WO2017133608A1 - 以多重dna片段为模板制备rna或dna探针的方法 - Google Patents

以多重dna片段为模板制备rna或dna探针的方法 Download PDF

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WO2017133608A1
WO2017133608A1 PCT/CN2017/072549 CN2017072549W WO2017133608A1 WO 2017133608 A1 WO2017133608 A1 WO 2017133608A1 CN 2017072549 W CN2017072549 W CN 2017072549W WO 2017133608 A1 WO2017133608 A1 WO 2017133608A1
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rna
dna
probe
polymerase
preparing
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徐学明
鲁隼
冯俊清
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广州复能基因有限公司
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    • C12Q1/6841—In situ hybridisation
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer

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  • the invention belongs to the field of nucleic acid diagnosis, and particularly relates to a method for labeling RNA or DNA probes and an application thereof.
  • Fluorescence in situ hybridization is an important non-radioactive in situ hybridization technique in which a fluorescent dye-labeled specific nucleic acid probe hybridizes with a corresponding target DNA or RNA molecule in a cell, if detected.
  • the chromosomal target DNA is homologously complementary to the nucleic acid probe used, and the two are subjected to denaturation-annealing-refolding. Since the DNA molecules are linearly arranged along the longitudinal axis of the chromosome on the chromosome, the probe can directly interact with the chromosome. Hybridization thus localizes a particular gene on the chromosome.
  • the qualitative or quantitative or relative localization analysis of the DNA to be tested is carried out by observing the fluorescent signal under a fluorescence microscope to determine the localization of the DNA region or RNA molecule bound to the fluorescent probe after hybridization with the specific probe in the chromosome or other organelle.
  • Fluorescence in situ hybridization has many advantages, mainly reflected in: (1) no need for radioisotope labeling, high safety; (2) short experimental period of FISH, strong probe stability; (3) multiple times Immunochemical reaction, the hybridization signal is amplified, the sensitivity is improved, and the sensitivity of detection is close to that of the isotope probe; (4) the resolution of FISH is as high as 3-20Mb; (5) FISH can be labeled with different modified nucleotide molecules.
  • the needle can be prepared into a multi-color probe, so that the positioning of several probes can be observed simultaneously on the same slice, and their relative positions and sequences can be directly obtained, and the functional gene localization and biological genome research can be accelerated.
  • FISH technology has been widely used in gene mapping, gene mapping, gene amplification and deletion detection, prenatal diagnosis, mammalian chromosome evolution research, etc. Especially in tumor diagnosis, FISH technology is widely used in breast cancer. Early diagnosis, efficacy detection, individualized treatment and prognosis of solid tumors such as bladder cancer, lung cancer and lymphoma. Studies have shown that the expression of some genes is closely related to the occurrence of tumors.
  • Fluorescence in situ hybridization is generally performed using DNA probes that detect DNA samples (genomic DNA) and RNA samples (mRNA or ncRNA). DNA probes are stable and easy to store. RNA probes are commonly used to detect RNA samples (mRNA or ncRNA). The advantage of the RNA probe is that it is a single-stranded probe, the hybridization efficiency is high, and the hybrid is more stable than the DNA probe. In fluorescence in situ hybridization, selection can be made according to the advantages and detection targets of the respective probes.
  • the object of the present invention is to provide a method for preparing an RNA or DNA probe using a multiplex DNA fragment as a template, which can efficiently prepare a large amount of RNA RNA probes with high specificity, high sensitivity and stability to meet production.
  • the method for preparing an RNA or DNA probe using the multiplex DNA fragment as a template comprises the following steps: A. screening a plurality of target sequences from a gene-specific cDNA transcribed in vitro or a DNA sequence of a specific gene in the genome, At least one pair of primers is designed for each target sequence, and the PCR amplification product of the primer is a multiplex DNA fragment, and the multiplex DNA fragment can include all target sequences; B. using the multiplex DNA fragment as a template, the following reaction is performed: a. Adding RNA polymerase and biotin-labeled UTP for reaction, incorporating UTP into the product, and purifying to obtain an RNA probe; or b. adding DNA polymerase and reacting with biotin or fluorescently labeled dUTP The dUTP is incorporated into the product and purified to obtain a DNA probe.
