Method for detecting single base mutation of nucleic acid
阅读说明：本技术 一种核酸单碱基突变检测的方法 (Method for detecting single base mutation of nucleic acid ) 是由 裴昊 杨海红 赖魏 李丽 于 2019-11-14 设计创作，主要内容包括：本发明公开了一种核酸单碱基突变的检测方法,通过锁式探针成环,通用引物1延伸,阻断剂和引物2特异性竞争引发超支化滚环扩增突变型核酸,从而达到检测突变的目的。所述方法从动力学角度解决了常规热力学方法检测核酸单碱基突变时的温度敏感性问题,克服了PCR需要变温及昂贵的缺点,同时提高了滚环扩增检测单碱基突变的灵敏度和特异性；将合理的竞争杂交反应与滚环等温扩增技术结合,使得可以灵敏地识别和选择性恒温扩增等位基因频率为0.1％的单碱基突变,可以对完整基因组DNA中包含的低频率单碱基未知突变进行简便、快速和高选择性等温扩增检测。可以用于癌症相关突变的检测,具有潜在的应用价值。(The invention discloses a method for detecting single base mutation of nucleic acid, which comprises the steps of looping through a padlock probe, extending a universal primer 1, and specifically competing with a blocking agent and a primer 2 to trigger hyperbranched rolling circle to amplify mutant nucleic acid so as to achieve the purpose of detecting mutation. The method solves the problem of temperature sensitivity when the conventional thermodynamic method is used for detecting the single base mutation of the nucleic acid from the aspect of dynamics, overcomes the defects of temperature change and high cost of PCR (polymerase chain reaction), and simultaneously improves the sensitivity and specificity of detecting the single base mutation by rolling circle amplification; the reasonable competitive hybridization reaction is combined with the rolling circle isothermal amplification technology, so that the single base mutation with the allele frequency of 0.1 percent can be sensitively identified and selectively amplified at constant temperature, and the low-frequency single base unknown mutation contained in the complete genome DNA can be simply, conveniently, quickly and highly selectively amplified and detected at isothermal. Can be used for detecting cancer-related mutation and has potential application value.)
1. A method for detecting single base mutation of nucleic acid is characterized by comprising the following specific steps:
(1) sample preparation: adding endonuclease into the genome DNA to carry out DNA fragmentation to obtain fragmented DNA;
(2) and (3) hybridization and connection: adding a lock probe, dNTPs, polymerase and ligase into the fragmented DNA obtained in the step (1) for target hybridization, filling and ligation to form a ring, and adding exonuclease I and exonuclease III for digestion to obtain a ring;
(3) blocking amplification: adding a blocking agent, a primer 1, a primer 2, dNTPs and polymerase into the loop obtained after digestion in the step (2) for selective amplification to obtain an amplification product;
(4) detection and analysis: and then carrying out qualitative analysis or quantitative analysis on the amplification product obtained in the step (3).
2. The detection method according to claim 1, wherein in the step (1), the sequence of the fragment to be detected in the genomic DNA is shown as SEQ ID No.1 or SEQ ID No. 2.
3. The detection method according to claim 1, wherein in the step (2), the polymerase is one or more of Taq DNA polymerase, T4 DNA polymerase, T7 DNA polymerase; and/or the ligase is one or more of Ampligase ligase, T4 DNA ligase and Taq DNA ligase; and/or, the concentration of the polymerase is 0.2-0.5U/μ L; and/or, the concentration of the ligase is 0.2-1.0U/muL; and/or, the concentration of the exonuclease I is 0.2-0.5U/mu L; and/or the concentration of the exonuclease III is 5-20U/mu L; and/or the sequence of the padlock probe is shown as SEQ ID NO. 3.
4. The assay of claim 1, wherein in step (2), the junction temperature is between 16 ℃ and 60 ℃; and/or, the connection time is 1-10 hours; and/or the temperature of the digestion is 37 ℃; and/or the digestion time is 1-4 h.
