Capture probe combination, kit and application

Abstract

The invention provides a capture probe combination, a kit and application. The capture probe combination provided by the invention can effectively improve the capture efficiency, the capture efficiency can reach 95% in a high-GC region and 85% in a low-GC region, the Tm value of the capture probe has the advantage of high consistency, simultaneous capture of the high-GC region and the low-GC region can be realized in one PCR reaction, and the detection sensitivity is high. And a basis is provided for high-throughput detection of human epidermal growth factor receptor coding gene mutation.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and more particularly to a capture probe assembly, kit, and application. Background Technology

[0002] The human epidermal growth factor receptor (EGFR) is a transmembrane protein containing an extracellular ligand-binding domain, a transmembrane domain, an intracellular cytoplasmic domain with tyrosine kinase activity, and an ATP-binding site. It is a member of the epidermal growth factor receptor family and plays an important role in physiological processes such as cell growth, proliferation, and differentiation.

[0003] Under normal circumstances, EGFR exists in its monomeric form without tyrosine kinase activity. During cell proliferation, EGFR monomers bind to ligands (such as epidermal growth factor EGF), prompting the formation of tyrosine kinase-active dimers. These dimers can bind to ATP, promoting autophosphorylation of the EGFR dimer and further activating downstream MAPK and mTOR signaling pathways, thereby promoting cell proliferation, differentiation, and migration. When the gene encoding EGFR is mutated, EGFR monomers can form phosphokinase-active dimers independently of ligands. These dimers bind to ATP, activating downstream MAPK and mTOR signaling pathways and promoting tumor cell proliferation, differentiation, and migration.

[0004] Current research has found that mutations in the EGFR-encoding gene are directly or indirectly associated with lung cancer, breast cancer, colorectal cancer, and head and neck cancer. Therefore, there is an urgent need to develop a product capable of detecting mutations in the human epidermal growth factor receptor (EGFR)-encoding gene. Summary of the Invention

[0005] The purpose of this invention is to provide a product capable of detecting mutations in the human epidermal growth factor receptor (HGF) gene, enabling the simultaneous capture of high-GC and low-GC regions in a single PCR reaction, thus providing a foundation for high-throughput detection of HGF gene mutation identification.

[0006] To achieve the above objectives, the present invention provides a capture probe assembly, wherein the capture probe assembly includes a first probe and a second probe;

[0007] The first probe is 16 bp in length, and its nucleotide sequence is shown in SEQ ID NO:1. The 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, and 16th bases in SEQ ID NO:1 are labeled with locked nucleic acids.

[0008] The second probe is 16 bp in length, and its nucleotide sequence is shown in SEQ ID NO:2. The 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, and 16th bases in SEQ ID NO:2 are locked nucleic acid labeled.

[0009] The capture probe assembly described above, wherein the 3' end of the first probe is labeled with a biotin-triethylene glycol group.

[0010] The capture probe assembly described above, wherein the 3' end of the second probe is labeled with a biotin-triethylene glycol group.

[0011] The capture probe combination described above, wherein the first probe is used to capture exon 19 of the human epidermal growth factor receptor encoding gene; and / or,

[0012] The second probe is used to capture exon 21 of the human epidermal growth factor receptor encoding gene.

[0013] The present invention provides a kit for detecting the human epidermal growth factor receptor encoding gene, wherein the kit includes the above-described capture probe combination.

[0014] The kit described above also includes at least one of a positive control and a negative control.

[0015] In the kit described above, the positive control is an artificial plasmid containing the human epidermal growth factor receptor encoding gene.

[0016] In the kit described above, the negative control is distilled water.

[0017] This invention provides the application of the above-described capture probe combination or the above-described kit in the preparation of products related to the capture, enrichment, or detection of the human epidermal growth factor receptor encoding gene.

[0018] This invention also provides a method for detecting mutations in the human epidermal growth factor receptor encoding gene, comprising the following steps:

[0019] DNA samples were extracted from the samples to be tested;

[0020] The target DNA sample is obtained by capturing the target DNA in the DNA sample using the above-described capture probe combination or the above-described kit;

[0021] The target DNA was analyzed to determine the mutation status of the gene encoding the human epidermal growth factor receptor.

[0022] The capture probe combination provided by this invention can effectively improve the capture efficiency, reaching 95% in high GC regions and 85% in low GC regions. Moreover, the Tm value of the capture probe has the advantage of high consistency, enabling simultaneous capture of high GC and low GC regions in a single PCR reaction, which lays the foundation for high-throughput detection of mutations in the human epidermal growth factor receptor encoding gene. Detailed Implementation

[0023] To enable those skilled in the art to better understand the present invention, the present invention will be further described in detail below. The specific embodiments listed below are merely descriptions of the principles and features of the present invention, and the examples are only for explaining the present invention and are not intended to limit the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] The gene encoding the human epidermal growth factor receptor (EGFR) is located on the short arm of chromosome 7, q22. It is a 110 kb DNA segment containing 28 exons. Studies have shown that mutations in the EGFR coding gene are mainly concentrated in the tyrosine kinase region, specifically exons 18-21, with mutations in exons 19 and 21 accounting for approximately 90% of all mutations; these are also known as classic mutations or hotspot mutations.

[0025] To overcome the problem of uneven capture efficiency of traditional capture probes in high-GC and low-GC regions of the EGFR gene, this invention optimizes the capture probe design using locked nucleic acid (LNA) modification technology for the high-GC region of exon 19 and the low-GC region of exon 21 of the EGFR gene, significantly improving the capture efficiency and specificity in regions with different GC contents.

[0026] In order to develop a product capable of detecting mutations in the human epidermal growth factor receptor (EGFR) encoding gene, this invention conducted a large number of experiments and found that exon 19 of the EGFR encoding gene has a high GC region and exon 21 has a low GC region, which affects the capture efficiency of the capture probe.

[0027] Specifically, high-GC regions refer to areas with a high GC content (generally ≥60%). Since GC base pairs contain three hydrogen bonds, and hydrogen bonds are crucial secondary bonds maintaining the stability of nucleic acid double-stranded structures, the higher the number of hydrogen bonds, the stronger the binding force between the double strands, and the higher the energy (e.g., temperature) required for melting. Therefore, high-GC regions, due to their greater number of hydrogen bonds and more stable double-stranded structures, have significantly higher melting temperatures (Tm values) than other regions. This makes them difficult to completely melt during polymerase chain reaction (PCR), resulting in the capture probe's inability to effectively bind to the target sequence in this region, thus reducing capture efficiency. During the PCR reaction, the difficulty in completely melting high-GC regions leads to the capture probe's inability to effectively bind to the target sequence in this region, affecting capture efficiency and amplification results. Furthermore, high-GC regions are prone to forming secondary structures, further increasing the difficulty of amplification.

[0028] Low-GC regions refer to areas with low GC content (generally GC percentage <30%). Due to the fewer hydrogen bonds in low-GC regions, double-stranded DNA in these regions exhibits poor stability and low Tm values, making it prone to probe unwinding during PCR reactions. This hinders the probe's ability to effectively capture the target sequence in these regions, thus reducing capture efficiency. Furthermore, the binding of the capture probe to the target sequence in low-GC regions is weak, making it difficult to distinguish the target sequence from other low-GC sequences with low homology. This leads to non-specific binding (such as binding to non-target sequences in low-GC regions), resulting in increased false positives and decreased specificity.

[0029] To address the aforementioned problems, a first aspect of the present invention provides a capture probe assembly, which includes a first probe and a second probe.

[0030] The first probe is 16 bp in length, and its nucleotide sequence is shown in SEQ ID NO:1. The 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, and 16th bases in SEQ ID NO:1 are labeled with locked nucleic acids.

