in the first aspect of the present invention.
在另一优选例中,所述的检测体系中,可编程核酸内切酶TbAgo的工作温度如本发明的第一方面所述。In another preferred embodiment, in the detection system, the working temperature of the programmable nuclease TbAgo is as described in the first aspect of the present invention.
在本发明的第三方面,提供了一种非诊断目的的靶标核酸分子的检测方法,包括步骤:In a third aspect of the present invention, a method for detecting a target nucleic acid molecule for non-diagnostic purposes is provided, comprising the steps of:
(a)提供一待测样本;(a) providing a sample to be tested;
(b)提供一种反应体系来处理待测样本,并形成反应液;所述反应体系即本发明第二方面所述的一种靶标核酸分子的检测体系;(b) providing a reaction system to process the sample to be tested and form a reaction solution; the reaction system is a target nucleic acid molecule detection system as described in the second aspect of the present invention;
(c)检测反应液的信号。(c) Detecting the signal of the reaction solution.
其中,若检出特定信号,则说明检测样本中包含特定的靶标核酸分子;若未检出特定信号,则说明检测样本中不包含特定靶标核酸分子。Among them, if a specific signal is detected, it means that the test sample contains a specific target nucleic acid molecule; if no specific signal is detected, it means that the test sample does not contain a specific target nucleic acid molecule.
在另一优选例中,所述反应体系包含对靶标核酸分子扩增的试剂。In another preferred embodiment, the reaction system comprises reagents for amplifying target nucleic acid molecules.
在另一优选例中,核酸扩增方法选自PCR扩增、RT-PCR扩增、LAMP扩增、RT-LAMP扩增、RPA扩增、RT-RPA扩增、连接酶链式反应、支链DNA 扩增、NASBA、SDA、转录介导扩增以及滚环扩增。In another preferred embodiment, the nucleic acid amplification method is selected from PCR amplification, RT-PCR amplification, LAMP amplification, RT-LAMP amplification, RPA amplification, RT-RPA amplification, ligase chain reaction, branched DNA amplification, NASBA, SDA, transcription-mediated amplification and rolling circle amplification.
在另一优选例中,所述的检测靶标核酸分子的方法检测的对象为SNP、点突变、删除或插入。In another preferred embodiment, the method for detecting a target nucleic acid molecule detects a SNP, a point mutation, a deletion or an insertion.
在另一优选例中,在步骤(c)中使用酶标仪读取信号。In another preferred embodiment, in step (c), a microplate reader is used to read the signal.
在另一优选例中,在步骤(c)中使用胶体金试纸条读取信号。In another preferred embodiment, a colloidal gold test strip is used to read the signal in step (c).
在另一优选例中,所述的检测靶标核酸分子的方法用于体外检测。In another preferred embodiment, the method for detecting target nucleic acid molecules is used for in vitro detection.
在另一优选例中,所述的检测靶标核酸分子的方法是非诊断性和非治疗性的。In another preferred embodiment, the method for detecting target nucleic acid molecules is non-diagnostic and non-therapeutic.
在本发明的第四方面,提供了Ago蛋白在靶标核酸分子的检测方法中的用途,所述Ago蛋白是TbAgo或与SEQ ID NO:1所示氨基酸序列具有至少80%同一性的相似蛋白。In the fourth aspect of the present invention, a use of an Ago protein in a method for detecting a target nucleic acid molecule is provided, wherein the Ago protein is TbAgo or a similar protein having at least 80% identity with the amino acid sequence shown in SEQ ID NO:1.
在另一优选例中,所述可编程核酸内切酶TbAgo包括来源于嗜热菌Thermusbrockianus的TbAgo;或具备类似切割活性的同源蛋白。In another preferred embodiment, the programmable endonuclease TbAgo includes TbAgo derived from thermophilic bacteria Thermus brockianus; or a homologous protein having similar cleavage activity.
在另一优选例中,所述可编程核酸内切酶TbAgo包括包括野生型和突变型 TbAgo。In another preferred example, the programmable nuclease TbAgo includes wild-type and mutant TbAgo.
在另一优选例中,所述可编程核酸内切酶TbAgo具有选自下组的氨基酸序列:In another preferred embodiment, the programmable endonuclease TbAgo has an amino acid sequence selected from the following group:
(i)如NCBI序列号WP_071678161.1所示的氨基酸序列;和(i) the amino acid sequence shown in NCBI sequence number WP_071678161.1; and
(ii)在如NCBI序列号WP_071678161.1所示序列的基础上,进行一个或多个氨基酸残基的替换、缺失、改变或插入,或在其N端或C端添加1至10个氨基酸残基(较佳地1至5个氨基酸残基,更佳地1至3个氨基酸残基),从而获得的氨基酸序列;并且所述获得的氨基酸序列与如NCBI序列号WP_071678161.1 所示序列具有≥80%(优选地≥90%,更优选地≥95%,例如≥96%、≥97%、≥98%或≥99%)的序列同一性;并且所获得的氨基酸序列具备与(i)相同或相似的功能。(ii) Based on the sequence shown in NCBI sequence number WP_071678161.1, one or more amino acid residues are replaced, deleted, changed or inserted, or 1 to 10 amino acid residues (preferably 1 to 5 amino acid residues, more preferably 1 to 3 amino acid residues) are added to its N-terminus or C-terminus to obtain an amino acid sequence; and the obtained amino acid sequence has ≥80% (preferably ≥90%, more preferably ≥95%, for example ≥96%, ≥97%, ≥98% or ≥99%) sequence identity with the sequence shown in NCBI sequence number WP_071678161.1; and the obtained amino acid sequence has the same or similar function as (i).
