Method for enriching mutation polymorphic site sequence
By using a method of enriching mutant polymorphic site sequences in SNP detection and single-base extension and ligation using selector primers, the problem of insufficient sensitivity and specificity of multi-site detection in the prior art is solved, and efficient mutation site enrichment and detection are achieved.
Patent Information
- Application Number
- CN202510469107.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-15
- Publication Date
- 2025-06-27
AI Technical Summary
The existing SNP detection technology lacks sensitivity and specificity in multi-site detection, especially when there are a large number of normal sequences in tumor gene samples, it is difficult to effectively detect samples with low mutation frequency.
Using a method of enriching the sequence of mutant polymorphic sites, a 5'-terminal phosphorylated selector is used as a primer to extend a single base using the nucleic acid sample to be tested as a template to form an intermediate that can be linked into a loop, and then ligation and later detection are performed.
This method can enrich a large number of targets in a single tube reaction, theoretically reaching 10,000 or even tens of thousands of targets, improving the sensitivity and specificity of detection, without the need to build a library in advance, and can achieve dozens of times the enrichment of mutation information through multiple rounds of single primer extension.
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Figure CN120210342A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of nucleic acid detection, and particularly relates to a method for enriching mutant polymorphic site sequences. Background Art
[0002] Single nucleotide polymorphism (SNP) mainly refers to DNA sequence polymorphism caused by the variation of a single nucleotide at the genomic level. It is the most common type of heritable variation in humans, accounting for more than 90% of all known polymorphisms. SNPs are widely present in the human genome, with an average of 1 in every 300 base pairs, and it is estimated that their total number can reach 3 million or more. They are the most representative DNA polymorphisms of individual differences in the population, and a considerable part is directly or indirectly related to individual phenotypic differences, susceptibility or resistance to diseases, and responsiveness to drugs. SNPs are considered to be early mutations that can be stably inherited.
[0003] SNP detection can be used for early screening, diagnosis, and prognosis judgment of various diseases. Currently, SNP detection is mainly carried out by high-throughput sequencing and fluorescence PCR methods. High-throughput sequencing is expensive and lacks sensitivity for detecting samples with low mutation frequencies. Fluorescence PCR is widely used in the detection of known mutant genes, using specific primers and probes to detect mutant genes. The number of mutant sites detected by fluorescence PCR is small and difficult to meet the requirements of existing multi-site detections. Although the number of detection sites can be increased by multiplex PCR, the fragment size of plasma DNA in cancer patients is about 160 bp, and there are certain challenges in the design of probes and primers for some detection sites. Multiplex PCR will also reduce the sensitivity and specificity of the detection results. There are a large number of normal sequences in tumor gene samples, and the existing fluorescence PCR detection methods have poor sensitivity for samples with low mutation frequencies. Summary of the Invention
[0004] To overcome the disadvantages and deficiencies of the prior art, the purpose of the present invention is to provide a method for enriching mutant polymorphic site sequences.
[0005] The present invention is realized as follows. A method for enriching mutant polymorphic site sequences, the method comprising the following steps:
[0006] (1) Using a 5'-end phosphorylated selector as a primer, under the condition of a mixture composed of a DNA polymerase and its corresponding buffer and single dNTP or reversible terminator dNTP, performing single-base extension using the nucleic acid sample to be tested as a template;
[0007] (2) Adding T4 DNA ligase buffer, heating at 90°C for 1 minute for denaturation, and placing on ice for 2 minutes for intramolecular renaturation to form a nucleic acid molecule with an open-loop structure;
[0008] (3) Add T4 DNA ligase to ligate into a loop when the bases extended are consistent with the base information on the selector;
[0009] (4) Add exonuclease to digest the DNA that has not been ligated into a loop.
[0010] Preferably, in step (1), the reversible terminator dNTP is selected from one or a mixture of more than one of the reversible terminator dNTPs of dATP, dTTP, dCTP, and dGTP.
[0011] Preferably, in step (1), if the mixture contains reversible terminator dNTP and it is reversible terminator 3'-ONH2-dNTP, pre-treat the reversible terminator dNTP to restore the 3'-terminal hydroxyl group.
[0012] Preferably, pre-treating the reversible terminator dNTP to restore the 3'-terminal hydroxyl group specifically is: treating with a deprotecting agent at room temperature for 10 minutes to restore the 3'-terminal hydroxyl group, purifying and recovering the intermediate product.