[0031] The second probe is 16 bp in length, and its nucleotide sequence is shown in SEQ ID NO:2. The 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, and 16th bases in SEQ ID NO:2 are locked nucleic acid labeled.

[0032] Locked nucleic acid (LNA) is an oligonucleotide derivative that has a similar phosphate backbone to DNA and RNA. Its ribose forms a double-loop structure between the 2'O and 4'C positions through a methylene bridge, thereby fixing the furanose conformation, reducing the flexibility of the ribose structure, and enhancing the stability of the local structure of the phosphate backbone.

[0033] Existing studies have shown that EGFR gene mutations are mostly concentrated in exons 18-21, with mutations in exons 19 and 21 accounting for approximately 90% of all mutations. These are hotspot regions for EGFR mutation detection. Therefore, this invention preferentially selects these two exons as detection targets, which has high clinical significance. Furthermore, using two separately designed probes allows for precise coverage and functional differentiation of regions with significantly different GC components, while also enhancing capture capabilities.

[0034] In this invention, the first and second probes are designed targeting exons 19 and 21 of the EGFR coding gene, respectively, and all bases of both probes are modified with locked nucleic acids (LNAs). Locked nucleic acids can significantly improve the thermostability and binding affinity of nucleic acid chains; therefore, introducing LNAs helps improve the probe's ability to recognize, stabilize, and specifically target the sequence. In the first probe (high GC region), LNA modification improves melting efficiency, making it easier for the probe to bind to the target sequence in complex structures, thus overcoming the difficulty of melting and binding double strands in high GC regions. In the second probe (low GC region), LNA modification compensates for its insufficient stability, easy melting, and poor specificity, thereby enhancing the binding force between the probe and the target sequence and reducing non-specific hybridization.

[0035] To achieve precise and coordinated capture of high-GC and low-GC regions, this invention employs two key strategies: (1) improving the consistency of the Tm values ​​of the two probes through fully locked nucleic acid modification, enabling them to be compatible with high-temperature conditions in a single capture reaction and avoiding probe bias or loss due to temperature differences; (2) designing dedicated probes for high-GC (exon 19) and low-GC (exon 21) regions respectively, to adapt to different sequence characteristics and leverage their specific binding advantages. These two strategies complement each other, achieving compatible and efficient capture of different GC regions, ensuring a dual improvement in overall capture efficiency and detection sensitivity.

[0036] In summary, the capture probe combination provided by this invention can effectively improve the capture efficiency, reaching 95% in high GC regions and 85% in low GC regions. Furthermore, the capture probes have the advantage of high consistency in Tm values, enabling simultaneous capture of both high GC and low GC regions in a single PCR reaction. This provides a foundation for high-throughput detection of mutations in the human epidermal growth factor receptor encoding gene.

[0037] In the above technical solution, the 3' end of the first probe is labeled with a biotin-triethylene glycol group; the 3' end of the second probe is labeled with a biotin-triethylene glycol group.

[0038] Biotin is a member of the B vitamins and has a high affinity for avidin (such as streptavidin); triethylene glycol acts as a spacer to separate biotin from the rest of the oligonucleotide to reduce potential steric hindrance.

[0039] In this invention, the 3' ends of the first and second probes are labeled with biotin-triethylene glycol groups, which can be used for magnetic bead fixation.

[0040] In one embodiment, streptavidin-modified magnetic beads (Dynabeads® M-280 Streptavidin) are used to bind probes with a biotin-triethylene glycol group labeled at the 3' end. The specific steps are as follows:

[0041] The synthesized first and second probes are reacted with magnetic beads to immobilize them on the bead surface. The target DNA sample is then incubated with the immobilized probes under suitable temperature and buffer conditions to achieve specific capture of the target sequence. After magnetic enrichment, unbound portions are removed to obtain high-purity target fragments, providing templates for subsequent PCR amplification or sequencing.

