Probe preparation method for improving fluorescence in situ hybridization probe specificity and application thereof

Abstract

The invention relates to the field of molecular biology, in particular to a probe preparation method for improving fluorescence in-situ hybridization probe specificity, 5%-10% of open-loop nucleotide triphosphate is added in the PCR probe preparation process to serve as a reaction substrate, the conformation of a probe is changed, and therefore the probe specificity and the binding force with a target are improved; the method provided by the invention is suitable for a plurality of disease diagnoses carried out by utilizing FISH, and background noise of a detection result is reduced after the specificity of the probe is improved, so that the accuracy of the detection result can be improved, and the difficulty of result analysis can be reduced.

Description

Technical Field

[0001] This application relates to the field of molecular biology, and specifically relates to a method for preparing a probe for improving the specificity of fluorescence in situ hybridization probes and its application. Background Art

[0002] Fluorescence in situ hybridization (FISH) is an important molecular biology technique widely used in fields such as genomics, cytogenetics, and pathology. This technique involves using specific probes to complementarily pair with target DNA sequences, and then using fluorescently labeled probes to detect the presence and location of the target sequences.

[0003] In genomics research, FISH is used for tasks such as chromosome painting, gene mapping, and quantitative analysis. It can reveal chromosomal structural variations, deletions, duplications, and rearrangements, providing important information for disease diagnosis and treatment. In addition, FISH can also be used to study tissue-specific gene expression and the spatial distribution of chromosomes, helping to understand gene function and regulatory mechanisms.

[0004] In cytogenetics, FISH is widely used in oncology research and clinical diagnosis. By analyzing chromosomal aberrations in tumor cells, some common tumor-related gene mutations and translocation events can be identified, helping to determine the type and prognosis of tumors. In addition, FISH can also be used to evaluate indicators such as the ploidy, proliferation ability, and apoptosis status of tumor cells, providing a basis for the selection and monitoring of treatment regimens.

[0005] In FISH experiments, specificity is very important as it determines the accuracy and reliability of experimental results. However, existing FISH probes have the phenomenon of binding to non-target sequences, resulting in low specificity, serious background noise, increasing the difficulty of analyzing detection results, reducing the accuracy of results, and at the same time, due to the presence of background noise, reducing the probe detection limit and sensitivity. Therefore, there is a great demand for improving the specificity of FISH probe compositions in clinical diagnosis applications. Summary of the Invention

[0006] In order to solve at least one of the above technical problems, a method for preparing a probe for improving the specificity of fluorescence in situ hybridization probes and its application are developed.

[0007] In the first aspect of this application, a method for preparing a probe for improving the specificity of fluorescence in situ hybridization probes is provided. The probe is prepared by PCR, and about 5% to about 10% of the total molar amount of nucleotide raw materials is added with UNA triphosphate during the process of preparing the probe by PCR.

[0008] In a preferred embodiment of the present application, during the process of preparing the probe by PCR, about 5%, about 6%, about 7%, about 8%, about 9% or about 10% of the total molar amount of nucleotide raw materials is added with unlocked nucleic acid (UNA) triphosphate.

[0009] In a preferred embodiment of the present application, the UNA triphosphate is selected from UNA-A triphosphate, UNA-T triphosphate, UNA-G triphosphate and UNA-C triphosphate.

[0010] In a preferred embodiment of the present application, the molar ratio of the UNA triphosphate selected from UNA-A triphosphate, UNA-T triphosphate, UNA-G triphosphate and UNA-C triphosphate is 1:1:1:1.

[0011] Unlocked nucleic acid (UNA) is a novel acyclic derivative of RNA, similar to DNA or RNA but with different compositions. UNA does not exist in any known natural organisms. UNA lacks the C2-C3 chemical bond of the RNA ribose ring, but it can still mimic unmodified RNA when bound to the RNA double-stranded structure. During the amplification labeling reaction in the probe preparation process, about 5% to about 10% of the total molar amount of nucleotide raw materials in the substrate is added with UNA triphosphate, so that the obtained probe sequence incorporates ring-opened nucleotides. The ring-opened nucleotides can change the conformation of the original sequence. The incorporation of a small amount of ring-opened nucleotides strengthens the binding affinity between the probe and the target, enhances the specificity, reduces the background noise, and at the same time enhances the stability of the probe.

[0012] In a preferred embodiment of the present application, the preparation method includes the following steps: (1) Determine the target gene sequence; (2) Use the perl plug-in program chunks.pl to split the target gene sequence in step (1) into 1-kb blocks and remove duplicate sequences; batch import the split blocks into the OligoArray software for probe design and probe screening, then export the screened probes to an EXCEL table, and add a 17-bp tag sequence to the 5' end and an 18-bp tag sequence to the 3' end of each probe respectively to obtain a series of probe sequences with tag sequences; (3) Use a DNA synthesizer to chemically synthesize the gene probe sequences with tag sequences obtained in step (2) respectively, and mix the chemically synthesized probes at the same locus to prepare a probe library of the target gene sequence; (4) Synthesize a universal primer with a fluorescent group at the 5' end, and use the universal primer to perform an amplification labeling reaction on the target gene sequence probe, wherein about 5% to about 10% of the total molar amount of nucleotide raw materials is optionally added with UNA triphosphate in the amplification labeling reaction; (5) Purify and dilute the amplified and labeled product of the probe library obtained in step (4) to obtain a fluorescently labeled probe library.