5. The detection method according to claim 1, wherein in the step (3), the ratio of the blocker to the primer 2 is 1-10: 1; and/or, the molar ratio of the blocking agent to the primer 1 is 1-10: 1; and/or the concentration of the primer 1 is 0.1-1 mu M; and/or the sequence of the primer 1 is shown as SEQ ID NO. 6; and/or the concentration of the primer 2 is 0.1-1 mu M; and/or the sequence of the primer 2 is shown as SEQ ID NO.7 or SEQ ID NO. 8; and/or the concentration of the dNTPs is 200-400 mu M.
6. The detection method according to claim 1, wherein in the step (3), the polymerase is one or more of vent (exo-) DNA polymerase, Bsu DNA polymerase, Bst 3.0DNA polymerase; and/or the concentration of the polymerase is 0.1-0.4U/. mu.L.
7. The detection method according to claim 1, wherein in the step (3), the blocker is designed to be completely complementary to a known wild-type target sequence, the 3' end of the primer 2 sequence has an overlapping region of 6-14 nt with the 5' end of the blocker sequence, and the 3' end of the blocker has a sequence of 12-30 nt which is not present in the primer 2, wherein the wild-type target sequence is shown as SEQ ID No.4 or SEQ ID No. 5; and/or the concentration of the blocking agent is 0.1-10 mu M; and/or the sequence of the blocker is shown as SEQ ID NO. 10.
8. The detection method according to claim 1, wherein in the step (3), the amplification temperature is 58-63 ℃; and/or the amplification time is 45-120 min.
9. Use of the detection method of claim 1 for the detection of single base mutations in extracted DNA or RNA.
10. Use of the assay of claim 1 for the detection of single base mutations in DNA or RNA in situ in a cell.
11. Use of the assay of claim 1 in the detection of DNA methylation.
Technical Field
The invention belongs to the field of gene mutation detection, relates to a method for detecting single base mutation of nucleic acid, and particularly relates to a method for detecting single base mutation in nucleic acid by inducing isothermal hyperbranched rolling circle amplification through selective competition of a blocking agent and enriching the single base mutation in the nucleic acid in an ultrasensitive and specific manner.
Background
Genetic mutations are important biomarkers for the diagnosis and monitoring of cancers (e.g., breast cancer, colon cancer). Small genetic variations, such as DNA point mutations, are important drivers of disease progression and also important indicators of diagnosis.
They are usually present in very low levels (< 1%) in humans due to local environment, cellular heterogeneity and migration. Analysis of rare nucleic acid variants with low frequency alleles, such as cancer mutations in extracellular DNA or drug resistance in pathogen subpopulations, presents challenges to current molecular diagnostic techniques.
The existing detection method has the following problems: I. existing methods require operating the reaction temperature in the "Goldilocks" region of each individual blocker or probe, relying on precise temperature control and primer design. Digital PCR detection is specific for both base position and specific base change. For recessive diseases, mutations in tumor suppressor genes, and even repeated mutations in many oncogenes, often require re-detection of the mutation at many base positions, and cannot detect both unknown and multiple mutations simultaneously. Deep sequencing methods are not economical because they waste most of the reading capacity when sequencing wild-type (WT) templates and amplicons.
The BDA system is unique in providing good mutation sensitivity, high mutation replication, fast turnaround, but still requires a PCR temperature swing process, resulting in some degree of economic cost. As an isothermal amplification technology, the rolling circle amplification can be realized in a common water bath without an expensive and complicated temperature change instrument, but is influenced by non-specific connection of the padlock probes, so that the background signal is high, and the sensitivity and specificity of detection are reduced; furthermore, padlock probes can only recognize known mutations, which are difficult to detect. Therefore, the invention provides a novel isothermal, economic and high-specificity method for detecting unknown nucleic acid mutation, which has important research significance and application value.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to solve the problem of temperature sensitivity when a conventional thermodynamic method is used for detecting single base mutation from the aspect of dynamics, overcome the defects of temperature change and high cost required by PCR (polymerase chain reaction), and simultaneously improve the sensitivity and specificity of detecting single base mutation by rolling circle amplification; the reasonable competitive hybridization reaction is combined with the rolling circle isothermal amplification technology, so that the single base mutation with the allele frequency of 0.1 percent can be sensitively identified and selectively amplified at constant temperature, and the low-frequency single base unknown mutation contained in the complete genome DNA can be simply, conveniently, quickly and highly selectively amplified and detected at isothermal.
The invention initiates isothermal hyperbranched rolling circle amplification through selective competition of a blocking agent, and the detection principle of ultra-sensitively and specifically enriching single base mutation in nucleic acid is shown in figure 1, and the specific principle is as follows:
the extracted genomic DNA is fragmented by a specific endonuclease, making it convenient for the subsequent positional ligation of padlock probes. Then adding a padlock probe with two ends matched with the target strand, hybridizing by a base complementary pairing principle, leaving a small section (10-20 nt) of gap (single base mutation existing region), filling and connecting by dNTPs in a 'gap-fill' mode. Two rings with only one base difference are obtained after the two rings are connected into a ring, and a
According to the principle, the invention adopts the following technical scheme:
the invention provides a detection method of single base mutation of nucleic acid (also called a detection method for initiating isothermal hyperbranched rolling circle amplification by selective competition of a blocking agent and enriching single base mutation in nucleic acid in an ultra-sensitive and specific manner), which comprises the following steps:
(1) sample preparation: adding endonuclease into genome DNA to carry out DNA fragmentation, and obtaining fragmented DNA.
(2) And (3) hybridization and connection: adding a lock probe, dNTPs, polymerase and ligase into the fragmented DNA obtained in the step (1) to perform target hybridization, filling and ligation cyclization; and then adding exonuclease I and exonuclease III for digestion to obtain the ring.
(3) Blocking amplification: and (3) adding a blocking agent, a
(4) Detection and analysis: and (4) carrying out qualitative analysis or quantitative analysis on the amplification product obtained in the step (3).
In the step (1), the genomic DNA is preferably extracted using a kit.
The kit can be used for extracting genomic DNA or RNA from blood, cells, tissues, urine, excrement and other substances.
In the step (1), the sequence of the segment to be detected in the genomic DNA is:
TATAAACTTGTGGTAGTTGGAGCTGTTGGCGTAGGCAAGAGTGCCTTGACGATACAGC (SEQ ID NO.1) or
In the step (1), the endonuclease is determined by a gene to be detected, the upstream and downstream nucleic acids of a fragment to be detected can be restrictively inscribed without affecting the position to be detected, and the enzyme digestion conditions refer to an enzyme instruction book.
In the step (2), the sequence of the padlock probe is as follows:
in the step (2), the concentration of the padlock probes is 1-100 nM; preferably, it is 100 nM.
In the step (2), the concentration of the dNTPs is 200-400 mu M; preferably 200. mu.M.
In the step (2), the polymerase is one or more of Taq DNA polymerase, T4 DNA polymerase, T7 DNA polymerase and the like; preferably, Taq DNA polymerase.
In the step (2), the concentration of the polymerase is 0.2-0.5U/mu L; preferably, it is 0.25U/. mu.L.
In the step (2), the ligase is one or more of Ampligase ligase, T4 DNA ligase, Taq DNA ligase and the like; preferably, it is Ampligase ligase.
In the step (2), the concentration of the ligase is 0.2-1.0U/mu L; preferably, it is 0.5U/. mu.L.
In the step (2), the connection temperature is 16-60 ℃; preferably, it is 60 ℃.
In the step (2), the connection time is 1-10 h; preferably, it is 2 h.
In the step (2), the concentration of the exonuclease I is 0.2-0.5U/mu L; preferably, it is 0.25U/. mu.L.
In the step (2), the concentration of the exonuclease III is 5-20U/mu L; preferably, it is 10U/. mu.L.
In the step (2), the digestion time is 1-4 h; preferably, it is 2 h.
In step (2), the temperature of the digestion is preferably 37 ℃.
Exonuclease I and exonuclease III are added in the step (2) for digesting redundant linear chains.
The purpose of adding the lock probe in the step (2) is to position the target region to be detected by a base complementary pairing principle so as to convert the target to be detected into the amplification of the lock probe.
The dNTPs are added in the step (2) in order to fill the gap in the form of 'gap-fill', and the gap formed after hybridization of the padlock probe and the target is extended by the action of polymerase, so that the padlock probe can be cyclized.
The polymerase is added in step (2) to act on the dNTPs to extend the space between the padlock probes so that the padlock probes can be circularized.
The ligase is added in step (2) in order to ligate the padlock probe into a loop.
And (3) in the step (2), the target-labeled hybrid padlock probe and the target to be detected form a stable duplex through a base complementary pairing principle.
The filling in step (2) means that the gap between the 3 'and 5' ends of the padlock probes is filled up by extension of dNTPs, and the two ends are adjacent to each other so that they can be ligated using a ligase.
In the step (3), the blocker is designed to be completely complementary with a known wild type target sequence, the 3' end of the primer 2 sequence and the 5' end of the blocker sequence have an overlapping region of 6-14 nt, and the 3' end of the blocker contains a sequence of 12-30 nt, which is not present in the primer 2.
Wherein, the known wild type target sequence specifically refers to:
TATAAACTTGTGGTAGTTGGAGCTGGTGGCGTAGGCAAGAGTGCCTTGACGATACAGC (SEQ ID NO.4) or
AGGGACCAGGTAAATATTTACCACGTCTTGGTGTTT ATTTTACCGTCTATATACAAG(SEQ ID NO.5) depending mainly on the target to be detected.
In the step (3), the sequence of the
In the step (3), the sequence of the primer 2 is as follows: CACTCTTGCCTACGCCA (SEQ ID NO.7) or CTTGTATATAGACGG TAAAATAAACACCAAGA (SEQ ID NO.8), which are designed according to the object.
In the step (3), the sequence of the blocker is as follows: GCCTACGCCACCAGCTCATTA (SEQ ID NO.9) or TAAACACCAAGACGTGGTAAATATTTACCTGGTAAAA (SEQ ID NO. 10).
In the step (3), the concentration of the blocking agent is 0.1-10 mu M; preferably, it is 1.5. mu.M.
In the step (3), the molar ratio of the blocking agent to the primer 2 is 1-10: 1; preferably, 3: 1.
in the step (3), the molar ratio of the blocking agent to the
in the step (3), the concentration of the
In the step (3), the concentration of the primer 2 is 0.1-1 mu M; preferably, it is 0.5. mu.M.
In the step (3), the concentration of the dNTPs is 200-400 mu M; preferably 200. mu.M.
In the step (3), the polymerase is one or more of vent (exo-) DNA polymerase, Bsu DNA polymerase, Bst 3.0DNA polymerase and the like; preferably, vent (exo-) DNA polymerase.
In the step (3), the concentration of the polymerase is 0.1-0.4U/mu L; preferably, it is 0.2U/. mu.L.
In the step (3), the amplification temperature is 58-63 ℃; preferably, it is 60 ℃.
In the step (3), the amplification time is 45-120 min; preferably, it is 90 min.
The polymerase is added in the step (3) to act on dNTPs to extend the
In step (4), the qualitative analysis is preferably performed by agarose gel electrophoresis.
Wherein, the concentration of the agarose gel is 1-3%; preferably, it is 3%.
Wherein the electrophoresis time is 2-3 h; preferably, it is 3 h.
Quantitative analysis was performed by adding SYBR Green I to the amplification product and incubating in step (4).
Wherein the concentration of SYBR Green I is 1-20 x; preferably, 20 x.
Wherein, the SYBR Green I can also be replaced by Gel red.
In the step (4), the incubation time is 10-60 min; preferably, it is 15 min.
In the step (4), the quantitative analysis is preferably carried out by measuring fluorescence in a fluorescence spectrophotometer.
Wherein the excitation wavelength of the spectrophotometer is 493 nm.
Wherein the emission wavelength of the spectrophotometer is 524 nm.
In a specific embodiment, the method specifically comprises the steps of:
(1) sample preparation:
1) genomic DNA extraction
a) Cultured monolayer human colon cancer cells SW48 and SW620 (adherent) were detached from the flask and collected in culture medium with pancreatin, and appropriate number of cells (not more than 5X 10) 6Cells) and normal chromosomes were transferred to a 1.5ml microcentrifuge tube and centrifuged at 300 Xg for 5 min. The supernatant was completely removed and discarded.
b) Cell particles were resuspended in PBS to a final volume of 200. mu.l.
c) Add 20. mu.L proteinase k.
d) Then 200. mu.L of buffer AL was added to the sample. Pulsed vortex mixing 15 s.
e) Incubate at 56 ℃ for 10 min.
f) A1.5 ml microcentrifuge tube was centrifuged to remove the droplets in the lid.
g) 200 mul of ethanol (96-100%) was added to the sample, followed by pulsed vortex stirring for 15 seconds. After mixing, a 1.5ml microcentrifuge tube was briefly centrifuged to remove the droplets in the cap.
h) Carefully transfer the mixture from step g to a QIAamp mini spin column (in a 2ml collection tube) without wetting the edges. The lid was closed and the centrifuge centrifuged at 8000rpm for 1 minute. The QIAamp mini spin column was placed in a clean 2ml collection tube and the collection tube containing the filtrate was discarded.
i) Open QIAamp mini spin columns, add 500. mu.L buffer AW1, without wetting the edges. The lid was closed and the centrifuge centrifuged at 8000rpm for 1 minute. The QIAamp mini spin column was placed in a clean 2ml collection tube and the collection tube containing the filtrate was discarded.
j) Open QIAamp mini spin column, add 500. mu.L buffer AW2 without wetting the edges. The lid and centrifuge were closed and centrifugation continued at full speed (20000rpm) for 3 minutes.
k) The QIAamp mini spin column was placed in a 1.5ml microcentrifuge tube, and the collection tube containing the filtrate was discarded. Open QIAamp mini spin column and add 200. mu.L of distilled water. Incubated at room temperature (15-25 ℃) for 5min, and then centrifuged at 8000rpm for 1 min.
l) UV measurement of A260/A280, quantification and determination of purity. The concentration is A260 x 50 ng/. mu.L, and the ratio of A260/A280 of pure DNA is 1.7-1.9. The length of the genomic DNA can be determined by agarose gel electrophoresis (PFGE).
2) Fragmentation of genomic DNA
The extracted DNA, Smart buffer (1X), BSA (0.3. mu.g/. mu.L), MseI enzyme (0.5U/. mu.L) were added to ice in this order, and then subjected to microcentrifugation, digestion in a water bath at 37 ℃ for 2 hours, inactivation in a water bath at 65 ℃ for 20min, and storage in a refrigerator at 4 ℃.
(2) And (3) hybridization and connection:
the fragmented DNA was denatured at 100 ℃ for 10min, and after 10min on ice, buffer (1X), padlock (100nM), dNTPs (200. mu.M), Taq polymerase (0.25U/. mu.L) and Ampligase ligase (0.5U/. mu.L) were added in this order, and they were allowed to vacancy-fill and ligated into a loop in a water bath at 60 ℃ for 2 h. Then adding exonuclease III (10U/. mu.L) and exonuclease I (0.25U/. mu.L), digesting redundant linear chains in water bath at 37 ℃ for 2h to obtain pure rings, inactivating enzyme activity in water bath at 80 ℃ for 20min, and storing in a refrigerator at 4 ℃.
(3) Blocking amplification
Water, ThermoPol buffer (1X), dNTPs (200. mu.M), the purified loop obtained in step (2), primer 1 (1. mu.M), primer 2 (0.5. mu.M), blocker (1.5. mu.M) and Vent (exo-) polymerase (0.2U/. mu.L) were sequentially added to ice, followed by microcentrifugation, denaturation in a water bath at 92 ℃ for 3min, amplification in a water bath at 60 ℃ for 90min, and storage in a refrigerator at 4 ℃.
(4) And (3) product detection:
electrophoresis on 3% agarose gel or fluorescence detection.
1) Electrophoresis: 3% agarose, 1 XTAE, 10. mu.L per well, electrophoresis at 100V for 3 h.
2) Fluorescence: mu.L of the sample was added to 195. mu.L of water and SYBR Green I was brought to a final concentration of 20X. Standing at room temperature in dark place for 15min, and measuring the maximum emission wavelength at 493nm excitation wavelength.
In step (1), please refer to the specific description for different kits.