  • RNA polymerases for probe labeling include: T7 RNA polymerase, SP6 RNA polymerase, T3 RNA polymerase, and Trc RNA polymerase.
  • DNA polymerase I DNA polymerase I
  • DNA polymerase Klenow fragment DNA polymerase Klenow fragment
  • Taq DNA polymerase DNA polymerase Klenow fragment
  • other DNA polymerases used in PCR include: DNA polymerase I, DNA polymerase Klenow fragment, Taq DNA polymerase, and other DNA polymerases used in PCR.
  • the screening comprises: searching for a specific mRNA to be detected, or searching for a specific gene DNA sequence in the genome, excluding other non-specific sequences Then, 2-50 intron-free and repeat-specific sequence regions are screened, and a homologous region sequence can be selected as a target sequence as needed.
  • the primer design comprises: designing at least one pair of primers for each region selected, such that the length of the amplification product is 100-1000 bp.
  • the RNA polymerase is T7 RNA polymerase, the 5' end of the primer plus the T7 promoter sequence.
  • the RNA polymerase is SP6 RNA polymerase, and the 5' end of the primer is added to the SP6 promoter sequence, respectively.
  • the RNA polymerase is T3 RNA polymerase, and the 5' end of the primer is added to the T3 promoter sequence, respectively.
  • the RNA polymerase is a Trc RNA polymerase, and the 5' end of the primer is added to the Trc promoter sequence, respectively.
  • T7 promoter sequence 5'-TAATACGACTCACTATAG-3'
  • SP6 promoter sequence 5'-CATACGATTTAGGTGACACTATA-3'
  • T3 promoter sequence 5'-AATTAACCCTCACTAAAG-3'
  • Trc promoter sequence 5'-TGTTGACAATTAATCATCCGGCTCGTATAAT-3'
  • RNA polymerases The combination of the above four RNA polymerases and the corresponding promoters is of the same type and can be used interchangeably.
  • the invention further provides a kit for rapidly detecting cancer-associated cytokines.
  • the kit for rapidly detecting cancer-associated cytokines of the present invention comprises an RNA or DNA probe prepared according to the method of the present invention.
  • the present invention uses a cDNA sequence as a template, and uses a bioinformatics technique to select a template sequence as a probe marker by comparison with a gene library.
  • a multiplex DNA fragment refers to a cDNA sequence in which two or more DNA fragments are used to cover and replace a full-length gene, and the plurality of DNA fragment sequences for a certain gene can not only fully reflect the specificity and representativeness of the gene, but also Avoid some repeats or homologous sequences to specifically enhance the specificity of the probe.
  • the present invention uses a multiplex DNA fragment as a template to design multiple pairs of primers for the target sequence to be detected, and a promoter or RNA polymerase binding sequence is added to the 5' end of the reverse primer to enable the final RNA probe to be completely covered. All target sequences.
  • the probe preparation method of the invention simplifies the probe preparation step, greatly shortens the entire experiment time, and more importantly, the probe specificity and stability are stronger, the background signal is lowered, and the sensitivity is higher. On this basis, the present invention provides new uses for a variety of rapid detection of tumors and related generally applicable kits.
  • RNA probes for tumor diagnosis have more advantages than DNA probes, for example, avoiding the possibility of double-stranded DNA probes being reproducible between two strands in a hybridization reaction, increasing the sensitivity of hybridization reactions.
  • the hybrid formed between RNA and RNA is more stable than the DNA-RNA hybrid; moreover, the hybrid formed between RNA-RNA is not affected by RNase, so the hybridization reaction can be treated with RNase to elute and remove Unbound probe can reduce this
  • the bottom signal therefore has the advantage of a low background. Therefore, the RNA probe prepared by the method of the present invention is advantageous for the development and preparation of related reagents and kits which are easy to store and transport, especially high quality probes and related kits for diagnosing and treating various tumors.
  • RNA or DNA probe is generally prepared by using a full-length cDNA cloning plasmid or a PCR product as a template, using RNA polymerase or DNA polymerase. However, most of the gene cDNA is above 1000 bp. If the hybridization reaction is carried out directly, the length of the probe is too long to enter the cell and hybridize, and a non-specific background signal is generated.
  • the prepared RNA or DNA probe is generally subjected to alkaline cleavage or DNase treatment to cleave the probe into fragments of 1000 bp or less. However, the process and extent of RNA alkaline lysis or DNase treatment are not easily controlled, so probe quality is difficult to guarantee.