在本发明的第五方面,提供了一种靶标核酸分子的检测试剂盒,包括Ago 蛋白、向导DNA、核酸探针。In a fifth aspect of the present invention, a detection kit for a target nucleic acid molecule is provided, comprising an Ago protein, a guide DNA, and a nucleic acid probe.
本发明通过对TbAgo蛋白的基因进行挖掘、序列比对后,构建了重组质粒pET28a—TbAgo,该重组质粒转化大肠杆菌BL21(DE3),实现了TbAgo的异源表达,后经Ni-NTA柱纯化得到了重组菌株所产TbAgo蛋白。The present invention constructed a recombinant plasmid pET28a-TbAgo by mining and aligning the gene of TbAgo protein, transformed Escherichia coli BL21 (DE3) with the recombinant plasmid to achieve heterologous expression of TbAgo, and then purified by Ni-NTA column to obtain the TbAgo protein produced by the recombinant strain.
本发明所得到的TbAgo蛋白分子量约为78.8kDa,该酶可利用5’-P gDNA 介导单链DNA靶标的切割。最适反应温度范围在50℃-75℃之间;可利用Mn2+作为活性离子,250μM-1000μM Mn2+可使其保持较高活性;该酶在100 mM-500mM的NaCl浓度范围时活性较高;该酶对gDNA具有较强的偏好性,仅在利用5’末端磷酸基团修饰的gDNA时有较高的活性,其他修饰如5’-OH修饰时活性较低;该酶可利用14nt-35nt的5’-P gDNA在靶标DNA的10-11位点发生切割;该酶可以区分靶标DNA与gDNA间的单点错配。The molecular weight of the TbAgo protein obtained by the present invention is about 78.8 kDa, and the enzyme can use 5'-P gDNA to mediate the cutting of single-stranded DNA targets. The optimal reaction temperature range is between 50°C and 75°C; Mn 2+ can be used as an active ion, and 250μM-1000μM Mn 2+ can keep it highly active; the enzyme has high activity in the NaCl concentration range of 100 mM-500mM; the enzyme has a strong preference for gDNA, and has high activity only when using gDNA modified with a 5' terminal phosphate group, and has low activity when other modifications such as 5'-OH modification are used; the enzyme can use 14nt-35nt 5'-P gDNA to cut at the 10-11 sites of the target DNA; the enzyme can distinguish single-point mismatches between the target DNA and the gDNA.
另一方面,本发明提供了一种基于可编程核酸内切酶TbAgo的靶标核酸的检测方法。该技术不仅具有高灵敏度、高特异性、高稳定性的特点,且可达到单一反应体系检测多种靶标核酸分子的要求。除此之外,此技术在SNV基因检测中也有应用前景。On the other hand, the present invention provides a method for detecting target nucleic acid based on programmable nuclease TbAgo. This technology not only has the characteristics of high sensitivity, high specificity and high stability, but also can meet the requirements of detecting multiple target nucleic acid molecules in a single reaction system. In addition, this technology also has application prospects in SNV gene detection.
应理解,在本发明范围内中,本发明的上述各技术特征和在下文(如实施例) 中具体描述的各技术特征之间都可以互相组合,从而构成新的或优选的技术方案。限于篇幅,在此不再一一累述。It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1是本发明所述的靶标核酸分子的检测方法的示意图。FIG1 is a schematic diagram of the method for detecting a target nucleic acid molecule according to the present invention.
图2是本发明实施例提供的部分已表征Argonaute蛋白的进化树结果图。FIG. 2 is a diagram showing the evolutionary tree of some characterized Argonaute proteins provided in an embodiment of the present invention.
图3是本发明实施例提供的部分已表征Ago蛋白的序列比对结果图。FIG. 3 is a diagram showing a sequence alignment result of some characterized Ago proteins provided in an embodiment of the present invention.
图4是本发明实施例提供的SDS-PAGE凝胶分析TbAgo纯度结果图。FIG. 4 is a graph showing the purity of TbAgo analyzed by SDS-PAGE gel according to an embodiment of the present invention.
图5是本发明实施例提供的测定TbAgo切割活性的测定结果。FIG. 5 is a result of measuring the cleavage activity of TbAgo provided in an embodiment of the present invention.
图6是本发明实施例提供的测定温度对TbAgo切割活性的影响的测定结果。FIG. 6 is a measurement result of the effect of measurement temperature on TbAgo cleavage activity provided in an embodiment of the present invention.
图7是本发明实施例提供的测定不同二价金属离子对TbAgo切割活性的影响的结果。FIG. 7 is a result of determining the effect of different divalent metal ions on the cleavage activity of TbAgo provided in an embodiment of the present invention.
图8是本发明实施例提供的测定不同Mn2+浓度对TbAgo切割活性的影响的结果。FIG8 is a result of determining the effect of different Mn 2+ concentrations on TbAgo cleavage activity provided in an embodiment of the present invention.
图9是本发明实施例提供的测定不同NaCl浓度对TbAgo切割活性的影响的结果。FIG. 9 is a result of determining the effect of different NaCl concentrations on TbAgo cleavage activity provided in an embodiment of the present invention.
图10是本发明实施例提供的测定适合TbAgo的最佳长度的引导DNA的结果。FIG. 10 is a result of determining the optimal length of guide DNA suitable for TbAgo provided in an embodiment of the present invention.
图11是本发明实施例提供的TbAgo切割单链DNA底物的动力学结果。FIG. 11 is a diagram showing the kinetic results of TbAgo cutting a single-stranded DNA substrate according to an embodiment of the present invention.
图12是本发明实施例提供的TbAgo针对gDNA与Target之间不同位点的单点错配的示意图。FIG12 is a schematic diagram of TbAgo provided in an embodiment of the present invention for single-point mismatches at different sites between gDNA and Target.
图13是本发明实施例提供的TbAgo针对gDNA与Target之间不同位点的单点错配可以区分切割的结果。FIG. 13 shows that TbAgo provided in an embodiment of the present invention can distinguish the cutting results for single-point mismatches at different sites between gDNA and Target.
图14是本发明实施例提供的使用TbAgo酶结合荧光核酸探针,检测目标基因的结果。FIG. 14 is a result of detecting a target gene using TbAgo enzyme combined with a fluorescent nucleic acid probe provided in an embodiment of the present invention.
图15是本发明实施例提供的使用TbAgo酶结合荧光核酸探针,在单一体系内检测多个目标基因的结果。FIG. 15 is a result of detecting multiple target genes in a single system using TbAgo enzyme combined with fluorescent nucleic acid probes provided in an embodiment of the present invention.
具体实施方式DETAILED DESCRIPTION
为了更清楚地说明本发明实施例的技术方案,下面将结合本发明实施例中的附图,对实施例中的技术方案进行清楚、完整的描述,显而易见地,所述实施例仅是本发明的一部分实施例,而不是全部的实施例。本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the technical solutions in the embodiments will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
实施例1:TbAgo基因序列的获得Example 1: Acquisition of TbAgo gene sequence
在NCBI数据库中,对已知的TtAgo的氨基酸序列进行相似性检索,选取部分序列一致性较高的氨基酸序列,采用MEGA软件进行分析,构建同源进化树,选取TbAgo作为候选酶。TbAgo氨基酸序列如SEQ ID NO:1所示。In the NCBI database, similarity search was performed on the known amino acid sequences of TtAgo, and some amino acid sequences with high sequence consistency were selected for analysis using MEGA software to construct a homology evolution tree, and TbAgo was selected as a candidate enzyme. The amino acid sequence of TbAgo is shown in SEQ ID NO:1.
SEQ ID NO:1SEQ ID NO:1
MMGDSRSLEVRLNRFLLRPLRPEEREPWLLVSELNPPPSREDVHALLA LLANRAGGRTARMGDSLLTWSPPESLLLEGTLSWRGNTYTYRLRPLARRVL NPRNPSERDALSALARRLLREVLEQFRREGFWVEGWAFYRKEHARGPGW RVLKGAALDLWVSAEGAMVLEVDPTYRILCDMTLEAWLAQGHPPPKRVKNA YNDRTWELLGLGEEDPQGILLPGGLNLVEYHASKGRIRDGGWGRVAWVAN PKDAKEKIPHLTSLLIPVLTLEDLHEEGGSNLALSIPWNQRQEETLKVALSVAR RLGVEHPKPVEAKAWRMRMPELRARRRVGKPADALRVGLYRAQETTLALLR LDGGRGWPDFLLKALENAFRASQARLHVREIHADPSQPLAFREALEEAKEA GVQAVLVLTPPLSWEERHRLKALFLKEGLPSQLLNVPIQREERHRLENALLGL LAKAGLQVVALEGAYPADLTVGFDAGGRKSFRFGGAACAVGSDGGHLLWSL PEAQAGERIPGEVVWDLLEEALLVFKRKRGRLPSRVLLLRDGRLPKDEFTLA LAKLRQLGIGFDLVSVRKSGGGRIYPTRGRLLDGLLVPVEERTFLLLTVHREF RGTPRPLKLVHEEGETPLEALAEQIYHLTRLYPASGFAFPRLPAPLHLADRLV KEVGRLGVRHLKEVDREKLFFV*MMGDSRSLEVRLNRFLLRPLRPEEEREPWLLVSELNPPPSREDVHALLA LLANRAGGRTARMGDSLLTWSPPESLLLEGTLSWRGNTYTYRLRPLARRVL NPRNPSERDALSALARRLLREVLEQFRREGFWVEGWAFYRKEHARGPGW RVLKGAALDLWVSAEGAMVLEVDPTYRILCDMTLEAWLAQGHPPPKRVKNA YNDRTWELLGLGEEDPQGPGGL NLVEYHASKGRIRDGGWGRVAWVAN PKDAKEKIPHLTSLLIPVLTLEDLHEEGGSNLALSIPWNQRQEETLKVALSVAR RLGVEHPKPVEAKAWRMRMPELRARRRVGKPADALRVGLYRAQETTLALLR LDGGRGWPDFLLKALENAFRASQARLHVREIHADPSQPLAFREALEEAKEA GVQAVLVLTPPLSWEERHRLKALFLKEGLPSQLLNVPIQREERHRLENALLGL LAKAGLQVVALEGAYPADLTVGFDAGGRKSFRFGGAACAVGSDGGHLLWSL PEAQAGERIPGEVVWDLLEEALLVFKRGRLPSRVLLLRDGRLPKDEFTLA LAKLRQLGIGFDLVSVRKSGGGRIYPTRGRLLDGLLVPVEERTFLLLTVHREF RGTPRPLKLVHEEGETPLEA LAEQIYHLTRLYPASGFAFPRLPAPLHLADRLV KEVGRLGVRHLKEVDREKLFFV*
进化树结果如图2所示。The evolutionary tree result is shown in Figure 2.
实施例2:TbAgo与其他已知pAgo序列比对Example 2: Alignment of TbAgo with other known pAgo sequences
在本实施例中,将TbAgo与部分已表征的pAgo进行了多重序列比对。In this example, multiple sequence alignment of TbAgo with some of the characterized pAgo was performed.
据文献报道,所有具有核酸酶活性的pAgo蛋白的靶向切割是通过一个保守的DEDX(X代表组氨酸,天冬氨酸或天冬酰胺)活性中心所介导。通过序列比对,可以发现TbAgo中存在DEDX活性中心(图3)。因此推测其可能具有核酸酶活性,需要进一步体外鉴定表征。According to literature reports, the targeted cleavage of all pAgo proteins with nuclease activity is mediated by a conserved DEDX (X represents histidine, aspartic acid or asparagine) active center. Through sequence alignment, it can be found that TbAgo has a DEDX active center (Figure 3). Therefore, it is speculated that it may have nuclease activity and needs further in vitro identification and characterization.
实施例3:TbAgo蛋白异源表达与纯化Example 3: Heterologous expression and purification of TbAgo protein
由实施例1获得TbAgo(WP_071678161.1)的氨基酸序列。将该基因序列经密码子优化合成后,克隆至pET28a表达载体,获得pET28a-TbAgo原核表达质粒,将之导入E.coliBL21(DE3)中,得到pET28a-TbAgo/E.coli BL21(DE3) 原核表达菌株。含重组质粒pET28a-TbAgo的表达菌株E.coli BL21(DE3)接种于含有50μg/mL卡那霉素的TB培养基中,37℃,220rpm摇床培养到OD600 至2.0-3.0之间,加入终浓度为0.1mM的IPTG,25℃,200rpm摇床继续培养16h-20h,诱导TbAgo蛋白的表达。离心收集菌体,使用重悬缓冲液(含20mM Tris-HCl、pH7.5、0.3M NaCl)重悬菌体,然后高压破碎菌体,离心获得上清。利用Ni-NTA柱亲和纯化蛋白,洗脱液经超滤浓缩、脱盐等步骤得到纯化的蛋白。纯化的蛋白保存于含20mM Tris-HCl、0.3M NaCl的缓冲液中,并通过BCA试剂盒测定蛋白浓度,测定步骤按照操作说明进行。以BSA作为标准品,配置标准溶液,绘制标准曲线,依此计算纯化的目的蛋白浓度,蛋白放于-80℃冰箱保存备用。SDS-PAGE电泳分析TbAgo蛋白。The amino acid sequence of TbAgo (WP_071678161.1) was obtained from Example 1. After the gene sequence was synthesized by codon optimization, it was cloned into the pET28a expression vector to obtain the pET28a-TbAgo prokaryotic expression plasmid, which was introduced into E. coli BL21 (DE3) to obtain the pET28a-TbAgo/E. coli BL21 (DE3) prokaryotic expression strain. The expression strain E. coli BL21 (DE3) containing the recombinant plasmid pET28a-TbAgo was inoculated in TB medium containing 50 μg/mL kanamycin, and cultured at 37°C, 220rpm shaking incubator until OD600 was between 2.0-3.0, and IPTG was added at a final concentration of 0.1mM, and cultured at 25°C, 200rpm shaking incubator for 16h-20h to induce the expression of TbAgo protein. The cells were collected by centrifugation, resuspended in a resuspension buffer (containing 20mM Tris-HCl, pH7.5, 0.3M NaCl), and then crushed by high pressure and centrifuged to obtain the supernatant. The protein was affinity purified using a Ni-NTA column, and the eluate was concentrated by ultrafiltration, desalted, and other steps to obtain the purified protein. The purified protein was stored in a buffer containing 20mM Tris-HCl and 0.3M NaCl, and the protein concentration was determined by a BCA kit. The determination steps were carried out according to the operating instructions. Using BSA as a standard, a standard solution was prepared, and a standard curve was drawn. The concentration of the purified target protein was calculated accordingly, and the protein was stored in a -80°C refrigerator for later use. TbAgo protein was analyzed by SDS-PAGE electrophoresis.
结果如图4所示。结果表明,目的蛋白TbAgo已得到纯化。The results are shown in Figure 4. The results show that the target protein TbAgo has been purified.
实施例4:TbAgo切割活性测定Example 4: TbAgo cleavage activity assay
设计带有荧光基团修饰的40nt单链DNA靶标核酸分子、带有荧光基团修饰的25nt的模拟切割产物单链DNA以及5’磷酸基团或5’羟基修饰的19nt gDNA,并送公司合成。Design a 40nt single-stranded DNA target nucleic acid molecule modified with a fluorescent group, a 25nt simulated cleavage product single-stranded DNA modified with a fluorescent group, and a 19nt gDNA modified with a 5’ phosphate group or 5’ hydroxyl group, and send them to the company for synthesis.
DNA靶标核酸分子序列(SEQ ID NO:2):DNA target nucleic acid molecule sequence (SEQ ID NO: 2):
5’-FAM-aaaataatttaatatactatacaacctactacctcttata-3’5’-FAM-aaaataatttaatatactatacaacctactacctcttata-3’
模拟切割产物序列(SEQ ID NO:3):Simulated cleavage product sequence (SEQ ID NO: 3):
5’-FAM-aaaataatttaatatactatacaac-3’5’-FAM-aaaataatttaatatactatacaac-3’
gDNA(SEQ ID NO:4):gDNA (SEQ ID NO:4):
5’-HO/P-tgaggtagtaggttgtata-3’5’-HO/P-tgaggtagtaggttgtata-3’
配置反应缓冲液(含10mM Tris-HCl pH7.5、150mM NaCl),在反应缓冲液中加入终浓度为0.5mM的MnCl2、2.5μM TbAgo、500nM合成的gDNA和500 nM 5’荧光基团修饰的序列互补单链DNA靶标核酸分子,在75℃反应60min,反应结束后,取10μL样品,按1:1比例加入上样缓冲液(含95%(去离子)甲酰胺,0.5mmol/L EDTA,0.025%溴酚蓝,0.025%二甲苯蓝),在14%的核酸 Urea-PAGE下进行电泳检测。Prepare reaction buffer (containing 10mM Tris-HCl pH7.5, 150mM NaCl), add MnCl 2 with a final concentration of 0.5mM, 2.5μM TbAgo, 500nM synthetic gDNA and 500nM 5' fluorescent group-modified sequence-complementary single-stranded DNA target nucleic acid molecule to the reaction buffer, react at 75°C for 60min, after the reaction, take 10μL sample, add loading buffer (containing 95% (deionized) formamide, 0.5mmol/L EDTA, 0.025% bromophenol blue, 0.025% xylene cyanol) at a ratio of 1:1, and perform electrophoresis detection under 14% nucleic acid Urea-PAGE.
结果如图5所示。结果表明,TbAgo可利用5’-P gDNA切割互补单链DNA。The results are shown in Figure 5. The results show that TbAgo can use 5'-P gDNA to cut complementary single-stranded DNA.
实施例5:TbAgo切割特性分析Example 5: Analysis of TbAgo cleavage characteristics
分别在不同温度(30℃、37℃、50℃、65℃、75℃、85℃、95℃) 下探究TbAgo的切割活性,在反应缓冲液中加入终浓度为0.5mM的MnCl2,终浓度为2.5μM的TbAgo,500nM合成的5’-P gDNA和500nM 40nt 5’荧光修饰的序列互补单链DNA靶标核酸分子,在不同温度下反应60min,反应产物在14%的核酸Urea-PAGE下进行电泳检测。The cleavage activity of TbAgo was investigated at different temperatures (30°C, 37°C, 50°C, 65°C, 75°C, 85°C, and 95°C). MnCl 2 with a final concentration of 0.5 mM, TbAgo with a final concentration of 2.5 μM, 500 nM synthetic 5'-P gDNA, and 500 nM 40 nt 5' fluorescently modified sequence-complementary single-stranded DNA target nucleic acid molecules were added to the reaction buffer. The reaction was carried out at different temperatures for 60 min, and the reaction products were detected by electrophoresis under 14% nucleic acid Urea-PAGE.
结果如图6所示。结果表明,TbAgo可在50℃-75℃的范围内利用5’-P gDNA切割互补靶标核酸分子。The results are shown in Figure 6. The results show that TbAgo can use 5'-P gDNA to cut complementary target nucleic acid molecules in the range of 50°C-75°C.
TbAgo、5’-P gDNA和靶标核酸分子浓度不变,在反应体系中分别加入终浓度0.5mM的CoCl2、CuCl2、MgCl2、MnCl2、ZnCl2、FeCl2、CaCl2溶液,在 75℃下,反应60min,在14%的核酸Urea-PAGE下进行电泳检测,分析金属离子对TbAgo切割活性的影响。The concentrations of TbAgo, 5'-P gDNA and target nucleic acid molecules remained unchanged, and CoCl 2 , CuCl 2 , MgCl 2 , MnCl 2 , ZnCl 2 , FeCl 2 and CaCl 2 solutions with a final concentration of 0.5 mM were added to the reaction system, respectively. The reaction was carried out at 75°C for 60 min, and electrophoresis was performed under 14% nucleic acid Urea-PAGE to analyze the effect of metal ions on the cleavage activity of TbAgo.
结果如图7所示。结果表明,TbAgo可以利用Mn2+、Mg2+或Co2+作为金属离子,去介导5’-P gDNA引导的DNA切割。The results are shown in Figure 7. The results show that TbAgo can use Mn 2+ , Mg 2+ or Co 2+ as metal ions to mediate 5'-P gDNA-guided DNA cleavage.
反应体系、缓冲液和条件不变,加入不同浓度的MnCl2:0mM、0.01mM、 0.05mM、0.1mM、0.25mM、0.5mM、1mM,测定在5’-P gDNA介导下TbAgo 的最适MnCl2浓度。The reaction system, buffer and conditions remained unchanged, and different concentrations of MnCl 2 were added: 0 mM, 0.01 mM, 0.05 mM, 0.1 mM, 0.25 mM, 0.5 mM, and 1 mM to determine the optimal MnCl 2 concentration of TbAgo mediated by 5'-P gDNA.
结果如图8所示。结果表明,0.25mM-1mM的Mn2+可使TbAgo保持较高活性。The results are shown in Figure 8. The results show that 0.25mM-1mM Mn 2+ can keep TbAgo highly active.
调整反应缓冲液成分,分别配置终浓度为10mM Tris-HCl pH7.5和不同浓度的NaCl(0mM、5 0mM、100mM、150mM、250mM、500mM、1000mM) 的反应缓冲液,其他反应体系不变,75℃反应60min,在14%的核酸 Urea-PAGE下进行电泳检测。Adjust the reaction buffer composition to prepare reaction buffers with a final concentration of 10mM Tris-HCl pH7.5 and different concentrations of NaCl (0mM, 50mM, 100mM, 150mM, 250mM, 500mM, 1000mM). Keep other reaction systems unchanged. React at 75℃ for 60min and detect by electrophoresis under 14% nucleic acid Urea-PAGE.
结果如图9所示。结果表明,TbAgo可以在NaCl浓度为0mM-500mM时发挥切割活性。The results are shown in Figure 9. The results showed that TbAgo can exert cleavage activity when the NaCl concentration is 0mM-500mM.
分别设计10nt-20nt以及25nt、35nt的5’-P gDNA(表1),探究不同长度gDNA对TbAgo切割活性的影响。5’-P gDNAs of 10nt-20nt, 25nt, and 35nt were designed (Table 1) to explore the effects of gDNAs of different lengths on the cutting activity of TbAgo.
表1Table 1
在反应缓冲液中加入终浓度为0.5mM的MnCl2,终浓度为2.5μM的TbAgo, 500nM合成的不同长度的5’-P gDNA和500nM 40nt 5’荧光修饰的序列互补单链DNA靶标核酸分子,在75℃反应60min,反应产物在14%的核酸 Urea-PAGE下进行电泳检测。MnCl 2 with a final concentration of 0.5 mM, TbAgo with a final concentration of 2.5 μM, 500 nM of synthetic 5'-P gDNA of different lengths and 500 nM of 40 nt 5' fluorescently modified sequence complementary single-stranded DNA target nucleic acid molecules were added to the reaction buffer, and the reaction was carried out at 75°C for 60 min. The reaction products were detected by electrophoresis under 14% nucleic acid Urea-PAGE.
结果如图10所示。结果表明,TbAgo可利用长度为14nt-35nt的5’-P gDNA,切割互补靶标核酸分子。The results are shown in Figure 10. The results show that TbAgo can use 5'-P gDNA with a length of 14nt-35nt to cut complementary target nucleic acid molecules.
在反应缓冲液中加入终浓度为0.5mM的MnCl2,终浓度为2.5μM的TbAgo, 500nM合成的19nt的5’-P gDNA和500nM 40nt 5’荧光修饰的序列互补单链 DNA靶标核酸分子,在75℃下分别反应0min、3min、6min、9min、12min、 15min,测定切割动力学。MnCl 2 with a final concentration of 0.5 mM, TbAgo with a final concentration of 2.5 μM, 500 nM synthetic 19 nt 5'-P gDNA and 500 nM 40 nt 5' fluorescently modified sequence complementary single-stranded DNA target nucleic acid molecules were added to the reaction buffer, and the reaction was carried out at 75°C for 0 min, 3 min, 6 min, 9 min, 12 min, and 15 min, respectively, to measure the cleavage kinetics.
结果如图11所示。结果表明,采用以上体系时,TbAgo在15min内可将目标DNA基本切割完全。The results are shown in Figure 11. The results show that when the above system is used, TbAgo can basically cut the target DNA completely within 15 minutes.
在5’-P gDNA的5’端开始第1-15位引入单碱基错配(图12,表2)的5’-P gDNA,反应体系不变,加入MnCl2、以及不同位点错配的5’-P gDNA和目标 DNA,测定TbAgo的区分单碱基错配的效果。75℃反应60min,在14%的核酸Urea-PAGE下进行电泳检测。5'-P gDNA with single base mismatches introduced at positions 1-15 starting from the 5' end of 5'-P gDNA (Figure 12, Table 2), the reaction system remained unchanged, MnCl 2 , 5'-P gDNA with mismatches at different positions and target DNA were added to determine the effect of TbAgo in distinguishing single base mismatches. The reaction was carried out at 75°C for 60 minutes, and electrophoresis was performed under 14% nucleic acid Urea-PAGE.
表2Table 2
结果如图13所示。结果表明,当错配发生在gDNA的7-14位,以及1、3、 5位时,TbAgo的切割效率显著降低。The results are shown in Figure 13. The results show that when the mismatch occurs at positions 7-14, and 1, 3, and 5 of the gDNA, the cleavage efficiency of TbAgo is significantly reduced.
实施例6:TbAgo对单个靶标核酸分子实现检测Example 6: TbAgo detects a single target nucleic acid molecule
本发明所述的靶标核酸分子的检测方法示意图如图1所示。具体的,首先对待测样本中的靶标核酸分子进行扩增;之后加入相应的检测体系,即Ago蛋白、 gDNA和核酸探针,Ago蛋白会结合gDNA,对靶标核酸分子切割,从而产生新的gDNA,新gDNA则会引导Ago蛋白切割核酸探针;最后检测核酸探针被切割后释放的信号。The schematic diagram of the detection method of the target nucleic acid molecule of the present invention is shown in Figure 1. Specifically, the target nucleic acid molecule in the sample to be tested is amplified first; then the corresponding detection system, i.e., Ago protein, gDNA and nucleic acid probe, is added, and the Ago protein will bind to the gDNA and cut the target nucleic acid molecule, thereby generating new gDNA, and the new gDNA will guide the Ago protein to cut the nucleic acid probe; finally, the signal released after the nucleic acid probe is cut is detected.
以模拟基因1、2、3(Gene1、Gene2、Gene3)为底物,设计引物,并送公司合成。采用TAKARA公司的PrimerSTAR试剂盒对模拟基因分别进行PCR扩增,具体按说明书操作。Using
根据Gene1、Gene2、Gene3的序列分别设计5’-P gDNA-gene1、5’-P gDNA-gene2、5’-P gDNA-gene3并送公司合成。目的是介导TbAgo对扩增后的靶标基因片段进行切割,从而产生新一轮的gDNA。According to the sequences of Gene1, Gene2, and Gene3, 5’-P gDNA-gene1, 5’-P gDNA-gene2, and 5’-P gDNA-gene3 were designed and sent to the company for synthesis. The purpose is to mediate TbAgo to cut the amplified target gene fragments, thereby generating a new round of gDNA.
根据新一轮gDNA的序列设计荧光基团和猝灭基团修饰的核酸探针 reporter-1~3。其中,Gene1取FAM修饰,Gene2取HEX修饰,Gene3取CY5 修饰。According to the sequence of the new round of gDNA, the fluorescent group and quenching group modified nucleic acid probes reporter-1~3 were designed. Among them, Gene1 was modified with FAM, Gene2 was modified with HEX, and Gene3 was modified with CY5.
本实施例所用模拟基因、引物、gDNA以及核酸探针序列见表3。The sequences of the simulated genes, primers, gDNA and nucleic acid probes used in this example are shown in Table 3.
表3Table 3
分别取三个模拟基因的PCR产物2μl或2μl的水(阴性对照)于反应缓冲液,加入终浓度为0.5mM的MnCl2、终浓度为2.5μM的TbAgo、相对应的250 nM合成的5’-P gDNA以及500nM reporter,75℃反应60min,酶标仪读取相应荧光值。2 μl of PCR products of three simulated genes or 2 μl of water (negative control) were taken into the reaction buffer, and MnCl 2 with a final concentration of 0.5 mM, TbAgo with a final concentration of 2.5 μM, the corresponding 250 nM synthetic 5'-P gDNA and 500 nM reporter were added. The reaction was carried out at 75°C for 60 min, and the corresponding fluorescence value was read by a microplate reader.
结果如图14所示,说明本发明的体系可有效检测靶标核酸分子。The results are shown in FIG14 , indicating that the system of the present invention can effectively detect target nucleic acid molecules.
实施例7:利用TbAgo进行单一体系检测多个靶标核酸分子Example 7: Detection of multiple target nucleic acid molecules using a single system using TbAgo
将实施例6的三对PCR扩增引物,即gene1-F/gene1-R、 Rgene2-F/gene2-R、gene3-F/gene3-R进行等量预混后,对检测样本进行PCR 扩增。The three pairs of PCR amplification primers in Example 6, namely gene1-F/gene1-R, gene2-F/gene2-R, and gene3-F/gene3-R, were premixed in equal amounts and then PCR amplified the test sample.
其中,检测样本中分别含有Gene1、Gene2、Gene3的零个、一个、两个或三个靶标核酸分子。Among them, the test sample contains zero, one, two or three target nucleic acid molecules of Gene1, Gene2 and Gene3 respectively.
取扩增产物2μl于反应缓冲液,加入终浓度为0.5mM的MnCl2、终浓度为5μM的TbAgo、终浓度均为250nM合成的5’-P gDNA-gene1、5’-P gDNA-gene2、5’-P gDNA-gene3以及终浓度均为500nM的reporter1、reporter2、 reporter3,75℃反应60min,酶标仪读取对应三种荧光基团的荧光值。Take 2 μl of the amplified product in the reaction buffer, add MnCl 2 with a final concentration of 0.5 mM, TbAgo with a final concentration of 5 μM, synthetic 5'-P gDNA-gene1, 5'-P gDNA-gene2, 5'-P gDNA-gene3 with a final concentration of 250 nM, and reporter1, reporter2, reporter3 with a final concentration of 500 nM, react at 75°C for 60 min, and read the fluorescence values of the corresponding three fluorescent groups with an enzyme reader.
结果如图15所示,说明本发明的体系可有效地在单一体系中进行多个靶标核酸分子的检测。The results are shown in FIG15 , which indicate that the system of the present invention can effectively detect multiple target nucleic acid molecules in a single system.
在本发明提及的所有文献都在本申请中引用作为参考,就如同每一篇文献被单独引用作为参考那样。此外应理解,在阅读了本发明的上述讲授内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.
序列表Sequence Listing
<110> 杭州孚斯泰生物技术有限公司<110> Hangzhou Fustai Biotechnology Co., Ltd.
<120> 一种基于TbAgo的核酸切割体系及靶标核酸分子的检测方法和试剂盒<120> A TbAgo-based nucleic acid cleavage system and a method and kit for detecting target nucleic acid molecules
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tgaggttgta ggttgtat 18tgaggttgta ggttgtat 18
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tgaggtacta ggttgtat 18tgaggtacta ggttgtat 18
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tgaggtagaa ggttgtat 18
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tgaggtagta ggatgtat 18tgaggtagta ggatgtat 18
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<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 29<400> 29
tgaggtagta ggtagtat 18tgaggtagta ggtagtat 18
<210> 30<210> 30
<211> 18<211> 18
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 30<400> 30
tgaggtagta ggttctat 18tgaggtagta ggttctat 18
<210> 31<210> 31
<211> 18<211> 18
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 31<400> 31
tgaggtagta ggttgaat 18tgaggtagta ggttgaat 18
<210> 32<210> 32
<211> 113<211> 113
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 32<400> 32
agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct 60agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct 60
tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag aaa 113tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag aaa 113
<210> 33<210> 33
<211> 120<211> 120
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 33<400> 33
ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg 60ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg 60
ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc 120ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc 120
<210> 34<210> 34
<211> 99<211> 99
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 34<400> 34
cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc 60cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc 60
gccgccgcaa ggaatggtgc atgcaaggag atggcgccc 99gccgccgcaa ggaatggtgc atgcaaggag atggcgccc 99
<210> 35<210> 35
<211> 16<211> 16
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 35<400> 35
taccgtcttc atggga 16
<210> 36<210> 36
<211> 16<211> 16
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 36<400> 36
tggtttgccc cagcag 16
<210> 37<210> 37
<211> 16<211> 16
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 37<400> 37
tagtaggttg aggccg 16
<210> 38<210> 38
<211> 18<211> 18
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 38<400> 38
agcgcgattt gctggtga 18
<210> 39<210> 39
<211> 24<211> 24
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 39<400> 39
tttcttgatg tctctgacca gaca 24tttcttgatg tctctgacca gaca 24
<210> 40<210> 40
<211> 16<211> 16
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 40<400> 40
ctgattgccc ttcacc 16
<210> 41<210> 41
<211> 22<211> 22
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 41<400> 41
gacagctcat gttatatccc gc 22gacagctcat gttatatccc gc 22
<210> 42<210> 42
<211> 20<211> 20
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 42<400> 42
cccttatgcg actcctgcat 20
<210> 43<210> 43
<211> 18<211> 18
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 43<400> 43
gggcgccatc tccttgca 18gggcgccatc tccttgca 18
<210> 44<210> 44
<211> 30<211> 30
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 44<400> 44
cgcaccctcc catgaagacg gtacggtgcg 30cgcaccctcc catgaagacg gtacggtgcg 30
<210> 45<210> 45
<211> 30<211> 30
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 45<400> 45
cgcacccctg ctggggcaaa ccagggtgcg 30cgcacccctg ctggggcaaa ccagggtgcg 30
<210> 46<210> 46
<211> 30<211> 30
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 46<400> 46
cgcaccacgg cctcaaccta ctacggtgcg 30cgcaccacgg cctcaaccta ctacggtgcg 30
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CN116024193A (en) * | 2021-10-27 | 2023-04-28 | 杭州孚斯泰生物技术有限公司 | TthAGO-based nucleic acid cleavage system, target nucleic acid molecule detection method and kit |
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CN113106087A (en) * | 2021-04-20 | 2021-07-13 | 上海交通大学 | Characterization and application of novel high-temperature Argonaute protein |
CN113201578A (en) * | 2021-04-29 | 2021-08-03 | 上海交通大学 | Novel high-temperature Argonaute protein TpsAgo characterization and application |
CN116024193A (en) * | 2021-10-27 | 2023-04-28 | 杭州孚斯泰生物技术有限公司 | TthAGO-based nucleic acid cleavage system, target nucleic acid molecule detection method and kit |
CN116606839A (en) * | 2023-05-09 | 2023-08-18 | 湖北大学 | Nucleic acid cutting system based on Argonaute protein Tcago and application thereof |
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CN113106087A (en) * | 2021-04-20 | 2021-07-13 | 上海交通大学 | Characterization and application of novel high-temperature Argonaute protein |
CN113201578A (en) * | 2021-04-29 | 2021-08-03 | 上海交通大学 | Novel high-temperature Argonaute protein TpsAgo characterization and application |
CN116024193A (en) * | 2021-10-27 | 2023-04-28 | 杭州孚斯泰生物技术有限公司 | TthAGO-based nucleic acid cleavage system, target nucleic acid molecule detection method and kit |
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CN116024193A (en) * | 2021-10-27 | 2023-04-28 | 杭州孚斯泰生物技术有限公司 | TthAGO-based nucleic acid cleavage system, target nucleic acid molecule detection method and kit |
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