Wherein, in the step (2), the specific amount of the DNA depends on the concentration of the extracted nucleic acid, and the total volume can be set by self.
The innovation point of the invention is mainly embodied in that the rolling circle isothermal amplification technology is combined with a competitive detection system, the defects of temperature change and high cost required by PCR are solved, and the sensitivity and specificity of rolling circle amplification are improved. The method is well suited for detecting hot spot mutations and mutations in circular nucleic acids.
The invention can simply, conveniently, quickly and selectively detect the unknown mutation of the low-frequency single base contained in the complete genome DNA by the detection method for the ultra-sensitively and specifically enriching the single base mutation in the nucleic acid by selectively competing and triggering the isothermal hyperbranched rolling circle amplification through the blocker, thereby solving the defects of temperature change and high cost required by PCR and simultaneously improving the sensitivity and specificity of the rolling circle amplification. The method is very suitable for the enrichment of hot spot mutation and the direct detection of circular nucleic acid mutation.
In the invention, the detection method for ultra-sensitively and specifically enriching single base mutation in nucleic acid by selectively competing and triggering isothermal hyperbranched rolling circle amplification through the blocker can be used as a mutation enrichment method.
In the invention, the detection method for ultra-sensitively and specifically enriching single base mutation in nucleic acid by selectively competing and triggering isothermal hyperbranched rolling circle amplification through the blocker can be used as a mutation detection method.
In the invention, the detection method for inducing isothermal hyperbranched rolling circle amplification through selective competition of the blocking agent and enriching single base mutation in nucleic acid with ultrasensitiveness and specificity can sensitively identify and selectively amplify single base mutation with allele frequency of 0.1% at constant temperature.
The method for detecting the single base mutation in the nucleic acid by inducing isothermal hyperbranched rolling circle amplification through selective competition of the blocking agent and enriching the single base mutation in the nucleic acid in an ultrasensitive and specific manner is used for detecting the KRAS mutation and has the characteristics of ① isothermal amplification, no need of a precise temperature control instrument, high specificity and sensitivity of ②, capability of detecting the single base mutation as low as 0.1 percent, capability of detecting unknown single base mutation of ③ and capability of directly detecting the mutation in the circular DNA of ④.
In the invention, the detection method for the ultra-sensitively and specifically enriching the single base mutation in the nucleic acid by selectively competing and triggering the isothermal hyperbranched rolling circle amplification through the blocker comprises but is not limited to the detection of DNA mutation and the detection of RNA mutation.
In the present invention, the detection method for ultra-sensitively and specifically enriching single base mutation in nucleic acid by selectively competing and triggering isothermal hyperbranched rolling circle amplification by a blocker includes but is not limited to the detection for KRAS mutation.
The invention also provides the application of the detection method in the detection of single base mutation of the extracted DNA or RNA.
The invention also provides the application of the detection method in the detection of the single base mutation of the in-situ DNA or RNA of the cell.
The invention also provides the application of the detection method in DNA methylation detection.
In the present invention, all sequences used can be obtained by extraction from an organism or by artificial synthesis. The invention has the beneficial effects that: the method for detecting the single base mutation in the nucleic acid by inducing isothermal hyperbranched rolling circle amplification through selective competition of the blocking agent and enriching the single base mutation in the nucleic acid in an ultrasensitive and specific manner has the advantages of simple and easily-operated required instruments and low cost. The detection method provided by the invention is used for detecting the single base mutation of the nucleic acid, and has the advantages of isothermal amplification, low requirement on equipment, economy, simplicity, low-frequency mutation detection, unknown mutation detection and hot spot enrichment realization. The detection method provided by the invention is used for detecting cancer-related mutation and has potential application value.
Drawings
FIG. 1 is an experimental schematic diagram of the detection method for ultra-sensitively and specifically enriching single base mutation in nucleic acid by using blocker selective competition to initiate isothermal hyperbranched rolling circle amplification.
FIG. 2 is a sensitivity analysis of the detection method.
FIG. 3 shows the fluorescence and sequencing results of KRAS mutation in genomic DNA extracted from human colon cancer cells.
Detailed Description
The present invention will be described in further detail with reference to the following specific examples and the accompanying drawings. The procedures, conditions, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited.