CN101659952B - Co-modified nucleic acid segment of locked nucleic acid and minor groove conjugation - Google Patents

Co-modified nucleic acid segment of locked nucleic acid and minor groove conjugation Download PDF

Info

Publication number
CN101659952B
CN101659952B CN 200910054805 CN200910054805A CN101659952B CN 101659952 B CN101659952 B CN 101659952B CN 200910054805 CN200910054805 CN 200910054805 CN 200910054805 A CN200910054805 A CN 200910054805A CN 101659952 B CN101659952 B CN 101659952B
Authority
CN
China
Prior art keywords
gt
lt
oligonucleotide
nucleic acid
lna
Prior art date
Application number
CN 200910054805
Other languages
Chinese (zh)
Other versions
CN101659952A (en
Inventor
倪卫琴
张彬彬
朱勤玮
朱旭平
沈步超
舒峰
赵红喜
邵俊斌
马丽丽
Original Assignee
上海之江生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 上海之江生物科技有限公司 filed Critical 上海之江生物科技有限公司
Priority to CN 200910054805 priority Critical patent/CN101659952B/en
Publication of CN101659952A publication Critical patent/CN101659952A/en
Application granted granted Critical
Publication of CN101659952B publication Critical patent/CN101659952B/en

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07HSUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813Hybridisation assays
    • C12Q1/6816Hybridisation assays characterised by the detection means
    • C12Q1/6818Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer

Abstract

The invention relates to a marking method of oligonucleotide and discloses a co-modified nucleic acid segment of a locked nucleic acid (LNA) and a minor groove binding agent (MGB), namely, both the LNA and the MGB are marked on the same oligonucleotide chain. The invention enables that the capacity of identifying nucleotide sequence mutation and SNP of the oligonucleotide is enhanced by applying the combined modification of the MGB and the LNA.

Description

锁核酸和小沟结合物共修饰核酸片段 Locked nucleic acids and minor groove binders were modified nucleic acid fragments

技术领域 FIELD

[0001] 本发明涉及生物技术领域,具体涉及寡核苷酸的标记方法。 [0001] The present invention relates to biotechnology, particularly relates to a method of labeled oligonucleotides.

[0002] 背景技术 [0002] BACKGROUND OF THE INVENTION

[0003] 为了保证在PCR扩增过程中,探针或引物与模板的结合效率,探针和引物都有一定的长度的限制。 [0003] In order to ensure during PCR amplification, the binding efficiency of the probe or primer and the template, the probes and primers have a certain length limit. 一般所设计的引物长度在1纩25个碱基,而像TaqMman探针的长度一般为2(Γ40个碱基。但在实际过程中,在一些突变位点,SNP和高变异病毒的检测中,一般的探针和引物往往检测效果不理想,灵敏度和特异性很差。 Primer length is generally designed in a fine cotton 25 bases, and as the length of the probe is generally TaqMman 2 (Γ40 bases, but in the actual process, some mutation site, the SNP detection and high variability of the virus general detection probes and primers often are not effective, poor sensitivity and specificity.

[0004] MGB全名minor groove binder即小沟结合物,是源于某些抗菌素分子的化学基团,其能嵌入DNA双螺旋结构中的小沟形成非共价结合,一般可以将寡核苷酸的退火温度提高10度以上,可以缩短引物或探针的长度,提高特异性和给设计带来方便。 [0004] MGB full name of the minor groove binder i.e., minor groove binder, a chemical group derived from certain antibiotic molecule, which can be embedded in the double helix DNA minor groove form a non-covalently bound oligonucleotides may be generally nucleoside acid annealing temperature increased by 10 degrees, you can shorten the length of the primer or probe, designed to improve the specificity and convenience. 锁核酸(Lockednucleic acid, LNA),是新型的核酸类似物,它包含了2,-氧4,碳亚甲基连接,这个连接限制了呋喃核糖环的灵活性,因而将其结构锁定成一个刚性的双环模式,因此而提高杂交效率和优越的稳定性。 Locked nucleic acids (Lockednucleic acid, LNA), a novel nucleic acid analog, which contains 2 - 4 oxygen, carbon methylene connection, which limits the flexibility of the ribofuranose ring, will thus lock into a rigid structure bicyclic mode, thereby raising the hybridization efficiency and superior stability. LNA单分子的化学结构决定了寡核苷酸序列与目标序列杂交后的稳定性,与未标记相比,其双链在相同的盐溶液中,每增加一个单体LNA分子将导致其退火温度提升8°C。 LNA monomolecular chemical structure determines the stability of the oligonucleotide sequence hybridizes with the target sequence, compared to unlabeled, in which the same double-stranded salt solution, each additional LNA monomer molecule will lead to the annealing temperature lifting 8 ° C. 可以缩短引物或探针的长度,提高特异性和给设计带来方便。 You can shorten the length of the primer or probe, designed to improve the specificity and convenience. 但是这两种方法由于其提高退火温度有限,因此有时也不能完全满足设计的要求。 However, because these two methods which increases the annealing temperature is limited, and therefore may not fully meet the design requirements.

