CN117230162A - Analysis method for realizing accurate indication of single nucleotide polymorphism by combining enzyme-assisted strand displacement with liquid chromatography - Google Patents

Abstract

Enzyme-assisted strand displacement combined with liquid chromatography is an analytical method to achieve precise indication of single nucleotide polymorphisms. First, aptamer-magnetic bead conjugates are prepared and stored for later use; hairpin probes, targets, and E. coli ligase Shake with nicotinamide adenine dinucleotide solution and mix thoroughly; then add deoxynucleoside triphosphate, DNA polymerase and nicking endonuclease and shake; add aptamer-magnetic bead conjugate to the centrifuge tube; then Add restriction endonuclease; use a magnet to separate the magnetic beads and unreacted probe, and inject the supernatant into HPLC for detection; use acetonitrile and triethylamine acetate solution as mobile phase A, and methanol as mobile phase B. , perform fluorescence detection of target substances in gradient elution mode. This method's negligible background signal and cumulative signal amplification make the target amplified, with a linear range of 0.1fM to 0.1nM. The invention has the advantages of high throughput, high sensitivity, high selectivity, high detection efficiency, etc.

Description

Enzyme-assisted strand displacement combined with liquid chromatography to achieve precise indication of single nucleotide polymorphisms analysis method

Technical field

The invention belongs to the field of analysis methods for precise indications of single nucleotide polymorphisms, and specifically relates to an analysis method for enzyme-assisted strand displacement combined with liquid chromatography to achieve precise indications of single nucleotide polymorphisms.

Background technique

Single nucleotide polymorphisms (SNPs) are the most common and stable form of genetic variation in the human genome, occurring on average once every 300 base pairs. Single nucleotide variations are associated with many human diseases and serve as biomarkers for the diagnosis of cancer initiation, progression, and metastasis. According to statistics, epidermal growth factor receptor (EGFR) mutations are the most common among non-small cell lung cancer (NSCLC) patients in my country, accounting for 40%-50% of all non-small cell lung cancer patients. Normally, EGFR function is short-lived and tightly controlled, and is turned off after it has completed its function. However, in some lung cancer cells, the EGFR gene is mutated, causing it to be unable to be shut down normally. Instead, it endlessly stimulates cell growth, eventually leading to the occurrence of cancer. Among all EGFR mutations in NSCLC, the incidence rate of point mutation in exon 21 (L858R) is 40%-45%. This mutation can cause activation of the tyrosine kinase domain and lead to tumor growth and metastasis. The T790M point mutation means that the 790th amino acid of the EGFR protein changes from T to M. This will cause the loss of killing ability against cancer cells and lead to drug resistance, which will lead to disease progression. NSCLC patients carrying the T790M drug resistance mutation generally have a poor prognosis. These two mutations account for approximately 60% of all EGFR mutations. For EGFR mutations, the higher the mutation proportion, the higher the degree of malignancy and the easier it is for recurrence and metastasis. However, analyzing only the mutation proportion of one mutant type is not enough to accurately determine the stage of the disease and drug resistance. Although the abnormal mutation of L858R can indicate the occurrence of NSCLC, it cannot determine the severity and development of the disease. On the other hand, relying solely on the increase in the abundance of T790M mutations in EGFR is not enough to determine the degree of progression and drug resistance of NSCLC, because excessive T790M mutations in EGFR are not only related to NSCLC, but also to malignant tumors such as breast cancer and ovarian cancer. related to the transfer. Furthermore, the abundance of mutants (∼fM) is very low. Therefore, it is crucial to accurately and sensitively measure the contents of multiple wild-type and mutant forms simultaneously and to perform clinical diagnosis, staging assessment, and prognosis monitoring of diseases through the proportion of single nucleotide mutations.

However, low selectivity and high limit of detection (LOD) are the main reasons that limit the accurate detection of SNPs. These reasons come from the following two aspects. First, mutant types usually coexist with large amounts of wild type, and the proportion of mutations in normal humans is less than 0.1%. Second, there is great similarity between the bases of wild-type and mutant types. Single nucleotide mutations include base substitutions, deletions, and insertions. Single base substitutions are called point mutations, and they are the most common type of mutation. DNA mutations caused by such deletions and insertions can be easily detected by traditional polymerase chain reaction (PCR) and next-generation sequencing methods. However, point mutations are difficult to detect using traditional techniques. To solve this problem, specific hybridization and specific enzymatic reactions have been developed to improve the accuracy of detecting SNPs. Specific hybridization relies on detecting subtle differences in stability caused by single-base variations. Wallace et al. systematically studied for the first time the possibility of exploiting subtle differences in thermal stability between perfectly matched and single-base mismatched sequences, and developed SNPs detection technology based on gene chip arrays. However, thermodynamically driven hybridization suffers from low specificity and sensitivity. Generally speaking, the performance of SNP quantification technology based on specific enzymatic reactions is better than that of specific hybridization technology. Wang et al. developed a SNPs detection technology based on enzyme ligation and enzyme cleavage. Although this technology is highly selective in identifying single-base mismatches, this detection technology cannot simultaneously detect multiple wild-type and Mutant type.

High-performance liquid chromatography (HPLC) is an efficient method for spatial and temporal separation of various analytes in a single run. However, because the content of wild-type and mutant types is very low and their sequences are very close, it is not ideal to directly separate and detect multiple mutants and wild-types by HPLC. In our previous work, the signals of different targets were converted into distinguishable signals of self-generated G-quadruplex fluorescent probes, which enabled the detection of the contents of multiple targets through HPLC fluorescence detectors in one run. . In this work, based on enzyme ligation, primer extension and enzyme cleavage strategies, a high selectivity, high throughput and low detection limit was developed based on the HPLC platform by integrating fluorescent probes of different bases and different lengths. SNPs detection strategy. This strategy uses DNA ligase and recognition probes (Probe 1, Probe 2, Probe 3 and Probe 4) to perform base recognition on the mutant and wild types. The strand displacement (SDA) reaction is triggered under the action of , and through continuous cyclic amplification, a large number of cleaved single strands are produced. After these single strands hybridize with the long and short probes on the magnetic beads, the nucleic acid cutting enzyme cuts off the partial sequences of the long and short probes at specific sites. These fluorescently labeled probe tail chains enter the HPLC. Achieve detection of two mutant types and the corresponding wild type in one injection. By simultaneously detecting the levels of L858R-MT, L858R-WT, T790M-MT and T790M-WT in the serum of multiple NSCLC patients with different stages and calculating the mutation proportion, the effectiveness of this method in disease diagnosis and staging assessment was verified.

Contents of the invention

Technical Problems Solved: In view of the above technical problems, the present invention provides an analysis method that combines enzyme-assisted strand displacement with liquid chromatography to achieve precise indications of single nucleotide polymorphisms.

Technical solution: An analysis method that uses enzyme-assisted strand displacement combined with liquid chromatography to achieve precise indications of single nucleotide polymorphisms. The steps are: (1) Streptavidin-coated magnetic beads are centrifuged and placed on on the magnetic stand, then discard the solvent, then use 1×B&W buffer to wash the magnetic beads, then place them on the magnetic stand, discard the solvent; resuspend the magnetic beads in 2×B&W buffer, add four different lengths of Biotin-labeled DNA fluorescent probes: P790M-MT, P790M-WT, P858R-MT, P858R-WT, mix at room temperature for 20 minutes, and the four biotin-labeled DNA fluorescent probes are coupled to streptavidin. After coating the magnetic bead surface, the obtained aptamer-magnetic bead conjugate was stored at 4°C for later use; all hairpin probes were heated to 95°C for 5 minutes, and then cooled to room temperature for 1 hour. , ready for use; (2) Add 20 μL NE buffer and 20 μL ThermoPol reaction buffer solution to the centrifuge tube; then add 5 μL of each 100 nM hairpin probe: Probe 1, Probe 2, Probe 3, Probe 4, and then add each 10μL of 100nM hairpin probes: Probe 5 and Probe 6. After mixing thoroughly, add 5μL of each 100pM target: L858R-MT, L858R-WT, T790M-MT and T790M-WT, 9U of E. coli ligase and 10μL of 10mM nicotinamide adenine dinucleotide solution, shake the reaction mixture to mix thoroughly; (3) After incubating at 37°C for 30 minutes, add 8μL of 10mM deoxynucleoside triphosphate and 6U of DNA to the centrifuge tube. The polymerase and 15 U of nicking endonuclease (Nt.BstNBI) were shaken for 10 seconds; after incubation at 60°C for two hours, 30 μL of aptamer-magnetic bead conjugate was added to the centrifuge tube; then 20 U of Restriction endonuclease (Nt.BbvCI); after incubating at 37°C for 1 hour, use a magnet to separate the magnetic beads and unreacted probes, and inject the supernatant into HPLC for detection; (4) Use 5 % acetonitrile and 100mM pH=7.0 triethylamine acetate (TEAA) solution as mobile phase A, methanol as mobile phase B, the column temperature was set to 35°C, the flow rate was 1.0mL/min, in gradient elution mode, 20min The proportion of internal methanol was increased from 10% to 30%. The excitation wavelength and emission wavelength of the fluorescence detector were set to 488nm and 520nm respectively, and the target object was detected by fluorescence.

The partial sequences near the 3' end of the above-mentioned Probe 1, Probe 2, Probe 3 and Probe 4 are designed to be complementary to their corresponding mutant or wild-type; there is a section of Nt.BbvCI at both ends of the four hairpin probe loops. The half-recognition site (5'-CCTCAGC-3') and a half-recognition site of Nt.BstNBI (5'-GACTC-3'); the partial sequence near the 5' end of Probe 5 and Probe 6 is consistent with the target part Sequence complementation.

The above NE buffer is prepared from 100mM NaCl, 50mM Tris-HCl, 10mM MgCl ₂ and 1mM DTT.

The above-mentioned ThermoPol reaction buffer solution includes 20mM Tris-HCl, 10mM (NH ₄ ) ₂ SO ₄ , 10mM KCl, 2mMgCl ₂ , and 0.1% (w/v) Triton X-100.

The above high performance liquid chromatography method uses a fully porous hybrid silica gel column Phenomenex The 3μm Oligo-RP chromatographic column is used for the separation of various nucleic acids, with an inner diameter of 50×4.6mm and a particle size of 3μm; use LCsolution software for data processing.

Beneficial effects: The negligible background signal and cumulative signal amplification of this method enable the LOD of the target objects L858R-MT, L858R-WT, T790M-MT and T790M-WT to be 26aM, 24aM, 19aM and 22aM respectively, with a linear range of 0.1fM to 0.1nM. The invention has the advantages of high throughput, high sensitivity, high selectivity, high detection efficiency, etc.

Description of drawings

Figure 1 is a schematic diagram of the analysis flow of the present invention.

Figure 2 shows the retention of long and short probes with different base sequences in HPLC.

Figure 3 shows the chromatograms of the four DNA probes after optimization of the orthogonal experiment.

Figure 4 is a feasibility analysis diagram, in which (A) a schematic diagram of the verification enzyme ligation reaction using L858R-MT as an example; (B) a fluorescence histogram of the verification enzyme ligation reaction using L858R-MT as an example.

Figure 5 is a feasibility analysis diagram. Among them (A) A schematic diagram of the verification strand displacement reaction using L858R-MT as an example; (B) A fluorescence histogram of the verification strand displacement reaction using L858R-MT as an example.

Figure 6 is a gel electrophoresis verification diagram taking L858R-MT as an example.

Figure 7 is a detection performance analysis diagram, in which (A) chromatograms of target substances at different concentrations; (B) resolution of four signal peaks in HPLC.