[0013] Preferably, the deprotecting agent is 0.7M NaOAc and 1.0M NaNO2, pH 5.2.
[0014] Preferably, in step (1), the single dNTP is selected from any one of dATP, dTTP, dCTP, and dGTP.
[0015] Preferably, in step (1), the DNA polymerase includes but is not limited to Taq DNA polymerase and Pfu DNA polymerase.
[0016] Preferably, step (4) specifically is: adding 10 units of exonuclease I (NEB) and 100 units of exonuclease III (NEB) and treating at room temperature for 15 minutes.
[0017] The present invention overcomes the deficiencies of the prior art and provides a method for enriching mutant polymorphic site sequences. The selector is a primer with a structural DNA, such as Figure 1As shown, it consists of the following parts: (1) a loop region at the 5' end, and the loop region sequence can be a common primer 1 sequence composed of 18 to 30 bases (i.e., the same for all SNPs to be detected), or a nucleic acid sequence encoding different SNP sites (i.e., each SNP corresponds to a nucleic acid sequence composed of 10 to 20 bases); (2) a stem region at the 5' end, and the stem contains at least a 5-base pairing region; (3) immediately followed by the base information of the SNP site; (4) then 4 to 8 bases, preferably 4 bases, which is the reverse complementary sequence at the 3' end of the selector; (5) then the common primer 2, and the common primer 2 can be missing depending on the final detection method; (6) immediately followed by a specific primer sequence, and the first base of its extension is the base information of the SNP.
[0018] In the present invention, the first base adjacent to the 5' end loop region on the selector is the mutant base information to be detected, and immediately followed by the reverse complementary sequence of the last 4 bases of the specific primer at the 3' end. When the selector performs single-base extension (using a set of reversible terminator dNTPs or a single ordinary dNTP) with the template to be detected, if the base extended by the selector is exactly complementary to the mutant base information to be detected on the selector, then the selector after single-base extension can form an intermediate that can be ligated into a loop, and then ligation and subsequent detection are carried out (detection can be performed using a gene chip or next-generation sequencing).
[0019] Compared with the disadvantages and deficiencies of the prior art, the present invention has the following beneficial effects:
[0020] (1) For multiplex PCR or the BDA method based on multiplex PCR, the limit of the detection multiplicity is greatly restricted. However, the present invention uses a single-primer extension reaction, and the multiplicity that can be enriched in a single-tube reaction can refer to the probe hybridization capture technology, and theoretically can reach tens of thousands or even hundreds of thousands of targets.
[0021] (2) The multiplicity of MAESTRO based on the probe capture technology has an advantage over the current popular technologies, but it requires library construction in advance and some information will be lost. However, the present invention (similar to MAESTRO, both through single-primer / probe hybridization) can enrich the mutant information by dozens of times through multiple rounds of single-primer extension without sacrificing the multiplicity and without the need for library construction in advance.
[0022] (3) In the early molecular inversion probe (MIP) genotyping technology, probes with a length exceeding 100 nt are required (the synthesis cost per base of primers exceeding 90 nt doubles). Moreover, for MIP to work effectively, both ends of the probe need to bind to the target simultaneously. However, there are ineffective bindings where only one end of the probe binds to the template or two different probes bind to the same target (considering that the length of cfDNA is only 160 nt, if the mutation site is at either end, then these templates cannot be enriched). More importantly, each round of gap filling in MIP takes thirty minutes, making it difficult to perform multiple rounds of amplification. In contrast, the probes to be synthesized in the present invention are about 70 nt in length, and only single-strand primer extension is involved, which can be completed within seconds and can be enriched multiplicatively by increasing the number of cycles. The subsequent extension or ligation reactions can all be completed within a few minutes. With the method of the present invention, multiplex analysis of more than 1000 probes can be carried out in a single PCR tube using standard laboratory equipment. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 is a schematic structural diagram of the selector; among them, Figure A is a schematic structural diagram of the linearized selector; Figure B is a schematic structural diagram of the selector during single-base extension (at a relatively high annealing temperature) (the loop structure in the figure may not necessarily form); Figure C is the secondary structure formed by the selector when the temperature is relatively low, with a single-base gap between the 3' end and the 5' end (i.e., the SNP base information);
[0024] Figure 2 is a schematic flow chart of the method of the present invention;
[0025] Figure 3 is a method for detecting circular nucleic acids. DETAILED DESCRIPTION OF THE EMBODIMENTS
[0026] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.
[0027] A method for enriching mutant polymorphic site sequences, as Figure 2 shown, the method includes the following steps:
[0028] (1) Using a 5'-end phosphorylated selector as a primer, under the condition of a mixture composed of a DNA polymerase and its corresponding buffer, single dNTP or reversible terminator dNTP, single-base extension is performed using the nucleic acid sample to be tested as a template.
[0029] In step (1), the reversible termination dNTP is selected from one or more mixtures of reversible termination dNTPs among dATP, dTTP, dCTP, and dGTP; the single dNTP is selected from any one of dATP, dTTP, dCTP, and dGTP; the DNA polymerase includes but is not limited to Taq DNA polymerase and Taq DNA polymerase.
[0030] The extension reaction system in step (1) consists of: 20 mM Tris-HCl (pH 8.3), 25 mM KCl, 10 mM MgCl2, 0.5 mM NAD, 0.01% Triton X-100 containing 1 unit of DNA polymerase, single dNTP (0.2 mM) or 3'-ONH2-dNTP (0.2 mM), 100 nM selector, 10 ng of DNA sample; the reaction process is: 94 °C for 15 seconds, 60 °C for 15 seconds, 35 cycles.
[0031] In step (1), if under the condition of a mixture composed of DNA synthase and its corresponding buffer reversible termination dNTP, and the reversible termination dNTP is reversible termination 3'-ONH2-dNTP, it is necessary to pretreat the reversible termination dNTP to restore the 3'-terminal hydroxyl group. Specifically, treat it with a deprotecting agent (0.7 M NaOAc and 1.0 M NaNO2, pH 5.2) at room temperature for 10 minutes to restore the 3'-terminal hydroxyl group, and purify and recover the intermediate product (recover according to the instructions of the oligonucleotide purification kit (Polymerase)).
[0032] (2) Add T4 DNA ligase buffer, heat-denature at 90 °C for 1 minute, and place on ice for 2 minutes for intramolecular renaturation to form a nucleic acid molecule with an open-ring structure.
[0033] In step (2), the 3'-terminal sequence of the extension product in step (1) forms an intramolecular completely complementary paired open-ring structure molecule with the 5'-terminal of the hairpin structure during the annealing process. Specifically, add 1×T4 DNA ligase buffer (NEB) to the extension product in step (1), heat-denature at 90 °C for 1 minute, and place on ice for 2 minutes for intramolecular renaturation to form a nucleic acid molecule with an open-ring structure.
[0034] (3) Add T4 DNA ligase, and ligate into a ring when the bases on the extended bases are consistent with the base information on the selector.
[0035] In step (3), based on the open-loop structured molecule formed in step (2), the substrate molecules can be ligated into a single-stranded closed-loop DNA molecule under the action of DNA ligase. Specifically, 100 active units of T4 DNA ligase (NEB) were added to the nucleic acid molecule with an open-loop structure in step (2) and ligated at 25 °C for 15 minutes to obtain a single-stranded closed-loop DNA molecule.
[0036] (4) Add exonuclease to digest the unligated circular DNA.
[0037] In step (4), exonuclease I and exonuclease III were used to remove the uncircularized nucleic acid. Specifically, 10 units of exonuclease I (NEB) and 100 units of exonuclease III (NEB) were added to the product obtained in step (3) and incubated at room temperature for 15 minutes.
[0038] To verify the effect of the present invention, the enrichment products of the above method were further detected in the examples of the present invention. The detection can be carried out by two methods: (1) According to the common primer sequence introduced when designing the selector, the information containing the SNP site was amplified by reverse PCR, and then the next step of high-throughput sequencing analysis was carried out ( Figure 3 ); (2) According to the nicking enzyme recognition site introduced when designing the selector and each SNP information being distinguished by the sequence of the coding region, and then through nicking enzyme-mediated nucleic acid amplification, the amplified product can be decoded for SNP information by gene chip or high-throughput sequencing method.
[0039] The present invention adopts a single primer extension reaction. The multiplicity that can be enriched by a single-tube reaction can refer to the probe hybridization capture technology, and theoretically can reach ten thousand or even tens of thousands of targets; the present invention can enrich the mutation information by dozens of times through multiple rounds of single primer extension without sacrificing the multiplicity and without the need to construct a library in advance; in addition, the probe that needs to be synthesized in the present invention is about 70 nt, and only involves the extension of a single-end primer, which can be completed within a few seconds and can be enriched by multiples by increasing the number of cycles. The subsequent extension or ligation reaction can be completed within a few minutes. By the method of the present invention, more than 1000 probes can be multiplex analyzed in a single PCR tube using standard laboratory equipment.
[0040] For example, the examples of the present invention provide the practical application of the above method. Specifically, after single-base extension of the mutant base information, it self-ligates into a ring for detecting the L194F mutation in the TP53 gene in human cells, which specifically includes the following steps:
[0041] (1) The total DNA of 293T and T47D cells was extracted using an animal genomic DNA extraction kit (Sangon Biotech, Shanghai) according to the method. In a 50-μL reaction system, 10 ng of genomic DNA, 0.5 pmol of primer P1, dATP (20 μmol / L), 5 units of Pfu DNA polymerase (Sangon Biotech, Shanghai), and the corresponding buffer (20 mM Tris-HCl (pH 8.8), 10 mM (NH4)2SO4, 0.1 mg / mL BSA, 10 mM KCl, 2 mM MgSO4, 0.1% Triton X-100) were added for PCR reaction (15 seconds at 94°C, 15 seconds at 56°C, for a total of 35 cycles). The PCR product was obtained using a PCR purification kit, which was a single-stranded DNA molecule that could be circularized.
[0042] Primer P1: 5’-P-GCATGGTTCGACAGATCCTAGAGCATGCtttatCGTGTGCTCTTCCGATCTttccttccactcggataa-3’;
[0043] (2) The PCR purified product was annealed (1 minute at 90°C, 3 minutes on ice) to obtain the annealed product.
[0044] (3) 0.2 μL of T4 DNA ligase and its buffer were added to the annealed product, and it was treated at 25°C for 30 minutes to obtain the ligation product. The uncircularized DNA in the ligation product was treated with exonuclease to obtain single-stranded circular DNA.
[0045] (4) Fluorescent quantitative PCR analysis was performed using primer F and primer R.
[0046] Primer F: 5’-CTCTAGGATCTGTCGAAC-3’;
[0047] Primer R: 5’-CGTGTGCTCTTCCGATCT-3’;
[0048] The results showed that the Ct value of 293T > 35, while the Ct value of T47D was 22.5, verifying that L194F mutation occurred in T47D, while no L194F mutation occurred in 293T cells.
[0049] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.
Claims
1. A method for enriching mutation polymorphic site sequences, characterized in that: The method comprises the following steps: (1) Using a 5'-terminal phosphorylated selector as a primer, single base extension is performed using a nucleic acid sample to be tested as a template under the conditions of a mixture consisting of a DNA synthesizer and its corresponding buffer, a single dNTP or a reversible termination dNTP; (2) Add T4 DNA ligase buffer, heat at 90°C for 1 minute, and place on ice for 2 minutes for intramolecular renaturation to form a nucleic acid molecule with an open ring structure; (3) adding T4 DNA ligase to connect the extended bases into a circle when the extended bases are consistent with the base information on the selector; (4) Add exonuclease to digest the DNA that has not been connected into a circle.
2. The method according to claim 1, characterized in that In step (1), the reversible termination dNTP is selected from one or more mixtures of the four reversible termination dNTPs of dATP, dTTP, dCTP and dGTP.
3. The method according to claim 2, characterized in that In step (1), if the mixture contains a reversibly terminated dNTP, and the reversibly terminated 3'-ONH2-dNTP, the reversibly terminated dNTP is pretreated to restore the 3' terminal hydroxyl group.
4. The method according to claim 3, characterized in that The reversibly terminated dNTP is pretreated to restore the 3' terminal hydroxyl group by treating it with a deprotecting agent at room temperature for 10 minutes to restore the 3' terminal hydroxyl group, and purifying and recovering the intermediate product.
5. The method according to claim 4, characterized in that The deprotecting agent is 0.7 M NaOAc and 1.0 M NaNO2, pH 5.
2.
6. The method according to claim 1, characterized in that In step (1), the single dNTP is selected from any one of dATP, dTTP, dCTP and dGTP.
7. The method according to claim 1, characterized in that In step (1), the DNA polymerase includes but is not limited to Taq DNA polymerase and Pfu DNA polymerase.
8. The method according to claim 1, characterized in that Step (4) specifically includes: adding 10 units of exonuclease I and 100 units of exonuclease III and treating at room temperature for 15 minutes.