[0005] 发明内容 [0005] SUMMARY OF THE INVENTION

[0006] 本发明的目的是克服现有技术中的缺陷,提供一种更好的标记寡核苷酸的方法, 用于DNA突变识别检测和SNP的识别。 [0006] The object of the present invention is to overcome the drawbacks of the prior art, there is provided a method of labeling an oligonucleotide better, for identifying DNA mutation detection and identification of SNP.

[0007] 本发明一方面提供了一种标记寡核苷酸的方法,为在同一条寡核苷酸链上即标记LNA又标记MGB。 [0007] In one aspect the present invention provides a method of labeled oligonucleotide, as the same one that is labeled oligonucleotide strand labeled LNA and MGB.

[0008] 所述MGB标记在所述寡核苷酸链的;T端;所述LNA标记在所述寡核苷酸链的个别核苷酸上。 [0008] The numerals in the MGB oligonucleotide chain; T terminal; LNA indicia on the individual nucleotides of the oligonucleotide chain.

[0009] 进一步的,所述个别核苷酸为发生突变的碱基。 [0009] Further, the individual is a nucleotide base mutations.

[0010] 在确定MGB及LNA在寡核苷酸链上的位置后,可通过寡核苷酸自动合成仪按亚磷酰胺合成方法自动合成并标记设计好的序列。 [0010] After determining the position in the MGB and the LNA oligonucleotide chain, it can be automatically synthesized by the phosphoramidite synthesis method and marker sequences designed automated oligonucleotide synthesizers.

[0011] 本发明适合标记的寡核苷酸链的长度为10-18个碱基。 The length of the [0011] present invention is suitable labeled oligonucleotide chain is 10-18 bases.

[0012] 本发明的标记寡核苷酸的方法可用于DNA突变和SNP的识别检测。 [0012] The method of the present invention is an oligonucleotide labeled nucleotides may be used to detect DNA mutations and to identify the SNP.

[0013]本发明第二方面公开 了一种LNA (Locked Nucleic Acid)和MGB (Minor GrooveBinder)共修饰的核酸片段,为既标记有LNA又标记有MGB的寡核苷酸链。 [0013] The second aspect of the present invention discloses a LNA (Locked Nucleic Acid) and MGB (Minor GrooveBinder) co-modified nucleic acid fragments is labeled with both the LNA and oligonucleotides labeled with a MGB chain.

[0014] 进一步的,所述MGB标记在所述寡核苷酸链的3、端;所述LNA标记在所述寡核苷酸链的个别碱基上。 [0014] Further, in the MGB marking the oligonucleotide strand 3, end; on the individual bases of the LNA oligonucleotide chain labeled.

[0015] 进一步的,所述经标记的核酸片段为Taqman探针,所述寡核苷酸链的5'端还标记有报告荧光基团,3'端标记有不发光的淬灭基团。 [0015] Further, the nucleic acid fragment is labeled by Taqman probes, the oligonucleotide strand 5 'end is further labeled with a reporter fluorescent group, 3' end with a non-luminescent quencher.

[0016] 较佳的,所述报告荧光基团为FAM或VIC,不发光的淬灭基团为NFQ。 [0016] Preferably, the reporter is a fluorophore FAM or VIC, no luminescence quenching groups NFQ.

[0017] 本发明第三方面公开了上述寡核苷酸链作为引物、Taqman探针的应用。 [0017] The third aspect of the present invention disclosed in the above-described oligonucleotide strand as a primer, application of the Taqman probe. [0018] 进一步的,本发明的寡核苷酸链可作为引物或Taqman探针用于DNA突变识别检测。 [0018] Further, the oligonucleotide chains of the invention may be identified as a DNA mutation detection primers or probes for Taqman.

[0019] 研究发现,将MGB与LNA同时标记修饰寡核苷酸,相比单个标记可进一步提高其稳定性,从而使其退火温度提高。 [0019] found that while the labeled MGB with the LNA modified oligonucleotide, a single mark can be further improved as compared to its stability, so that it annealing temperature increased. 这样不仅让实验者在设计中拥有更大的选择空间,还可以提高寡核苷酸序列的特异性。 This will not only allow experimenters to have greater choice in the design, but also can improve the specificity of oligonucleotide sequences.

[0020] 本发明的新型寡核苷酸通过运用MGB(minor groove binder)和LNA(Locked nucleicacid, LNA)的组合修饰:1)使Tm值提高了12_25°C,比只运用一种修饰技术的Tm 值提高8-15°C提高了10°C左右。 [0020] The novel oligonucleotides of the present invention by using a nucleotide MGB (minor groove binder) and LNA (Locked nucleicacid, LNA) modified combination of: 1) reacting a Tm value improves 12_25 ° C, than use only one modified technique Tm is increased by 8-15 ° C increased about 10 ° C. 这可以保证寡核苷酸在满足Tm值的条件下,比单修饰或不修饰的序列更短,幻寡核苷酸序列中对A,G,C,T的组成要求降低。 This ensures that the oligonucleotide under conditions Tm value, than a single modified or not modified sequence shorter oligonucleotide sequence in phantom reduced A, G, C, T, required composition. 这些改变使得寡核苷酸:对核酸序列突变和SNP的识别能力增强。 These changes make oligonucleotides: the ability to identify nucleic acid sequences and SNP mutation enhanced.

[0021] 本专利运用非常广泛,可以有效的运用到引物,Taqman探针等。 [0021] The present Patent Application is very wide, it can be effectively applied to the primer, the Taqman probes.

附图说明 [0022] 图 1 : 反应条件1反应体系1 -样品1的PCR结果 [0023] 图 2 : 反应条件1反应体系1 -样品2的PCR结果 [0024] 图 3 : 反应条件1反应体系1 -样品3的PCR结果 [0025] 图 4 : 反应条件1反应体系1 -样品4 [的PCR结果[0026] 图 5 : 反应条件1反应体系2 -样品1的PCR结果 [0027] 图 6 : 反应条件1反应体系2 -样品2的PCR结果 [0028] 图 7 : 反应条件1反应体系2 -样品3的PCR结果 [0029] 图 8 : 反应条件1反应体系2 -样品4 [的PCR结果[0030] 图 9 : 反应条件1反应体系3 -样品1的PCR结果 [0031] 图 10 :反应条件 1反应体系 3-样品 2的PCR结果[0032] 图 11 :反应条件 1反应体系 3-样品 3的PCR结果[0033] 图 12 :反应条件 1反应体系 3-样品 4的PCR结果[0034] 图 13 :反应条件 2反应体系 1-样品 1的PCR结果[0035] 图 14 :反应条件 2反应体系 1-样品 2的PCR结果[0036] 图 15 :反应条件 2反应体系 1-样品 3的PCR结果[003 BRIEF DESCRIPTION [0022] Figure 1: Reaction Conditions Reaction System 1 1 - PCR results of Sample 1 [0023] FIG. 2: 1 Reaction conditions Reaction System 1 - PCR results of Sample 2 [0024] Figure 3: Reaction conditions Reaction System 1 1 - sample PCR results 3 [0025] Figure 4: reaction conditions 1 reaction system 1 - sample PCR results 4 [in [0026] Figure 5: the reaction conditions a reaction system 2 - PCR results of sample 1 [0027] Figure 6: reaction conditions 1 reaction system 2 - PCR results of sample 2 [0028] Figure 7: reaction condition 1 reaction system 2 - PCR results of sample 3 [0029] Figure 8: reaction condition 1 reaction system 2 - sample PCR results 4 [of [ 0030] FIG 9: 1 reaction conditions reaction system 3 - PCR results of sample 1 [0031] Figure 10: reaction conditions of the PCR results of 3- reaction system 1 sample 2 [0032] FIG. 11: 1 reaction conditions reaction system 3 sample 3- the PCR results [0033] FIG. 12: 1 reaction conditions reaction system 3- PCR results of sample 4 [0034] Figure 13: reaction conditions for the PCR reaction was 1- 2 results sample 1 [0035] Figure 14: reaction conditions reaction system 2 PCR results 1- sample 2 [0036] Figure 15: PCR reaction conditions results sample 1- [0032 reaction system 3 7] 图 16 :反应条件 2反应体系 1-样品 4的PCR结果[0038] 图 17 :反应条件 2反应体系 2-样品 1的PCR结果[0039] 图 18 :反应条件 2反应体系 2-样品 2的PCR结果[0040] 图 19 :反应条件 2反应体系 2-样品 3的PCR结果[0041] 图 20 :反应条件 2反应体系 2-样品 4的PCR结果[0042] 图 21 :反应条件 2反应体系 3-样品 1的PCR结果[0043] 图 22 :反应条件 2反应体系 3-样品 2的PCR结果[0044] 图 23 :反应条件 2反应体系 3-样品 3的PCR结果[0045] 图 24 :反应条件 2反应体系 3-样品 4的PCR结果[0046] 图 25 :反应条件 3反应体系 1-样品 1的PCR结果[0047] 图 26 :反应条件 3反应体系 1-样品 2的PCR结果[0048] 图 27 :反应条件 3反应体系1- 样品 3 的 PCR 结果[0049] 图 28 :反应条件 3反应体系1- 样品 4 的 PCR 结果[0050] 图 29 :反应条件 3反应体系2- 样品 1 的 PCR 结果[0051] 图 30 :反应条件 3反应体系2- 样品 2 的 PCR 结果[0 7] FIG. 16: Reaction conditions Reaction System 2 sample PCR results 1- 4 [0038] Figure 17: Reaction conditions Reaction System 2 2- PCR results of Sample 1 [0039] Figure 18: Reaction conditions Reaction System 2 Sample 2 2- the PCR results [0040] FIG. 19: reaction conditions reaction system 2 2- 3 sample PCR results [0041] Figure 20: reaction conditions for PCR 2 reaction system 4 results sample 2- [0042] Figure 21: reaction conditions reaction system 2 3- PCR results of sample 1 [0043] Figure 22: reaction conditions reaction system 2 3- PCR results of sample 2 [0044] Figure 23: reaction conditions reaction system 2 3 3- sample PCR results [0045] Figure 24: reaction condition 2 reaction system 3- PCR results of sample 4 [0046] FIG. 25: the results of PCR reaction conditions of the reaction system 3 1- sample 1 [0047] Figure 26: reaction conditions for PCR reaction system results 1- 3 sample 2 [0048] PCR conditions of the reaction system 3 2- sample 1: Figure 27: PCR reaction 3 reaction conditions results sample 1- system 3 [0049] Figure 28: reaction conditions for reaction 3 PCR results 1- system sample 4 [0050] 29 FIG. results [0051] Figure 30: reaction conditions for the reaction system 3 2- PCR results sample 2 [0 052] 图 31 :反应条件 3反应体系2- 样品 3 的 PCR 结果[0053] 图 32 :反应条件 3反应体系2- 样品 4 的 PCR 结果[0054] 图 33 :反应条件 3反应体系3- 样品 1 的 PCR 结果[0055] 图 34 :反应条件 3反应体系3- 样品 2 的 PCR 结果[0056] 图 35 :反应条件 3反应体系3- 样品 3 的 PCR 结果[0057] 图 36 :反应条件 3反应体系3- 样品 4 的 PCR 结果 052] Figure 31: Reaction conditions for the reaction system 3 2- PCR results Sample 3 [0053] Figure 32: Reaction conditions for the reaction system 3 2- PCR results of Sample 4 [0054] Figure 33: Reaction conditions for the reaction system 3 3- Sample 1 the PCR results [0055] Figure 34: reaction conditions for the reaction system 3 3- PCR results of sample 2 [0056] Figure 35: reaction conditions for the reaction system 3 3- 3 of sample PCR results [0057] Figure 36: reaction conditions reaction system 3 PCR results of sample 4 3-

