WO2019080595A1 - 一种原位杂交探针的制备方法 - Google Patents

一种原位杂交探针的制备方法

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WO2019080595A1
WO2019080595A1 PCT/CN2018/099530 CN2018099530W WO2019080595A1 WO 2019080595 A1 WO2019080595 A1 WO 2019080595A1 CN 2018099530 W CN2018099530 W CN 2018099530W WO 2019080595 A1 WO2019080595 A1 WO 2019080595A1
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dna
label
situ hybridization
product
hybridization probe
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PCT/CN2018/099530
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French (fr)
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蒋晓群
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厦门龙进生物科技有限公司
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Priority to EP18871629.4A priority Critical patent/EP3702455B1/en
Priority to US16/758,283 priority patent/US11359193B2/en
Publication of WO2019080595A1 publication Critical patent/WO2019080595A1/zh

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Definitions

  • the invention relates to the field of biotechnology DNA probes, in particular to a preparation method of in situ hybridization probes.
  • DNA hybridization is a process of renaturation after denaturing into a single strand with both a DNA fragment carrying a label (ie, a DNA probe) and a DNA having the same base sequence (ie, a target sequence) according to the principle of base complementation. The process of combining to form a double strand (ie, DNA hybridization).
  • Qualitative and quantitative measurements of target sequences are performed by detection of labels on probes.
  • the probe is typically in the solution as a mobile phase, while the target sequence is typically in the stationary phase.
  • Southern hybridization the comparative genomic hybridization of DNA on the chip (CGH), in situ hybridization on cell droplets and tissue sections, chromosome or Hybridization of cells in solution or in a microfluidic system.
  • CGH DNA on the chip
  • in situ hybridization probes are exemplified below.
  • in situ hybridization is a small fragment of DNA with a marker, ie hybridization probe, and tissue Or a molecular cytogenetic technique in which intracellular DNA homologous target sequences are hybridized to detect changes in DNA (or genes) or chromosome numbers or positions in the specimen by detecting signals generated by the markers.
  • the in situ hybridization probe is a nucleotide sequence with a label (small fragment DNA), which has strict requirements on its length, too long to penetrate to the position of the target sequence, and too short to be insufficient in specificity. Non-specific signal or high background.
  • Fluorescence in situ hybridization is the hybridization of fluorescently labeled DNA probes to DNA homologous sequences in tissues or cells, and the results are recorded and analyzed by observing and counting the number and distribution of various fluorescent signals under a fluorescence microscope. It combines the high sensitivity and intuitiveness of fluorescent signals with the localization of in situ hybridization in cells and tissues to detect and diagnose chromosomal or genetic abnormal cell and tissue samples for the classification of various genetically related diseases. Prognosis provides an accurate basis and shows significant advantages over some traditional techniques. (1. Raap AK: Advances in fluorescence in situ hybridization. Mutat Res 400: 287–298 (1998). 2.
  • Raap AK Overview of fluorescence in situ hybridization techniques for molecular cytogenetics. Curr Protoc Cytom Chapter 8: Unit 8.1 (2001 ).3.Wang N: Methodologies in cancer cytogenetics and molecular cytogenetics.Am J Med Genet.115:118–124(2002).4.Wolff DJ,Bagg A,Cooley LD,Dewald GW,Hirsch BA, et al:Guidance For fluorescence in situ hybridization testing in hematologic disorders. J Mol Diagn 9: 134–143 (2007)).
  • in situ hybridization probes for clinical detection of gene amplification, gene deletion, and chromosomal ectopic are generally prepared by using a large amount of BAC DNA as a template, and most of the labeling methods used are notch translation methods or random primer methods (for example, the name of the invention)
  • notch translation methods or random primer methods for example, the name of the invention
  • the preparation of the probe by these two methods requires a large amount of BAC DNA to be prepared, and the cost is high.
  • the labeling method is not easy to accurately control the length range of the probe, resulting in a large variation in the products produced by different batches (reproducible), product performance.
  • the indicator is unstable.
  • the object of the present invention is to provide a method for solving the problem that the large-scale preparation of in situ hybridization probes in the prior art is high in cost, the probe length range is not easy to control, and the product quality is unstable, and a DNA in situ hybridization probe is provided. Preparation method.
  • the present invention provides a method for preparing a DNA in situ hybridization probe, which comprises the following steps,
  • Fragmentation and selective recovery of target DNA Fragmentation of target DNA, selective recovery of small fragments of 150-600 bp;
  • Enzyme modification treatment enzyme modification treatment on both ends of 150-600 bp small fragment DNA, so that both ends are blunt end, 5' end plus phosphoric acid and 3' end plus dA;
  • the above-described treated small fragment DNA is ligated to a DNA adaptor having a restriction endonuclease site sequence to form a DNA macrocycle and a long strand; the restriction endonuclease site is included
  • the DNA adaptor of the sequence adds phosphoric acid to the 5' end and dT to the 3' end;
  • A isothermal amplification, adding a substrate with a label upon amplification to obtain a labeled DNA product
  • the obtained amplification product is incorporated into the single nucleotide substrate with the label by nick translation or random primer method to obtain a DNA product with a label;
  • the label comprises a direct label and an indirect label, the direct label being a label detectable by the detection system, including but not limited to a radioisotope, biotin, antigen or hapten such as digoxin, fluorescein;
  • Indirect labels are certain chemical groups that cannot be detected by detection systems. These chemical groups include, but are not limited to, aliphatic primary amine groups, thiols, and need to pass through
  • the direct label of the reactive group (via the reactive group) is coupled, for example, the amine group (aa, aminoallyl) and the succinimidyl ester (SE, succinimidyl ester) are reacted under alkaline conditions to be connected, but will be directly
  • SE succinimidyl ester
  • the DNA product with the label is digested with a DNA restriction endonuclease corresponding to the base sequence of the above DNA adaptor, and the length of the digested product is 150-600 bp;
  • the obtained digested product is an in situ hybridization probe
  • the label in the labeled DNA product is an indirect label
  • the resulting digested product is coupled to a reactive direct label to provide an in situ hybridization probe.
  • the obtained in situ hybridization probe was further purified and quantified.
  • the target DNA is a purified target DNA.
  • the primer used in the isothermal amplification is an oligonucleotide or a random sequence oligonucleotide containing an adaptor sequence of a restriction endonuclease site.
  • the enzyme used for isothermal amplification is a DNA polymerase having strand displacement activity.
  • the enzyme used for the isothermal amplification is a large fragment of Phi29 DNA polymerase and Bst DNA polymerase.
  • the label is a direct label or an indirect label.
  • the target DNA is DNA comprising a probe target sequence.
  • the sequence of the cleavage site on the inserted adaptor of the present invention is based on the principle of not impeding the digestion of the DNA incorporated into the label, for example, incorporating the dUTP with a label, and the endonuclease cannot be cleaved after the substitution of T by U. , the sequence containing T and A should not be selected; the principle that the inserted restriction site sequence is as rare as possible in the target sequence reduces the influence on the design length range of the probe product.
  • the invention adopts DNA polymerase with strand displacement activity (such as Phi29 DNA polymerase and large fragment of Bst DNA polymerase), is a highly efficient DNA polymerase, has special chain displacement and continuous synthesis characteristics, and works at a constant temperature, and is often applied to trace DNA. Massive amplification, such as the preparation of single-cell whole-genome sequencing samples.
  • DNA polymerase with strand displacement activity such as Phi29 DNA polymerase and large fragment of Bst DNA polymerase
  • the invention uses a small amount of target DNA as a starting material, and prepares a small fragment library within a design length range, and is connected to a DNA adaptor having a restriction endonuclease site sequence to form a DNA macrocycle. And long-chain templates, which are extensively amplified by Loop mediated isothermal amplification (LAMP) (for circular DNA) and thermostatic multiple displacement amplification (MDA) (for linear DNA). Amplification is carried out simultaneously with the labeling or subsequent incorporation of the label by nick translation (or random primer method and its variant), and then the labeled DNA is cleaved by restriction endonuclease to reduce the length of the original library fragment.
  • LAMP Loop mediated isothermal amplification
  • MDA thermostatic multiple displacement amplification
  • the present invention integrates a probe fragment of a designed length into a large interval by an enzyme cleavage site sequence.
  • the loop and long-stranded DNA are probe templates, and the inserted restriction endonuclease sequence (or random sequence) is used as a primer sequence to perform isothermal amplification of DNA, and other methods are used to incorporate the labeled substance at the same time as or after the amplification.
  • the substrate is labeled with DNA, and the inserted cleavage site is a cleavage site for the probe to cleave the labeled DNA into a design length range, which provides a more convenient method for mass production of the probe of the design length range, increasing the yield and accuracy.
  • the length of the probe is controlled, the consumption of raw materials in the production process is reduced, the cost is saved, and the performance index of the product is improved.
  • In situ hybridization probes prepared by the methods of the invention can be used, but are not limited to, in situ hybridization or CGH chip hybridization of interphase or metaphase nuclei (on cell sheets or tissue sections), and analysis of hybridization results can be used to understand specific genes or The presence or absence, number change, positional change of a DNA fragment in a particular cell/cell population/tissue/plant/animal/human body is associated with early detection of the disease, diagnosis, selection of a therapeutic drug, or prognosis.
  • Example 1 is an agarose gel electrophoresis pattern of the HER2 gene detection FISH probe of Example 2;
  • FIG. 2 is a diagram showing the results of FISH experiments of the HER2 gene detection FISH probe of Example 2 and CEN17 of Example 1 on normal human metaphase cell chromosomes;
  • Fig. 3 is a graph showing the results of FISH experiments of the HER2 gene detection FISH probe of Example 2 and CEN17 of Example 1 on normal human interphase cells.
  • Figure 4 is a graph showing the results of FISH experiments on the chromosomes of normal human metaphase cells of the chromosome 13 specific FISH probe of Example 3.
  • Enzyme modification treatment is carried out on both ends of the small fragment DNA recovered in the second step, so that the ends are flattened, the 5' end is added with phosphoric acid, and the 3' end is added with dA;
  • the small fragment DNA after the step three enzyme modification treatment is connected with the DNA adaptor having the restriction endonuclease site sequence of the 5' end plus phosphoric acid and the 3' end plus dT. DNA macrocycles and long chains;
  • the DNA macrocycle and the long strand obtained in the fourth step are subjected to isothermal rolling circle (LAMP) and multi-point displacement amplification (MDA), and simultaneously incorporated with a label.
  • LAMP isothermal rolling circle
  • MDA multi-point displacement amplification
  • a deoxynucleotide such as dUTP-fluorescein or aa-dUTP; obtaining a DNA with a label after amplification;
  • the above-obtained amplified DNA with the label is digested, and the restriction enzyme corresponding to the inserted adaptor sequence is used to obtain a digested product having a length of 150-600 bp;
  • the target DNA described in the first step is a double-stranded DNA, which may be linear or circular, may be in a supercoiled state, may be continuous or nicked, and includes, but is not limited to, plasmid, BAC, YAC, PCR product. , RT-PCR product, DNA digestion product or ligation product, artificial amplification product, artificial synthesis product;
  • step 5 If the label incorporated in step 5 is the indirect label aa, it is also necessary to add a coupling step between steps 6 and 7 to mark the direct label.
  • the method for preparing the third in situ hybridization probe of the present invention differs from the above two methods in the following steps, and the other steps are the same:
  • Step 5 performing isothermal amplification without incorporating a deoxynucleotide with a label
  • Step 6 The DNA obtained in the fifth step is incorporated into the single nucleotide substrate with the label by nick translation, and the DNA is labeled;
  • Step 7 Digesting the labeled DNA product with a restriction endonuclease corresponding to the restriction site on the adaptor to obtain a digested product having a length of 150-600 bp;
  • Step 8 Purify and quantify the obtained digested product, and obtain the probe to complete the preparation of the DNA in situ hybridization probe.