  • An advantage of the method of the present invention is that a 100-500 nt ideal length RNA probe can be prepared by a single reaction using a plurality of DNA fragments comprising the sequence of interest, and can be directly used in the next hybridization test.
  • the probe preparation process does not require alkali cleavage and the like, and a probe of uniform length is obtained, which is a fragment of a desired length of the hybridization test, thereby enhancing probe specificity, reducing background signal interference, and saving a large amount of experimental time and improving. Test efficiency.
  • the ideal length of the probe in DNA and RNA hybridization is 100-500 bp (Michael R. Green and Joseph Sambrook, Molecular Cloning: A Lab or at or y Manual. Cold Spring Harb or Lab or at or y Press; 4th edition (June 15, 2012)). If the probe is too short, the hybridization specificity is not high, and the hybridization is unstable; if the probe is too long, it is not easy to permeate the cells, the hybridization efficiency is lowered, and a non-specific background signal is easily generated.
  • RNA probe alkaline lysis
  • DNA probe DNase
  • the PCR reaction can precisely control the length of the product, but the efficiency of directly labeling the probe with PCR is not high, and many enzymes used in the PCR reaction are not effective in incorporating the labeled dUTP into the product. PCR reactions also cannot be used to label RNA probes.
  • the invention utilizes the characteristics that the PCR reaction can precisely control the length of the product, and uses the preparation of a plurality of ideal length DNA fragments as a template to delabel the probe, thereby effectively controlling the length of the labeled probe at the DNA template level, and preparing the probe.
  • RNA or DNA probes do not require RNA base lysis or DNase treatment The process is used directly for the hybridization reaction.
  • a plurality of ideal length DNA fragments as templates, it is also possible to effectively cover a specific target gene sequence according to the needs of the detection purpose.
  • an RNA polymerase binding sequence is added at the end of each primer so that a plurality of ideal length DNA fragments can be simultaneously used as a template in the same labeling reaction to prepare an RNA probe.
  • Table 1 compares the similarities and differences between the conventional method of preparing probes and the methods described herein.
  • the methods described herein facilitate the screening and preparation of highly specific probes and can eliminate the effects of non-coding sequences and other non-specific sequences.
  • the design of multiple DNA fragments is more targeted to the detection sequence, which not only excludes some non-coding sequences and other non-specific sequences, but also greatly improves the specificity of the probe.
  • the methods described herein facilitate the discrimination of homologous sequences and different splicing forms of RNA.
  • the multiplex DNA fragment of the present invention can be designed to determine whether to add or delete some homologous sequences according to the test subject, or to include only different splicing forms for the purpose of detecting a specific target sequence.
  • a promoter sequence or an RNA polymerase binding sequence is added to the 5' end of the reverse primer to ensure that each fragment can be transcribed into a probe, and multiple pairs of primers can be designed for the target sequence to ensure the probe. Covers all specific regions of the target gene.
  • FIG. 1 is a flow chart of a method for preparing an RNA probe using a multiplex DNA fragment as a template, and shows an RNA probe labeling process using a multiplex DNA fragment as a template.
  • Figure 2 is an experimental result of an RNA probe prepared by the method of the present invention in FISH.
  • FIG. 3 is a flow chart showing a method for preparing a DNA probe using a multiplex DNA fragment as a template according to the present invention, which shows a DNA probe prepared by random primer labeling using a multiplex DNA fragment as a template.
  • FIG. 1 The method for preparing an RNA probe using a multiplex DNA fragment as a template according to the present invention is shown in FIG.
  • Her2 human epidermal growth factor receptor 2 gene RNA probe
  • Her2 human epidermal growth factor receptor 2 gene RNA probe
  • Primer design is as follows:
  • primers referred to in the above table are merely examples, and those skilled in the art can design more suitable primers according to the principles of the present invention.
  • 10 ⁇ NTPs were prepared, and the concentrations of ATP, CTP, and GTP were 10 mM, and the concentration of TTP was 5 mM.
  • the reaction solution was mixed and reacted at 37 ° C for 2-3 hours.
  • the supernatant was transferred to a new EP tube, an equal volume of isopropanol was added, and the mixture was allowed to stand for 10 min, and then centrifuged at 12,000 rpm for 10 min to remove the supernatant.
  • RNA probe prepared by using a multiplex DNA fragment as a template was obtained.
  • the biotin-labeled Her2 RNA probe obtained by the above method was used to detect the expression level of Her2 mRNA of human breast cancer cell MCF-7 (purchased from ATCC).
  • FIG. 1 The method for preparing a DNA probe using the multiplex DNA fragment as a template according to the present invention is shown in FIG.
  • Each of the selected regions is designed with at least one pair of primers so that the amplification product is 100-1000 bp in length.
  • a random primer a random 6 nucleotide oligonucleotide fragment
  • They can be randomly complementary (annealed) to denatured double-stranded DNA to provide a 3' hydroxyl terminus, in the absence of a 5' ⁇ 3' exonuclease activity of a large fragment of a DNA polymerase (eg, a large Klenow fragment), in a primer
  • the 3' hydroxyl terminus is added nucleotides one by one until the next primer.
  • the 6 nucleotide mixture appears with all possible binding sequences, and the binding of the primer to the template occurs in a random manner, so the label uniformly covers the entire length of the DNA of the target fragment.
  • the fragment of interest was amplified by PCR from a cDNA plasmid template (from the GeneCopoeia cDNA plasmid library).
  • 10 ⁇ dNTPs were prepared in which the concentrations of dATP, dCTP, and GTP were both 2 mM, and the concentration of dTTP was 1 mM.
  • the reaction mixture was mixed and reacted at 37 ° C for 5 h.
  • the supernatant was transferred to a new EP tube, an equal volume of isopropanol was added, and the mixture was allowed to stand for 10 min, and then centrifuged at 12,000 rpm for 10 min to remove the supernatant.

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Abstract

公开了一种以多重DNA片段为模板制备RNA或DNA探针的方法，包括以下步骤：从体外转录的基因特异性cDNA或是基因组中特定基因DNA序列中筛选出多个靶序列，对于每个靶序列设计至少一对引物，所述引物的PCR扩增产物为多重DNA片段，所述多重DNA片段能包括所有靶序列；以所述多重DNA片段为模板，进行以下反应：加入RNA聚合酶和带有生物素标记的UTP进行反应，将UTP掺入产物，纯化后得到RNA探针；或者加入DNA聚合酶和带有生物素或荧光标记的dUTP进行反应，将dUTP掺入产物，纯化后得到DNA探针。

Description

以多重DNA片段为模板制备RNA或DNA探针的方法 技术领域
本发明属于核酸诊断领域，具体涉及一种RNA或DNA探针标记的方法及其应用。
背景技术
荧光原位杂交技术(Flu或escence in situ hybridization，FISH)是一种重要的非放射性原位杂交技术，荧光染料标记的特异核酸探针与细胞内相应的靶DNA或RNA分子杂交，如果被检测的染色体靶DNA与所用的核酸探针是同源互补的，二者经变性-退火-复性，由于DNA分子在染色体上是沿着染色体纵轴呈线性排列，因而探针可以直接与染色体进行杂交从而将特定的基因在染色体上定位。通过在荧光显微镜下观察荧光信号，以确定与特异探针杂交后结合了荧光探针的DNA区域或RNA分子在染色体或其他细胞器中的定位，对待测DNA进行定性、定量或相对定位分析。
荧光原位杂交技术(FISH)具有很多优点，主要体现在：(1)无需放射性同位素标记，安全性较高；(2)FISH的实验周期短，探针稳定性强；(3)通过多次免疫化学反应，使其杂交信号放大，提高灵敏度，检测的灵敏度接近同位素探针杂交；(4)FISH的分辨率高达3-20Mb；(5)FISH可以用不同修饰核苷酸分子标记不同的探针，即可以制备成多色探针，因此可以在同一张切片上同时观察几种探针的定位，直接得到它们的相关位置和顺序，加速功能基因定位和生物基因组的研究。
FISH技术已经在基因定位、基因作图、基因扩增和缺失的检测、产前诊断、哺乳动物染色体进化研究等领域得到了广泛应用，特别是在肿瘤诊断方面，FISH技术被广泛用于乳腺癌、膀胱癌、肺癌、淋巴瘤等实体瘤的早期诊断、疗效检测、个体化治疗和预后判断等几个方面。研究表明，一些基因的表达与肿瘤的发生密切相关。
荧光原位杂交(FISH)一般是应用DNA探针进行检测，DNA探针可以检测DNA样本(基因组DNA)，也可以检测RNA样本(mRNA或ncRNA)。DNA探针稳定性好，易于保存。RNA探针一般用于检测RNA样本(mRNA或ncRNA)。 RNA探针的优点在于是单链探针，杂交效率高，且杂交体较DNA探针稳定。在荧光原位杂交中，可以跟据各自探针的优点及检测目标进行选择。
发明内容
本发明的目的是提供一种以多重DNA片段为模板制备RNA或DNA探针的方法，该方法能有效地大量制备特异性强、灵敏度高和稳定性强的RNA或DNA探针，以满足生产FISH肿瘤检测试剂盒的需求。
本发明所述的以多重DNA片段为模板制备RNA或DNA探针的方法，包括以下步骤：A.从体外转录的基因特异性cDNA或是基因组中特定基因DNA序列中筛选出多个靶序列，对于每个靶序列设计至少一对引物，所述引物的PCR扩增产物为多重DNA片段，所述多重DNA片段能包括所有靶序列；B.以所述多重DNA片段为模板，进行以下反应：a.加入RNA聚合酶和带有生物素标记的UTP进行反应，将UTP掺入产物，纯化后得到RNA探针；或者b.加入DNA聚合酶和带有生物素或萤光标记的dUTP进行反应，将dUTP掺入产物，纯化后得到DNA探针。
探针标记常用的RNA聚合酶包括：T7RNA聚合酶、SP6RNA聚合酶、T3RNA聚合酶和Trc RNA聚合酶。
探针标记常用的DNA聚合酶包括：DNA聚合酶I、DNA聚合酶Klenow片段、Taq DNA聚合酶及其他用于PCR中的DNA聚合酶。
根据本发明所述的制备RNA或DNA探针的方法的进一步特征，所述的步骤A中，所述筛选包括：查找待检测特定mRNA，或者查找基因组中特定基因DNA序列，排除其它非特异性序列；然后，筛选2-50个无内含子和重复序列特定序列区域，根据需要可选择同源区域序列，作为靶序列。
根据本发明所述的制备RNA或DNA探针的方法的进一步特征，所述的步骤A中，所述引物设计包括：所选的每个区域各设计至少一对引物，使扩增产物长度为100-1000bp。
根据本发明所述的制备RNA或DNA探针的方法的进一步特征，所述RNA聚合酶是T7RNA聚合酶，引物的5'端加上T7启动子序列。
根据本发明所述的制备RNA或DNA探针的方法的进一步特征，所述RNA聚合酶是SP6RNA聚合酶，引物的5'端分别加上SP6启动子序列。
根据本发明所述的制备RNA或DNA探针的方法的进一步特征，所述RNA聚合酶是T3RNA聚合酶，引物的5'端分别加上T3启动子序列。