具体实施方式 Detailed ways

[0058] 以下列举具体实例以进一步阐述本发明,应理解实例并非用于限制本发明的保护范围。 [0058] Specific examples are listed below to further illustrate the present invention, it should be appreciated that examples are not intended to limit the scope of the present invention.

[0059] 实施例1 :以Taqman探针为例 [0059] Example 1: In Example Taqman Probe

[0060] 1.引物、探针的设计与合成 [0060] 1. Primer design and synthesis of probes

[0061] 以乙型肝炎病毒的阿德福韦(ADV)突变为检测模板,设计引物和探针。 [0061] In HBV adefovir (ADV) mutation detection template, primer and probe design.

[0062] 设计原理:1)引物以能够扩增出包含阿德福韦(ADV)突变的一段核苷酸序列为目的,按照引物设计原则进行设计. [0062] The design principles: 1) a primer capable of amplifying a nucleotide sequence comprising adefovir (ADV) mutations, is designed for the purpose in accordance with the rules for primer design.

[0063] 2)探针以突变序列为目标序列进行设计。 [0063] 2) the mutant sequence as probe target sequence design.

[0064] 设计的目标:在乙肝阳性标本中,未发生阿德福韦(ADV)突变的样本由于探针不能与之结合,所以不能释放出荧光信号;相反突变样本与目的核苷酸片段结合产生荧光信号。 [0064] The design goal: hepatitis B positive samples, not adefovir (ADV) Since mutated probe can not bind to the sample, it can not release a fluorescent signal; contrary sample binds to the mutant nucleotide fragment fluorescent signal.

[0065] 引物=Forward primer :ADV-236_1 :5,-TGGGCCTCAGTCCGTTTC-3,(SEQ ID NO :1) [0065] Primer = Forward primer: ADV-236_1: 5, -TGGGCCTCAGTCCGTTTC-3, (SEQ ID NO: 1)

[0066] Reverse primer. :ADV-236_2 :5' -CCAATTACATATCCCATGAAGTTAAG-3,(SEQ IDNO : 2) [0066] Reverse primer:. ADV-236_2: 5 '-CCAATTACATATCCCATGAAGTTAAG-3, (SEQ IDNO: 2)

[0067] 1)普通I^aqman探针,2) MGB探针,3) LNA探针,4)新探针. [0067] 1) I ^ aqman ordinary probe, 2) MGB probe, 3) LNA probes, 4) a new probe.

[0068] 各种探针的制备: [0068] Preparation of various probes:

[0069] 1.普通Taqman 探针:ADV-236_3 :5,-FAM-TGGGTATACATTTAACCCC-BHQ1 (SEQIDN0 : 3) [0069] 1. Normal Taqman probes: ADV-236_3: 5, -FAM-TGGGTATACATTTAACCCC-BHQ1 (SEQIDN0: 3)

[0070] 2. MGB 探针:MGB-ADV-236-3 :5,-FAM-TGGGTATACATTTAACCC-MGBNFQ (SEQID NO :4) (Minor groove binder/Non-f luorescent quencher), ^w]Hft^MGB 设计原则。 [0070] 2. MGB Probe: MGB-ADV-236-3: 5, -FAM-TGGGTATACATTTAACCC-MGBNFQ (SEQID NO: 4) (Minor groove binder / Non-f luorescent quencher), ^ w] Hft ^ MGB Design in principle.

[0071] 1)以MGB 为3、端设计TGGGTATACATTTAACCC 核苷酸序列; [0071] 1) In MGB to 3, TGGGTATACATTTAACCC end designed nucleotide sequence;

[0072] 2)通过寡核苷酸自动合成仪按亚磷酰胺合成方法合成设计好的序列。 [0072] 2) automated oligonucleotide synthesizer by the phosphoramidite synthesis method for synthesizing the designed sequence.

[0073] 3. LNA 探针:LNA-ADV-236_4 :5,-FAM-tatacAtttaaCcCc-BHQl (SEQ ID NO :5),该探针序列符合EXIQON公司提供的LNA设计原则。 [0073] 3. LNA probes: LNA-ADV-236_4: 5, -FAM-tatacAtttaaCcCc-BHQl (SEQ ID NO: 5), the probe sequence corresponds LNA design principles EXIQON companies.

[0074] 1)设计tatacAtttaaCcCc核苷酸序列,在突变的位置用LNA进行标记; [0074] 1) Design tatacAtttaaCcCc nucleotide sequence, labeled with a mutation at a position LNA;

[0075] 2)通过寡核苷酸自动合成仪按亚磷酰胺合成方法合成设计好的序列。 [0075] 2) automated oligonucleotide synthesizer by the phosphoramidite synthesis method for synthesizing the designed sequence. [0076] 4.新探针:MGB-LNA-ADV-236-5 -FAM-acatttaaCccc-MGBNFQ(Minor groovebinder/Non-fluorescent quencher)(SEQ ID NO :6) [0076] 4. New probe: MGB-LNA-ADV-236-5 -FAM-acatttaaCccc-MGBNFQ (Minor groovebinder / Non-fluorescent quencher) (SEQ ID NO: 6)

[0077] (注:如A,C表示为这个碱基是用LNA特殊单体标记的) [0078] 该探针序列符合ABI公司提供的MGB设计原则和EXIQON公司提供的LNA设计原 则。 [0077] (Note: A, C represents the nucleotide is LNA for the labeled specific monomer) [0078] The LNA design MGB probe sequence corresponds to the design provided by ABI principles and the principles EXIQON companies. [0079] 1)以MGB为3、端设计acatttaaCccc核苷酸序列; [0080] 2)通过寡核苷酸自动合成仪按亚磷酰胺合成方法合成设计好的序列。 [0079] 1) In MGB to 3, acatttaaCccc end designed nucleotide sequence; [0080] 2) Synthesis of sequences designed by synthesis by the phosphoramidite automated oligonucleotide synthesizers. [0081] 实验样本:4个不同浓度的乙型肝炎阿德福韦(ADV)变异模板为样本。 [0081] The test samples: four different concentrations of HBV adefovir (ADV) mutated template sample. [0082] 反应体系1 : [0083] 模板 4μ 1[0084] IOX PCR reaction buffer 4μ 1[0085] MgCl2 (25mM) 3 μ 1[0086] Forward primer(20 μ Μ) 0. 5μ 1[0087] Reverse primer(20 μ Μ) 0. 5μ 1[0088] probe(20 μ Μ) 0. 3μ 1[0089] dNTP(10mM each) 0. 8μ 1[0090] Taq DNA polymerase(5U) 0. 4μ 1[0091] 双蒸水 26. 5μ 1[0092] 总体积40 μ 1 [0093] 反应体系2 : [0094] 模板 4μ 1[0095] 10Χ PCR reaction buffer 4μ 1[0096] MgCl2 (25mM) 4μ 1[0097] Forward primer(20 μ Μ) 0. 5μ 1[0098] Reverse primer(20 μ Μ) 0. 5μ 1[0099] probe(20 μ Μ) 0. 3μ 1[0100] dNTP(10mM each) 0. 8μ 1[0101] Taq DNA polymerase(5U) 0. 4μ 1[0102] 双蒸水 25. 5μ 1[0103] 总体积40 μ 1 [0104] 反应体系3 : [0105] 模板 4μ 1[0106] 10Χ PCR reaction buffer 4μ 1[0107] MgCl2 (25mM) 6 μ 1[0108] Forward primer(20 μ Μ) 0. 5μ 1[0109] Reverse primer(20 μ Μ) 0. 5μ 1[0110] probe(20 μ Μ) 0. 3μ 1[0111] dNTP(10mM each) 0. 8μ 1[0112] Taq DNA polymerase(5U) 0. 4μ 1[0113]双蒸水 23·5μ1 [0082] The reaction system was 1: [0083] Template 4μ 1 [0084] IOX PCR reaction buffer 4μ 1 [0085] MgCl2 (25mM) 3 μ 1 [0086] Forward primer (20 μ Μ) 0. 5μ 1 [0087] Reverse primer (20 μ Μ) 0. 5μ 1 [0088] probe (20 μ Μ) 0. 3μ 1 [0089] dNTP (10mM each) 0. 8μ 1 [0090] Taq DNA polymerase (5U) 0. 4μ 1 [0091 ] double distilled water 26. 5μ 1 [0092] total volume of 40 μ 1 [0093] reaction system 2: [0094] template 4μ 1 [0095] 10Χ PCR reaction buffer 4μ 1 [0096] MgCl2 (25mM) 4μ 1 [0097] Forward primer (20 μ Μ) 0. 5μ 1 [0098] Reverse primer (20 μ Μ) 0. 5μ 1 [0099] probe (20 μ Μ) 0. 3μ 1 [0100] dNTP (10mM each) 0. 8μ 1 [0101] Taq DNA polymerase (5U) 0. 4μ 1 [0102] distilled water 25. 5μ 1 [0103] total volume of 40 μ 1 [0104] reaction system 3: [0105] template 4μ 1 [0106] 10Χ PCR reaction buffer 4μ 1 [0107] MgCl2 (25mM) 6 μ 1 [0108] Forward primer (20 μ Μ) 0. 5μ 1 [0109] Reverse primer (20 μ Μ) 0. 5μ 1 [0110] probe (20 μ Μ) 0. 3μ 1 [0111] dNTP (10mM each) 0. 8μ 1 [0112] Taq DNA polymerase (5U) 0. 4μ 1 [0113] 23 · 5μ1 double distilled water