  • the method for preparing the fourth in situ hybridization probe of the present invention differs from the preparation method of the third in situ hybridization probe in the following steps, and the other steps are the same:
  • Step 6 The DNA obtained in the step 5 is labeled with a single nucleotide substrate with a label by a random primer method to label the DNA;
  • the preparation method of the fifth in situ hybridization probe of the present invention is different from the preparation method of the fourth in situ hybridization probe by the following steps, and the other steps are the same:
  • Step 6 The DNA obtained in the fifth step is replaced with a random primer by using an oligonucleotide of the adaptor sequence as a primer to label the DNA;
  • the PCR product was purified using the QIAquick PCR Purification Kit.
  • the enzyme modification treatment using New England Biolabs Ultra End Repair/dA-Tailing Module, follow the instructions in the instructions, trim the end of the DNA fragment recovered in step 2, phosphorylate the 5' end, add dA at the 3' end, and then use Qiagen PCR purification kit. (QIAquick PCR Purification Kit) recovers the modified DNA fragment;
  • the adaptor is double-stranded, and its 5' end is phosphorylated.
  • the sequence is as follows:
  • the ligated product was purified and recovered using Qiagen's QIAamp DNA Blood Kits, the unligated adaptor was removed, and the OD value was determined to obtain a macrocyclic and long-chain DNA probe template.
  • the product was purified and recovered using Qiagen's QIAamp DNA Blood Kits to determine the OD value.
  • Enzyme digestion The restriction enzyme N was obtained by restriction endonuclease SacII to obtain a digested product having a length of 150-600 bp.
  • the digestion reaction system is (can increase or decrease the reaction volume proportionally):
  • the above digestion reaction system was placed at 37 ° C for 16 hours;
  • the digested product was purified using Qiagen's PCR Purification Kit and the OD value was determined to obtain a green fluorescent fluorescein FITC-labeled chromosome 17 count FISH probe (denoted as CEN17).
  • the DNA is derived from BAC, and the amplification and labeling are carried out separately.
  • the amplification is carried out using a 6-base random primer (included in the kit), and after amplification, the label is incorporated by the nick translation method.
  • the incorporated label is a direct label or an indirect label.
  • BAC Bacterial Artificial Chromosome
  • the terminal sequencing method was used to determine the sequence of hundreds of bases at both ends of the BAC clone insert, and compared with the known sequence published by NCBI, and the next experiment was carried out correctly;
  • Example 2 taking 1 ⁇ g of BAC DNA, fragmentation treatment by focused ultrasound method and recovering 150-600bp DNA fragment, the same method as in Example 1;
  • the adaptor containing the DNA restriction endonuclease SmaI site sequence is ligated to the DNA fragment recovered in step four;
  • the adaptor is double-stranded, and its 5' end is phosphorylated.
  • the sequence is as follows:
  • the DNA obtained in the fourth step was used as a template for large-scale amplification, using Qiagen's REPLI-g Single Cell Kit (the principle is LAMP and MDA, using Phi29 DNA polymerase, with a random sequence of six)
  • the base oligonucleotide is a primer
  • the amplified product is purified and recovered by Qiagen QIAamp DNA Blood Kits according to the instructions;
  • the amplification product obtained in step 5 is used as a template, and the direct label dUTP-TAMRA (carboxytetramethylrhodamine) or the indirect label aa-dUTP is incorporated by nick translation method as follows:
  • the amplified product was purified and recovered using Qiagen's QIAamp DNA Blood Kits, and the OD value was determined.
  • step 6 the DNA obtained in step 6 is digested with restriction endonuclease SmaI to obtain a digested product having a length of 150-600 bp;
  • the enzyme digestion reaction system is:
  • the above digestion reaction system was placed at 37 ° C for 16 hours;
  • step 7 Purify the digested product of step 7 using Qiagen's PCR purification kit to determine the OD value.
  • step 6 incorporates a direct label, an orange-red fluorescent TAMRA-labeled HER2 probe is obtained; if step 6 incorporates an indirect label, then:
  • the product was purified by ethanol precipitation and the OD value was determined to obtain an orange-red fluorescein TAMRA-labeled HER2 probe.
  • Example 2 The nick translation labeling method is characterized by rapid, simple, uniform and high specificity of the labeled probe, and the labeling rate of the probe is higher than that of the Bst DNA polymerase-incorporated label; the indirect labeling ratio is directly Marking saves costs.
  • the target DNA is derived from the entire chromosome, using random primers, isothermal amplification and labeling are performed simultaneously, and the incorporated label is an indirect label.
  • step 3 Obtaining a large amount of DNA: The chromosome obtained in step 2 is used as a DNA source, and a large amount of DNA is amplified by Qiagen's REPLI-g Single Cell Kit. According to the instructions, Qiagen QIAamp DNA Blood Kits are used for amplification. The product is purified and recovered;
  • the adaptor containing the DNA restriction endonuclease site sequence is ligated with the DNA fragment recovered after the step 5 modification to obtain a DNA probe template, and the method is the same as in the first embodiment;
  • a 6-base random oligonucleotide (sequence NNNNNN, SEQ ID NO: 4, where N represents A or T or G or C) is used as a primer for amplification, and the reaction system and process are as follows (the reaction can be proportionally increased) volume):
  • the product was purified and recovered using Qiagen's QIAamp DNA Blood Kits to determine the OD value.
  • the purified product was purified by Qiagen PCR Purification Kit, and the OD value was determined.
  • the product was purified by ethanol precipitation and the OD value was determined to obtain a chromosome 13-specific FISH probe labeled with orange-red fluorescein TAMRA.
  • FIG. 1 is an agarose gel electrophoresis pattern of the obtained HER2 probe, wherein lane 1 For the 100bp DNA ladder, Lane 2 is the probe, showing that the probe length is in the 150-600bp design range, meeting the design requirements.
  • the composition of the FISH probe hybridization solution per 10 ⁇ l of the HER2 gene was as follows: the orange-red fluorescein TAMRA-labeled HER2 gene obtained in Example 2 was detected as 20 ng of the FISH probe, and the chromosome 17 counting probe CEN17 obtained in Example 1 was 10 ng, human COT-1 DNA. (purchased from ThermoFisher Scientific) 1 ⁇ g, 50% by volume deionized formamide, 2X SSC, 10% by weight of dextran sulfate.