根据本发明所述的制备RNA或DNA探针的方法的进一步特征，所述RNA聚合酶是Trc RNA聚合酶，引物的5'端分别加上Trc启动子序列。
T7启动子序列：5’-TAATACGACTCACTATAG-3′
SP6启动子序列：5’-CATACGATTTAGGTGACACTATA-3′
T3启动子序列：5’-AATTAACCCTCACTAAAG-3′
Trc启动子序列：5’-TGTTGACAATTAATCATCCGGCTCGTATAAT-3′
上述四种RNA聚合酶与相应启动子的组合为同类型的设计，可以替换使用。
本发明进一步提供了一种快速检测癌症相关的细胞因子的试剂盒。
本发明所述的快速检测癌症相关的细胞因子的试剂盒包括根据本发明所述的方法所制备的RNA或DNA探针。
本发明是以cDNA序列为模板，利用生物信息学技术，通过与基因文库进行比对而鳞选出来的作为探针标记的模板序列。多重DNA片段是指用2个或以上的DNA片段去涵盖及取代全长基因的cDNA序列，这些针对某个基因的多个DNA片段序列不仅能充分体现该基因的特异性及代表性，而且能避免一些重复序列或同源序列以有针对性地增强探针的特异性。
本发明首次用多重DNA片段作为模板，针对待检测目标序列设计多对引物，并在反向引物5’端加上启动子或RNA聚合酶结合序列，以使最终得到的RNA探针能够全部覆盖所有靶序列。本发明探针制备方法简化了探针制备步骤，大幅度缩短整个实验时间，更重要的是，探针特异性和稳定性更强，降低背景信号，灵敏度更高。在此基础上，本发明提供了多种肿瘤初诊快速检测方面的新用途和相关的普遍适用的试剂盒。
本发明所述的制备RNA或DNA探针的方法能有效地大量制备特异性强、灵敏度高和稳定性强的RNA或DNA探针。用于肿瘤诊断的RNA探针比DNA探针有更多的优势，例如：避免了双链DNA探针在杂交反应中两条链之间易复性的可能性，提高了杂交反应的敏感性；RNA与RNA之间形成的杂交体比DNA-RNA杂交体更稳定；而且，RNA-RNA之间形成的杂交体不受RNA酶的影响，因此杂交反应后可用RNA酶处理，以洗脱除去未结合的探针，可降低本 底信号，因此有本底低的优点。因此，利用本发明所述方法制备的RNA探针有利于研发和制备出容易保存和运输的相关试剂及试剂盒，尤其是用于诊断和治疗多种肿瘤的高品质探针及相关试剂盒。
传统的探针标记方法直接以cDNA作为标记模板。制备检测mRNA的探针时，一般是应用全长cDNA克隆质粒或PCR产物作为模板，利用RNA聚合酶或DNA聚合酶制备RNA或DNA探针。但是，大部分基因cDNA在1000bp以上，如直接进行杂交反应会因探针长度太长而不利于进入细胞和杂交，且会产生非特异性背景信号。制备的RNA或DNA探针一般需碱裂解或DNA酶处理，使探针裂解成1000bp以下的片段。然而，RNA碱裂解或DNA酶处理过程和程度不易控制，因此探针质量难以保证。
本发明所述的方法的优势在于：采用包含所述目的序列的多条DNA片段通过一步反应即可制备100-500nt理想长度的RNA探针，并且可直接用于下一步的杂交试验。探针制备过程不需要碱裂解等步骤，就可得到长度均一性好的探针，为杂交试验理想长度的片段，因此增强探针特异性，降低背景信号干扰，并节约大量实验时间，提高了试验效率。
在DNA和RNA杂交反应中，探针的理想长度为100-500bp(Michael R.Green and Joseph Sambrook，Molecular Cloning：A Lab或at或y Manual.Cold Spring Harb或Lab或at或y Press；4th edition(June 15，2012))。如果探针太短，杂交特异性不高，且杂交不稳定；如果探针太长，它不易透过细胞，杂交效率降低，且易产生非特异性背景信号。
现有的RNA或DNA探针标记的方法都很难有效地将标记好的探针长度控制在这范围内。因此，现有技术必须借助碱裂解(RNA探针)或DNA酶(DNA探针)去降解标记好的探针以达到上述理想长度。
PCR反应可以精确地控制产物的长度，但用PCR直接标记探针的效率不高，而且很多用于PCR反应的酶都不能有效地将标记的dUTP掺入到产物中去。PCR反应也不能用于标记RNA探针。
本发明是利用PCR反应可以精确控制产物长度的特点，用其制备多个的理想长度的DNA片段作为模板去标记探针，这样在DNA模板水平就有效地控制了标记的探针长度，制备的RNA或DNA探针不用RNA碱裂解或DNA酶处理 过程而直接用于杂交反应。此外，用多个的理想长度的DNA片段作为模板，还能根据检测目的的需要而有效地覆盖特定的目标基因序列。同时在每一个引物末端加上RNA聚合酶结合序列，使多个的理想长度的DNA片段可同时在同一标记反应中作为模板可以制备RNA探针。
下表1比较了常规制备探针的方法与本发明所述的方法的异同。
Figure PCTCN2017072549-appb-000001
本发明所述的方法有利于筛选和制备高特异性探针，并可消除非编码序列及其它非特异性序列的影响。多重DNA片段的设计对检测序列更有针对性，不仅排除了一些非编码序列及其它非特异性序列，也能使探针的特异性大大提高。
本发明所述的方法有利于区分同源性序列及RNA的不同剪切形式。本发明多重DNA片段的设计可根据检测对象来确定加入或删除一些同源性序列，也可以只包含或不包含不同剪切形式而达检测特异性目标序列的目的。
本发明中设计引物时，在反向引物5'端加上启动子序列或RNA聚合酶结合序列，可确保每个片段都能转录成探针，针对目标序列设计多对引物，能够保证探针覆盖靶基因所有特异的区域。
附图说明
图1为本发明所述的以多重DNA片段为模板制备RNA探针的方法的流程图，显示以多重DNA片段为模板的RNA探针标记过程。
图2是采用本发明所述方法制备的RNA探针在FISH中的实验结果。
图3为本发明所述的以多重DNA片段为模板制备DNA探针的方法的流程图，显示以多重DNA片段为模板，用随机引物标记(Random Priming)制备DNA探针。
具体实施方式
实施例一：以多重DNA片段为模板制备RNA探针
本发明所述的以多重DNA片段为模板制备RNA探针的方法参见图1。
以下以Her2(人类表皮生长因子受体2)基因RNA探针为例，具体说明如何采用上述方法制备得到生物素标记的Her2RNA探针。
一、PCR引物的设计：
(1)查找Her2基因mRNA序列，排除其它非特异性RNA序列。
例如，在GenBank(http://www.ncbi.nlm.nih.gov/genbank/)中查找Her2mRNA序列，用NCBI BLAST(http://blast.ncbi.nlm.nih.gov/Blast.cgi)排除其它非特异性RNA序列。
(2)筛选2-50个无内含子和重复序列特定序列区域，根据需要可选择同源区域序列，作为靶序列。
(3)对于每个靶序列各设计至少一对引物，使扩增产物长度为100-1000bp。引物设计如下：
Figure PCTCN2017072549-appb-000002
Figure PCTCN2017072549-appb-000003
上表所涉及的引物仅仅是举例，本领域技术人员可以根据本发明的原理设计出更多的合适的引物。
(4)根据需要将T7启动子或其他RNA聚合酶结合序列加在反向引物的5'端，以确保PCR产物与目的片段互补并包含全部靶序列(见上表中小写字母)。
二、标记模板的制备：通过PCR从cDNA质粒模板(来自GeneCopoeia cDNA质粒库)扩增带有T7启动子的目的片段即Her2DNA模板。
三、RNA探针的制备：
配制10×NTPs，配制的NTPs中，使ATP、CTP、GTP的浓度均为10mM，TTP的浓度为5mM。
在一个无RNase的EP管中，加入以下成分：
Figure PCTCN2017072549-appb-000004
Figure PCTCN2017072549-appb-000005
上述反应在无RNase的环境中操作。
混匀反应液，于37℃下反应2-3小时。
加入1μL DNase I和5μL DNase I缓冲液，混匀后于37℃下15分钟，以除去模板DNA。
加入2μL 0.5M EDTA(pH 8.0)，65℃下反应10min以终止反应。
取2-3μL的产物电泳检测，缓冲液用1×MOPS(3-吗啉丙磺酸)。
加入100μL DEPC水(焦碳酸二乙酯)，150μL苯酚和200μL氯仿至EP管中，震荡混匀，10000rpm离心10min。
上清液转移至新的EP管中，加入等体积的异丙醇，混匀静置10min后，12000rpm离心10min，去上清。
75％的酒精洗涤沉淀后，加DEPC水溶解，测产物浓度并电泳检测，保存于-20℃。
由此，得到以多重DNA片段为模板制备的RNA探针。
将上述方法得到的生物素标记的Her2RNA探针用于检测人乳腺癌细胞MCF-7(购自ATCC)Her2mRNA的表达水平。
在荧光显微镜下观察，实验结果如图2所示。左图：用DAPI即4',6-二脒基-2-苯基吲哚(4',6-diamidino-2-phenylindole)染色，蓝色荧光显示细胞核。中图：用所述探针与目的mRNA杂交，红色荧光显示人乳腺癌细胞MCF-7Her2mRNA的分布及表达水平。右图为左图与中图的合成图像，直观地显示细胞核与Her2mRNA相对位置。
实施例二：以多重DNA片段为模板制备DNA探针
本发明所述的以多重DNA片段为模板制备DNA探针的方法参见图3。
一、PCR引物的设计：
(1)查找待检测特定mRNA，或者查找基因组中特定基因DNA序列，排除其它非特异性RNA序列。
(2)筛选2-50个无内含子和重复序列特定序列区域，根据需要可选择同源区域序列。
(3)所选的每个区域各设计至少一对引物，使扩增产物长度为100-1000bp。
在一个特定实施例中，可设计随机引物(Random primer)，即随机的长6个核苷酸的寡核苷酸片段。它们能与变性的双链DNA随机互补结合(退火)，以提供3’羟基端，在无5’→3’外切酶活性的DNA聚合酶大片段(如Klenow大片段)作用下，在引物的3’羟基末端逐个加上核苷酸直至下一个引物处。6个核苷酸混合物出现所有可能结合序列，引物与模板的结合以一种随机的方式发生，所以标记可均匀覆盖目标片段DNA的全长。
二、标记模板的制备：通过PCR从cDNA质粒模板(来自GeneCopoeia cDNA质粒库)扩增目的片段。
三、DNA探针的制备：
配制10×dNTPs，其中，dATP、dCTP、GTP的浓度均为2mM，dTTP的浓度为1mM。
在一个EP中，加入以下成分：
Figure PCTCN2017072549-appb-000006
混匀反应液，于37℃下反应5h。
加入2μL 0.5M EDTA(PH 8.0)，65℃下反应10min以终止反应。
取2-3μL的产物电泳检测，缓冲液用1×TAE。
加入100μL水，150μL苯酚和200μL氯仿至EP管中，震荡混匀，10000rpm离心10min。
上清液转移至新的EP管中，加入等体积的异丙醇，混匀静置10min后，12000rpm离心10min，去上清。
75％的酒精洗涤沉淀后，加水溶解，测产物浓度并电泳检测，保存于-20℃。
由此，得到以多重DNA片段为模板制备的DNA探针。

Claims (9)

  1. 一种以多重DNA片段为模板制备RNA或DNA探针的方法，其特征在于，包括以下步骤：
    A.从体外转录的基因特异性cDNA或是基因组中特定基因DNA序列中筛选出多个靶序列，对于每个靶序列设计至少一对引物，所述引物的PCR扩增产物为多重DNA片段，所述多重DNA片段能包括所有靶序列；
    B.以所述多重DNA片段为模板，进行以下反应：
    a.加入RNA聚合酶和带有生物素标记的UTP进行反应，将UTP掺入产物，纯化后得到RNA探针；或者
    b.加入DNA聚合酶和带有生物素或萤光标记的dUTP进行反应，将dUTP掺入产物，纯化后得到DNA探针。
  2. 根据权利要求1所述的制备RNA或DNA探针的方法，其特征在于，所述RNA聚合酶是T7RNA聚合酶，引物的5'端加上T7启动子序列。
  3. 根据权利要求1所述的制备RNA或DNA探针的方法，其特征在于，所述RNA聚合酶是SP6RNA聚合酶，引物的5'端分别加上SP6启动子序列。
  4. 根据权利要求1所述的制备RNA或DNA探针的方法，其特征在于，所述RNA聚合酶是T3RNA聚合酶，引物的5'端分别加上T3启动子序列。
  5. 根据权利要求1所述的制备RNA或DNA探针的方法，其特征在于，所述RNA聚合酶是Trc RNA聚合酶，引物的5'端分别加上Trc启动子序列。
  6. 根据权利要求1所述的制备RNA或DNA探针的方法，其特征在于，所述DNA聚合酶是选自：DNA聚合酶I、DNA聚合酶Klenow片段、Taq DNA聚合酶及其他用于PCR中的DNA聚合酶。
  7. 根据权利要求1所述的制备RNA或DNA探针的方法，其特征在于：所述步骤A中，所述筛选包括：查找待检测特定mRNA，或者查找基因组中特定基因DNA序列，排除其它非特异性序列；然后，筛选2-50个无内含子和重复序列特定序列区域，根据需要可选择同源区域序列，作为靶序列。
  8. 根据权利要求2所述的制备RNA或DNA探针的方法，其特征在于：所述步骤A中，所述引物设计包括：所选的每个区域各设计至少一对引物，使扩增产 物长度为100-1000bp。
  9. 一种快速检测癌症相关的细胞因子的试剂盒，其特征在于：包括根据权利要求1所述的方法所制备的RNA探针或DNA探针。
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