CN114606293A - High-specificity nucleic acid hybridization method based on double-stranded nucleic acid target - Google Patents
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- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 claims description 3
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 claims description 3
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Abstract
The invention relates to a high specificity nucleic acid hybridization method based on a double-stranded nucleic acid target, which comprises the following steps: (1) obtaining a double-stranded target gene with hybridization activity by UDG-PCR; (2) the double-chain target is detected by the combination of the fuel chain, the adjusting chain and the probe chain. The invention provides a more specific nucleic acid hybridization method by taking double-stranded nucleic acid as a target and utilizing the property of double copies of the double-stranded nucleic acid on the premise of not damaging sensitivity. Compared with the prior art, the nucleic acid hybridization technology based on the method has the advantages of specificity, sensitivity, stability and the like, and has good application prospects in the fields of genotyping, mutation detection and the like.
Description
Technical Field
The invention belongs to the field of nucleic acid hybridization, and particularly relates to a high-specificity nucleic acid hybridization method based on a double-stranded nucleic acid target.
Background
Specific hybridization of complementary sequences is an essential property of nucleic acids. Due to its high predictability and specific Watson-Crick base complementary pairing, nucleic acid hybridization plays an important role in the fields of medical diagnosis, DNA nanotechnology, and the like. For example, artificially synthesized nucleic acid hybridization probes are widely used in diagnostic tests such as genotyping, DNA microarray, mutation detection, and the like.
However, the accuracy of nucleic acid hybridization analysis methods is greatly limited by the unintended binding of similar sequences. At the thermodynamic gain provided by many bases pairing correctly, the thermodynamic penalty due to few base mismatches can often be neglected. By raising the reaction temperature to around the melting temperature, the specificity of nucleic acid hybridization can be increased, but it is difficult to predict and control with precision. In addition, divide intoThe subsbeacon probes further increase the specificity of nucleic acid hybridization by introducing secondary structures. Recently, it has been demonstrated that sticky end hybridization probes (toehold hybridization probes) can robustly recognize single-base changes in nucleic acid sequences over a wide range of temperatures and salinity. These probes were designed to react at standard free energy (. DELTA.G)ο rxn) Close to zero react with their intended target, resulting in a hybridization rate close to 50% with the intended target. Whereas a single nucleotide change of the target significantly reduced its hybridization rate to the probe (median 2%).
Based on the current nucleic acid hybridization method, it is difficult to satisfy both high sensitivity and high specificity. How to further improve the specificity of hybridization without impairing the sensitivity is a challenge in practical applications of nucleic acid probes. The nucleic acid hybridization technology with high specificity and high sensitivity has wide application prospect. For example, circulating tumor DNA, which is the most promising biomarker for current cancer diagnosis, is usually present at trace levels and is surrounded by large amounts of wild-type circulating free DNA. Therefore, the development of specific and sensitive nucleic acid hybridization methods can provide new aids for the detection of circulating tumor DNA.
An ideal nucleic acid hybridization method should satisfy three conditions simultaneously: high specificity (no binding to sequences containing mismatches), high sensitivity (robust binding to the target), high stability (ensuring consistency of results under different circumstances). In practical applications, the current nucleic acid hybridization techniques still have some drawbacks.
Disclosure of Invention
The invention provides a high-specificity nucleic acid hybridization method based on a double-stranded nucleic acid target, aiming at the problem that the traditional nucleic acid hybridization method is difficult to combine sensitivity and specificity.
The invention relates to a high specificity nucleic acid hybridization method based on a double-stranded nucleic acid target, which comprises the following steps:
(1) preparing a double-stranded nucleic acid target with hybridization activity by UDG-PCR;
partial dTTP in the upstream and downstream primers of PCR is replaced by UTP, and the substrates of PCR reaction are dATP, dTTP and dCTP and dGTP and high fidelity DNA polymerase; according to PCR Master Mix (Vazyme, China) 10. mu.l, template 1. mu.l, upstream and downstream primers 1. mu.l each, ddH was used20 is supplemented to 20 mul to prepare a reaction system; PCR was performed under the following conditions: step one, pre-denaturation at 95 ℃ for 30 s; step two, repeating the step two 39 times at the temperature of 95 ℃ for 10s and the temperature of 60 ℃ for 30 s; 2.5U of UDG enzyme (NEB, UK) was added to the PCR product and incubated at 37 ℃ for 10 min; then incubating at 95 ℃ for 5 min; finally, purifying the product by using an EZ-10 column type DNA purification kit (raw, China) to obtain a double-chain target gene with hybridization activity;
(2) detecting the double-stranded target obtained in the step (1) with high specificity through the combination of a plurality of nucleic acid chains;
adding a Fuel chain (Fuel strand), a regulating chain (regulated strand) and a Probe chain (Probe strand) into the system; reaction in TE Mg2+In a buffer solution; the fluorescence change was then monitored on a Rotor-Gene Q real-time fluorescent quantitative PCR analyzer (QIAGEN, Germany); finally, the existence of the target is determined by analysis.
The invention provides a nucleic acid hybridization method with sensitivity equivalent to that of the former method and with more specificity by taking a double-stranded nucleic acid target as a breakthrough. Double-stranded nucleic acid targets contain twice as much information as single-stranded nucleic acid targets relative to single-stranded nucleic acid targets. By utilizing the property of double copy of the double-stranded nucleic acid target, in one nucleic acid hybridization reaction, the single reaction can be completed only after the upper strand and the lower strand of the double-stranded nucleic acid target are successfully proofread, thereby improving the specificity of the reaction.
First, we obtained a target gene having a hybridization activity by UDG-PCR. The principle is shown in FIG. 1, in which some T bases in the upstream and downstream primers of PCR are replaced with U bases, and cyclic amplification is performed. Subsequently, the specific cleavage ability of the UDG enzyme for U base is utilized to generate a cohesive end, which is a binding site for nucleic acid hybridization, thereby obtaining hybridization activity. We verified the cleavage activity of the UDG enzyme by polyacrylamide gel electrophoresis. As shown in FIG. 3, when the gel was not stained, only a band appeared in lane 2 (without UDG enzyme treatment, the fluorophore on the primer was not cleaved), and no band appeared in lane 3 (the fluorophore on the primer was cleaved by UDG enzyme). After gel staining, marker in lane 1, and similar bands in both lanes 2 and 3 (evidence the presence of PCR product in lane 3). In addition, we performed capillary electrophoresis characterization. As shown in FIG. 4, a single peak was observed in the PCR product without UDG enzyme treatment. Whereas there was almost no peak at the same position after UDG enzyme treatment. This demonstrates the presence of the PCR product and successful cleavage of the product by the UDG enzyme.
Aiming at the obtained double-stranded target gene with hybridization activity, a reaction system is designed to detect the upper strand and the lower strand of a target. As shown in FIG. 2, 3 strands are added in the system in total, and under the combined action of the fuel strand and the regulatory strand, the double-stranded nucleic acid target is opened to generate FT and CT strands. On the other hand, if a mismatch occurs in the target, the reaction is difficult to proceed because the free energy Δ G >0 of the reaction. In this step, we performed a single proofreading of the single strand on the underside of the target. Wherein the fuel strand serves to correct the presence of mismatches and the regulatory strand serves to regulate the exposed single-stranded region of the product and to assist the fuel strand in opening the double strand. After the double-stranded target is opened, the generated CT chain can react with the probe chain, and the fluorescent group marked at the tail end of the probe chain is separated from the quenching group, so that a fluorescent signal is generated. And, complete identity of the product PT strand to the target strand sequence can again participate in similar reactions, thereby achieving recycling of the target. However, the mismatch target is difficult to perform in the first step reaction, and even if the first step reaction is completed a few times, the product CS chain gene still carries a mutant base and is difficult to react with the probe. Here, we proofread the upper single strand of the double stranded nucleic acid target using the probe strand and achieve recycling of the matched target by a second reaction.
To achieve the best reaction performance, we have optimized the length of the cohesive ends (toehold) of the nucleic acid strand, as shown in fig. 5, when the toehold length is 8nt, the reaction rate is the fastest. In general, sticky-end hybridization probes are less effective at identifying mismatches at locations remote from the toehold. The method has a good recognition for the mismatches at different positions, as shown in FIG. 6, the signals generated by the targets with the mismatches at different positions are very low compared with the signals generated by the matched targets. To demonstrate the superiority of the hybridization method based on double-stranded nucleic acid targets, we tested mutations of different abundance. As shown in FIG. 7, even if the mutation abundance was as low as 0.01%, it could be visually distinguished from 100% of Wild Type (WT). Finally, in order to verify the potential of the method in clinic, primers are designed aiming at EGFR-L858R mutation which is difficult to detect clinically, and products of the primers are detected after templates with different mutation abundances are subjected to PCR amplification. FIG. 8 is a melting curve of PCR amplification products, and it can be seen that all the curves are single-peak and the peak positions are consistent, thus proving that the target gene is successfully amplified by PCR. FIG. 9 shows the detection results of PCR products, and it can be seen that even after PCR amplification, mutations with different abundances can be clearly distinguished.
In conclusion, the double-stranded nucleic acid is taken as a target, and a new idea and a new method are provided for nucleic acid hybridization. First, we provide a method (UDG-PCR) that can produce a double-stranded target gene having hybridization activity; in addition, a nucleic acid hybridization reaction system aiming at the double-chain target is successfully designed, and the sensitivity of the reaction is further improved through target self-circulation on the premise of ensuring the specificity. By means of the method, more specific and sensitive nucleic acid hybridization can be realized, and the application of the nucleic acid hybridization technology in practice is enriched.
Drawings
FIG. 1 is a schematic diagram of the principle of UDG-PCR
FIG. 2 is a schematic diagram of the principle of nucleic acid hybridization reaction
FIG. 3 is a representation of polyacrylamide gel electrophoresis of UDG-PCR
FIG. 4 is a representation of the capillary electrophoresis of UDG-PCR
FIG. 5 is a graph of the results of toehold length optimization
FIG. 6 is a graph showing the effect of the method on the recognition of mismatches occurring at different positions
FIG. 7 is a graph of the effect of detection on different abundance mutations
FIG. 8 is a melting curve of PCR products of EGFR-L858R template at different concentrations
FIG. 9 is a graph showing the effect of PCR detection on EGFR-L858R mutant templates with different abundances
Detailed Description
The high specificity nucleic acid hybridization method based on the double-stranded nucleic acid target comprises the following steps:
(1) preparing a double-stranded nucleic acid target with hybridization activity by UDG-PCR;
partial dTTP in the PCR upstream and downstream primers is replaced by UTP, and the PCR reaction substrates are dATP, dTTP, dCTP, dGTP and high-fidelity DNA polymerase; according to PCR Master Mix (Vazyme, China) 10. mu.l, template 1. mu.l, upstream and downstream primers 1. mu.l each, ddH was used20 is supplemented to 20 mul to prepare a reaction system; PCR was performed under the following conditions: step one, pre-denaturation at 95 ℃ for 30 s; step two, repeating the step two 39 times at the temperature of 95 ℃ for 10s and the temperature of 60 ℃ for 30 s; 2.5U of UDG enzyme (NEB, UK) was added to the PCR product and incubated at 37 ℃ for 10 min; then incubating at 95 ℃ for 5 min; finally, purifying the product by using an EZ-10 column type DNA purification kit (raw, China) to obtain a double-chain target gene with hybridization activity;
(2) detecting the double-stranded target obtained in the step (1) by combining a plurality of nucleic acid strands with high specificity;
adding a fuel chain (10 mu mol/L, 1 mu L), a regulating chain (10 mu mol/L, 1 mu L) and a probe chain (200nmol/L, 1 mu L) into the system; reaction in TE Mg2+(11.5mmol/L) in buffer; the fluorescence change (temperature 37 ℃) was subsequently monitored in a Rotor-Gene Q real-time fluorescent quantitative PCR analyzer (QIAGEN, Germany); finally, the existence of the target is determined through analysis.
Claims (9)
1. The invention relates to a high specificity nucleic acid hybridization method based on a double-stranded nucleic acid target, which comprises the following steps:
(1) preparing a double-stranded nucleic acid target with hybridization activity by UDG-PCR;
partial dTTP in the PCR upstream and downstream primers is replaced by UTP, and the PCR reaction substrates are dATP, dTTP, dCTP, dGTP and high-fidelity DNA polymerase; according to PCR Master Mix (Vazyme, China) 10. mu.l, template 1. mu.l, upstream and downstream primers 1. mu.l each, ddH was used20 is supplemented to 20 mul to prepare a reaction system; PCR was performed under the following conditions: step one, pre-denaturation at 95 DEG C30 s; step two, 10s at 95 ℃ and 30s at 60 ℃, and repeating the step two 39 times; 2.5U of UDG enzyme (NEB, UK) was added to the PCR product and incubated at 37 ℃ for 10 min; then incubating at 95 ℃ for 5 min; finally, purifying the product by using EZ-10 column type DNA purification kit (raw, China) to obtain double-chain target gene with hybridization activity;
(2) detecting the double-stranded target obtained in the step (1) with high specificity through the combination of a plurality of nucleic acid chains;
adding a Fuel chain (Fuel strand), a regulating chain (regulated strand) and a Probe chain (Probe strand) into the system; reaction in TE Mg2+In a buffer solution; the fluorescence change was then monitored on a Rotor-Gene Q real-time fluorescent quantitative PCR analyzer (QIAGEN, Germany); finally, the existence of the target is determined through analysis.
2. The method for hybridizing double-stranded nucleic acid target-based high specificity nucleic acids according to claim 1, wherein the product obtained by UDG-PCR in step (1) is double-stranded nucleic acid with both sticky ends (toehold).
3. The method for hybridizing double-stranded nucleic acid target-based nucleic acids with high specificity according to claim 1, wherein the distance between U bases in the primer in step (1) is preferably less than 9 nt.
4. The method for hybridizing double-stranded nucleic acid target-based high specificity nucleic acids according to claim 1, wherein the polymerase used in the PCR in step (1) is a non-archaeal polymerase, such as taq DNA polymerase.
5. The method for hybridizing double-stranded nucleic acid target-based high specificity nucleic acids according to claim 1, wherein the final concentration of the fuel chain in the step (2) is 1 μmol/L.
6. The method for hybridizing high specificity nucleic acids based on double-stranded nucleic acid targets according to claim 1, wherein the final concentration of the regulatory strand in step (2) is 1. mu. mol/L.
7. The method for hybridizing double-stranded nucleic acid target-based high specificity nucleic acids according to claim 1, wherein the final concentration of the probe strand in the step (2) is 20 nmol/L.
8. The method for hybridizing double-stranded nucleic acid target-based high specificity nucleic acid according to claim 1, wherein TE Mg is added in step (2)2+Mg in buffer2+The concentration of (2) was 11.5 mmol/L.
9. The method for hybridizing double-stranded nucleic acid target-based high specificity nucleic acids according to claim 1, wherein the temperature for fluorescence detection in step (2) is 37 ℃.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110294689A1 (en) * | 2010-05-27 | 2011-12-01 | Affymetrix, Inc | Multiplex Amplification Methods |
| CN106244703A (en) * | 2016-08-26 | 2016-12-21 | 山东大学 | Strand replacement reaction based on sticky end mediation combines the method for polymerization nicking isothermal amplification technique detection UDG activity |
| CN106755451A (en) * | 2017-01-05 | 2017-05-31 | 苏州艾达康医疗科技有限公司 | Nucleic acid is prepared and analyzed |
| CN110195099A (en) * | 2019-05-28 | 2019-09-03 | 西安交通大学 | A kind of application of more target gene parallel detection combination probes and its kit |
| CN111778317A (en) * | 2020-07-24 | 2020-10-16 | 重庆医科大学 | Gene mutation fluorescence detection method based on raised loop strand displacement probe and application thereof |
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Patent Citations (5)
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|---|---|---|---|---|
| US20110294689A1 (en) * | 2010-05-27 | 2011-12-01 | Affymetrix, Inc | Multiplex Amplification Methods |
| CN106244703A (en) * | 2016-08-26 | 2016-12-21 | 山东大学 | Strand replacement reaction based on sticky end mediation combines the method for polymerization nicking isothermal amplification technique detection UDG activity |
| CN106755451A (en) * | 2017-01-05 | 2017-05-31 | 苏州艾达康医疗科技有限公司 | Nucleic acid is prepared and analyzed |
| CN110195099A (en) * | 2019-05-28 | 2019-09-03 | 西安交通大学 | A kind of application of more target gene parallel detection combination probes and its kit |
| CN111778317A (en) * | 2020-07-24 | 2020-10-16 | 重庆医科大学 | Gene mutation fluorescence detection method based on raised loop strand displacement probe and application thereof |
Non-Patent Citations (3)
| Title |
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| YOUNA KIM等: "Colorimetric Assay for Uracil DNA Glycosylase Activity Based on Toehold-Mediated Strand Displacement Circuit", 《BIOTECHNOL J》 * |
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Application publication date: 20220610 |