[0114]总体积 40 μ 1 [0114] The total volume of 40 μ 1

[0115] 反应条件1: [0115] Reaction conditions 1:

[0116] [0116]

Figure CN101659952BD00071

[0117] 反应条件2: [0117] Reaction conditions 2:

[0118] [01]

Figure CN101659952BD00072

[0119] 反应条件3: [0119] Reaction conditions 3:

[0120] [0120]

Figure CN101659952BD00073

[0121] 对比结果(结果参见图1-9): [0121] comparative results (results see FIG. 1-9):

[0122] [0122]

Figure CN101659952BD00074

[0123] 通过不同反应体系和反应条件的对比实验,我们可以看出,普通探针无法对突变位点进行检测,MGB和LNA的探针虽然有时也能检测到突变位点,但是与新探针相比,其在检测灵敏度,特异性,荧光释放以及稳定性都比较差。 [0123] By comparing the different reaction system and experimental conditions, we can see that the ordinary probe can not detect point mutations, although MGB probes and LNA may also be detected mutation point, but with the new Probe compared needle, in which the detection sensitivity, specificity, stability and relatively poor fluorescent release. 反应条件和反应体系的稍微改变就会引起检测结果的差异,重复性也较差。 Slightly changing the reaction conditions and the reaction system will cause differences in test results, reproducibility is also poor.

[0124] 序列表 [0124] SEQUENCE LISTING

[0125] <110>上海之江生物科技有限公司 [0125] <110> Shanghai Jiang Biotechnology Co., Ltd.

[0126] <120>锁核酸和小沟结合物共修饰核酸片段 [0126] <120> locked nucleic acids and minor groove binders were modified nucleic acid fragments

[0127] <130>ZJ090336 [0127] <130> ZJ090336

[0128] <160>6 [0128] <160> 6

[0129] <170>PatentIn version 3. 1 [0129] <170> PatentIn version 3. 1

[0130] <210>1 [0130] <210> 1

[0131] <211>18 [0131] <211> 18

[0132] <212>DNA[0133] <213>artificial sequence [0132] <212> DNA [0133] <213> artificial sequence