[0013] On the one hand, most of the design and screening work of the probes in this application is completed by software, which greatly reduces the workload of probe design. On the other hand, in the above preparation method, by adding tag sequences to both ends of each probe, it can cooperate with universal primers, and further, a large and diverse probe library can be synthesized by PCR amplification method, so that the realization of PCR amplification of the probe library is achieved.

[0014] In a preferred embodiment of the present application, the target gene sequences in step (1) are selected from different detection sites of the same mutant type and / or different detection sites of different mutant types.

[0015] In a preferred embodiment of the present application, different detection sites of the same mutant type are labeled with fluorescent groups of the same color.

[0016] In a preferred embodiment of the present application, the different mutant types are selected from two or more different gene mutant types.

[0017] In a preferred embodiment of the present application, different detection sites of different gene mutant types are labeled with different fluorescent groups.

[0018] By adopting the above technical solution, the probes for different sites of the same mutant type obtained are monochromatic probes, which can be used to detect the deletion, amplification and splitting of the gene mutant type. When the chromosome or gene is deleted, the detection signal shows the disappearance of the signal. When the chromosome or gene is amplified or split, the detection signal shows an increase in the number of signals. The probes for different sites of different mutant types obtained are multi-color probes, such as two-color or three-color probes. A group of probes can simultaneously detect multiple different gene mutant types that may exist in the diagnosis and / or disease, which can not only save samples, but also improve the detection rate and the comprehensiveness of the detection results.

[0019] In a preferred embodiment of the present application, the method for determining the target gene in step (1) includes the following steps: (1) Determine the mutant type corresponding to the disease; (2) Determine the gene region corresponding to each mutant type; (3) Determine the probe sequence according to the determined gene region, and the determination of the probe sequence includes downloading the BAC sequence of the corresponding region on UCSC or manually screening specific sequences.

[0020] In a preferred embodiment of the present application, the diagnosis is selected from the diagnosis of myelodysplastic syndrome, the diagnosis of multiple myeloma, prenatal screening diagnosis, lymphoma diagnosis, breast cancer diagnosis, lung cancer diagnosis or soft tissue tumor diagnosis.

[0021] In a preferred embodiment of the present application, the same gene mutation type for myelodysplastic syndrome diagnosis is selected from one of del(5q), del(7q), amp(8), del(20q), and del(Y), and the different gene mutation types for myelodysplastic syndrome diagnosis are selected from two or more of del(5q), del(7q), amp(8), del(20q), and del(Y).

[0022] In a preferred embodiment of the present application, the same gene mutation type for multiple myeloma diagnosis is selected from one of del(17p13), del(13q14), del(1p32), amp(1q21), IGH rearrangement probe, t(11;14), t(6;14), t(4;14), t(14;16), and t(14;20), and the different gene mutation types for multiple myeloma diagnosis are selected from two or more of del(17p13), del(13q14), del(1p32), amp(1q21), IGH rearrangement probe, t(11;14), t(6;14), t(4;14), t(14;16), and t(14;20).

[0023] In a preferred embodiment of the present application, the same gene mutation type for prenatal screening diagnosis is selected from one of trisomy 21, trisomy 18, trisomy 13, 45X, and 47XXY, and the different gene mutation types for prenatal screening diagnosis are selected from two or more of trisomy 21, trisomy 18, trisomy 13, 45X, and 47XXY.

[0024] In a preferred embodiment of the present application, the same gene mutation type for lymphoma diagnosis is selected from one of ALK rearrangement probe, Kappa chain rearrangement probe, Lambda chain rearrangement probe, BCL2 rearrangement probe, BCL6 rearrangement probe, MYC rearrangement probe, CCND1 rearrangement probe, CCND2 rearrangement probe, MALT1 rearrangement probe, del(17p13), t(9;14), t(11;14), t(11;18), t(6;14), t(11;14), t(4;14), t(14;16), and t(14;20), and the different gene mutation types for lymphoma diagnosis are selected from two or more of ALK rearrangement probe, Kappa chain rearrangement probe, Lambda chain rearrangement probe, BCL2 rearrangement probe, BCL6 rearrangement probe, MYC rearrangement probe, CCND1 rearrangement probe, CCND2 rearrangement probe, MALT1 rearrangement probe, del(17p13), t(9;14), t(11;14), t(11;18), t(6;14), t(11;14), t(4;14), t(14;16), and t(14;20).

[0025] In a preferred embodiment of the present application, the same gene mutation type for breast cancer diagnosis is selected from one of amp(HER2), amp(TOP2A), amp(8q24), amp(CCND1), del(PTEN), t(12;15), and the different gene mutation types for breast cancer diagnosis are selected from two or more of amp(HER2), amp(TOP2A), amp(8q24), amp(CCND1), del(PTEN), t(12;15).

[0026] In a preferred embodiment of the present application, the same gene mutation type for lung cancer diagnosis is selected from one of ALK rearrangement probe, ROS1 rearrangement probe, RET rearrangement probe, NTRK1 rearrangement probe, NTRK2 rearrangement probe, NTRK3 rearrangement probe, amp(MET), amp(CCND1), amp(PIK3CA), t(2;22), t(12;15), and the different gene mutation types for lung cancer diagnosis are selected from two or more of ALK rearrangement probe, ROS1 rearrangement probe, RET rearrangement probe, NTRK1 rearrangement probe, NTRK2 rearrangement probe, NTRK3 rearrangement probe, amp(MET), amp(CCND1), amp(PIK3CA), t(2;22), t(12;15).

[0027] By adopting the above technical solution, the background noise of the color development result for the diagnosis of myelodysplastic syndrome, multiple myeloma, prenatal screening, lymphoma, breast cancer or lung cancer is low, indicating that the probe composition prepared by the preparation method of the present application has high specificity. For the above different diagnostic requirements, the probe composition prepared by the preparation method of the present application can effectively reduce the background noise, indicating that the method of the present application has universality and can be used for the preparation of FISH probes for the diagnosis of various diseases.

[0028] In a preferred embodiment of the present application, the 17bp tag sequence in step (2) is TGTAAAACGACGGCCAG, and the 18bp tag sequence is GGTCATAGCTGTTTCCTG.

[0029] Adding tag sequences at both ends of the probe can facilitate PCR amplification using universal primers, greatly reducing the primer design and screening work, enabling the above-mentioned probe amplification by PCR. The above tag sequences are carefully designed and screened by the applicant and have been verified to be applicable to the amplification of BAC clone sequence probes corresponding to multiple target genes.

[0030] In a preferred embodiment of the present application, the probe screening conditions in step (2) are as follows: the probe length is 50 - 150 bp, the TM value is 85 - 99 °C, the GC ratio is 40 - 80%, it does not contain TTTT / GGGG / AAAA / CCCC, and the minimum interval between probes is 5 bp.

[0031] The above parameters for probe screening can be adjusted according to specific experimental purposes and requirements. Among them, the probe length of 50 - 150 bp is to ensure that the probe length is within a suitable range, neither too short to reduce specificity nor too long to increase synthesis costs. The TM value of 85 - 99 °C is to ensure that the melting temperature (Tm) of the probe is within a suitable range to guarantee the specificity and stability of the probe in the experiment. The GC ratio of 40 - 80% is to ensure that the GC content of the probe is within a suitable range to guarantee the stability and specificity of the probe. Not containing TTTT / GGGG / AAAA / CCCC is to avoid the occurrence of repetitive sequences in the probe, thereby reducing the possibility of non-specific binding.

[0032] In a preferred embodiment of the present application, the universal primer sequences in step (4) are TGTAAAACGACGGCCAGT and CAGGAAACAGCTATGACC.

[0033] Using the above universal primers in combination with the above tag sequences can specifically recognize each probe sequence and perform efficient amplification to obtain a probe library suitable for FISH detection.

[0034] In a preferred embodiment of the present application, the polymerase used in the amplification labeling reaction is Therminator DNA polymerase.

[0035] Therminator DNA polymerase is a DNA polymerase widely used in molecular biology experiments, which has the characteristics of high fidelity, high stability and high efficiency. Therefore, it is widely used in experiments such as DNA replication, PCR amplification, and gene cloning. Therminator DNA polymerase can simultaneously recognize natural deoxynucleoside triphosphate substrates and UNA triphosphate substrates and perform high-fidelity PCR amplification. By using the above polymerase and adding a certain amount of 5% - 10% UNA triphosphate substrate to the reaction substrate, a probe sequence incorporating a small amount of open-ring nucleotides can be obtained.

[0036] In a preferred embodiment of the present application, the fluorescent groups in step (4) are selected from green fluorescent groups, red fluorescent groups and cyan fluorescent groups.

[0037] Adopting the above technical solution, first, the excitation and emission wavelengths of the red, green, and cyan fluorescent groups are different and can be observed separately through different filters, thus reducing the problems of spectral overlap and crosstalk; second, red and cyan or red and green are considered complementary colors because their positions on the color wheel are very different, so they can be clearly distinguished, and this characteristic can be used for image processing to improve the accuracy of analysis; finally, there is a rich variety of fluorescent dyes for red, green, and cyan, and appropriate dyes can be selected according to experimental requirements.

[0038] In the second aspect of the present application, a probe composition prepared by the preparation method defined in any of the above technical solutions is provided.

[0039] In a preferred embodiment of the present application, the probe is selected from chromosome painting probes, repetitive sequence probes, and locus-specific probes.

[0040] In a preferred embodiment of the present application, the probe is selected from locus-specific probes, and the locus-specific probe is selected from single-color counting probes, two-color counting probes, two-color separation probes, two-color dual-fusion probes, two-color but-fusion probes, extra signal probes, and three-color probes.

[0041] There is no limitation on the specific type of the probe in the probe composition prepared by the preparation method of the present application. Therefore, existing FISH probe types can all be prepared by the method of the present application to improve the specificity of the above probes.

[0042] In the third aspect of the present application, the application of the above probe composition in the preparation of diagnostic products for mutant gene diseases is provided.

[0043] In a preferred embodiment of the present application, the diagnosis of mutant gene diseases is selected from the diagnosis of myelodysplastic syndrome, multiple myeloma, prenatal screening diagnosis, lymphoma diagnosis, breast cancer diagnosis, lung cancer diagnosis, or soft tissue tumor diagnosis.