  • composition of each 10 ⁇ l of chromosome 13-specific FISH probe hybridization solution was as follows: the orange-red fluorescein TAMRA-labeled chromosome 13-specific FISH probe obtained in Example 3 was 20 ng, and human COT-1 DNA (purchased from Thermo Fisher Scientific) 1 ⁇ g, 50 % by volume of deionized formamide, 2X SSC, 10% by weight of dextran sulfate.
  • slides are dehydrated by gradient ethanol, and dried; conventional pepsin digestion, 10 ⁇ l of the above HER2 gene detection FISH probe hybrid solution or chromosome 13 specific FISH probe hybridization solution
  • the slide was applied to the hybridization area of the slide, covered with a cover glass, and sealed with a rubber water, denatured at 78 ° C for 5 minutes, sealed in a wet box, and hybridized overnight at 37 ° C.
  • the tablets were washed in the following order: 4 ⁇ SSC solution, shaking and rinsing for 5 minutes; 2 ⁇ SSC, 0.1% by volume of Tween-20 solution, shaking and rinsing for 4 ⁇ 2.5 minutes; 0.1 ⁇ SSC solution, shaking and rinsing for 5 minutes Dehydrated in a gradient ethanol and dried. 20 ⁇ l of DAPI counterstain was added dropwise to the hybridization site and immediately covered with a coverslip. The results were observed and photographed using a suitable filter under a fluorescence microscope.
  • Figure 2 is a diagram showing the results of FISH experiments on the chromosomes of normal human metaphase cells (derived from normal human peripheral blood mononuclear cells) cultured in vitro by the HER2 gene detection FISH probe hybridization solution; wherein 1 is the HER2 gene detection FISH probe Signal, 2 is the signal generated by chromosome 17 in situ hybridization probe CEN17.
  • Figure 3 is a diagram showing the results of FISH experiments on the normal human interphase cells (derived from normal human peripheral blood mononuclear cells) cultured in vitro by the HER2 gene detection FISH probe hybridization solution; wherein 1 is the HER2 gene detection FISH probe production
  • the signal, 2 refers to the signal generated by the in situ hybridization probe CEN17 on chromosome 17.
  • the CEN17 signal is located on the centromere of chromosome 17, and the HER2 signal is located at the centromere of the long arm of chromosome 17 (region 1 and band 2, 17q12), in normal cells.
  • Each has 2 signals to meet the design requirements and intended purpose.
  • Figure 4 is a diagram showing the results of FISH experiments on chromosomes of normal human metaphase cells (derived from normal human peripheral blood mononuclear cells) cultured on chromosome 13 specific FISH probe hybridization solution, and a pair of chromosomes 13 showed fluorescence signals as a whole. Meet design requirements and intended goals.

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Abstract

提供了一种原位杂交探针的制备方法。该制备方法步骤如下：将目标DNA片段化，回收150-600bp的片段，经过酶修饰处理，与含有限制性内切酶位点序列的DNA衔接子间隔连接成DNA大环和长链；再采用A等温扩增，扩增时加入带有标记物的单核苷酸底物，得到带有标记物的DNA产物；或B等温扩增，所得产物以缺口平移法或随机引物法掺入带有标记物的单核苷酸底物，得到带有标记物的DNA产物；对所得带有标记物的DNA产物用对应的限制性内切酶进行消化，得到长度为150-600bp的原位杂交探针。

Description

一种原位杂交探针的制备方法 技术领域
本发明涉及生物技术DNA探针领域，尤其涉及一种原位杂交探针的制备方法。
背景技术
DNA杂交是依据碱基互补原理，以带有标记物的DNA片段(即为DNA探针)，与具有相同碱基序列的DNA(即靶序列)两者变性成单链后在复性的过程中互相结合形成双链的过程(即DNA杂交)。通过对探针上标记物的检测，用于靶序列的定性和定量测量。探针通常处于溶液中为流动相，而靶序列通常处于固定相。依据靶序列所处的状态和载体不同，如DNA在膜上的杂交称为Southern杂交，DNA在芯片上的比较基因组杂交(CGH)，在细胞滴片和组织切片上的原位杂交，染色体或细胞在溶液中或微流控系统中的杂交等。在商业运用上，需要大量地制备DNA探针，下面以原位杂交探针为例进行说明。
原位杂交技术(In-Situ Hybridization简称ISH)和荧光原位杂交技术(Florescence In-Situ Hybridization简称FISH)；原位杂交技术是以带有标记物的DNA小片段，即杂交探针，与组织或细胞内的DNA同源靶序列进行杂交，通过检测标记物所产生的信号，来检测标本中DNA(或基因)或染色体数目或位置变化的一种分子细胞遗传学技术。原位杂交探针即是带有标记物的核苷酸序列(小片段DNA)，对于其长度有较严格的要求，太长不易穿透到达靶序列所在位置，太短则特异性不足而产生非特异信号或高背景。荧光原位杂交(FISH)是以荧光标记的DNA探针与组织或细胞内的DNA同源序列进行杂交，通过在荧光显微镜下观察和计数各种荧光信号的数量和分布来记录和分析结果。它将荧光信号的高灵敏度、直观性和原位杂交在细胞和组织内的定位结合起来，从而对染色体或基因异常的细胞、组织样本进行检测和诊断，为各种基因相关疾病的分型和预后提供准确的依据，显示出与一些传统技术相比的明显优势。(1.Raap AK:Advances in fluorescence in situ hybridization.Mutat Res 400:287–298(1998).2.Raap AK:Overview of fluorescence in situ hybridization techniques for molecular cytogenetics.Curr Protoc Cytom Chapter 8:Unit 8.1(2001).3.Wang N:Methodologies in cancer cytogenetics and molecular cytogenetics.Am J Med Genet.115:118–124(2002).4.Wolff DJ,Bagg A,Cooley LD,Dewald GW,Hirsch BA, et al:Guidance for fluorescence in situ hybridization testing in hematologic disorders.J Mol Diagn 9:134–143(2007))。
目前临床检测基因扩增、基因缺失、染色体异位的原位杂交探针一般是以大量的BAC DNA为模板制备而来，使用的标记方法大多为缺口平移法或随机引物法(例如，发明名称为：乳腺癌分子标志物相关探针的制备方法及其应用，申请号：201010284090.3中山大学达安基因股份有限公司；发明名称为：用于检测前列腺癌的检测剂及其用途，申请号201010113005.7，北京金菩嘉医疗科技有限公司)。采用这两种方法制备探针都需要制备大量的BAC DNA，成本较高，标记方法不易准确控制探针的长度范围，导致不同批次生产的产品变化较大(重复性不好)，产品性能指标不稳定。
发明内容
本发明的目的在于提供一种解决现有方法大规模制备原位杂交探针成本较高，探针长度范围不易控制而致产品质量不稳定的问题，而提供了一种DNA原位杂交探针的制备方法。
为实现上述目的，本发明提供一种DNA原位杂交探针的制备方法，其特征在于，包括以下步骤，
目标DNA的片段化和选择性回收：将目标DNA片段化，选择性回收150-600bp的小片段；
酶修饰处理：对150-600bp的小片段DNA的两端进行酶修饰处理，使两端成平末端、5'端加磷酸和3'端加dA；
连接成大环和长链：将上述处理后的小片段DNA与含有限制性内切酶位点序列的DNA衔接子间隔连接成DNA大环和长链；所述含有限制性内切酶位点序列的DNA衔接子的5'端加了磷酸和3'端加了dT；
大量DNA的获得和标记：
A，等温扩增，扩增时加入带有标记物的底物，得到带有标记的DNA产物；
或B，等温扩增，所得扩增产物以缺口平移法或随机引物法掺入带有标记物的单核苷酸底物，得到带有标记物的DNA产物；
所述的标记物包括直接标记物和间接标记物，直接标记物为能够被检测系统检测到的标记物，包括但不限于放射性同位素、生物素、抗原或半抗原如地高辛、荧光素；间接标记物为某些化学基团，不能够被检测系统检测到，这些化学基团包括但不限于脂肪族伯胺基(aliphatic primary amine group)、硫醇(thiols)，需要经过与带有对应反应基团的直接标记物(通过反应基团)进行偶联，比如胺基(aa,aminoallyl)与琥珀酰亚胺酯 (SE,succinimidyl ester)在碱性条件下发生反应而连接，而将直接标记物标记上去。
酶切：采用与上述DNA衔接子上碱基序列对应的DNA限制性内切酶消化带有标记物的DNA产物，酶切产物长度范围150-600bp；
得到探针：
若所述带有标记物的DNA产物中的标记物为直接标记物，则所得到的酶切产物即为原位杂交探针；
若所述带有标记物的DNA产物中的标记物为间接标记物，则所得到的酶切产物经过与具有反应活性的直接标记物进行偶联，从而得到原位杂交探针。
进一步，所述得到的原位杂交探针进一步纯化，定量。
进一步，所述目标DNA为纯化后的目标DNA。
进一步，所述A或B步骤中，等温扩增时所用的引物为含有限制性内切酶位点的衔接子序列的寡核苷酸或随机序列寡核苷酸。
进一步，所述A或B步骤中，等温扩增所用的酶为具有链置换活性的DNA多聚酶。
进一步，所述等温扩增所用的酶为Phi29DNA多聚酶、Bst DNA多聚酶大片段。
进一步，所述A或B步骤中，所述标记物为直接标记物或间接标记物。
所述目标DNA即包含探针靶序列的DNA。
本发明插入的衔接子上的酶切位点序列：插入序列以不影响酶切掺入标记物的DNA为原则，例如掺入带有标记物的dUTP，T被U替代后内切酶不能切割，则不应选择含有T和A的序列；以插入的酶切位点序列在靶序列中尽量稀少为原则，减少对探针产品设计长度范围的影响。
本发明采用具有链置换活性的DNA多聚酶(如Phi29DNA多聚酶、Bst DNA多聚酶大片段)，是高效的DNA多聚酶，具有特殊的链置换和连续合成特性，并且在恒温下工作，常应用于微量DNA的大量扩增，比如单细胞全基因组测序样本的制备。
本发明包含以下有益效果：
本发明以小量的目标DNA为起始物，将其制成设计长度范围内的小片段文库，与含有限制性内切酶位点序列的DNA衔接子(adaptor)互相间隔连接成DNA大环和长链模板，通过恒温滚环扩增(Loop mediated isothermal amplification，LAMP)(针对环状DNA)和恒温多点置换扩增(Multiple displacement amplification，MDA)(针对线性DNA)进行大量扩增，在扩增的同时掺入标记物或随后以缺口平移法(或随机引物法及其变种)掺入标记物，再以限制性内切酶将标记了的DNA切开还原成原来文库片段长度的小片段，即得到大量的设 计长度范围的探针。与通常直接以BAC、质粒或PCR产物等为模板采用缺口平移法或随机引物法标记制备探针的技术比较，本发明通过将设计长度的探针文库片段以酶切位点序列间隔连接成大环和长链DNA为探针模板，以插入的酶切位点序列(或随机序列)为引物序列，进行DNA等温扩增，在扩增的同时或之后采用其他方法掺入带有标记物的底物标记DNA，插入的酶切位点是将标记DNA切割成设计长度范围的探针的切割位点，提供了更为方便地大量生产设计长度范围探针的制备方法，增加了产量，准确地控制了探针的长度范围，降低了生产过程中原材料的消耗，节省成本，提高了产品的性能指标。
采用本发明的方法制备的原位杂交探针可用于但不限于(细胞片或组织切片上的)间期或中期细胞核的原位杂交或CGH芯片杂交,对杂交结果的分析能了解特定基因或DNA片段在特定细胞/细胞群/组织/植物/动物/人体中的存在与否、数目变化、位置变化，与疾病的早期发现，诊断，选择治疗用药，或判断预后相关。
附图说明
图1为实施例2的HER2基因检测FISH探针的琼脂糖凝胶电泳图；
图2为实施例2的HER2基因检测FISH探针和实施例1的CEN17在正常人中期细胞染色体上的FISH实验结果图；
图3为实施例2的HER2基因检测FISH探针和实施例1的CEN17在正常人间期细胞上的FISH实验结果图。
图4为实施例3的13号染色体特异性FISH探针在正常人中期细胞染色体上的FISH实验结果图。
具体实施方式
下面详细描述本发明的实施例，所述实施例的示例在附图中示出，其中自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附图描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
本发明的第一种原位杂交探针的制备方法：
一、将纯化了的目标DNA片段化处理成长度为300bp左右的小片段；
二、回收150-600bp长度范围的小片段DNA；
三、对步骤二回收的小片段DNA两端进行酶修饰处理，使两端成平末端、5'端加磷酸和3'端加dA；
四、在DNA连接酶的作用下，将步骤三酶修饰处理后的小片段DNA与含有限制性内切酶位点序列的5'端加磷酸和3'端加dT的DNA衔接子间隔连接成DNA大环和长链；
五、采用衔接子序列的寡核苷酸为引物，将步骤四所得的DNA大环和长链进行等温滚环(LAMP)和多点置换扩增(MDA)，同时掺入带有标记物的脱氧核苷酸，如dUTP-荧光素或aa-dUTP；得到扩增后带有标记物的DNA；
六、对上述所得扩增后带有标记物的DNA进行酶切，采用与插入的衔接子序列对应的限制性内切酶，得到长度为150-600bp的酶切产物；
七、对上述所得酶切产物进行纯化、定量，所得即为探针，完成DNA杂交探针的制备。
步骤一中所述的目标DNA是双链DNA，可以是线性或环状，可以是超螺旋状态，可以是连续的也可以是有缺口的，来源包括但不限于质粒、BAC、YAC、PCR产物、RT-PCR产物、DNA酶切产物或连接产物、人工扩增产物、人工合成产物；
所述的连接满足DNA衔接子上3’末端的dT与小片段DNA 3’末端的dA互补；
步骤五中如果掺入的标记物为间接标记物aa，则还需要在步骤六和七之间增加一个偶联步骤，将直接标记物标记上去。
本发明的第二种原位杂交探针的制备方法，其中除上述步骤五所述的引物为随机引物外，其他均相同。
本发明的第三种原位杂交探针的制备方法，与上述两种方法的不同点在于以下步骤，其他步骤均相同：
步骤五、进行等温扩增时不掺入带有标记物的脱氧核苷酸；
步骤六、将步骤五得到的DNA，以缺口平移法掺入带有标记物的单核苷酸底物，对DNA进行标记；
步骤七、采用与衔接子上酶切位点对应的限制性内切酶消化标记了的DNA产物，得到长度为150-600bp的酶切产物；
步骤八、对上述所得酶切产物进行纯化、定量，所得即为探针，完成DNA原位杂交探针的制备。
本发明的第四种原位杂交探针的制备方法，与第三种原位杂交探针的制备方法的不同点在于以下步骤，其他步骤均相同：
步骤六、将步骤五得到的DNA，以随机引物法掺入带有标记物的单核苷酸底物，对DNA 进行标记；
本发明的第五种原位杂交探针的制备方法，与第四种原位杂交探针的制备方法的不同点在于以下步骤，其他步骤均相同：
步骤六、将步骤五得到的DNA，采用衔接子序列的寡核苷酸为引物替代随机引物，对DNA进行标记；
实施例1：人17号染色体计数FISH探针(CEN17)的制备
一、以正常人基因组DNA(可以采购得到)为模板，按照PETER E.WARwRToN,St GILLIAN M.GREIG,THOMAS HAAF,AND HUNTINGTON F.WILLARD.PCR Amplification of Chromosome-Specific Alpha Satellite DNA:Definition of Centromeric STS Markers and Polymorphic Analysis.GENOMICS 11,324-333(1991)里描述的方法得到0.85kb的17号染色体着丝粒α卫星重复序列片段PCR产物。
采用QIAquick PCR Purification Kit纯化PCR产物。
二、取1μg的以上步骤获得的DNA，以聚焦超声波法进行破碎至300bp左右的长度，通过2％的琼脂糖凝胶电泳，以100bp DNA ladder为参照，切割回收150-600bp长度范围内的DNA片段；采用Qiagen公司的凝胶提取试剂盒(QIAquick Gel Extraction Kit)回收片段；
三、酶修饰处理：采用New England Biolabs公司的
Figure PCTCN2018099530-appb-000001
Ultra End Repair/dA-Tailing Module，按照说明书的操作步骤将步骤二回收的DNA片段末端修齐，将其5’端磷酸化，并在3’端加dA，然后采用Qiagen公司的PCR纯化试剂盒(QIAquick PCR Purification Kit)对修饰后的DNA片段进行回收；
四、连接成大环和长链：将上述所得含有DNA限制性内切酶SacII位点序列的衔接子(adaptor)与步骤三回收的修饰后的DNA片段连接；
衔接子(adaptor)为双链，其5’端均磷酸化。序列如下所示：
5’-pCCGCGGT-3'
3’-TGGCGCCp-5'                  SEQ ID NO：1。
衔接子(adaptor)连接的具体操作步骤如下：
采用如下连接体系，将微离心管置于冰上，加入如下50μl的反应液(可以按比例扩大反应体积)，用加样器轻柔地上下吹打混合，短暂离心，然后置于16℃条件下过夜。
Figure PCTCN2018099530-appb-000002
Figure PCTCN2018099530-appb-000003
采用Qiagen公司的QIAamp DNA Blood Kits，对连接后的产物进行纯化回收，去除未连接的衔接子(adaptor)，测定OD值，获得大环和长链DNA探针模板；
五、大量标记DNA的获得：将步骤四得到的探针模板DNA大量扩增，同时掺入标记物dUTP-FITC(fluorescein isothiocyanate)(也可以是间接标记物aa-dUTP，如此，则需要在第六步之后增加一步与FITC SE的偶联反应，具体参见后述实施例)，以插入的序列CCGCGGT(SEQ ID NO：2，即SEQ ID NO:1的单链)为引物，反应体系及过程如下(可以按比例增加反应体积)：
Figure PCTCN2018099530-appb-000004
先96℃，2分钟，再室温10分钟，然后置于冰上，再加入下列成分，终体积50μl，混匀；
Figure PCTCN2018099530-appb-000005
然后，先60℃，6小时，再80℃，15分钟。
采用Qiagen公司的QIAamp DNA Blood Kits对产物进行纯化回收，测定OD值。
六、酶切：以限制性内切酶SacII将步骤五所得进行酶切，得到长度为150-600bp的酶切产物。
酶切反应体系为(可以按比例增加或减少反应体积)：
步骤五所得                     100μg
Sac II                         1000u(单位)
去离子水                       使达到1000μl
将上述酶切反应体系置于37℃持续16小时；
七、采用Qiagen公司的PCR纯化试剂盒(QIAquick PCR Purification Kit)纯化酶切产物，测定OD值，获得绿色荧光素FITC标记的17号染色体计数FISH探针(记为CEN17)。
实施例2：人类表皮生长因子受体2(HER2)基因检测FISH探针的制备
与以上实施例1不同的是DNA来源于BAC，扩增和标记分开进行，扩增时采用6个碱基的随机引物(包含在试剂盒中)，扩增后采用缺口平移法掺入标记物，掺入的标记物为直接标记物或间接标记物。
一、根据HER2基因在染色体上的位置，自ThermoFisher Scientific公司购买细菌人工染色体(Bacterial Artificial Chromosome,BAC)克隆：RP11-909L6,其中含有HER2基因及其左右的染色体DNA，长度约为185kb；
采用末端测序方法测定BAC克隆插入片段两末端数百个碱基的序列，与NCBI发表的已知序列进行对比核实，正确的进行下一步实验；
二、取1μg BAC DNA，以聚焦超声波法进行片段化处理并回收150-600bp的DNA片段，方法同实施例1；
三、酶修饰处理：方法同实施例1；
四、将含有DNA限制性内切酶SmaI位点序列的衔接子(adaptor)与步骤四回收的DNA片段连接；
衔接子(adaptor)为双链，其5’端均磷酸化。序列如下所示：
5’-pCCCGGGT-3'
3’-TGGGCCCp-5'          SEQ ID NO：3；
与衔接子(adaptor)连接:方法同实施例1：
五、大量DNA的获得：以步骤四得到的DNA为模板大量扩增，采用Qiagen公司的REPLI-g Single Cell Kit试剂盒(其原理为LAMP和MDA，，采用Phi29DNA多聚酶，以随机序列的六个碱基寡核苷酸为引物)，按照说明书操作，采用Qiagen公司的QIAamp DNA Blood Kits对扩增产物进行纯化回收；
六、DNA的标记：以步骤五获得的扩增产物为模板，以缺口平移法掺入直接标记物dUTP-TAMRA(carboxytetramethylrhodamine)或间接标记物aa-dUTP，方法如下：
Figure PCTCN2018099530-appb-000006
Figure PCTCN2018099530-appb-000007
将上述混合物置于30℃持续16小时；
采用Qiagen公司的QIAamp DNA Blood Kits对扩增产物进行纯化回收，测定OD值。
七、酶切：以限制性内切酶SmaI将步骤六所得DNA进行酶切，得到长度为150-600bp的酶切产物；
酶切反应体系为：
DNA                           100μg
SmaI                          1000u(单位)
去离子水                      使达到1000μl
将上述酶切反应体系置于37℃持续16小时；
八、采用Qiagen公司的PCR纯化试剂盒纯化步骤七的酶切产物，测定OD值，
如果步骤六掺入的是直接标记物，则获得橘红色荧光TAMRA标记的HER2探针；如果步骤六掺入的是间接标记物，则：
九、DNA中掺入的aa与TAMRA SE的偶联，参照以下文献或者产品说明书(反应体积可以按比例相应扩大)：
W.Gregory Cox and Victoria L.Singer.Fluorescent DNA hybridization probe preparation using amine modification and reactive dye coupling.BioTechniques36:114-122(January 2004)
十、产物经乙醇沉淀法纯化后测定OD值，获得橘红色荧光素TAMRA标记的HER2探针。
关于实施例2的说明：缺口平移标记法的特点是快速、简便、标记的探针均匀、特异性高，探针标记率比Bst DNA多聚酶掺入标记的探针标记率高；间接标记比直接标记节省成本。
实施例3：人13号染色体特异FISH探针
与以上实施例1和例2不同的是：目标DNA来源于整条染色体，采用随机引物，等温扩增和标记同时进行，掺入的标记物为间接标记物。
一、采购或自备(方法略)正常人外周血中期淋巴细胞滴片；
二、采用细胞遗传学染色体显带技术，在显微镜下辨别和编号染色体，从玻片上刮取和收集十条完整的13号染色体；
三、大量DNA的获得：以步骤二得到的染色体为DNA来源，采用Qiagen公司的REPLI-g Single Cell Kit试剂盒进行DNA大量扩增，按照说明书操作，采用Qiagen公司的QIAamp DNA Blood Kits对扩增产物进行纯化回收；
四、取1μg以上步骤获得的DNA，以聚焦超声波法进行破碎并回收150-600bp的DNA片段，方法同实施例1；
五、酶修饰处理：方法同实施例1；
六、将含有DNA限制性内切酶位点序列的衔接子(adaptor)与步骤五修饰后回收的DNA片段连接获得DNA探针模板，方法同实施例1；
七、大量标记DNA的获得：以步骤六得到的DNA为模板大量等温扩增，同时掺入间接标记物aa-dUTP，
扩增时采用6个碱基的随机寡核苷酸(序列为NNNNNN，SEQ ID NO:4，其中N表示A或T或G或C)为引物，反应体系及过程如下(可以按比例增加反应体积)：
Figure PCTCN2018099530-appb-000008
先96℃，2分钟，再室温10分钟，然后置于冰上，再加入下列成分，终体积50μl，混匀；
Figure PCTCN2018099530-appb-000009
然后，先60℃，6小时，再80℃，15分钟。
采用Qiagen公司的QIAamp DNA Blood Kits对产物进行纯化回收，测定OD值。
八、以限制性内切酶将步骤七所得进行酶切，得到150-600bp的片段，方法同实施例1；
九、采用Qiagen公司的PCR纯化试剂盒(QIAquick PCR Purification Kit)纯化酶切产物，测定OD值，
十、DNA中掺入的aa与TAMRA SE的偶联，方法同实施例2；
十一、产物经乙醇沉淀法纯化后测定OD值，获得橘红色荧光素TAMRA标记的13号染色体特异性FISH探针。
实施例4：效果验证实验
将所述实施例2所得橘红色荧光素TAMRA标记的HER2基因检测FISH探针进行琼脂糖凝胶电泳，结果见图1，图1为所得HER2探针的琼脂糖凝胶电泳图，其中泳道1为100bp DNA ladder，泳道2为探针，显示探针长度在150-600bp的设计范围，达到设计要求。
配制HER2基因检测FISH探针杂交液
每10μl HER2基因检测FISH探针杂交液的成分如下：实施例2所得橘红色荧光素TAMRA标记的HER2基因检测FISH探针20ng，实施例1所得17号染色体计数探针CEN17 10ng，人COT-1DNA(购自ThermoFisher Scientific)1μg，50％体积比的去离子甲酰胺，2XSSC，10％重量体积比的硫酸葡聚糖。
配制13号染色体特异性FISH探针杂交液
每10μl 13号染色体特异性FISH探针杂交液的成分如下：实施例3所得橘红色荧光素TAMRA标记的13号染色体特异性FISH探针20ng，人COT-1DNA(购自ThermoFisher Scientific)1μg，50％体积比的去离子甲酰胺，2XSSC，10％重量体积比的硫酸葡聚糖。
原位杂交：
采用细胞悬液，常规制作染色体玻片标本，玻片过梯度乙醇依次脱水，晾干；常规胃蛋白酶消化，将10μl上述HER2基因检测FISH探针杂交液或13号染色体特异性FISH探针杂交液加于玻片杂交区域，盖上盖玻片，以橡胶水封片，78℃变性5分钟，封片后置于湿盒中，37℃杂交过夜。再按照如下顺序洗片：4×SSC溶液中，振荡漂洗5分钟；2×SSC,0.1％体积比的Tween-20溶液中，振荡漂洗4×2.5分钟；0.1×SSC溶液中，振荡漂洗5分钟；在梯度乙醇中依次脱水，晾干。将20μl DAPI复染剂滴加于杂交区域位置，立即盖上盖玻片。在荧光显微镜下选用合适的滤光片观察和照相记录结果。
试验结果数据见图2、3和4所示。图2为HER2基因检测FISH探针杂交液在体外培养的正常人中期细胞(来源于正常人外周血单核细胞)染色体上的FISH实验结果图；其中，1为HER2基因检测FISH探针产生的信号，2为17号染色体计数原位杂交探针CEN17产生的信号。
图3为HER2基因检测FISH探针杂交液在体外培养的正常人间期细胞(来源于正常人外周血单核细胞)上的FISH实验结果图；其中，1所指为HER2基因检测FISH探针产生的信号， 2所指为17号染色体计数原位杂交探针CEN17产生的信号。
从图2和图3可以看出，CEN17信号定位于17号染色体的着丝粒上，HER2信号位于17号染色体长臂近着丝粒处(1区2带，17q12)，在正常的细胞中各有2个信号，达到设计要求和预期目的。
图4为13号染色体特异性FISH探针杂交液在体外培养的正常人中期细胞(来源于正常人外周血单核细胞)染色体上的FISH实验结果图，一对13号染色体整体显示荧光信号，达到设计要求和预期目的。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，也不能理解为对依据本发明而获得探针产物的应用范围的限制，本领域的普通技术人员在不脱离本发明的原理和宗旨的情况下在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (7)

  1. 一种原位杂交探针的制备方法，其特征在于，包括以下步骤，
    目标DNA的片段化和选择性回收：将目标DNA片段化，选择性回收150-600bp的小片段；
    酶修饰处理：对150-600bp的小片段的两端进行酶修饰处理，使两端成平末端、5'端加磷酸和3'端加dA；
    连接成大环和长链：将上述处理后的小片段与含有限制性内切酶位点序列的DNA衔接子间隔连接成DNA大环和长链；所述含有限制性内切酶位点序列的DNA衔接子的5'端加了磷酸和3'端加了dT；
    大量DNA的获得和标记：
    A，等温扩增，扩增时掺入带有标记物的单核苷酸底物，得到带有标记物的DNA产物；
    或B，等温扩增，所得扩增产物以缺口平移法或随机引物法掺入带有标记物的单核苷酸底物，得到带有标记物的DNA产物；
    酶切：采用与上述DNA衔接子上碱基序列对应的DNA限制性内切酶消化带有标记物的DNA产物，酶切产物长度范围150-600bp；
    得到探针：
    若所述带有标记物的DNA产物中的标记物为直接标记物，则所得到的酶切产物即为原位杂交探针；
    若所述带有标记物的DNA产物中的标记物为间接标记物，则所得到的酶切产物经过与具有反应活性的直接标记物进行偶联，从而得到原位杂交探针。
  2. 根据权利要求1所述原位杂交探针的制备方法，其特征在于，所述得到的原位杂交探针进一步纯化，定量。
  3. 根据权利要求1所述原位杂交探针的制备方法，其特征在于，所述目标DNA为纯化后的目标DNA。
  4. 根据权利要求1所述原位杂交探针的制备方法，其特征在于，所述A或B步骤中，等温扩增时所用的引物为含有限制性内切酶位点的衔接子序列的寡核苷酸或随机序列寡核苷酸。
  5. 根据权利要求1所述原位杂交探针的制备方法，其特征在于，所述A或B步骤中，等温扩增所用的酶为具有链置换活性的DNA多聚酶。
  6. 根据权利要求5所述原位杂交探针的制备方法，其特征在于，所述等温扩增所用的 酶为Phi29DNA多聚酶、Bst DNA多聚酶大片段。
  7. 根据权利要求1所述原位杂交探针的制备方法，其特征在于，所述A或B步骤中，所述标记物为直接标记物或间接标记物。
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