[0134] <220> [0134] <220>

[0135] <223> 引物 [0135] <223> Primer

[0136] <400>1 [0136] <400> 1

[0137] tgggcctcag tccgtttc 18 [0137] tgggcctcag tccgtttc 18

[0138] <210>2 [0138] <210> 2

[0139] <211>26 [0139] <211> 26

[0140] <212>DNA [0140] <212> DNA

[0141] <213>artificial sequence [0141] <213> artificial sequence

[0142] <220> [0142] <220>

[0143] <223> 引物 [0143] <223> Primer

[0144] <400>2 [0144] <400> 2

[0145] ccaattacat atcccatgaa gttaag 26 [0145] ccaattacat atcccatgaa gttaag 26

[0146] <210>3 [0146] <210> 3

[0147] <211>19 [0147] <211> 19

[0148] <212>DNA [0148] <212> DNA

[0149] <213>artificial sequence [0149] <213> artificial sequence

[0150] <220> [0150] <220>

[0151] <223> 引物 [0151] <223> Primer

[0152] <400>1 [0152] <400> 1

[0153] tgggcctcag tccgtttc 18 [0153] tgggcctcag tccgtttc 18

[0154] <210>2 [0154] <210> 2

[0155] <211>26 [0155] <211> 26

[0156] <212>DNA [0156] <212> DNA

[0157] <213>artificial sequence [0157] <213> artificial sequence

[0158] <220> [0158] <220>

[0159] <223> 引物 [0159] <223> Primer

[0160] <400>2 [0160] <400> 2

[0161] ccaattacat atcccatgaa gttaag 26 [0161] ccaattacat atcccatgaa gttaag 26

[0162] <210>3 [0162] <210> 3

[0163] <211>19 [0163] <211> 19

[0164] <212>DNA [0164] <212> DNA

[0165] <213>artificial sequence [0165] <213> artificial sequence

[0166] <220> [0166] <220>

[0167] <223>Ί^απι&η 探针 [0167] <223> Ί ^ απι & η probe

[0168] <400>3 [0168] <400> 3

[0169] tgggtataca tttaacccc 19 [0169] tgggtataca tttaacccc 19

[0170] <210>4 [0170] <210> 4

[0171] <211>18[0172] <212>DNA [0171] <211> 18 [0172] <212> DNA

[0173] <213>artificial sequence [0173] <213> artificial sequence

[0174] <220> [0174] <220>

[0175] <223>MGB 探针 [0175] <223> MGB probe

[0176] <400>4 [0176] <400> 4

[0177] tgggtataca tttaaccc 18 [0177] tgggtataca tttaaccc 18

[0178] <210>5 [0178] <210> 5

[0179] <211>15 [0179] <211> 15

[0180] <212>DNA [0180] <212> DNA

[0181] <213>artificial sequence [0181] <213> artificial sequence

[0182] <220> [0182] <220>

[0183] <223>LNA 探针 [0183] <223> LNA probe

[0184] <400>5 [0184] <400> 5

[0185] tatacattta acccc 15 [0185] tatacattta acccc 15

[0186] <210>6 [0186] <210> 6

[0187] <211>12 [0187] <211> 12

[0188] <212>DNA [0188] <212> DNA

[0189] <213>artificial sequence [0189] <213> artificial sequence

[0190] <220> [0190] <220>

[0191 ] <223>MGB与LNA共修饰探针 [0191] <223> MGB probes and LNA modifications were

[0192] <400>6 [0192] <400> 6

[0193] acatttaacc cc 12 [0193] acatttaacc cc 12

Claims (2)

1. 一种LNA和MGB共修饰的寡核苷酸作为乙型肝炎病毒的阿德福韦突变识别检测中Taqman探针的用途,所述MGB标记在所述寡核苷酸链的3端;所述LNA标记在所述寡核苷酸链的个别核苷酸上,所述个别核苷酸为发生突变的碱基,所述寡核苷酸的序列为SEQ ID NO :6。 Adefovir and MGB An LNA modified oligonucleotides were as detecting HBV mutations identified uses Taqman probes, the MGB mark the end of the oligonucleotide chain 3; the marks on the individual LNA nucleotide of the oligonucleotide strands, the individual is a nucleotide base mutation, the nucleotide sequence of the oligonucleotide SEQ ID NO: 6.
2.如权利要求1所述寡核苷酸的用途,其特征在于,所述寡核苷酸链的5'端还标记有报告荧光基团,3'端标记有不发光的淬灭基团。 2. Use of an oligonucleotide according to claim 1, wherein the oligonucleotide strand 5 'end is further labeled with a reporter fluorescent group, 3' end with a non-luminescent quencher .
CN 200910054805 2009-07-14 2009-07-14 Co-modified nucleic acid segment of locked nucleic acid and minor groove conjugation CN101659952B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN 200910054805 CN101659952B (en) 2009-07-14 2009-07-14 Co-modified nucleic acid segment of locked nucleic acid and minor groove conjugation

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN 200910054805 CN101659952B (en) 2009-07-14 2009-07-14 Co-modified nucleic acid segment of locked nucleic acid and minor groove conjugation
PCT/CN2009/072848 WO2011006306A1 (en) 2009-07-14 2009-07-21 Nucleic acid fragments co-modified by both locked nucleic acid and minor groove binder

Publications (2)

Publication Number Publication Date
CN101659952A CN101659952A (en) 2010-03-03
CN101659952B true CN101659952B (en) 2012-07-18

Family

ID=41788205

Family Applications (1)

Application Number Title Priority Date Filing Date
CN 200910054805 CN101659952B (en) 2009-07-14 2009-07-14 Co-modified nucleic acid segment of locked nucleic acid and minor groove conjugation

Country Status (2)

Country Link
CN (1) CN101659952B (en)
WO (1) WO2011006306A1 (en)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102433376B (en) * 2011-10-19 2013-07-31 上海千友生物科技有限公司 Fluorescence quenching-based genetic variation detection method and probe
WO2013075079A1 (en) * 2011-11-17 2013-05-23 Rheonix, Inc. System and methods for selective molecular analysis
CN104293937B (en) * 2014-09-28 2016-12-07 广东省妇幼保健院 A set of probes based on liquid chip thalassemia detection point sensitizing locked nucleic acid mutation detection method and detection kit
CN104531852A (en) * 2014-12-12 2015-04-22 广东省妇幼保健院 Probe and primer sensitized by locking nucleic acid and used for detecting C677T mutation of MTHFR gene, kit and detection method
CN105671204B (en) * 2016-03-15 2019-07-02 山东省动物疫病预防与控制中心 NDV/H9-AIV/IBV triple fluorescent PCR detection reagent

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1833034A (en) 2003-06-20 2006-09-13 埃克斯魁恩公司 Probes, libraries and kits for analysis of mixtures of nucleic acids and methods for constructing the same

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7205105B2 (en) * 1999-12-08 2007-04-17 Epoch Biosciences, Inc. Real-time linear detection probes: sensitive 5′-minor groove binder-containing probes for PCR analysis
US7348146B2 (en) * 2003-10-02 2008-03-25 Epoch Biosciences, Inc. Single nucleotide polymorphism analysis of highly polymorphic target sequences
US7381818B2 (en) * 2003-10-28 2008-06-03 Epoch Biosciences, Inc. Fluorescent probes containing 5′-minor groove binder, fluorophore and quenching moieties and methods of use thereof
GB2413796B (en) * 2004-03-25 2006-03-29 Global Genomics Ab Methods and means for nucleic acid sequencing
AT541945T (en) * 2006-04-11 2012-02-15 Univ Wien Tech Detection and quantification of fecal contamination in environmental samples

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1833034A (en) 2003-06-20 2006-09-13 埃克斯魁恩公司 Probes, libraries and kits for analysis of mixtures of nucleic acids and methods for constructing the same

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Letertre C.et al..Evaluation of the performance of LNA and MGB probes in 5"-nuclease PCR assays.《Molecular and cellular probes》.2003,第17卷(第6期),307-311.
郑夔等.一种无RNA纯化步骤TaqMan RT-PCR方法用于快速检测登革病毒.《华南预防医学》.2008,第34卷(第3期),6-9,14.

Also Published As

Publication number Publication date
WO2011006306A1 (en) 2011-01-20
CN101659952A (en) 2010-03-03

Similar Documents

Publication Publication Date Title
Schweitzer et al. Combining nucleic acid amplification and detection
KR100230718B1 (en) Isothermal strand displacement nucleic acid amplification
US8192937B2 (en) Methods for quantification of microRNAs and small interfering RNAs
JP3514630B2 (en) Amplification and detection of nucleic acid sequences
AU2001242634B2 (en) Hybridisation beacon and method of rapid sequence detection and discrimination
JP5619702B2 (en) Method using a dual specificity oligonucleotide
CA2377508C (en) Pcr primer constructs for use in an integrated signalling system
EP0777747B1 (en) Nucleotide sequencing method
JP6434932B2 (en) The composition of the toe hold primer duplex and methods of use thereof
ES2380923T3 (en) Quantization Procedures microRNA and small interfering RNA
JP3897805B2 (en) Amplification and detection of variant nucleic acid using the same nucleic acid
JP3843215B2 (en) Primers with high specificity, amplification and kits
JP4972541B2 (en) Quantitative amplification method using a labeled probe and 3 &#39;→ 5&#39; exonuclease activity
AU2006221202B2 (en) Processes using dual specificity oligonucleotide and dual specificity oligonucleotide
WO2002055740A2 (en) Method for detecting disease associated mutations by means of primer extension
WO1992011390A1 (en) Nucleic acid sequence detection by triple helix formation
US8198027B2 (en) Methods and compositions for nucleic acid amplification
AU2002348612A1 (en) Hybridization portion control oligonucleotide and it&#39;s uses
JP4263612B2 (en) Annealing regulation primer and use its
CN101815789B (en) Enrichment of a target sequence
EP1117839B1 (en) Multi-loci genomic analysis by a method of improved cycle sequencing
CN101878315A (en) Diagnosis kit and chip for bladder cancer using bladder cancer specific methylation marker gene
US8679788B2 (en) Methods for the detection of nucleic acid differences
CN106434857A (en) TD probe and its uses
EP2789689B1 (en) Chimeric primers with hairpin conformations and methods of using same

Legal Events

Date Code Title Description
C06 Publication
C10 Request of examination as to substance
C14 Granted
C56 Change in the name or address of the patentee