[0044] In the fourth aspect of the present application, a kit is provided, and the kit contains (1) A mixture of the probe composition defined in any of the above technical solutions and a hybridization buffer; (2) A DAPI counterstain.

[0045] Definitions and explanations Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings. A specific term or phrase should not be considered indeterminate or unclear without a special definition, but should be understood according to the meaning understood by those of ordinary skill in the art. When a trade name appears in this article, it is intended to refer to the corresponding product or its active ingredient.

[0046] In the present invention, unless otherwise specified, the terms "comprising", "including" and "containing" or their equivalents are open-ended expressions, meaning that in addition to the listed elements, components or steps, other unspecified elements, components or steps may also be covered.

[0047] Unless otherwise specified, the "multiple" in the term "multiple" of the present invention refers to an integer greater than or equal to 2, including but not limited to 2, 3, 4, 5, 6, 7, 8, 9 and 10, up to the maximum number of items in the options.

[0048] Unless otherwise specified, the "optional" and / or "optionally" in the terms "optional" and / or "optionally" of the present application means that it can be selected or not selected. When not selected, it means that this step, this limitation or this component does not exist. When selected, it means that the relevant operations or limitations are carried out according to the subsequent description.

[0049] Unless otherwise specified, the term "UNA" in the present application represents a novel acyclic RNA derivative, similar to DNA or RNA, and the general structural formula of UNA is where B represents the natural bases adenine, thymine, guanine and cytosine.

[0050] Unless otherwise specified, the general structural formula of UNA triphosphate is

[0051] Unless otherwise specified, the structural formula of the term "UNA-A triphosphate" in the present application is

[0052] Unless otherwise specified, the structural formula of the term "UNA-T triphosphate" in the present application is

[0053] Unless otherwise specified, the structural formula of the term "UNA-G triphosphate" in the present application is

[0054] Unless otherwise specified, the structural formula of the term "UNA-C triphosphate" in the present application is

[0055] In summary, the present invention includes at least one of the following beneficial effects: 1. In the probe preparation method of the present application, the screening of probes mainly relies on programs, and the more stable PCR method is used to amplify and label probes. The probe labeling rate is higher and more uniform. Since fluorescence labeling is only carried out at the 5' end of the probe, it does not affect the hybridization pairing reaction of the probe, and the batch-to-batch stability is high.

[0056] 2. The present application adds tag sequences to both ends of each probe, and then cooperates with universal primers for PCR amplification to synthesize probes, enabling the PCR synthesis of a large number of different probes to be realized.

[0057] 3. In the preparation method of the probe of the present application, during the amplification process, about 5%-10% of the ring-opening nucleotide equivalent to the total molar amount of nucleosides is added. On the basis that the fluorescence of the prepared probe is slightly enhanced after binding, the specificity and stability of the probe can be further improved, and the background noise can be reduced. Description of the Drawings

[0058] Figure 1 : Color development comparison of the probes prepared in Example 1 with the probes prepared in Comparative Examples 1-1 and 1-2. (a) Color development result of the CSF1R probe of Example 1 of the present application for normal cells; (b) Color development result of the CSF1R probe of Comparative Example 1-1 for normal cells; (c) Color development result of the CSF1R probe of Comparative Example 1-2 for normal cells; Figure 2 : Color development comparison of the probes prepared in Example 2 with the probe prepared in Comparative Example 2. (a) Color development result of the probe of Example 2 of the present application for normal cells; (b) Color development result of the probe of Comparative Example 2 for normal cells; Figure 3 : Color development comparison of the probes prepared in Example 3 with the probe prepared in Comparative Example 3. (a) Color development result of the probe of Example 3 of the present application for normal cells; (b) Color development result of the probe of Comparative Example 3 for normal cells. Detailed Description of the Invention

[0059] The present invention will be described in detail below by way of examples, but this does not mean any adverse limitation to the present invention. The present invention has been described in detail herein, and its specific embodiments have also been disclosed. It will be obvious to those skilled in the art that various changes and improvements can be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention.

[0060] Example 1 Preparation of a Diagnostic Probe for Myelodysplastic Syndrome - Preparation of a CSF1R Probe Containing UNA (1) Download the BAC clone gene sequences RP11-100O5 and RP11-432O16 corresponding to the locus EGR1 from the UCSC Genome Browser.

[0061] (2) The RP11-100O5 and RP11-432O16 sequences obtained in step (1) were respectively split into 1-kb blocks using the perl plug-in program chunks.pl and duplicate sequences were removed; the split blocks were batch-imported into the OligoArray software for probe design and probe screening, and then the screened probes were exported to an EXCEL spreadsheet. Then, a 17-bp tag sequence was added to the 5' end and an 18-bp tag sequence was added to the 3' end of each probe to obtain a series of probe sequences with tag sequences; the probe design and probe screening methods were that the probe length was 50-150 bp, the TM value was 85-99 °C, the GC ratio was 40-80%, it did not contain TTTT / GGGG / AAAA / CCCC, and the minimum interval between probes was 5 bp.

[0062] (3) The gene probe sequences with tag sequences obtained in step (2) were chemically synthesized using a DNA synthesizer, and the chemically synthesized probes at the same locus were mixed to respectively prepare an EGR1 probe library; (4) A universal primer with a red fluorescent group at the 5' end was synthesized and used to perform an amplification labeling reaction on the CSF1R probe library synthesized in step (3), and 5% of UNA triphosphate was added to the total molar amount of nucleotide raw materials in the amplification labeling reaction; the universal primer sequences were TGTAAAACGACGGCCAGT and CAGGAAACAGCTATGACC.

[0063] (5) The amplification labeling products of the probe library obtained in step (4) were purified and diluted to obtain a fluorescently labeled CSF1R probe library with a probe concentration of 40 ng / mL.

[0064] The difference between the preparation method of the probe in Comparative Example 1-1 and the preparation method of Example 1 was that UNA triphosphate was not added to the nucleotide raw materials in the amplification labeling reaction in step (4).

[0065] The difference between the preparation method of the probe in Comparative Example 1-2 and the preparation method of Example 1 was that 20% of UNA triphosphate was added to the total molar amount of nucleotide raw materials in the amplification labeling reaction in step (4).

[0066] Example 2 Preparation of a multiple myeloma diagnostic probe - Preparation of the P53 / CEP17 probe Part 1: Preparation of the P53 probe (1) Download the BAC clone gene sequences RP11-1081A10 and RP11-107F4 corresponding to the locus P53 from the UCSC Genome Browser.

[0067] (2) The RP11-1081A10 and RP11-107F4 sequences obtained in step (1) were respectively segmented into 1-kb blocks using the perl plug-in program chunks.pl and duplicate sequences were removed; the segmented blocks were batch-imported into the OligoArray software for probe design and probe screening, and then the screened probes were exported to an EXCEL table. Then, a 17-bp tag sequence was added to the 5' end of each probe and an 18-bp tag sequence was added to the 3' end, obtaining a series of probe sequences with tag sequences; the probe design and probe screening methods were that the probe length was 50-150 bp, the TM value was 85-99 °C, the GC ratio was 40-80%, it did not contain TTTT / GGGG / AAAA / CCCC, and the minimum interval between probes was 5 bp.

[0068] (3) The gene probe sequences with tag sequences obtained in step (2) were chemically synthesized respectively using a DNA synthesizer, and the probes chemically synthesized at the same locus were mixed to prepare a P53 probe library; (4) A universal primer with a red fluorescent group at the 5' end was synthesized and used to perform an amplification labeling reaction on the P53 probe library synthesized in step (3), and 8% of UNA triphosphate of the total molar amount of nucleotide raw materials was added in the amplification labeling reaction; the universal primer sequences were TGTAAAACGACGGCCAGT and CAGGAAACAGCTATGACC.

[0069] (5) The amplification labeling product of the probe library obtained in step (4) was purified and diluted to obtain a fluorescently labeled P53 probe library.

[0070] Part Two: Preparation of CEP17 Probes (1) The centromeric region sequence of chromosome 17, such as the sequence shown in SEQ ID No 1, was segmented into 1-kb blocks using the perl plug-in program chunks.pl and duplicate sequences were removed; the segmented blocks were batch-imported into the OligoArray software for probe design and probe screening, and then the screened probes were exported to an EXCEL table. Then, a 17-bp tag sequence was added to the 5' end of each probe and an 18-bp tag sequence was added to the 3' end, obtaining a series of probe sequences with tag sequences; the probe design and probe screening methods were that the probe length was 50-150 bp, the TM value was 85-99 °C, the GC ratio was 40-80%, it did not contain TTTT / GGGG / AAAA / CCCC, and the minimum interval between probes was 5 bp.

[0071] (2) The gene probe sequences with tag sequences obtained in step (1) were chemically synthesized respectively using a DNA synthesizer, and the probes chemically synthesized at the same locus were mixed to prepare a CEP17 probe library; (3) Synthesize a universal primer with a green fluorescent group at the 5' end and use this universal primer to perform an amplification labeling reaction on the CEP17 probe library synthesized in step (2), wherein 8% of the total molar amount of nucleotide raw materials is added with UNA triphosphate in the amplification labeling reaction; the universal primer sequences are TGTAAAACGACGGCCAGT and CAGGAAACAGCTATGACC.

[0072] (4) Purify and dilute the amplification labeling product of the probe library obtained in step (3) to obtain a fluorescence-labeled CEP17 probe library.

[0073] Mix the P53 probes prepared in Part 1 and the CEP17 probes prepared in Part 2 at the same concentration. The concentrations of both probes are 30 ng / μL.

[0074] The difference between the P53 probe and the CEP17 probe in Comparative Example 2 and those in Example 2 is that UNA triphosphate is not added to the nucleotide raw materials in the amplification labeling reaction in the corresponding step (4).

[0075] Example 3 Preparation of prenatal screening and diagnosis probes - Preparation of 13q14.2 / 21q22.13 dual-color probes Part 1: Preparation of 13q14.2 probes (1) Download the BAC clone gene sequences RP11-1150C12, RP11-798D7, and RP11-795G6 corresponding to the 13q14.2 locus from the UCSC Genome Browser.