Figure 8 shows the standard curves of L858R-MT, L858R-WT, T790M-MT and T790M-WT. Error bars represent the standard deviation of three independent experiments.

Figure 9 is a chromatogram for accurate detection and analysis of single nucleotide mutations. The concentration of (A) the mutant type is 1% of the wild-type concentration; (B) the concentration of the mutant type is 0.1% of the wild-type concentration.

Figure 10 is a comparison chart between the method of the invention and the qRT-PCR method. (A) Relative expression level and qRT-PCR of L858R-MT in serum samples; (B) Relative expression level and qRT-PCR of T790M-MT in serum samples.

Figure 11 shows the chromatograms of original and spiked serum samples. Among them (A) sample1; (B) sample2; (C) sample3.

Detailed ways

The following examples further illustrate the content of the present invention, but should not be understood as limiting the present invention. Without departing from the spirit and essence of the present invention, any modifications and substitutions made to the method, steps or conditions of the present invention shall fall within the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

Example 1: Detection of L858R-MT, L858R-WT, T790M-MT and T790M-WT extracted from whole blood samples of NSCLC patients at different stages

1. Extraction of target substances from actual samples

DNA was extracted from whole blood of NSCLC patients using Tiangen Bio DNA extraction kit. Mix 200 μL of blood with 20 μL of proteinase K. Then inject 200 μL of GB buffer, mix completely by inverting, and place at 70°C for about ten minutes. After the solution is clear, centrifuge briefly and remove the water beads inside the tube cap. Add 200 μL of absolute ethanol and shake completely for about 15 seconds. At this time, flocculent sediment may form. After a short centrifugation, remove the water beads inside the tube cap. Put the solution in the above steps together with the floc sediment into the absorption column CB3 (put the absorption column into the absorption tube), centrifuge at 12000 rpm for 30 seconds, pour out the mother liquor, and put the absorption column CB3 back into the absorption catheter. Pour 500 μL of buffer GD into the absorption column CB3, centrifuge at 12,000 rpm for 30 seconds, discard the mother liquor, and place the absorption column CB3 in the recovery conduit again. In the absorption column CB3, add 600 μL of rinse solution PW, centrifuge at 12000 rpm for about 30 seconds, discard the waste liquid, and then place the absorption column CB3 in the recovery conduit. Repeat the previous step. Store the adsorption column CB3 at room temperature for 1 minute and completely dry the remaining solution in the absorbent material. The main purpose of this step is to completely remove the remaining rinse liquid in the absorption column, because the residual alcohol in the rinse liquid will interfere with the experimental results of subsequent enzyme reactions (enzyme digestion, PCR, etc.). Put the adsorption column CB3 into a clean centrifuge tube, drop 50-200 μL of buffer TE for elution into the center of the absorption membrane, leave it in the room for 2-5 minutes, centrifuge at 12000 rpm for 2 minutes, and remove the solution completely Collect in centrifuge tubes and store at -20°C for later use.

2. Actual sample analysis

First, after coupling the biotin-labeled aptamer probe to the surface of streptavidin-coated magnetic beads, the resulting aptamer-magnetic bead conjugate was stored at 4°C for later use. All hairpin probes were heated to 95°C for 5 minutes and then slowly cooled to room temperature (25°C) for 1 hour before use. Then add 20 μL NE buffer and 20 μL ThermoPol reaction buffer solution to the brown centrifuge tube. Then add 5 μL each of 100 nM hairpin probes (Probe 1, Probe2, Probe 3, and Probe 4) in sequence, then add 10 μL each of 100 nM hairpin probes (Probe 5 and Probe 6), mix thoroughly, and then add 20 μL of serum extract, 9 U of E.coli ligase and 10 μL of 10 mM nicotinamide adenine dinucleotide solution, shake the reaction mixture for 10 seconds to mix thoroughly. After incubating at 37°C for 30 minutes, add 8 μL of 10 mM deoxynucleoside triphosphates (dNTPs), 6 U of DNA polymerase and 15 U of nicking endonuclease (Nt.BstNBI) to the centrifuge tube and shake for 10 seconds. After incubation at 60°C for two hours, the magnetic bead conjugates with long and short fluorescent probes were added to the brown centrifuge tube. Then 20 U of restriction endonuclease (Nt.BbvCI) was added. After one hour of incubation at 37°C, use a magnet to separate the magnetic beads and unreacted DNA probes, and inject the supernatant into HPLC for detection.

A solution of 5% acetonitrile and 100 mM triethylamine acetate (TEAA, pH=7.0) was used as the mobile phase (A), and methanol was used as the mobile phase (B). The column temperature was set to 35°C and the flow rate was 1.0 mL/min. In gradient elution mode, the proportion of methanol was increased from 10% to 30% within 20 min. The excitation wavelength and emission wavelength of the fluorescence detector were set to 488nm and 520nm respectively, and the actual sample was subjected to fluorescence detection. The detection results were finally applied to the standard curve in Figure 8 to obtain the actual content of the target substance in the actual sample.

The present invention detects L858R-MT, L858R-WT, T790M-MT and T790M-WT extracted from NSCLC patients. The results are shown in Table 1 and Table 2. It can be seen that 5.364fM of L8585R-MT and 4.598fM of T790M-MT were detected in sample 1 respectively. The mutation proportions of L858R-MT and T790M-MT were 1.02% and 1.08% respectively. In sample 2, 6.073fM of L858R-MT and 5.049fM of T790M-MT were detected respectively. The mutation proportions of L858R-MT and T790M-MT were 1.19% and 1.20% respectively. In sample 3, 20.649fM of L858R-MT and 18.697fM of T790M-MT were detected respectively. The mutation proportions of L858R-MT and T790M-MT were 4.13% and 4.18% respectively.

Subsequently, 0.5fM and 5fM L858R-MT and T790M-MT, 0.5pM and 5pM L858R-WT and T790M-WT standard solutions were added to sample 1, sample 2 and sample 3 respectively, as shown in Figure 11AB. Chromatograms of NSCLC samples before and after. The relative recoveries obtained ranged from 100.7% to 104.6%. The relative recovery (RSD) of this method is between 1.6% and 4.7%. The above results all show that this method has good practicability for detecting various mutation ratios in actual samples.

4. Comparison of methods

We also used the qRT-PCR method to amplify and detect the same samples. As shown in Figure 10: AB, the results of this method are basically consistent with the results detected after amplification using qRT-PCR. At the same time, we also compared the detection performance of this method with some common methods for detecting SNPs. As can be seen from Table 4, other methods cannot detect multiple mutant types and wild types in one run, while our method can detect multiple mutant types and wild types in one run. Excellent sensitivity and selectivity were obtained by this method, indicating that it has strong signal amplification ability and excellent recognition ability of single nucleotide mutations.

Table 1 Nucleic acid sequences

Table 2 Detection of L858R-MT and L8580M-WT isolated from serum samples of NSCLC patients at different stages

^aRelative recovery = (total concentration - blank concentration)/spiked concentration

Table 3 Detection of T790M-MT and T790M-WT isolated from serum samples of NSCLC patients at different stages

^aRelative recovery = (total concentration - blank concentration)/spiked concentration

Table 4 Comparison of different single nucleotide polymorphism detection strategies

Claims (5)

1. The analytical method for realizing the accurate indication of the single nucleotide polymorphism by combining the enzyme-assisted strand displacement with the liquid chromatography is characterized by comprising the following steps: (1) Centrifuging the magnetic beads with the streptavidin coating, placing the magnetic beads on a magnetic frame, discarding the solvent, then cleaning the magnetic beads by using a 1 XB & W buffer solution, placing the magnetic beads on the magnetic frame, and discarding the solvent; the beads were resuspended in 2 XB & W buffer and four different lengths of biotin-labeled DNA fluorescent probes were added: P790M-MT, P790M-WT, P858R-MT and P858R-WT are uniformly mixed at room temperature for 20min, and after the four DNA fluorescent probes marked by biotin are coupled to the surfaces of the magnetic beads coated with streptavidin, the obtained aptamer-magnetic bead conjugate is preserved at 4 ℃ for later use; all hairpin probes were heated to 95 ℃ for 5 minutes, then cooled to room temperature for 1 hour for use; (2) Add 20. Mu.L NE buffer and 20. Mu.L Thermopol reaction buffer to the centrifuge tube; then 5 μl of 100nM hairpin probe each was added: probe 1, probe 2, probe 3, probe 4, then 10. Mu.L of each of the 100nM hairpin probes were added: after Probe 5 and Probe 6 were thoroughly mixed, 5. Mu.L of each target of 100/pM was added: L858R-MT, L858R-WT, T790M-MT and T790M-WT, E.coli ligase of 9U and 10. Mu.L 10mM of nicotinamide adenine dinucleotide solution were thoroughly mixed by shaking the reaction mixture; (3) After incubation for 30 min at 37 ℃, 8 μl of 10mM deoxynucleotide triphosphate, 6U DNA polymerase and 15U nicking endonuclease (Nt. BstNBI) were added sequentially to the centrifuge tube and shaken for 10 seconds; after incubation at 60 ℃ for two hours, 30 μl of the aptamer-magnetic bead conjugate was added to the centrifuge tube; followed by addition of 20U restriction endonuclease (Nt. BbvCI); after incubation for 1 hour at 37 ℃, magnetic beads and unreacted probes are separated by a magnet, and the supernatant is injected into HPLC for detection; (4) The target was fluorescence detected using 5% acetonitrile and 100mM triethylamine acetate (TEAA) solution at ph=7.0 as mobile phase a, methanol as mobile phase B, column temperature set at 35 ℃, flow rate 1.0mL/min, and in gradient elution mode, the proportion of methanol increased from 10% to 30% over 20min, excitation wavelength and emission wavelength of fluorescence detector set at 488nm and 520nm, respectively.

2. The method for analyzing accurate single nucleotide polymorphism by combining enzyme-assisted strand displacement with liquid chromatography according to claim 1, wherein the method comprises the following steps: the partial sequences of the Probe 1, the Probe 2, the Probe 3 and the Probe 4 near the 3' end are designed to be complementary with the corresponding mutant type or wild type; at each end of the four hairpin probe loops, a segment of Nt. BbvCI half recognition site (5 '-CCTCC GC-3') and a segment of Nt. BstNBI half recognition site (5 '-GACTC-3'); the partial sequences near the 5' end of Probe 5 and Probe 6 are complementary to the partial sequences of the target.

3. The method for analyzing accurate single nucleotide polymorphism by combining enzyme-assisted strand displacement with liquid chromatography according to claim 1, wherein the method comprises the following steps: the NE buffer solution is prepared from 100mM NaCl,50mM Tris-HCl and 10mM MgCl ₂ 1mM DTT.

4. The method for analyzing accurate single nucleotide polymorphism by combining enzyme-assisted strand displacement with liquid chromatography according to claim 1, wherein the method comprises the following steps: the Thermopol reaction buffer solution comprises 20mM Tris-HCl,10mM (NH) ₄ ) ₂ SO ₄ ，10 mM KCl，2 mM MgCl ₂ ，0.1% (w/v) Triton X-100。

5. The method for analyzing accurate single nucleotide polymorphism by combining enzyme-assisted strand displacement with liquid chromatography according to claim 1, wherein the method comprises the following steps: the high performance liquid chromatography adopts a full-porous hybrid silica gel column Phenomenex Clarity [ mu ] m Oligo-RP chromatographic column for separating various nucleic acids, wherein the inner diameter is 50 multiplied by 4.6mm, and the particle diameter is 3 [ mu ] m; data processing was performed using LCsolution software.