CN106755284B - Cascade amplification DNA machine based on label-free molecular beacon and application - Google Patents
Cascade amplification DNA machine based on label-free molecular beacon and application Download PDFInfo
- Publication number
- CN106755284B CN106755284B CN201611063378.1A CN201611063378A CN106755284B CN 106755284 B CN106755284 B CN 106755284B CN 201611063378 A CN201611063378 A CN 201611063378A CN 106755284 B CN106755284 B CN 106755284B
- Authority
- CN
- China
- Prior art keywords
- rmb
- smb
- dna
- region
- tep
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
- C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/91—Transferases (2.)
- G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- G01N2333/91205—Phosphotransferases in general
- G01N2333/91245—Nucleotidyltransferases (2.7.7)
- G01N2333/9125—Nucleotidyltransferases (2.7.7) with a definite EC number (2.7.7.-)
- G01N2333/9128—RNA-directed DNA polymerases, e.g. RT (2.7.7.49)
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a cascade amplification DNA machine based on a label-free molecular beacon and application thereof, wherein the cascade amplification DNA machine comprises T7Exo, RMB and SMB, and the RMB and SMB contain a protruded 5' end and are protected from being cut by T7 Exo. First, telomerase extends the TS primer, generating a DNA containing tandem repeats (TTAGGG)nThe TEP of (1). Then, TEP hybridizes with RMB, activating the DNA machinery, unwinding RMB containing a blunt or concave 5' end, leaving it unprotected, initiating cyclic cleavage of T7Exo, releasing the intact TEP and a large number of DNA fragments (priming DNA), and performing primary amplification. Next, DNA-specific opening SMB was primed and cycled by T7Exo, releasing large amounts of G4 sequence, enabling secondary amplification. Finally, the TEP and released G4 sequences strongly interact with NMM, together producing significantly enhanced fluorescence.
Description
Technical Field
The invention relates to a label-free molecular beacon-based cascade amplification DNA machine and application.
Background
Human telomerase is a ribonucleoprotein reverse transcriptase consisting of telomerase rna (htr) and telomerase reverse transcriptase. Telomerase is produced by a three-step process: hTR binds to the 3 'end of telomeric DNA, nucleotide incorporation, separation of the extended DNA strands terminates synthesis or migrates to generate yet another cycle of DNA synthesis responsible for the addition of tandem repeats at the 3' end of the telomere. Telomerase function has an important role in the proliferation and differentiation of cells, and enhancement of its activity can lead to continued division and even immortalization of cells. Most human cancers express high telomerase activity, while normal somatic cells express low telomerase activity. Sensitive detection of telomerase activity is of great significance for cancer diagnosis and development of anti-cancer drugs.
The traditional method for detecting telomerase activity is the Polymerase Chain Reaction (PCR) -based Telomeric Repeat Amplification Protocol (TRAP). However, the radioactive hazards and poorly quantifiable electrophoretic consequences of TRAP limit their widespread use. In addition, a number of PCR-free isothermal amplification assays have been proposed for the detection of telomerase activity. In particular, due to the safety and simplicity of fluorescence methods, a variety of fluorescence-based analytical methods have been developed. Of these assays, nuclease-assisted signal amplification has attracted considerable attention due to its high amplification efficiency. Nuclease-assisted assays can be broadly divided into two categories. One is based on polymerase-assisted cyclic amplification. After the primer of the Telomerase Substrate (TS) is extended, the primer probe can be bound with the template to generate polymerization reaction under the action of polymerase. However, polymerase-based methods often produce a non-specific background, since non-specific probes may be able to act as priming probes, where the amplification reaction occurs at the high DNA replication activity of the polymerase. To avoid the above-mentioned deficiencies, another class of exonuclease-assisted based cycling amplifications has received much attention. After the TS primer extension reaction, the fluorescent probe can be cleaved by an exonuclease, moving the fluorophore and the quencher away from each other, resulting in an enhanced fluorescent signal. Unfortunately, the above-described assay methods involve labeling of the DNA probe, not only to reduce the probe binding efficiency due to steric effects, but also to high background due to incomplete quenching of the fluorophore by the quencher.
Disclosure of Invention
In order to solve the defects of the prior art, the invention provides a cascade amplification DNA machine based on an unmarked molecular beacon and application thereof, which can improve the sensitivity of telomerase activity detection.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a label-free molecular beacon group comprises a label-free identification molecular beacon (RMB) and a label-free Signaling Molecular Beacon (SMB),
the RMB comprises, in order from a 5 ' end to a 3 ' end, a RMB overhang foothold region, a RMB first stem region, a RMB spacer strand, and a RMB second stem region, the RMB first stem region being fully complementary to the RMB second stem region to form a RMB stem strand, such that the RMB overhang foothold region forms a protruding 5 ' end of the RMB, the RMB overhang foothold region and the RMB first stem region form a region capable of hybridizing to a Telomerase Extension Product (TEP), and a portion of the RMB spacer strand forms a priming DNA region with a portion of the RMB second stem region;
the SMB includes SMB overhang standing point region, the first stem region of SMB, RMB interval chain and SMB second stem region by 5 ' end to 3 ' end in proper order, the first stem region of SMB with SMB second stem region complete complementary formation SMB stem chain makes SMB overhang standing point region form the convex 5 ' end of SMB, SMB overhang standing point region and the first stem region of SMB form can with the region that initiates DNA hybridization, part SMB interval chain and part SMB second stem region form G4 sequence.