[0042] In the above technical solution, the first probe is used to capture exon 19 of the human epidermal growth factor receptor (HGF) gene; the second probe is used to capture exon 21 of the HGF gene.

[0043] Based on the above research, a second aspect of the present invention provides a kit for detecting the human epidermal growth factor receptor encoding gene, comprising the above-described capture probe combination.

[0044] Furthermore, the above-mentioned kit also includes at least one of a positive control and a negative control. The positive control may be an artificial plasmid containing the human epidermal growth factor receptor encoding gene. The negative control may be distilled water.

[0045] A third aspect of the present invention provides the application of the above-described capture probe combination or the above-described kit in the preparation of capture, enrichment or detection products for human epidermal growth factor receptor encoding genes.

[0046] It is understood that the capture probe combination provided in the first aspect of the present invention or the kit provided in the second aspect can be used to capture or enrich the human epidermal growth factor receptor encoding gene. The enriched human epidermal growth factor receptor encoding gene sample can then be used to detect whether there is a mutation, thereby laying the foundation for high-throughput detection of human epidermal growth factor receptor encoding gene mutation identification.

[0047] A fourth aspect of this invention provides a method for detecting mutations in the human epidermal growth factor receptor encoding gene, comprising the following steps:

[0048] DNA samples were extracted from the samples to be tested;

[0049] The target DNA sample is obtained by capturing the target DNA in the DNA sample using the above-described capture probe combination or the above-described kit;

[0050] The target DNA was analyzed to determine the mutation status of the gene encoding the human epidermal growth factor receptor.

[0051] First, a DNA sample can be extracted from the sample to be tested; for example, a peripheral blood sample to be tested can be obtained from a non-small cell carcinoma patient, and then a DNA sample can be extracted from the peripheral blood sample.

[0052] Before capturing target DNA using the capture probe combo or detection kit, the first or second probe in the capture probe can be coupled with streptavidin-coated magnetic beads to facilitate subsequent capture.

[0053] In one embodiment, a capture probe assembly can be added to 200 μL of phosphate-buffered saline (PBS), with 50 pmol of the first probe and 50 pmol of the second probe added. Then, 55 µL of streptavidin-coated magnetic beads are added, and the mixture is incubated at room temperature for 30 min. After incubation, the mixture is washed with 200 μL of PBS to remove the free probes, thus obtaining capture probe-coupled magnetic beads.

[0054] Subsequently, the target DNA in the DNA sample was captured by coupling the aforementioned capture probe with magnetic beads, thus obtaining the target DNA sample.

[0055] In one embodiment, 10 ng of DNA sample and 10 μL of 10 mg / mL capture probe-conjugated magnetic beads can be hybridized in a hybridization buffer, wherein the hybridization buffer can consist of 5× sodium citrate buffer (SSC), 0.1% SDS, 0.1 mg / mL deproteinized salmon sperm DNA, and 10 mM EDTA. After hybridization, unbound DNA can be removed by washing with a washing buffer, wherein the washing buffer can consist of 1× SSC and 0.1% SDS. Then, the target DNA is eluted with an elution buffer, wherein the elution buffer can consist of 10 mM Tris-HCl (pH 8.5).

[0056] After obtaining the target DNA, it can be sequenced or subjected to mutation detection using methods such as qPCR to determine the mutation status of the human epidermal growth factor receptor encoding gene.

[0057] The technical solution of this application will be further explained below with reference to specific embodiments. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise specified, all reagents used are commercially available or obtained through public channels.

[0058] Example 1:

[0059] This embodiment provides a locked nucleotide (LNA) modified capture probe assembly, including a first probe and a second probe. The first probe is used to capture exon 19 of the human epidermal growth factor receptor (EGFR) gene, which is 16 bp in length and has the nucleotide sequence shown in SEQ ID NO:1, with LNA labeling for the first 16 bases. The second probe is used to capture exon 21 of the EGFR gene, which is also 16 bp in length and has the nucleotide sequence shown in SEQ ID NO:2, with LNA labeling for the first 16 bases, as detailed in Table 1. Both the first and second probes are labeled with biotin-TEG at their 3' ends for immobilization with magnetic beads.

[0060] Table 1

[0061]

[0062] This embodiment provides a method for preparing the first and second probes described above, comprising the following steps: Solid-phase synthesis is performed using an ABI 394 DNA synthesizer according to the nucleotide sequence designed in Example 1. Each base site requiring modification is introduced with an LNA nucleotide monomer to ensure that the entire sequence of both the first and second probes is LNA modified. The synthesis program is performed according to the cycle set in the instrument manual. In the last synthesis cycle, Biotin-TEG-Phosphoramidite reagent is added for coupling, labeling the 3' end of the probe with Biotin-TEG. The coupling reaction is carried out on a solid-phase support, and the reaction time and conditions are as specified in the reagent instructions to ensure labeling efficiency and uniformity. After synthesis, a deprotection reaction is performed according to a standard procedure to remove the protecting group. The oligonucleotide chain is released by alkaline treatment and collected in the reaction solution. Purification is performed using a C18 reversed-phase high-performance liquid chromatography column, with acetonitrile / water gradient elution as the mobile phase. The purified peak is collected to remove incomplete synthesis or other impurities, ensuring a purity of ≥90%. The purified probe sample was analyzed for molecular weight using a MALDI-TOF mass spectrometer to confirm the sequence length and the correctness of the biotin labeling. Finally, the purified probe was dissolved in RNase-free water to prepare a 100 μM storage solution, which was then aliquoted into low-adsorption tubes and stored at -20°C to avoid repeated freeze-thaw cycles.

[0063] Example 2:

[0064] To test and compare the capture effect of the LNA-modified capture probe combination in Example 1, a control group was set up in this example as follows:

[0065] Control group 1: A capture probe assembly comprising a third probe and a fourth probe. The third probe is 16 bp in length, with the nucleotide sequence shown in SEQ ID NO:1; the fourth probe is 16 bp in length, with the nucleotide sequence shown in SEQ ID NO:2. Both the third and fourth probes are labeled with Biotin-TEG at their 3' ends for immobilization with magnetic beads.

[0066] Control group 2: A capture probe combination comprising a fifth probe and a sixth probe. The fifth probe, 18 bp in length, is used to capture exon 19 of the EGFR coding gene; the first 18 bases of its sequence are LNA-labeled, and the nucleotide sequence is shown in SEQ ID NO:3. The sixth probe, 18 bp in length, is used to capture exon 21 of the EGFR coding gene; the first 18 bases of its sequence are LNA-labeled, and the nucleotide sequence is shown in SEQ ID NO:4. See Table 2 for details. Both the fifth and sixth probes are labeled with Biotin-TEG at their 3' ends for immobilization with magnetic beads.

[0067] Table 2

[0068]

[0069] Control group 3: A capture probe combination comprising a seventh probe and an eighth probe. The seventh probe is 18 bp in length, with the nucleotide sequence shown in SEQ ID NO:3; the eighth probe is 18 bp in length, with the nucleotide sequence shown in SEQ ID NO:4. Both the seventh and eighth probes are labeled with Biotin-TEG at their 3' ends.

[0070] The capture probe combination from Example 1 was added to 200 μL of phosphate-buffered saline (PBS), with 50 pmol of each of the first and second probes added. Then, 55 µL of 10 mg / mL streptavidin-coated magnetic beads (Dynabeads™ M-280 Streptavidin, Thermo Fisher) were added. The mixture was incubated at room temperature (25°C) for 30 min, gently inverting to maintain the magnetic beads in suspension during incubation. After incubation, the beads were washed three times with 200 μL of PBS to remove free probes, yielding capture probe-coupled magnetic beads. Control groups 1, 2, and 3 were prepared using the same method to obtain their corresponding capture probe-coupled magnetic beads.

[0071] The sample to be tested was a positive plasmid containing exon 19 and exon 21 of the human EGFR gene, with GC contents of 65% (exon 19, i.e., high GC region) and 28% (exon 21, i.e., low GC region), respectively. 10 ng of the sample to be tested and 10 μL of magnetic beads coupled with a 10 mg / mL capture probe were hybridized in 100 μL of hybridization buffer at 65°C for 1 h. The hybridization buffer consisted of 5× sodium citrate buffer (SSC), 0.1% SDS, 0.1 mg / mL deproteinized salmon sperm DNA, and 10 mM EDTA. After hybridization, the sample was washed three times with washing buffer (1×SSC and 0.1% SDS) to remove unbound positive plasmids. The target DNA was then eluted at 95°C for 5 min using elution buffer (10 mM Tris-HCl, pH 8.5).

[0072] The target DNA was detected by quantitative real-time PCR (qPCR). The Ct values ​​of exon 19 and exon 21 were calculated. The amount of captured DNA was then converted using a standard curve, and the capture efficiency was calculated as A / B×100%, where A is the amount of captured DNA and B is the amount of positive plasmid DNA initially added. The results are shown in Table 3.

[0073] Table 3

[0074]

[0075] Table 3 shows that compared with control group 3, control group 1 has a lower capture efficiency; however, compared with control group 2, Example 1 has a significantly higher capture efficiency in both high GC region and low GC region. This indicates that the LNA-modified capture probe combination of the present invention has a synergistic effect between its sequence and LNA modification. Therefore, using the LNA-modified capture probe combination of the present invention can effectively enhance the capture efficiency in both high GC region and low GC region, and significantly improve the consistency of Tm value, thereby improving probe stability.

[0076] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A capture probe assembly, characterized in that, The capture probe assembly includes a first probe and a second probe; The first probe is 16 bp in length, and its nucleotide sequence is shown in SEQ ID NO:

1. The 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, and 16th bases in SEQ ID NO:1 are labeled with locked nucleic acids. The second probe is 16 bp in length, and its nucleotide sequence is shown in SEQ ID NO:

2. The 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, and 16th bases in SEQ ID NO:2 are locked nucleic acid labeled.

2. The capture probe assembly according to claim 1, characterized in that, The 3' end of the first probe is labeled with a biotin-triethylene glycol group.

3. The capture probe assembly according to claim 1 or 2, characterized in that, The 3' end of the second probe is labeled with a biotin-triethylene glycol group.

4. The capture probe assembly according to claim 1 or 2, characterized in that, The first probe is used to capture exon 19 of the human epidermal growth factor receptor encoding gene; and / or, The second probe is used to capture exon 21 of the human epidermal growth factor receptor encoding gene.

5. A kit for detecting the human epidermal growth factor receptor encoding gene, characterized in that, Includes the capture probe combination as described in any one of claims 1-3.

6. The reagent kit according to claim 5, characterized in that, It also includes at least one of positive control samples and negative control samples.

7. The reagent kit according to claim 6, characterized in that, The positive control is an artificial plasmid containing the human epidermal growth factor receptor encoding gene.

8. The kit according to claim 6 or 7, characterized in that, The negative control sample is distilled water.

9. The use of a capture probe combination according to any one of claims 1-4 or a kit according to any one of claims 5-8 in the preparation of products related to the capture, enrichment or detection of the human epidermal growth factor receptor encoding gene.

10. A method for detecting mutations in the human epidermal growth factor receptor encoding gene, characterized in that, Includes the following steps: DNA samples were extracted from the samples to be tested; The target DNA sample is obtained by capturing the target DNA in the DNA sample using the capture probe combination according to any one of claims 1-4 or the kit according to any one of claims 5-8. The target DNA was analyzed to determine the mutation status of the human epidermal growth factor receptor encoding gene.

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