[0076] (2) Use the perl plug-in program chunks.pl to split the sequences of RP11-1150C12, RP11-798D7, and RP11-795G6 obtained in step (1) into 1-kb-sized blocks and delete the repetitive sequences; batch import the split blocks into the OligoArray software for probe design and probe screening, then export the screened probes to an EXCEL spreadsheet, and add a 17-bp tag sequence to the 5' end and an 18-bp tag sequence to the 3' end of each probe to obtain a series of probe sequences with tag sequences; the probe design and probe screening methods are that the probe length is 50-150 bp, the TM value is 85-99 °C, the GC ratio is 40-80%, it does not contain TTTT / GGGG / AAAA / CCCC, and the minimum interval between probes is 5 bp.

[0077] (3) Use a DNA synthesizer to chemically synthesize the gene probe sequences with tag sequences obtained in step (2) respectively, and mix the chemically synthesized probes at the same locus to prepare a 13q14.2 probe library; (4) Synthesize a universal primer with a green fluorescent group at the 5' end and use this universal primer to perform an amplification labeling reaction on the 13q14.2 probe library synthesized in step (3), wherein 10% of the total molar amount of nucleotide raw materials is added with UNA triphosphate in the amplification labeling reaction; the sequence of the universal primer is TGTAAAACGACGGCCAGT and CAGGAAACAGCTATGACC.

[0078] (5) Purify and dilute the amplification labeling product of the probe library obtained in step (4) to obtain a fluorescently labeled 13q14.2 probe library.

[0079] Part Two: Preparation of 21q22.13 Probes The difference between the preparation method of the 21q22.13 probe and the preparation method of the probe in Part One is that the BAC clone gene sequences used in step (1) are RP11-980O13 and RP11-95G19, and a red fluorescent group is used in step (4).

[0080] Mix the 13q14.2 probe prepared in Part One and the 21q22.13 probe prepared in Part Two at equal concentrations, and the concentrations of both probes are 30 ng / μL.

[0081] The difference between the 13q14.2 / 21q22.13 dual-color probe of Comparative Example 3 and Example 3 is that UNA triphosphate is not added to the nucleotide raw materials in the amplification labeling reaction described in the corresponding step (4).

[0082] Test Example 1 Color Development Comparison of the Probes Prepared in Example 1 and the Probes Prepared in Comparative Examples 1-1 and 1-2 I. Test Method: (1) Sample treatment: Put the bone marrow cell smear sample into a container containing 2×SSC, heat it in a microwave oven at high power for 3 min until the liquid boils, and then continue to heat it at medium-low power for 10 min. After the treatment is completed, immediately place the slide in gradient alcohol pre-cooled at -20°C for dehydration and air drying, and divide the sample into 3 portions numbered 1-3 for standby; (2) Preparation of probe hybridization mixture: Mix the fluorescently labeled CSF1R probe composition prepared in Example 1 and the hybridization buffer in a volume ratio of 1:9; (3) Preparation of control probe hybridization mixture: Mix the fluorescently labeled CSF1R probe composition without UNA prepared in Comparative Examples 1-1 and 1-2 and the hybridization buffer in a volume ratio of 1:9; (4) Probe sample co-variation: Sample No. 1 is hybridized and mixed with 10 μL of the CSF1R probe hybridization mixture in step (2), Sample No. 2 is hybridized and mixed with 10 μL of the CSF1R probe hybridization mixture of Comparative Example 1-1 in step (3), and Sample No. 3 is hybridized and mixed with 10 μL of the CSF1R probe hybridization mixture of Comparative Example 1-2 in step (3). Cover slips of 22×22 mm are used respectively, and the slides are sealed with rubber glue. After sealing, the slides are placed in a hybridization instrument for denaturation at 90 °C for 1 min and hybridization at 37 °C for 30 - 60 min; (5) Post-hybridization washing: After hybridization is completed, remove the cover slip, and place the slide in a pre-warmed washing solution at 60 °C to wash away the unbound probes; (6) Counterstaining and microscopic examination: Drop 10 μL of anti-quenching mounting medium on the air-dried slide for sealing, and then observe the hybridization results under a fluorescence microscope respectively.

[0083] II. Experimental results are as shown Figure 1 below Figure 1 (a) shows the color development result of the CSF1R probe of Example 1 of the present application for normal cells, Figure 1 (b) shows the color development result of the CSF1R probe of Comparative Example 1-1 for normal cells; Figure 1 (c) shows the color development result of the CSF1R probe of Comparative Example 1-2 for normal cells.

[0084] III. Experimental conclusions It can be seen from Figure 1 that Figure 1 (b) has obvious background noise, while Figure 1 (a) has significantly reduced background noise. At the same time, Figure 1 (c) has unclear signals and obvious tailing, while Figure 1 (a) has strong fluorescence intensity and clear boundaries, and good color development effect. Incorporating a certain amount of UNA into the probe sequence can improve the probe specificity and enhance the binding ability of the probe to the target. Therefore, a fluorescence signal with significantly reduced background noise and clear signals without tailing can be obtained. However, when the amount of UNA incorporated into the probe sequence exceeds a certain content, the binding force between the probe and the target becomes weak, and the probe is easily dissociated from the target, resulting in unclear fluorescence signal boundaries and tailing. Therefore, the probe composition prepared in the present application has strong binding ability, good color development effect, strong probe specificity, and low background noise. Using the probe composition prepared by the preparation method of the present application for the diagnosis of myelodysplastic syndrome can improve the accuracy of the detection results and reduce the difficulty of FISH result analysis.

[0085] Comparison of color development between the probe prepared in Example 2 and the probe prepared in Comparative Example 2 in Test Example 2 I. Test method: (1) Sample treatment: Put the bone marrow cell smear sample into a container containing 2×SSC, heat it in a microwave oven at high power for 3 min until the liquid boils, and then continue to heat it at medium-low power for 10 min. Immediately after the treatment, place the slide in gradient alcohol pre-cooled to -20 °C for dehydration and air drying. Divide the sample into two parts, numbered 1 and 2, for standby; (2) Preparation of probe hybridization mixture: Mix the fluorescently labeled P53 / CEP17 probe composition prepared in Example 2 and the hybridization buffer in a volume ratio of 1:9; (3) Preparation of control probe hybridization mixture: Mix the fluorescently labeled P53 / CEP17 probe composition without UNA prepared in Comparative Example 2 and the hybridization buffer in a volume ratio of 1:9 respectively; (4) Probe-sample co-denaturation: Hybridize and mix Sample No. 1 with 10 μL of the P53 / CEP17 probe hybridization mixture in step (2), and hybridize and mix Sample No. 2 with 10 μL of the fluorescently labeled P53 / CEP17 probe hybridization mixture without UNA in step (3). Cover each with a 22×22 mm coverslip, seal the slide with rubber cement. After sealing, place the slide in a hybridization instrument for denaturation at 90 °C for 1 min and hybridization at 37 °C for 30 - 60 min; (5) Post-hybridization washing: After hybridization is completed, remove the coverslip, and place the slide in a pre-warmed washing solution at 60 °C to wash away the unbound probes; (6) Counterstaining and microscopic examination: Drop 10 μL of anti-quenching mounting medium on the air-dried slide for sealing, and then observe the hybridization results under a fluorescence microscope respectively.

[0086] II. Experimental results are as shown in the appendix Figure 2 as follows Figure 2 (a) shows the color development result of the P53 / CEP17 probe composition prepared in Example 2 of the present application for normal cells, Figure 2 (b) shows the color development result of the P53 / CEP17 probe composition of Comparative Example 2 for normal cells.

[0087] III. Experimental conclusions The P53 / CEP17 probe compositions prepared in Example 2 of the present application and the probe composition of Comparative Example 2 both screen the BAC clone gene sequences on chromosome 17. Therefore, for normal cells, their color development results show two red and two green signals, indicating that both the P53 / CEP17 probe composition of the present application and the probe composition of Comparative Example 2 are effective and reliable. However, by comparing Figure 2 (a) and Figure 2 (b), it can be clearly found that Figure 2 the signal background noise of Figure 2(b) Background noise, and the reduction of background noise benefits from the improvement of the specificity of the probe composition. The reduction of background noise can greatly reduce the difficulty of analyzing diagnostic results. At the same time, the improvement of the specificity of the probe composition can also reduce the sample usage. The CEP17 probe is labeled with a green fluorescent group at the centromeric region of chromosome 17. During the use of the probe, on the one hand, it can be used to detect whether there are abnormalities in the whole chromosome 17, and on the other hand, it can also be used as an internal reference for the P53 probe to a certain extent to exclude the influence of the probe itself on the results during the detection process.

[0088] Color development comparison between the probe prepared in Example 3 and the probe prepared in Comparative Example 3 in Test Example 3 I. Test method: (1) Sample treatment: Put the amniotic fluid cell smear sample into a container containing 2×SSC, heat it in a microwave oven at high fire for 3 min until the liquid boils, and then continue to heat it at medium-low fire for 10 min. After the treatment is completed, immediately place the slide in gradient alcohol pre-cooled at -20°C for dehydration and air drying. Divide the sample into two parts, No. 1-2, for standby; (2) Preparation of the probe hybridization mixture: Mix the fluorescently labeled 13q14.2 / 21q22.13 probe composition prepared in Example 3 and the hybridization buffer in a volume ratio of 1:9; (3) Preparation of the control probe hybridization mixture: Mix the fluorescently labeled 13q14.2 / 21q22.13 probe composition without UNA prepared in Comparative Example 3 and the hybridization buffer in a volume ratio of 1:9 respectively; (4) Probe-sample co-denaturation: Hybridize and mix the No. 1 sample with 10 μL of the 13q14.2 / 21q22.13 probe hybridization mixture in step (2), and hybridize and mix the No. 2 sample with 10 μL of the fluorescently labeled 13q14.2 / 21q22.13 probe hybridization mixture without UNA in step (3). Cover the samples with 22×22 mm coverslips respectively, seal the slides with rubber glue. After sealing, place the slides in a hybridization instrument at 90°C for denaturation for 1 min and hybridize at 37°C for 30-60 min; (5) Post-hybridization washing: After hybridization is completed, remove the coverslip, and place the slide in a pre-warmed washing solution at 60°C to wash away the unbound probes; (6) Counterstaining and microscopic examination: Drop 10 μL of anti-quenching mounting medium on the air-dried slide for sealing, and then observe the hybridization results under a fluorescence microscope respectively.

[0089] II. The experimental results are as shown in the Figure 3 appendix Figure 3 (a) is the color development result of the 13q14.2 / 21q22.13 probe composition prepared in Example 3 of the present application for normal cells,Figure 3 (b) is the color development result of the 13q14.2 / 21q22.13 probe composition prepared in Comparative Example 3 on normal cells.

[0090] III. Experimental Conclusions The 13q14.2 / 21q22.13 probe is used to detect whether there are abnormalities in chromosome 13 and chromosome 21. For normal cells, both the probe composition prepared in Example 3 of the present application and the probe composition of Comparative Example 3 show detection signals of two red and two green, indicating that both the probe composition of the present application and the probe composition of Comparative Example 3 are normal and reliable. According to Figure 3 (a) and Figure 3 (b) signal comparative analysis, it can be known that the detection signal of the probe composition of the present application is clear and bright, and the background is clean, while the detection signal of the probe composition of Comparative Example 3 is scattered and not focused, and there is background noise. The reason for the analysis is that the probe composition of the present application has strong specificity, strong binding to the target region, and is not easily dissociated after binding, while the probe composition of Comparative Example 3 has weak binding force to the target, is easily dissociated after binding, and thus the signal is scattered and not concentrated.

[0091] In summary, the probe composition prepared in the present application has strong binding ability, good color development effect, strong probe specificity, and low background noise. Using the probe composition prepared by the preparation method of the present application for the diagnosis of myelodysplastic syndrome can improve the accuracy of the detection result and reduce the difficulty of FISH result analysis.

[0092] The above are all preferred embodiments of the present application, and the protection scope of the present application is not limited accordingly. Therefore, all equivalent changes made according to the structure, shape, and principle of the present application shall be covered within the protection scope of the present application.

Claims

1. A method for preparing a probe to improve the specificity of fluorescence in situ hybridization probe, characterized in that, The probe is prepared by PCR, and about 5% to about 10% of the total molar amount of nucleotide raw materials is added with UNA triphosphate during the process of preparing the probe by PCR.

2. The preparation method according to claim 1, wherein The UNA triphosphate is selected from UNA-A triphosphate, UNA-T triphosphate, UNA-G triphosphate and UNA-C triphosphate. Preferably, the molar mass ratio of UNA-A triphosphate, UNA-T triphosphate, UNA-G triphosphate and UNA-C triphosphate is 1:1:1:

1.

3. The preparation method according to claim 1, characterized in that, The preparation method includes the following steps: (1) Determine the target gene sequence; (2) Use the perl plug-in program chunks.pl to split the target gene sequence in step (1) into 1-kb blocks respectively and remove the repetitive sequences; batch import the split blocks into the OligoArray software for probe design and probe screening, then export the screened probes to an EXCEL table, and add a 17-bp tag sequence to the 5' end and an 18-bp tag sequence to the 3' end of each probe respectively to obtain a series of probe sequences with tag sequences; (3) Use a DNA synthesizer to chemically synthesize the gene probe sequences with tag sequences obtained in step (2) respectively, and mix the chemically synthesized probes at the same locus to prepare a probe library of the target gene sequence; (4) Synthesize a universal primer with a fluorescent group at the 5' end, and use the universal primer to perform an amplification labeling reaction on the target gene sequence probe, and about 5% to about 10% of the total molar amount of nucleotide raw materials is optionally added with UNA triphosphate during the amplification labeling reaction; (5) Purify and dilute the amplification labeling product of the probe library obtained in step (4) to obtain a fluorescently labeled probe library.

4. The preparation method according to claim 3, characterized in that, The target gene sequence in step (1) is selected from different detection sites of the same mutant type and / or different detection sites of different mutant types.

5. The preparation method according to claim 3, characterized in that, The 17-bp tag sequence in step (2) is TGTAAAACGACGGCCAG, the 18-bp tag sequence is GGTCATAGCTGTTTCCTG, and the universal primer sequences in step (4) are TGTAAAACGACGGCCAGT and CAGGAAACAGCTATGACC.

6. The preparation method according to claim 3, wherein Among them, the probe screening conditions in step (2) are that the probe length is 50-150 bp, the TM value is 85-99 °C, the GC ratio is 40-80%, it does not contain TTTT / GGGG / AAAA / CCCC, and the minimum interval between probes is 5 bp.

7. The preparation method according to claim 3, wherein, Among them, the polymerase used in the amplification labeling reaction is selected from Therminator DNA polymerase.

8. A probe composition prepared by the preparation method according to any one of claims 1-7.

9. The probe composition according to claim 8, wherein The probe is a site-specific probe, and the site-specific probe is selected from a single-color counting probe, a two-color counting probe, a two-color separation probe, a two-color double fusion probe, a two-color single fusion probe, an extra signal probe and a three-color probe.

10. Use of the probe composition according to claim 8 or 9 in the preparation of a diagnostic product for a gene mutation disease. Preferably, the diagnosis of the gene mutation disease is selected from the diagnosis of myelodysplastic syndrome, the diagnosis of multiple myeloma, prenatal screening diagnosis, the diagnosis of lymphoma, the diagnosis of breast cancer, the diagnosis of lung cancer, or the diagnosis of soft tissue tumors.

Images