The TEP is a product obtained by performing extension reaction on the TS primer by using telomerase.
The extension reaction is a reaction for extending the TS primer by using the activity of telomerase and generating a plurality of TTAGGG repeated sequences by extending the 3' end of the TS primer.
The G4 sequence is a DNA single strand rich in guanine base (G), and can be folded into a G-tetraploid structure under certain ionic strength. The structure consists of stacked planes of G-quartets, with the guanine bases of each plane interacting through Hoogsteen hydrogen bonds.
Preferably, the nucleotide sequence of RMB is: the nucleotide sequence of RMB-10. The sequence is (5 '-3'), CTAACC CTA ACC CTA ACT CTG CTC TTT ACG GGT TGG GGC TGA GTT AGGG.
Wherein, the structure subregion is:
CC CTA ACT CTG CTCTTT ACG GGT TG
wherein the italic portion is the RMB overhang foothold area, the underlined portion is the RMB first stem area, the normal portion is the RMB spacer chain, and the shaded portion is the RMB second stem area.
The functional partition is as follows:CTA ACC CTA ACC CTA ACT CTG CTCTTT
g G, wherein the underlined sections are the RMB overhang foothold region and the RMB first stem region for hybridization to TEP and the italic section is the priming DNA region for priming secondary amplification.
Preferably, the nucleotide sequence of SMB is: nucleotide sequence of SMB-1. The sequence is CTA ACT CAGCCC CAA CCC GTG GGT AGG GCG GGT TGG GGC T.
Wherein, the structure subregion is:
AG CCC CAA CCC GTG GGT A
wherein, the italic part is SMB suspension foothold area, and the underline part is the first stem region of SMB, and normal part is SMB interval chain, and the shadow part is SMB second stem region.
The functional partition is as follows:CTA ACT CAG CCC CAA CCC GT
GC T, with the underlined sections being the SMB overhang foothold region and the SMB first stem region for hybridization to the priming DNA and the italics section being the G4 sequence for signal export.
An application of the label-free molecular beacon group in telomerase activity detection.
A cascade DNA amplification machine based on label-free molecular beacons comprises the label-free molecular beacon group and exonuclease.
Preferably, the exonuclease is T7 exonuclease (T7 Exo).
An application of the above cascade amplification DNA machine based on the marker-free molecular beacon in telomerase activity detection.
A method for detecting telomerase activity by a cascade amplification DNA machine based on a label-free molecular beacon comprises the steps of initiating an extension reaction of a TS primer by telomerase in a cell extracting solution to obtain TEP, adding the TEP into the DNA machine to carry out the cascade amplification reaction, and then carrying out fluorescence detection on an amplification system to obtain fluorescence intensity so as to establish a linear equation between the fluorescence intensity and the concentration of the cell extracting solution; and finally substituting the obtained fluorescence intensity of the sample to be detected into the linear equation to calculate the cell concentration of the sample to be detected.
The 'process' in the process of obtaining the fluorescence intensity of the sample to be detected by carrying out the process on the sample to be detected is to carry out extension reaction on the sample to be detected to obtain TEP, then add the TEP into the DNA machine to carry out cascade amplification reaction, and then carry out fluorescence detection on a mixed system obtained after the cascade amplification reaction to obtain the fluorescence intensity.
The amplification system is a mixed system obtained after cascade amplification reaction.
The cascade amplification reaction of the invention connects two or more signal amplification processes in a certain way, namely, the previous amplification reaction can excite the next amplification reaction after the previous amplification reaction is carried out, thereby achieving the effect of improving the amplification efficiency.
Preferably, the mixed system obtained after the cascade amplification reaction is mixed with N-methylporphyrindipropionic acid IX (NMM) and then subjected to fluorescence detection.
Preferably, the telomerase is taken from a HeLa cell.
Among them, HeLa cell is a cell used in biological and medical research, and is derived from the cervical cancer cell line of Haelietta-Lax (Henrietta locks), a U.S. female.
Preferably, the steps are as follows:
(1) respectively mixing cell extracting solutions with different concentrations with TS primers, and incubating at constant temperature to enable telomerase to extend the TS primers to respectively obtain TEPs with different cell numbers;
(2) respectively adding TEP generated by reaction of cell extracting solutions with different concentrations obtained in the step (1) into the cascade amplification DNA machine, and incubating at constant temperature to enable the TEP to perform cascade amplification reaction in the cascade amplification DNA machine;
(3) obtaining the fluorescence intensity of different mixed systems incubated in the step (3) through fluorescence detection;
(4) establishing a linear equation between the fluorescence intensity and the concentration of the cell extracting solution according to the fluorescence intensity obtained in the step (3);
(5) and (4) substituting the fluorescence intensity obtained after the step (1) to the step (3) of the sample to be detected into the linear equation in the step (4), and calculating to obtain the cell concentration of the sample to be detected.
The nucleotide sequence of TEP is:AAT CCG TCG AGC AGA G 
wherein the underlined part is the TS primer sequence and the tilted part is the primer extension sequence, as reported in the literature (X.ren, H.Li, R.W.Clarke, D.A.Alves, L.ying, D.Klenerman, S.Balasubramanian, J.Am.Chem.Soc.2006,128, 4992-5000; G.Zhu, K.Yang, C.Zhang, Chem.Commun.2015,51,6808-6811), the value of n is 1,2, and 3 accounts for 90% in all cases.
The telomerase extract in step (1) can be prepared according to the method described in the literature (X.xu, L.Wang, Y.Huang, W.Gao, K.Li, W.Jiang, anal.chem.2016,88, 9885-containing 9889).
Further preferably, the incubation conditions in step (1) are: incubate at 37 ℃ for 2 h.
More preferably, the system mixed in the step (1) is: 10 μ L of 5 XTAP buffer, 5 μ L of dNTPs at a concentration of 10mM, 5 μ L of BSA at a concentration of 1mg/mL, 5 μ L of TS primer at a concentration of 5 μ M, 5 μ L of telomerase extract and 20 μ L of DEPC (diethylpyrocarbonate) -treated ultrapure water.
The telomerase extracting solution is a diluent of a cell extracting solution by adopting a 1 XCHAPS lysis buffer solution.
The 1 XCAPS lysis buffer was 10mM Tris-HCl, pH 7.5, 1mM MgCl21mM EGTA, 5 mMB-mercaptoethanol, 0.1mM PMSF, 0.5% CHAPS, 10% glycerol.
Still more preferably, the 5 XTAP buffer is 100mM Tris-HCl, 7.5mM MgCl2315mM KCl, 0.025% Tween-20, 5mM EGTA, pH 8.3.
Further preferably, the machine for cascade amplification of DNA in step (2) is: mu.L of RMB at a concentration of 1. mu.M, 5. mu.L of SMB at a concentration of 10. mu.M, 3. mu.L of T7Exo at a concentration of 10U/. mu.L, 4. mu.L of 10 XT 7Exo reaction buffer and 18. mu.L of DEPC-treated ultrapure water.
Still more preferably, the 10 × T7Exo reaction buffer is: 20mM Tris-Ac, pH7.9, 50mM KAc,10mM Mg (Ac)2And 1mM DTT.
Further preferably, RMB and SMB require annealing by heating at 95 ℃ for 5min before preparing the tandem amplification DNA machine. The annealing by heating at 95 ℃ for 5min comprises the following steps: keeping at 95 deg.C for 5min, and slowly cooling to room temperature for at least 1 h.
Further preferably, in the step (3), the mixed system obtained in the step (2) is added with 5. mu.L of NMM with a concentration of 25. mu.M and 5. mu.L of KCl with a concentration of 100mM, and after incubating together at 37 ℃ for 0.5h, fluorescence detection is performed.
Further preferably, in step (3), the fluorescence detection parameters are: lambda [ alpha ]ex＝399nm，λemThe voltage of the PMT detector was 700V at 612 nm. Wherein λexAt the maximum excitation wavelength, λemIs the maximum emission wavelength.
Further preferably, in step (4), the linear equation is established as follows: Δ F ═ 0.4707 × C + 38.87.
Wherein, F-F0F is the fluorescence intensity of the sample to be measured, F0The fluorescence intensity of the sample without the telomerase extracting solution, C is the concentration of HeLa cells in the sample to be detectedBit: cell/mL, linear range is: 50 cells/mL-2000 cells/mL, R2＝0.993。
The application of the present invention to the detection of telomerase activity and the method for detecting telomerase activity can be aimed at either disease diagnosis or treatment or non-disease diagnosis or treatment.
A kit for detecting telomerase activity comprises a TS primer, the label-free molecular beacon group, an extension reaction solution and a cascade amplification solution.
Preferably, the extension reaction solution comprises 5 × TRAP buffer, dNTPs and BSA.
Further preferably, the 5 XTAP buffer is 100mM Tris-HCl, 7.5mM MgCl2315mM KCl, 0.025% Tween-20, 5mM EGTA, pH 8.3.
Preferably, the cascade amplification solution comprises T7Exo and 10 × T7Exo reaction buffers.
Further preferably, the 10 XT 7Exo reaction buffer is 20mM Tris-Ac, pH7.9, 50mM KAc,10mM Mg (Ac)2And 1mM DTT.
The principle of the telomerase activity detection method based on the label-free molecular beacon-mediated cascade amplification DNA machine is as follows:
the RMB contains a pendant toehold region and its stem strand attached to it for hybridization with the TEP, and priming DNA partially enclosed in the stem for priming secondary amplification. SMB comprises a toehold region with its attached stem strand for hybridization to priming DNA, and a partially blocked G4 sequence for signal export. First, telomerase triggers an extension reaction of TS primer, producing a DNA with tandem repeat sequence (TTAGGG)nThe TEP of (1). Then, the toehold regions of TEP and RMB and the linked stem strands hybridize, unfolding the hairpin structure, resulting in a flat or concave 5' end. T7Exo is a sequence-independent nuclease capable of specifically catalyzing the stepwise cleavage of single nucleotides from the blunt or concave 5' end of double-stranded DNA. Under the action of T7Exo, TEP is circulated to release a large amount of priming DNA, and primary amplification is realized. The released priming DNA hybridizes to SMB, producing a DNA duplex with a concave 5' end. In double strands under the action of T7ExoSMB was cleaved to release the G4 sequence, allowing secondary amplification. Finally, the TEP and G4 sequences strongly interact with NMM, together resulting in significantly enhanced fluorescence. The schematic diagram of the detection is specifically shown in fig. 1.
The invention has the beneficial effects that:
1. the method does not need to carry out fluorescence labeling, and can be used for sensitive detection of telomerase activity.
2. According to the invention, telomerase activity in the natural HeLa cell extracting solution which is equivalent to 50 cells/mL is successfully detected, and the linear change range is from 50 cells/mL to 2000 cells/mL.
3. The method is successfully used for the inhibition effect analysis of the telomerase inhibitory drug, and proves that the strategy has the potential of telomerase inhibitor screening.
Drawings
FIG. 1 is a schematic diagram of a fluorescence analysis method for the specific and sensitive detection of telomerase activity;
FIG. 2 is a fluorescence spectrum of a system comprising (a) TS primer, RMB, SMB and T7 Exo; (b) CSTP, RMB, SMB and T7 Exo;
fig. 3 is a graph of detection of telomerase activity in natural lysates of HeLa cells, a: fluorescence spectrum, a: negative, b: first-order amplification, c: cascade amplification, B: non-denaturing polyacrylamide gel (15%) electrophoresis analysis of the principle of fluorescence analysis of telomerase activity, channel 1: TS primer, channel 2: TS primer and HeLa cell extract, channel 3: RMB, channel 4: SMB, channel 5: RMB and T7Exo, channel 6: SMB and T7Exo, channel 7: RMB, T7Exo, TS primers and HeLa cell extracts, channel 8: RMB, T7Exo, TS primers, HeLa cell extract and SMB, channel 9: standard band (marker);
FIG. 4 shows F/F of different RMBs and different SMBs0Wherein F and F0Fluorescence intensity of the samples with and without HeLa cell extract, a: effect of RMBs of different foothold areas, B: the effect of SMBs containing a different complementary nucleotide base from the priming DNA;
FIG. 5, the left graph shows fluorescence curves obtained for different concentrations of HeLa cell extracts, and the right graph shows a linear relationship between fluorescence intensity and concentration of HeLa cell extracts;
FIG. 6 shows the specificity of the telomerase activity detection strategy;
FIG. 7 shows the relative fluorescence intensity with different AZT concentrations (0,0.5,1.0,2.0,5.0,10.0 and 15.0. mu.M).
Detailed Description
The present invention will be further described with reference to the following examples and accompanying drawings.
Reagent
The oligonucleotides were synthesized and purified by Shanghai Biotech, Inc. T7Exo was supplied by new england biosciences. Bovine Serum Albumin (BSA) and 3 '-azido-3' -deoxythymidine (AZT) were supplied by Sigma-Alrich. Deoxynucleotide triphosphates (dNTPs) and ethylene glycol ethylene ether diamine tetraacetic acid (EGTA) were obtained from Shanghai Producer, Inc. NMM is provided by J & K science, inc. 1 × CHAPS lysis buffer was purchased from Millipop. All water used in the experiment was nuclease-free ultrapure water (18.25 M.OMEGA.. multidot.cm), and all other reagents were of analytical grade.
Telomerase extension reaction
Telomerase extracts from HeLa cells were prepared according to the reported method (X.xu, L.Wang, Y.Huang, W.Gao, K.Li, W.Jiang, anal.chem.88(2016) 9885-. Then, 10. mu.L of 5 XTAP buffer (100mM Tris-HCl, 7.5mM MgCl)2315mM KCl, 0.025% Tween-20, 5mM EGTA, pH 8.3), 5. mu.L of dNTPs at a concentration of 10mM, 5. mu.L of BSA at a concentration of 1mg/mL, 5. mu.L of TS primer at a concentration of 5. mu.M, 5. mu.L of HeLa cell lysate and 20. mu.L of DEPC-treated ultrapure water to obtain an extension reaction mixture system, and incubating at 37 ℃ for 2 hours. In the control experiment, HeLa cell lysate was first heat treated at 90 ℃ for 10 min. In an inhibition experiment, AZT solutions with different dosages are added into a telomerase extension reaction buffer solution for reaction for 6 hours.
Cascade amplification reaction of DNA machine
RMB and SMB were first annealed by heating at 95 ℃ for 5 min. Then, 5. mu.L of the extension reaction mixture and 5. mu.L of 1. mu.M RMB, 5. mu.L of 10. mu.M SMB, 3. mu.L of concentrated solutionT7Exo at a concentration of 10U/. mu.L, 10 XT 7Exo reaction buffer (20mM Tris-Ac, pH7.9, 50mM KAc,10mM Mg (Ac) 4. mu.L2And 1mM DTT) and 18. mu.L of DEPC treated ultrapure water. The mixture was reacted at 25 ℃ for 2h to allow a cascade amplification reaction of the DNA machinery to occur. For the fluorescent response of single-stage amplification, an unlabeled Molecular Beacon (MBP) with a TEP recognition sequence and a signal export sequence was designed. mu.L of the extension reaction mix, 5. mu.L of 1. mu.M MBP and 30U T7Exo in 1 XT 7Exo reaction buffer at 25 ℃ for 2h, allowing single first stage amplification to occur.
Fluorescence test and gel electrophoresis
After the cascade amplification reaction in the DNA machine, 5. mu.L of NMM at a concentration of 25. mu.M and 5. mu.L of KCl at a concentration of 100mM were added to the resulting mixed system, and incubated together at 37 ℃ for 0.5 h. Fluorescence was measured with an F-7000 spectrometer (Hitachi, Japan). The spectrometer parameters were set as follows: lambda [ alpha ]ex＝399nm,λemThe voltage of the PMT detector was 700V at 612 nm.
15% polyacrylamide gel electrophoresis (PAGE) was performed in 1 XTBE buffer running at 30mA current for approximately 1.0 h. Subsequently, the gel was stained with Ethidium Bromide (EB) and imaged with a UV imaging system.
Principle of telomerase activity analysis method
The telomerase activity detection method based on the marker-free molecular beacon-mediated cascade amplification DNA machine is shown in FIG. 1. The RMB contains a pendant foothold region and stem strands linked for hybridization to the TEP, and priming DNA partially enclosed in the stem to prime secondary amplification. SMB consists of a foothold area with its attached stem strand for hybridization with priming DNA, and a G4 sequence partially enclosed in the stem for signal export. First, telomerase triggers an extension reaction of TS primer, producing a DNA with tandem repeat sequence (TTAGGG)nThe TEP of (1). Then, the TEP and RMB toehold regions and their associated stem strands hybridize, spreading the hairpin structure of RMB, resulting in a blunt or concave 5' end. T7Exo is a sequence-independent nuclease capable of specifically catalyzing the stepwise cleavage of single nucleotides from the blunt or concave 5' end of double-stranded DNA. Under the action of T7Exo, TEP is cycled, releasing large amounts of priming DNA, enabling first-order amplification. The released priming DNA hybridizes to SMB, producing a DNA duplex with a concave 5' end. Under the action of T7Exo, SMB in the double strand is cut to release G4 sequence, so as to realize secondary amplification. Finally, the TEP and G4 sequences strongly interact with NMM, together resulting in significantly enhanced fluorescence.
Feasibility study
Firstly, the feasibility is verified by using a Chemically Synthesized Telomerase Product (CSTP), wherein four telomere repetitive sequences are designed on the basis of a TS primer, namely the nucleotide sequence of the CSTPAAT CCG TCG AGC AGA G(TTA GGG)nWherein n is 4, and 4 TTAGGG repetitive sequences are shown in the table 1. FIG. 2 shows that the system containing TS primer, RMB, SMB and T7Exo showed very low fluorescence intensity, while the fluorescence intensity was significantly increased when TS primer was replaced with CSTP. The above results show that the unlabeled molecular beacon is cleaved by T7Exo only after hybridizing with the synthesized TTAGGG repetitive sequence, so that a large amount of G4 structures are released, and a remarkably enhanced fluorescence signal is obtained.
The proposed strategy was then used to detect telomerase activity in native lysates of HeLa cells. As shown in fig. 3A, the mixture containing TS primer, RMB, SMB and T7Exo (negative) showed very low fluorescence intensity, demonstrating that RMB and SMB with 5' footholds are not cleaved by T7Exo in the absence of telomerase, ensuring low background. However, the fluorescence intensity was significantly enhanced after adding the HeLa cell extract. Meanwhile, compared with single-stage amplification, the cascade amplification shows higher fluorescence enhancement, and shows the high amplification efficiency of the cascade strand displacement amplification in the strategy.
The reaction principle was further verified using PAGE (15%), as shown in fig. 3B. TEP was generated after incubation of TS primers with HeLa cell extract (channel 2). There was no significant change between channels 3 and 5, and channels 4 and 6, indicating that RMB and SMB were stable against T7 Exo. The observed mobility retarded band and the missing RMB band in channel 7 indicate that RMB is degraded after addition of cell extract and that primary amplification is successfully performed in the presence of telomerase. The strong band of mobility retardation and the band of significantly shallower SMB observed in channel 8, demonstrate that further SMB is degraded and secondary amplification proceeds successfully.
Probe optimization
The reaction conditions usually have a large influence on the sensing performance, so F/F is used0Values (F and F)0The fluorescence intensity of the sample with and without the HeLa cell extract, respectively) as a standard.
In this cascade DNA amplification machine, hybridization between TEP and RMB, priming DNA and SMB play an important role in generating amplification signals. First, hybridization between TEP and RMB was studied. On the one hand, if the 5' foothold area of the RMB is too short, it will increase the difficulty of the TEP opening the RMB. On the other hand, since the number of extended repeat units is typically 1,2 and 3, which are in each case 90% in proportion, the excessively long toehold area of the RMB may in turn result in the RMB still having a protruding 5' end after bonding with the TEP. Four RMBs (RMB-7, RMB-10, RMB-13 and RMB-16, see Table 1 for sequence) were thus prepared with different length 5' footholds. FIG. 4A shows that RMB-10 has the largest F/F0Value, therefore, RMB-10 is preferred for subsequent experiments. In addition, in order to obtain efficient hybridization between SMB and the priming DNA released in the primary amplification, four SMBs (SMB-0, SMB-1, SMB-2 and SMB-3, sequences shown in Table 1) having different numbers of complementary nucleotide bases from the priming DNA were prepared. FIG. 4B shows that SMB-1 has the largest F/F0Value, therefore SMB-1 is preferred for subsequent experiments.
TABLE 1 oligonucleotide sequences used in this experiment
Labeling: the foothold region and stem complement of the probe are italicized and underlined, respectively.
Analytical Performance of the telomerase Activity assay
Under the optimal conditions, the sensitivity and the detection range are researched by using HeLa cell extracting solutions with different concentrations. As shown in the left panel of FIG. 5, the fluorescence intensity gradually increased as the concentration of the HeLa cell extract was increased from 50HeLa cells/mL to 50000HeLa cells/mL. FIG. 5 shows, on the right, that good linearity is obtained in the range of 50 cells/mL to 2000 cells/mL. The linear equation is Δ F ═ 0.4707 × C +38.87, where C is the concentration of HeLa cell extract and R is2＝0.993，ΔF＝F-F0F and F0The fluorescence intensity of the samples with and without HeLa cell extract, respectively.
Fig. 6 shows that heat-inactivated HeLa cell extracts have low intensity close to background levels, demonstrating that the fluorescence response is dependent only on telomerase activity. In addition, for the somatic cell extract, typically, the HL-7702 cell extract had only weak fluorescence enhancement, but the HeLa cell extract and the HepG2 cell extract had significant fluorescence enhancement. The difference in the fluorescence response between the extracts from HeLa cells and HepG2 cells was due to the difference in the expression of telomerase activity. The results show that the cascade amplification DNA machine has high specificity and universality and has wide prospect of further clinical application.
Precision and reproducibility
Precision and reproducibility were studied in order to evaluate the authenticity and reliability of the experimental results. The Relative Standard Deviations (RSD) of the same batches in HeLa cell extracts of 400 cells/mL, 1200 cells/mL and 2000 cells/mL were 4.3%, 2.0% and 3.1%, respectively. The RSD of the same samples were measured for the different batches over three days to be 5.7%, 2.6% and 3.2%, respectively. The above results demonstrate that the cascade DNA amplification machine of the present invention has satisfactory precision and reproducibility.
Inhibition assay
Inhibition experiments were studied using AZT as a model inhibitor. As shown in fig. 7, the relative fluorescence intensity gradually decreased with increasing AZT dose, demonstrating the potential application of the strategy in inhibitor screening and drug studies related to telomerase activity. It was also further confirmed that the cleavage of the probe by T7Exo was dependent only on telomerase activity.
Conclusion
In summary, the cascade amplification DNA machine of the present invention develops a fluorescence strategy for sensitive detection of telomerase activity. The cascade DNA amplification machine of the present invention is characterized in the following aspects: (1) the telomerase activity in the natural HeLa cell extracting solution which is equivalent to 50 cells/mL is successfully tested by the ingenious design of a marker-free molecular beacon mediated cascade amplification DNA machine; (2) unlike exonuclease III, T7Exo catalyzes the removal of mononucleotides from the flat or concave 5 'end of double-stranded DNA, protects the extended repeat sequence from non-specific cleavage under the protection of the 5' end of the TS primer, and ensures the detection accuracy; (3) in the label-free homogeneous phase design, the probe does not need any chemical modification, and is simple, economic and easy to automate. The results prove that the cascade amplification DNA machine has the potential of detecting the activity of telomerase and developing telomerase related anti-cancer drugs.
Although the embodiments of the present invention have been described with reference to the accompanying drawings, it is not intended to limit the scope of the invention, and it should be understood by those skilled in the art that various modifications and variations can be made without inventive faculty, based on the technical solutions of the present invention.
SEQUENCE LISTING
<110> Shandong university
<120> cascade amplification DNA machine based on label-free molecular beacon and application
<130>2016
<160>11
<170>PatentIn version 3.5
<210>1
<211>18
<212>DNA
<213> primer
<400>1
aatccgtcga gcagagtt 18
<210>2
<211>40
<212>DNA
<213> Artificial sequence
<400>2
aatccgtcga gcagagttag ggttagggtt agggttaggg 40
<210>3
<211>46
<212>DNA
<213> Artificial sequence
<400>3
accctaaccc taactctgct ctttacgggt tggggctgag ttaggg 46
<210>4
<211>49
<212>DNA
<213> Artificial sequence
<400>4
ctaaccctaa ccctaactct gctctttacg ggttggggct gagttaggg 49
<210>5
<211>52
<212>DNA
<213> Artificial sequence
<400>5
accctaaccc taaccctaac tctgctcttt acgggttggg gctgagttag gg 52
<210>6
<211>55
<212>DNA
<213> Artificial sequence
<400>6
ctaaccctaa ccctaaccct aactctgctc tttacgggtt ggggctgagt taggg 55
<210>7
<211>39
<212>DNA
<213> Artificial sequence
<400>7
ctaactcagc cccaacccgg ggtagggcgg gttggggct 39
<210>8
<211>40
<212>DNA
<213> Artificial sequence
<400>8
ctaactcagc cccaacccgt gggtagggcg ggttggggct 40
<210>9
<211>41
<212>DNA
<213> Artificial sequence
<400>9
ctaactcagc cccaacccgt agggtagggc gggttggggc t 41
<210>10
<211>42
<212>DNA
<213> Artificial sequence
<400>10
ctaactcagc cccaacccgt aagggtaggg cgggttgggg ct 42
<210>11
<211>36
<212>DNA
<213> Artificial sequence
<400>11
ctaaccctaa ccctaacccg ggtagggcgg gttggg 36
Claims (7)
1. A kit for detecting telomerase activity for non-disease diagnosis or treatment is characterized by comprising a TS primer, a label-free molecular beacon group, an extension reaction solution and a cascade amplification solution; the label-free molecular beacon group comprises a label-free recognition molecular beacon RMB and a label-free signal molecular beacon SMB,
the RMB comprises, in order from a 5 ' end to a 3 ' end, an RMB overhang foothold region, an RMB first stem region, an RMB spacer strand, and an RMB second stem region, wherein the RMB first stem region is fully complementary to the RMB second stem region to form an RMB stem strand, such that the RMB overhang foothold region forms a protruding 5 ' end of the RMB, the RMB overhang foothold region and the RMB first stem region form a region capable of hybridizing to a telomerase extension product TEP, and a portion of the RMB spacer strand overlaps a portion of the RMBThe second stem region forms a priming DNA region; the nucleotide sequence of the RMB is as follows: the nucleotide sequence of RMB-10 is SEQ ID NO. 4; the nucleotide sequence of the TEP is as follows: (TTAAT CCG TCG AGC AGAG AGGG)nwherein the underlined part is a TS primer sequence and the inclined part is a primer extension sequence;
the SMB sequentially comprises an SMB suspension foot point region, an SMB first stem region, an SMB interval chain and an SMB second stem region from 5 ' end to 3 ' end, wherein the SMB first stem region and the SMB second stem region are completely complementary to form the SMB stem chain, the SMB suspension foot point region forms a protruded 5 ' end of the SMB, the SMB suspension foot point region and the SMB first stem region form a region capable of hybridizing with initiation DNA, and part of the SMB interval chain and part of the SMB second stem region form a G4 sequence; the nucleotide sequence of the SMB is as follows: the nucleotide sequence SEQ ID NO.8 of SMB-1;
the extension reaction solution comprises 5 xTRAP buffer solution, dNTPs and BSA;
the cascade amplification solution included T7Exo and 10 xt 7Exo reaction buffer.
2. Use of the kit of claim 1 for the detection of telomerase activity for non-disease diagnostic or therapeutic purposes.
3. A method for detecting telomerase activity for non-disease diagnostic or therapeutic purposes based on the kit of claim 1, comprising the steps of: initiating an extension reaction of a TS primer by telomerase in a cell extracting solution to obtain TEP, adding the TEP into a cascade amplification solution containing a label-free molecular beacon group to perform a cascade amplification reaction, and performing fluorescence detection on an amplification system to obtain fluorescence intensity so as to establish a linear equation between the fluorescence intensity and the concentration of the cell extracting solution; and finally substituting the obtained fluorescence intensity of the sample to be detected into the linear equation to calculate and obtain the cell concentration of the sample to be detected.
4. The method according to claim 3, wherein the fluorescence detection is carried out after mixing the mixed system obtained after the cascade amplification reaction with NMM.
5. A method as claimed in claim 3, characterized by the steps of:
(1) respectively mixing cell extracting solutions with different concentrations with TS primers, and incubating at constant temperature to enable telomerase to extend the TS primers to respectively obtain TEPs with different cell numbers;
(2) respectively adding TEP generated by the reaction of the cell extracting solution with different concentrations obtained in the step (1) into a cascade amplification solution containing an unmarked molecular beacon group, and incubating at constant temperature to enable the TEP to perform cascade amplification reaction;
(3) obtaining the fluorescence intensity of different mixed systems incubated in the step (2) through fluorescence detection;
(4) establishing a linear equation between the fluorescence intensity and the concentration of the cell extracting solution according to the fluorescence intensity obtained in the step (3);
(5) and (4) substituting the fluorescence intensity of the sample to be detected, which is obtained after the steps (1) to (3) are carried out, into the linear equation in the step (4), and calculating to obtain the cell concentration of the sample to be detected.
6. The method of claim 5, wherein the cascade amplification solution containing the label-free set of molecular beacons in step (2) is: 5 μ L of RMB at a concentration of 1 μ M, 5 μ L of SMB at a concentration of 10 μ M, 3 μ L of T7Exo at a concentration of 10U/. mu.L, 4 μ L of 10 XT 7Exo reaction buffer and 18 μ L of DEPC-treated ultrapure water;
the 10 × T7Exo reaction buffer was: 20mM Tris-Ac, pH7.9, 50mM KAc,10mM Mg (Ac)2And 1mM DTT;
RMB and SMB required a 5min heating anneal at 95 ℃ prior to preparation of the tandem amplification solution containing the label-free set of molecular beacons, said 5min heating at 95 ℃ anneal being: keeping at 95 deg.C for 5min, and slowly cooling to room temperature for at least 1 h.
7. The method according to claim 5, wherein in step (3), the product obtained in step (2) is subjected toAdding 5 μ L NMM with concentration of 25 μ M and 5 μ L KCl with concentration of 100mM into the mixed system, incubating at 37 deg.C for 0.5h, and performing fluorescence detection; in the step (3), the fluorescence detection parameters are as follows: lambda [ alpha ]ex＝399nm，λemVoltage of PMT detector 700V at 612 nm; in the step (4), the established linear equation is as follows: Δ F is 0.4707 × C +38.87,
wherein, F-F0F is the fluorescence intensity of the sample to be measured, F0The fluorescence intensity of the sample without the telomerase extracting solution is shown, and C is the concentration of the HeLa cells in the sample to be detected, unit: cell/mL, R2＝0.993。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611063378.1A CN106755284B (en) | 2016-11-28 | 2016-11-28 | Cascade amplification DNA machine based on label-free molecular beacon and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611063378.1A CN106755284B (en) | 2016-11-28 | 2016-11-28 | Cascade amplification DNA machine based on label-free molecular beacon and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106755284A CN106755284A (en) | 2017-05-31 |
| CN106755284B true CN106755284B (en) | 2020-04-24 |
Family
ID=58904601
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611063378.1A Active CN106755284B (en) | 2016-11-28 | 2016-11-28 | Cascade amplification DNA machine based on label-free molecular beacon and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106755284B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109266721B (en) * | 2018-10-08 | 2021-11-09 | 山东师范大学 | Method for detecting telomerase activity based on non-quenching molecular beacon |
| CN109652502B (en) * | 2019-01-18 | 2022-05-10 | 广东省生态环境技术研究所 | Method and kit for label-free fluorescence detection of gene |
| CN109797200B (en) * | 2019-02-13 | 2022-07-19 | 中国科学院苏州生物医学工程技术研究所 | Ratio type telomerase activity quantitative detection method |
| CN112795627A (en) * | 2021-02-21 | 2021-05-14 | 中南大学 | Method for improving sensitivity of detecting circular RNA and detection kit |
| CN112941154B (en) * | 2021-02-21 | 2022-09-09 | 中南大学 | Molecular beacon probe, application of molecular beacon probe in preparation of circBART2.2 detection preparation and kit |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102827836A (en) * | 2012-06-11 | 2012-12-19 | 中国科学院成都生物研究所 | Oligonucleotide probe, and method for detecting target molecule through using it |
| CN105154563A (en) * | 2015-09-30 | 2015-12-16 | 陕西师范大学 | Triple-amplification-technology-based homogeneous-phase label-free method for detecting activity of telomerase |
| CN105802963A (en) * | 2016-04-01 | 2016-07-27 | 中国科学院成都生物研究所 | Oligonucleotide probe |
-
2016
- 2016-11-28 CN CN201611063378.1A patent/CN106755284B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102827836A (en) * | 2012-06-11 | 2012-12-19 | 中国科学院成都生物研究所 | Oligonucleotide probe, and method for detecting target molecule through using it |
| CN105154563A (en) * | 2015-09-30 | 2015-12-16 | 陕西师范大学 | Triple-amplification-technology-based homogeneous-phase label-free method for detecting activity of telomerase |
| CN105802963A (en) * | 2016-04-01 | 2016-07-27 | 中国科学院成都生物研究所 | Oligonucleotide probe |
Non-Patent Citations (3)
| Title |
|---|
| Exonuclease-assisted cascaded recycling amplification for label-free detection of DNA;Sai Bi 等;《Chem. Commun.》;20111121;第48卷;第1018页摘要、右栏第2段和Scheme 1 * |
| Label-free molecular beacons-based cascade amplification DNA machine for sensitive detection of telomerase activity;Kan Li 等;《Talanta》;20170304;第167卷;第645-650页 * |
| PCR-free and label-free fluorescent detection of telomerase activity at single-cell level based on triple amplification;Yanfang Gao 等;《Biosensors and Bioelectronics》;20160312;第81卷;第415页摘要、第416页左栏第2段、第417-418页第3.1节和Scheme 1 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106755284A (en) | 2017-05-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106755284B (en) | Cascade amplification DNA machine based on label-free molecular beacon and application | |
| JP6571895B1 (en) | Nucleic acid probe and genomic fragment detection method | |
| US9611506B2 (en) | Reaction mixtures for forming cDNA from an RNA template | |
| JP5798917B2 (en) | Isothermal nucleic acid amplification | |
| JamesáYang | A T7 exonuclease-assisted cyclic enzymatic amplification method coupled with rolling circle amplification: a dual-amplification strategy for sensitive and selective microRNA detection | |
| JP4999460B2 (en) | Methods and kits for nucleic acid primer-based amplification | |
| US20060211000A1 (en) | Methods, compositions, and kits for detection of microRNA | |
| US20080194416A1 (en) | Detection of mature small rna molecules | |
| EP2615171B1 (en) | Method for inhibiting nucleic acid amplification using light and highly sensitive method for selective nucleic acid amplification | |
| US10689718B2 (en) | HEV Assay | |
| Ma et al. | Rapid, sensitive and highly specific label-free fluorescence biosensor for microRNA by branched rolling circle amplification | |
| Lan et al. | Linear-hairpin variable primer RT-qPCR for MicroRNA | |
| JP2015522292A (en) | Collaborative primers, probes, and their uses | |
| CZ14998A3 (en) | Hiv-1 detection method | |
| JP2012508571A (en) | RNA detection method | |
| Xu et al. | Twin target self-amplification-based DNA machine for highly sensitive detection of cancer-related gene | |
| US20230304081A1 (en) | Primer and probe design method, detection composition, and kit for mirna detection | |
| US20200370107A1 (en) | Beacon-mediated exponential amplification reaction (bear) using a single enzyme and primer | |
| Jia et al. | Signal amplification of microRNAs with modified strand displacement-based cycling probe technology | |
| CN114134208A (en) | Fluorescent quantitative PCR kit, reaction system and nucleic acid quantitative detection method | |
| JP2010535525A5 (en) | ||
| AU2008286254A1 (en) | Method for synthesizing a cDNA in a sample in an enzymatic reaction | |
| Liu et al. | A sensitive isothermal fluorescence biosensor for microRNA detection coupling primer exchange reaction with catalytic hairpin assembly | |
| CN110951919A (en) | Oligonucleotide for detection, kit and detection method using same | |
| CN109486932A (en) | Medication related gene parting kit and classifying method for thiopurine drugs |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |