CN101250590A - Method for detecting DNA base mutation - Google Patents

Method for detecting DNA base mutation Download PDF

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CN101250590A
CN101250590A CNA2008101034584A CN200810103458A CN101250590A CN 101250590 A CN101250590 A CN 101250590A CN A2008101034584 A CNA2008101034584 A CN A2008101034584A CN 200810103458 A CN200810103458 A CN 200810103458A CN 101250590 A CN101250590 A CN 101250590A
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dna
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base mutation
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CN101250590B (en
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王树
贺芳
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Institute of Chemistry CAS
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Abstract

The invention discloses a process for detecting base mutation in DNA. The process for detecting base mutation in DNA which is provided by the invention comprises firstly synthesizing compound in formula (I), and then generating energy transmission with marked dye after forming composite through conducting electrostatic interaction for compound in formula (I) and single strain oligonucleotide, and detecting base mutation in DNA through DNA conformational change. The process which is provided by the invention overcomes the weaknesses existing in current process for detecting base mutation in DNA of complicated step, higher cost, low sensitivity and low selectivity, and has the advantages of simplicity, economical, high sensitivity and reliability.

Description

A kind of method that detects the DNA base mutation
Technical field
The present invention relates to whether exist among a kind of DNA of detection the method for base mutation.
Background technology
In recent years, the DNA base mispairing has been subjected to everybody extensive concern, studies show that, Human genome approximately contains 10 9Individual base pair.All might cause DNA in reproduction process, to produce base mispairing or mismatch the physics of DNA and chemical depletion,, still have the part mispairing not to be repaired though organism self exists powerful repair system to be used to repair mispairing.Because a base mutation all might cause fatal cancer and gene-correlation disease, thus for the detection of DNA base mispairing for clinical diagnosis, gene expression analysis and treatment and biological medicine research all have great importance.
At present existing several different methods can be used for the detection of DNA base mispairing, and wherein major part is based on the principle that dna mismatch can produce considerable influence to the stability of dna double spiral, utilizes the specific hybrid of oligonucleotide to detect the DNA base mispairing.Method based on above-mentioned principle has the simple advantage of principle, but has complex steps, cost is higher, sensitivity is low shortcoming, and because the influence of non-specific hybridization causes the aforesaid method selectivity to reduce, is difficult to realize the detection of single base mutation.
Summary of the invention
The purpose of this invention is to provide the method that whether has base mutation among a kind of DNA of detection.
Whether there is the method for base mutation among the detection DNA provided by the invention, may further comprise the steps successively:
1) with fluorescein-labelled strand polynucleotide M; The normal sequence of described M and DNA to be measured is complementary fully and can form the G-tetramer structure;
2) make fluorescein-labeled M form the G-tetramer structure;
3) M and the ethidium bromide that will form the G-tetramer structure mixes, add the compound of DNA to be measured and following formula (I), simultaneously the control systems of the compound of dna fragmentation that adding is made up of the normal sequence of DNA to be measured and following formula (I) be set, determine as follows whether DNA to be measured exists sudden change: if the I of DNA reaction system to be measured 600nm/ I 422nmSmaller or equal to 1.5 ± 0.08 of control systems, there is base mutation in DNA to be measured; If the I of DNA reaction system to be measured 600nm/ I 422nmEqual 2.0 ± 0.07 of control systems, there is not base mutation in DNA to be measured; Described I 600nmFor emission wavelength is the fluorescence intensity of the emission peak of 600nm; Described I 422nmFor emission wavelength is the fluorescence intensity of the emission peak of 422nm;
In the formula (I), m=1～10, n=2～100; R 1, R 2, R 3, R 4=C XH 2X+1Or-O (C XH 2X+1), x=1～10; R 5=C XH 2X+1, x=1～3; R 6=Br, Cl, I, CF 3COO or CH 3CO0.
In the described formula (I), m=6, n=6, R 1=H, R 2=H, R 3=H, R 4=H, R 5=CH 3, R 6=Br.
In the described formula (I), m=6, n=6, R 1=H, R 2=H, R 3=H, R 4=H, R 5=CH 2CH 3, R 6=I.
Whether there is the method for base mutation among the detection DNA provided by the invention, also can may further comprise the steps successively:
1) preparation strand polynucleotide D1, the normal sequence of described D1 and DNA to be measured is complementary fully;
2) preparation strand polynucleotide D2 is with fluorescein-labelled sequence D 2; Described D2 can form hairpin dna, and stem ring and D1 are complementary fully, and stem and D1 have at least 6nt not complementary, and the restriction enzyme site of restriction enzyme is contained in the stem district;
3) D1 and D2 are mixed, add DNA to be measured again, add the corresponding restriction enzyme of restriction enzyme site with D2 stem district then, add the compound of following formula (I) again; System that the dna fragmentation of forming with the normal sequence of DNA to be measured replaces DNA to be measured is set simultaneously in contrast;
Determine as follows whether DNA to be measured exists sudden change: if the I of DNA reaction system to be measured 424nm/ I 527nmSmaller or equal to 2.57 ± 0.05 of control systems, there is base mutation in DNA to be measured; If the I of DNA reaction system to be measured 424nm/ I 527nmEqual 3.8 ± 0.11 of control systems, there is not base mutation in DNA to be measured; Described I 424nmFor emission wavelength is the fluorescence intensity of the emission peak of 424nm; Described I 527nmFor emission wavelength is the fluorescence intensity of the emission peak of 527nm;
In the formula (I), m=1～10, n=2～100; R 1, R 2, R 3, R 4=C XH 2X+1Or-O (C XH 2X+1), x=1～10; R 5=C XH 2X+1, x=1～3; R 6=Br, Cl, I, CF 3COO or CH 3COO.
In the described formula (I), m=6, n=6, R 1=H, R 2=H, R 3=H, R 4=H, R 5=CH 3, R 6=Br.
In the described formula (I), m=6, n=6, R 1=H, R 2=H, R 3=H, R 4=H, R 5=CH 2CH 3, R 6=I.
Whether there is the method for base mutation among the detection DNA provided by the invention, also can may further comprise the steps successively:
1) preparation strand polynucleotide K1, the normal sequence of described K1 and DNA to be measured is complementary fully;
2) preparation strand polynucleotide K2, the middle portion of described K2 has DNAzyme activity (wherein the sequence of active part is 5 '-AGGCTAGCTACAACGA-3 '), K2 and K1 have at least 15nt complementary fully, and include the DNAzyme of 10nt (DNAzyme) sequence at least, K2 and K1 at least respectively have 6nt not complementary end to end;
3) preparation DNAzyme substrate S uses fluorescein-labelled S; The DNA part of substrate S is complementary fully with K2, and the restriction enzyme site that comprises two special RNA bases is arranged in the middle of the substrate S;
4) K1 and K2 are mixed, add substrate S then, the compound that adds DNA to be measured and following formula (I), the control systems of the compound of dna fragmentation that adding is made up of the normal sequence of DNA to be measured and following formula (I) is set simultaneously, determines as follows whether DNA to be measured exists sudden change: if the I of DNA reaction system to be measured 424nm/ I 527nmSmaller or equal to 1.595 ± 0.06 of control systems, there is base mutation in DNA to be measured; If the I of DNA reaction system to be measured 424nm/ I 527nmEqual 3.08 ± 0.1 of control systems, there is not base mutation in DNA to be measured; Described I 424nmFor emission wavelength is the fluorescence intensity of the emission peak of 424nm; Described I 527nmFor emission wavelength is the fluorescence intensity of the emission peak of 527nm.
Figure S2008101034584D00041
In the formula (I), m=1～10, n=2～100; R 1, R 2, R 3, R 4=C XH 2X+1Or-O (C XH 2X+1), x=1～10; R 5=C XH 2X+1, x=1～3; R 6=Br, Cl, I, CF 3COO or CH 3COO.
In the described formula (I), m=6, n=6, R 1=H, R 2=H, R 3=H, R 4=H, R 5=CH 3, R 6=Br.
In the described formula (I), m=6, n=6, R 1=H, R 2=H, R 3=H, R 4=H, R 5=CH 2CH 3, R 6=I.
Method provided by the invention has overcome that existing detection DNA base mutation method steps is loaded down with trivial details, cost is higher, sensitivity is low and shortcoming such as selectivity is low, has simple, economical, highly sensitive and reliable advantage.
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.
Description of drawings
Fig. 1 is for using the figure as a result of G-tetramer DNA detection base mutation.
Fig. 2 is for using the figure as a result of hairpin structure DNA detection base mutation.
Fig. 3 is for using the figure as a result of DNAzyme DNA detection base mutation.
Embodiment
The present invention realizes by the following technical solutions:
At first, synthesize and gather 2,7-[9,9-two (6 '-N, N, N--trimethylammonium amine bromide) hexane-fluorenes]-altogether-(1,4-benzene) (PFP).
According to reference (Stork, M.; Gaylord, B.S.; Heeger, A.J.; Bazan, G.C.Adv.Mater.2002,14,361-366.) the positively charged ion luminescent conjugated polymers of synthetic above-mentioned formula (I), called after PFP.PFP is a kind of novel fluorescent probe, compare with micromolecular fluorescent probe, it has height conjugated molecular structure, is made up of many repeating units, this structure causes it to have the effect that fluorescence amplifies, and improves detection sensitivity greatly thereby can high-sensitively detect optical signalling.The obtained the maximum absorption of PFP is 380nm, and maximum emission peak is at 427nm.Foundation
Figure S2008101034584D00042
Energy shifts theoretical, and it can shift (maximum absorption band is 480nm, and maximum emission peak is 527nm) with fluorescein generation energy under the certain distance condition.Fluorescein is marked on the oligonucleotide, and positively charged PFP and electronegative oligonucleotide form mixture by electrostatic interaction.The distance of the fluorescein of PFP and mark is furthered, thereby energy shifts generation.Because dna structure or length variations influence electric density, thereby electrostatic interaction intensity changes and causes the variation of energy transfer efficiency.
Then, utilize PFP and oligonucleotide to form behind the mixture and the dyestuff generation energy of mark shifts and the conformational change of DNA detects base mutation among the DNA by electrostatic interaction.
DNA has multiple conformation, and wherein more common have a double-spiral structure, also has the triple-helix structure and the special G-tetramer, hair fastener type structure etc. in addition.The invention provides three kinds of detection methods, the G-tetramer structure that method one mainly is based on DNA has influenced from PFP to the fluorescein again to double-stranded conversion that the double energy to ethidium bromide (EB) shifts; Method two mainly is protection and the release that utilizes hairpin structure DNA (beacon), exchange by adding target dna driving DNA, thereby discharge the substrate DNA of the hairpin structure of restriction enzyme, the fluorescein fragment that becomes short chain after substrate is digested reduces energy transfer efficiency, because base mutation hinders exchange, detect the DNA enzyme and cut and exchange the base mutation that can detect DNA so shift by energy; Method three mainly is based on the protection and the release of DNAzyme (DNAzyme); exchange by adding target dna driving DNA; discharging DNAzyme carries out enzyme to the substrate S that is marked with fluorescein and cuts; the fluorescein fragment that becomes short chain after substrate is digested reduces energy transfer efficiency; because base mutation hinders exchange, detect the DNA enzyme and cut and exchange the base mutation that can detect DNA so shift by energy.The result proves that three kinds of above methods have simple, economical, highly sensitive and reliable advantage for the base mutation detection of DNA.
Three kinds of methods provided by the invention, specific as follows:
Method one, utilize G-tetramer structure DNA and PFP to detect base mutation among the DNA
Present method mainly arrives double-stranded conversion based on the G-tetramer structure of DNA.The G-tetramer structure of DNA is the special conformation of a class.Some DNA that are rich in bases G can be induced to form the G-tetramer structure when potassium ion or zymoplasm exist.
1) synthetic PFP.
2) design and preparation G-tetramer dna sequence dna M.
Design section of DNA sequence M, M can form G-tetramer structure and complementary fully with the normal sequence of DNA to be measured, at end (5 ' or 3 ') the mark fluorescent element of M.
3) base mutation among the detection DNA to be measured.
1. fluorescein-labeled M and PFP, EB are mixed, add potassium ion or blood coagulation enzyme dna, M forms the G-tetramer structure.
2. add dna sequence dna to be measured, obtain reaction system; Simultaneously, in step mixing solutions 1., the normal sequence of adding and DNA equivalent to be measured, system in contrast.For the DNA that does not contain base mutation (promptly complementary fully with M), the G-tetramer is destroyed, and duplex structure forms, and EB embeds dsDNA; For the DNA that contains base mutation (promptly can not be complementary fully with M), the base of sudden change can influence the balance of the G-tetramer and double-stranded DNA greatly.
3. behind the 10min, under exciting, 380nm obtains excitation spectrum.For the DNA that does not contain base mutation, can take place from PFP to the fluorescein again to shift to the double energy of EB; For the DNA that contains base mutation, the energy transfer efficiency to EB reduces again from PFP to the fluorescein, and along with the increase of mutating alkali yl number, the energy transfer efficiency reduction amplitude to EB increases again from PFP to the fluorescein.So, if the I of DNA reaction system to be measured 600nm/ I 422nmSmaller or equal to 1.5 ± 0.08 of control systems, there is base mutation in DNA to be measured; If the I of DNA reaction system to be measured 600nm/ I 422nmEqual 2.0 ± 0.07 of control systems, there is not base mutation in DNA to be measured.I 600nmFor emission wavelength is the fluorescence intensity of the emission peak of 600nm; I 422nmFor emission wavelength is the fluorescence intensity of the emission peak of 422nm.
In this method, double-stranded DNA intercalator ethidium bromide (EB) is introduced into.The maximum absorption band of EB is at 520nm, and maximum emission peak is 610nm.Form the G-tetramer structure when adding potassium ion or blood coagulation enzyme dna, the electric density of DNA increases, cause the very high energy transfer efficiency between PFP and the fluorescein, but owing to the EB molecule can not be embedded in the G-tetramer, so the energy that can not observe from the fluorescein to EB shifts.And after adding with G-tetramer sequence complementary DNA, the G-tetramer is destroyed and duplex structure forms.EB embeds and will take place from PFP to the fluorescein again that the double energy to EB shifts behind the dsDNA.The base mutation of DNA can influence the balance of the G-tetramer and double-stranded DNA greatly, and the fluorescence intensity of the EB that is exaggerated by observation just can detect the base mutation of DNA.Utilize this method can realize the detection of base mutation among the DNA, and low DNA concentration that can be detected can reach 50nM.Therefore, present method can realize that highly sensitive and highly selective detect the base mutation among the DNA.
Method two, utilize hairpin structure DNA and PFP to detect base mutation among the DNA
Mainly be protection and the release that utilizes hairpin structure DNA (beacon).Hairpin structure DNA generally contains 25～35 Nucleotide, contains two portions: (1) ring district: be the strand state, generally be made up of 10～30 Nucleotide, be used for and the target dna specific combination; (2) stem district: be duplex structure, be used for stablizing hairpin structure.The number of the stable general stem of hairpin structure DNA district base pair should be greater than 8.
1) synthetic PFP.
2) design and preparation dna sequence dna D1 and D2.
Design two segment DNA sequence D 1 and D2, the normal sequence of D1 and DNA to be measured is complementary fully; D2 is a hairpin dna, and contains the restriction enzyme site of restriction enzyme in the stem district, and the stem ring of D2 and D1 are complementary fully, and the stem of D2 and D1 have at least 6nt not complementary, use fluorescein-labelled D2;
3) base mutation among the detection DNA to be measured.
1. D1 and D2 are mixed, destroy the hairpin structure of D2, form stable dsDNA.
2. add DNA to be measured, simultaneously, in the mixing solutions of step 1, the normal sequence of adding and DNA equivalent to be measured, system in contrast.Add the corresponding restriction enzyme of restriction enzyme site again with D2 stem district.For the DNA that does not contain base mutation, exchange takes place and the dsDNA of formation D1 and DNA to be measured with D2 then, D2 is released, and forms hairpin structure again, produces restriction enzyme site, and the stem of D2 is cut off from restriction enzyme site, discharges the short chain dna fragmentation that has fluorescein; For the DNA that contains base mutation, the exchange energy between the DNA is prohibited, and the restriction enzyme site of D2 is protected by D1.
3. add PFP, under 380nm excites, obtain excitation spectrum.For the DNA that does not contain base mutation,, have only weak energy to shift between PFP and the fluorescein because the electric density of short chain dna fragmentation is less; For the DNA that contains base mutation, stronger energy takes place between PFP and the fluorescein shift, along with the increase I of mutating alkali yl number 424nm/ I 527nmReduce gradually, energy transfer efficiency increases gradually.So, if the I of DNA reaction system to be measured 424nm/ I 527nmSmaller or equal to 2.57 ± 0.05 of control systems, there is base mutation in DNA to be measured; If the I of DNA reaction system to be measured 424nm/ I 527nmEqual 3.80 ± 0.11 of control systems, there is not base mutation in DNA to be measured; Described I 424nmFor emission wavelength is the fluorescence intensity of the emission peak of 424nm; Described I 527nmFor emission wavelength is the fluorescence intensity of the emission peak of 527nm.
Utilize this method can realize that highly sensitive and highly selective detect the base mutation among the DNA.The minimal detectable concentration of target dna can reach 75nM.This method also has good universality in addition, can detect the DNA of a plurality of sequences.
Method three, utilize DNAzyme (DNAzyme) and PFP to detect base mutation among the DNA
Mainly be based on the protection and the release of DNAzyme.DNAzyme is a kind of single stranded DNA fragment with high-efficiency catalytic activity and structure recognition capability of utilizing external molecular evolution technique to obtain.What mainly select for use in present method is the DNAzyme with RNA nicking activity.
1) synthetic PFP.
2) design and preparation dna sequence dna K1 and K2.
Design two segment DNA sequence K1 and K2, the normal sequence of K1 and DNA to be measured is complementary fully; The middle portion of K2 has DNAzyme activity (wherein the sequence of active part is 5 '-AGGCTAGCTACAACGA-3 '), K2 and K1 have at least 15nt complementary fully, and include the DNAzyme of 10nt (DNAzyme) sequence at least, K2 and K1 at least respectively have 6nt not complementary end to end;
3) design DNAzyme substrate S, the DNA part of substrate S is complementary fully with K2, in the middle of the substrate S restriction enzyme site that comprises two special RNA bases is arranged, and uses fluorescein-labelled S;
4) base mutation among the detection DNA to be measured.
1. K1 and K2 are mixed, form partially double strandedly, respectively have the Nucleotide of small portion not match end to end.
2. add substrate S, add DNA to be measured again, in the mixing solutions of step 1, add and the normal sequence of DNA equivalent to be measured system in contrast, 37 ℃ of reactions simultaneously.For the DNA that does not contain mutating alkali yl, exchange takes place and the dsDNA of formation K1 and DNA to be measured with K2 then, K2 is released, and combines the digested short chain DNA that is marked with fluorescein that discharges of substrate S with substrate S; For the DNA that contains mutating alkali yl, the exchange energy between the DNA is prohibited, thereby can not discharge DNAzyme K2, and substrate S can not be cut.
3. add PFP, under 380nm excites, obtain excitation spectrum.For the DNA that does not contain mutating alkali yl, can only observe weak energy and shift; For the DNA that contains base mutation, can observe strong energy and shift.So, if the I of DNA reaction system to be measured 424nm/ I 527nmSmaller or equal to 1.595 ± 0.06 of control systems, there is base mutation in DNA to be measured; If the I of DNA reaction system to be measured 424nm/ I 527nmEqual 3.08 ± 0.1 of control systems, there is not base mutation in DNA to be measured; Described I 424nmFor emission wavelength is the fluorescence intensity of the emission peak of 424nm; Described I 527nmFor emission wavelength is the fluorescence intensity of the emission peak of 527nm.
Utilize this method can realize that equally highly sensitive and highly selective detect the base mutation among the DNA.The minimal detectable concentration of target dna can reach 20nM.This method does not have strict restriction to the sequence of target dna yet, and more convenient to operate, simple.
Experimental technique among the following embodiment if no special instructions, is ordinary method.
All marks and unlabelled oligonucleotide are all synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd and precious biotechnology (Dalian) company limited.
The ultraviolet spectrometer model that the present invention uses is Jasco V-550 type.The model of fluorescence spectrophotometer is Hitachi F-4500, and excitation light source is an xenon lamp.Used fluorescence cuvette is the disposable polystyrene cuvette of 3mL.Water used in the experiment all need filter through the Millipore purification system.
The concentration of the oligonucleotide among the present invention all is to obtain by the absorption value that detects the 260nm place.
Embodiment 1, application PFP and the G-tetramer detect the base mutation among the DNA
One, PFP1's is synthetic
Figure S2008101034584D00081
The structure of PFP1 is suc as formula shown in (II), and synthetic method is as follows:
Single port bottle to 100mL adds 50mL toluene, 5.2 gram neopentyl glycol and 0.5 gram tosic acid, refluxes 24 hours, and steaming desolventizes the back silicagel column and separates (eluent: obtain 4.8 grams 1 methylene dichloride), new penta diester of 4-benzene hypoboric acid. 1H?NMR(400MHz，CDCl 3)：δ(ppm)7.78(4H，s)，3.77(8H，s)，1.02(12H，s)。
In two mouthfuls of bottles of 25mL, add 5.4mL toluene and 3.6mL 2M salt of wormwood, blast nitrogen after 30 minutes, add 325 milligram 2,7-two bromo-9,9-two (6-bromine hexyl) fluorenes, 165 milligram 1, new penta diester of 4-benzene hypoboric acid and 20 milligrams of tetrakis triphenylphosphine palladiums, in following 95 ℃ of stirring reactions of nitrogen 24 hours, the cooling back adds chloroform and water, gets chloroform layer washing back and uses anhydrous magnesium sulfate drying, revolves inspissation and contracts, acetone precipitation obtains 210 milligrams of light yellow solids.Solid is dissolved in 20 milliliters of methylene dichloride, and it is centrifugal after 24 hours in stirring at room to add 1 milliliter of Trimethylamine 99, and collecting precipitation is also dry, obtains 208 milligrams of polymer P FP1, M n=3.5 * 10 4 1H?NMR(400MHz，DMSO)：δ(ppm)7.3-8.0(10H，m)，2.9-3.2(16H，m)，2.2-2.3(4H，m)，1.5-1.6(4H，m)，0.9-1.2(30H，m)。
Two, the preparation of sample to be tested
The synthetic DNA sequence is as follows:
Sequence 1 (no base mutant DNA sequence ssDNA C): 5 '-CCCTAACCCTAACCCTAACCC-3 ';
Sequence 2 (single base mutation dna sequence dna ssDNA 1NC): 5 '-CCCTAACCCTAACACTAACCC-3 ';
Sequence 3 (three base mutation dna sequence dna ssDNA 3NC): 5 '-CCCAAACCCAAACCCAAACCC-3 ';
Sequence 4 (six basic mutant DNA sequence ssDNA 6NC): 5 '-CCCAATCCCAATCCCAATCCC-3 '.
Three, detect the base mutation of sequence
1) implementation sequence M uses fluorescein-labelled M.
Sequence M:5 '-FAM-GGGTTAGGGTTAGGGTTAGGG-3 '.
2) be that to add 100 μ L concentration in the phosphate buffer solution (pH=7.4) of 50mM be the KCl of 1M in 1900 μ L ionic strengths, the final concentration that makes KCl is 50mM.
3) will add steps 2 at the fluorescein-labeled M of 4 ℃ of inductive) in the solution that obtains, to final concentration be [G-quadruplex-F1]=5.0 * 10 -8M.
4) PFP and EB are added in the solution that step 3) obtains, to final concentration be: [PFP]=1.25 * 10 -6M, repeating unit concentration; [EB]=1.5 * 10 -6M.
5) in the solution that step 4) obtains, add DNA to be measured, preparation feedback liquid, divide following two kinds of situations:
1. reaction solution A: in the solution that step 4) obtains, add the DNA shown in the sequence 1, to final concentration be [ssDNAc]=5.0 * 10 -8M is the control systems first.
2. reaction solution B: in the solution that step 4) obtains, add the DNA shown in the sequence 2, to final concentration be [ssDNA 1NC]=5.0 * 10 -8M.
3. reaction liquid C: in the solution that step 4) obtains, add the DNA shown in the sequence 3, to final concentration be [ssDNA 3NC]=5.0 * 10 -8M.
4. reaction solution D: in the solution that step 4) obtains, add the DNA shown in the sequence 4, to final concentration be [ssDNA 6NC]=5.0 * 10 -8M.
5. reaction solution E: the solution that step 4) obtains, second in contrast.
6) mensuration of fluorescence spectrum
Behind the 10min, get two kinds of reaction solutions that 10 μ L step 5) obtain respectively, under 380nm excites, obtain excitation spectrum.
Carry out revision test 3 times, data and result among the figure are mean value.
Fig. 1 is for using the figure as a result of G-tetramer DNA detection base mutation; 1a is the fluorescence spectrum of reaction solution A and reaction solution E, and solid line is reaction solution A, and dotted line is reaction solution E; 1b is the FRET efficient histogram of reaction solution A, B, C and D, [ssDNAc]=[ssDNA 1NC]=[ssDNA 3NC]=[ssDNA 6NC]=5.0 * 10 -8M.
As seen from the figure, reaction solution E can only observe the fluorescence of PFP and fluorescein and can not observe the fluorescence of EB; The emission peak of EB appearred in reaction solution A, taken place from PFP to the fluorescein again that the double energy to EB shifts, and I 600nm/ I 422nm=1.6 ± 0.08; Reaction solution B occurs from PFP to the fluorescein again, and the energy to EB shifts I 600nm/ I 422nm=1.2 ± 0.07, efficiency ratio reaction solution A has reduced by 25%; Reaction liquid C occurs from PFP to the fluorescein again, and the energy to EB shifts I 600nm/ I 422nm=0.8 ± 0.06, efficiency ratio reaction solution A has reduced by 50%; Reaction solution D occurs from PFP to the fluorescein again, and the energy to EB shifts I 600nm/ I 422nm=0.8 ± 0.04, efficiency ratio reaction solution A has reduced by 50%.
Embodiment 2, utilize PFP and the G-tetramer to detect base mutation among the DNA
One, PFP2's is synthetic
Figure S2008101034584D00101
The structure of PFP2 is suc as formula shown in (III), and synthetic method is as follows:
Single port bottle to 100mL adds 50mL toluene, 5.2 gram neopentyl glycol and 0.5 gram tosic acid, refluxes 24 hours, and steaming desolventizes the back silicagel column and separates (eluent: obtain 4.8 grams 1 methylene dichloride), new penta diester of 4-benzene hypoboric acid. 1H?NMR(400MHz，CDCl 3)：δ(ppm)7.78(4H，s)，3.77(8H，s)，1.02(12H，s)。
Single port bottle to 50mL adds 10mL exsiccant tetrahydrofuran (THF) and 0.65 gram 2,7-dibromo fluorenes (source company is foreignized in the Zhengzhou peace), subcooling adds 2mL 2M butyl lithium solution (Alfa Aesar company) low temperature stirring 1 hour after-78 ℃, add 1.0 grams 1,6-two iodohexanes (Beijing coupling company) spend the night in room temperature reaction, finish reaction.Add 10mL water and 15mL chloroform to reaction solution, separatory, organic layer steam behind washing, anhydrous magnesium sulfate drying and desolventize, and the silicagel column separation (petrol ether/ethyl acetate: 20/1, v/v) obtain 0.8 gram 2,7-two bromo-9,9-two (6-iodine hexyl) fluorenes. 1H?NMR(400MHz，CDCl 3)：δ(ppm)7.82(d，2H)，7.42(m，4H)，3.39(m，4H)，2.06(m，4H)，1.67(m，4H)，1.22(m，8H)，0.69(m，4H)。
In two mouthfuls of bottles of 25mL, add 6mL tetrahydrofuran (THF) and 3mL 2M yellow soda ash, blast nitrogen after 30 minutes, add 390 milligram 2,7-two bromo-9,9-two (6-iodine hexyl) fluorenes, 182 milligram 1,4-benzene hypoboric acid peopentyl ester and 23 milligrams of tetrakis triphenylphosphine palladiums, in following 80 ℃ of stirring reactions of nitrogen 48 hours, the cooling back adds chloroform and water, gets chloroform layer washing back and uses anhydrous magnesium sulfate drying, revolves inspissation and contracts, acetone precipitation obtains 230 milligrams of light yellow solids.Solid is dissolved in 25 milliliters of methylene dichloride, and it is centrifugal after 24 hours in stirring at room to add 1 milliliter of 33% Trimethylamine 99 alcoholic solution, and collecting precipitation is also dry, obtains 222 milligrams of polymer P FP2, Mn=4.1 * 10 4 1HNMR(400MHz，DMSO)：δ(ppm)7.1-8.0(10H，m)，3.2(4H，m)，3.1(12H，m)，2.1(4H，m)，1.2(16H，m)。
Two, the preparation of sample to be tested
The synthetic DNA sequence is as follows:
Sequence 5 (no base mutant DNA sequence ssDNA C): 5 '-CCAACCACACCAACC-3 ';
Sequence 6 (single base mutation dna sequence dna ssDNA 1NC): 5 '-CCTACCACACCAACC-3 '.
Three, detect the single base mutation of sequence
1) implementation sequence M uses fluorescein-labelled M.
Sequence M:5 '-FAM-GGTTGGTGTGGTTGG-3 '.
2) be that to add 100 μ L concentration in the phosphate buffer solution (pH=7.4) of 50mM be the KCl of 1M in 1900 μ L ionic strengths, the final concentration that makes KCl is 50mM.
3) will add steps 2 at the fluorescein-labeled M of 4 ℃ of inductive) in the solution that obtains, to final concentration be [G-quadruplex-F1]=5.0 * 10 -8M.
4) PFP and EB are added in the solution that step 3) obtains, to final concentration be: [PFP]=1.25 * 10 -6M, repeating unit concentration; [EB]=1.5 * 10 -6M.
5) in the solution that step 4) obtains, add DNA to be measured, preparation feedback liquid, divide following two kinds of situations:
1. reaction solution A: in the solution that step 4) obtains, add the DNA shown in the sequence 5, to final concentration be [ssDNAc]=5.0 * 10 -8M, system first in contrast.
2. reaction solution B: in the solution that step 4) obtains, add the DNA shown in the sequence 6, to final concentration be [ssDNA NC]=5.0 * 10 -8M.
3. reaction liquid C: the solution that step 4) obtains, second in contrast.
6) mensuration of fluorescence spectrum
Behind the 10min, get two kinds of reaction solutions that 10 μ L step 5) obtain respectively, under 380nm excites, obtain excitation spectrum.
Carry out revision test 3 times, result data is a mean value.
Reaction liquid C can only be observed the fluorescence of PFP and fluorescein and can not observe the fluorescence of EB; The emission peak of EB appearred in reaction solution A, taken place from PFP to the fluorescein again that the double energy to EB shifts, and I 600nm/ I 422nm=1.86 ± 0.05; Reaction solution B occurs from PFP to the fluorescein again, and the energy to EB shifts I 600nm/ I 422nm=1.56 ± 0.08, efficiency ratio reaction solution A has reduced by 16%.
Embodiment 3, utilize hairpin structure DNA and PFP to detect base mutation among the DNA
One, PFP's is synthetic
Step 1 with embodiment 1.
Two, the preparation of sample to be tested
The synthetic DNA sequence is as follows:
Sequence 7 (no base mutant DNA sequence Tc):
5’-GATTGCGGAAAGAAGGTATGAGATAATGTCAC-3’；
Sequence 8 (single base mutation dna sequence dna T 1NC):
5’-GATTGCGGAAAGAAGGTATGAGATAATATCAC-3’：
Sequence 9 (2 base mutation dna sequence dna T 2NC):
5’-GATTGCGTAAAGAAGGTATGAGATAATATCAC-3’；
Sequence 10 (3 base mutation dna sequence dna T 3NC):
5’-GATTGCGTAAAGAAGGTATGAGACAATATCAC-3’；
Sequence 11 (5 base mutation dna sequence dna T 5NC):
5’-GATTACGGAAAGTAGGTCTGAGATCATGCCAC-3’。
Three, detect the base mutation of sequence
1) design D1 and D2 use fluorescein-labelled D2, and the sequence of D1 and D2 is as follows:
D1：5’-GTGACATTATCTCATACCTTCTTTCCGCAATCGGA-3’；
D2：5’-FAM-CTGGCCTCCGATTGCGGAAAGAAGGTATGAGATCGGAGGCCAG-3’。
2) reduce to room temperature behind the 80 ℃ of hydridization 20min of D2 (10 μ M) with the D1 (10 μ M) of 7.5 μ L and 5.0 μ L and obtain duplex structure.
3) preparation feedback liquid
Reaction solution A: with the restriction enzyme damping fluid of 2.5 μ L, 1.5 the restriction enzyme restriction endonuclease HaeLLI of μ L (10unit/ μ L) (NEB company), 7.5 the Millipore water of DNA of μ L sequence 7 (10 μ M) and 1.0 μ L together is incorporated in 37 ℃ of reaction 50min, is the control systems first.
Reaction solution B: with the restriction enzyme damping fluid of 2.5 μ L, the restriction enzyme restriction endonuclease HaeIII of 1.5 μ L (10unit/ μ L) (NEB company), the Millipore water of DNA of 7.5 μ L sequences 8 (10 μ M) and 1.0 μ L together are incorporated in 37 ℃ of reaction 50min.
Reaction liquid C: with the restriction enzyme damping fluid of 2.5 μ L, the restriction enzyme restriction endonuclease HaeIII of 1.5 μ L (10unit/ μ L) (NEB company), the Millipore water of DNA of 7.5 μ L sequences 9 (10 μ M) and 1.0 μ L together are incorporated in 37 ℃ of reaction 50min.
Reaction solution D: with the restriction enzyme damping fluid of 2.5 μ L, the restriction enzyme restriction endonuclease HaeIII of 1.5 μ L (10unit/ μ L) (NEB company), the Millipore water of DNA of 7.5 μ L sequences 10 (10 μ M) and 1.0 μ L together are incorporated in 37 ℃ of reaction 50min.
Reaction solution E: with the restriction enzyme damping fluid of 2.5 μ L, the restriction enzyme restriction endonuclease HaeIII of 1.5 μ L (10unit/ μ L) (NEB company), the Millipore water of DNA of 7.5 μ L sequences 11 (10 μ M) and 1.0 μ L together are incorporated in 37 ℃ of reaction 50min.
Reaction solution F: with the restriction enzyme damping fluid of 2.5 μ L, the Millipore water of the restriction enzyme restriction endonuclease HaeIII of 1.5 μ L (10unit/ μ L) (NEB company) and 1.0 μ L together is incorporated in 37 ℃ of reactions 50min, second in contrast.
Reaction solution G: with the restriction enzyme damping fluid of 2.5 μ L, the Millipore water of DNA of 7.5 μ L sequences 7 (10 μ M) and 1.0 μ L together is incorporated in 37 ℃ of reaction 50min, in contrast third.
4) mensuration of fluorescence spectrum
Get four kinds of reaction solutions that 10 μ L step 3) obtain respectively, join respectively 2.0mL the HEPEs damping fluid (contain HEPEs 25mM, pH=8.0) in, add the PFP (2 * 10 of 1.5 μ L respectively -3M) after, under 380nm, excite, detect fluorescence spectrum.
Carry out revision test three times, data among the figure and result are the mean value of 3 tests.
Fig. 2 is for using the figure as a result of hairpin structure DNA detection base mutation; 2a is the fluorescence spectrum of reaction solution A, B, F, G; 2b is the FRET efficient histogram of reaction solution A, B, C, D and E.[Tc]＝[T 1NC]＝[T 2NC]＝[T 3NC]＝[T 5NC]]＝15nM。
The result shows, the I of reaction solution G 424nm/ I 527nmBe about 0.42 ± 0.05 (mean+SD); The I of reaction solution A 424nm/ I 527nmBe about 1.597 ± 0.107; The I of reaction solution B 424nm/ I 527nmBe about 1.08 ± 0.046, reduced by 32% than reaction solution A; The I of reaction liquid C 424nm/ I 527nmBe about 0.69 ± 0.046, reduced by 57% than reaction solution A; The I of reaction solution D 424nm/ I 527nmBe about 0.503 ± 0.006, reduced by 69% than reaction solution A; The I of reaction solution E 424nm/ I 527nmBe about 0.46 ± 0.01, reduced by 72% than reaction solution A.
Embodiment 4, utilize hairpin structure DNA and PFP to detect base mutation among the DNA
One, PFP2's is synthetic
Step 1 with embodiment 2.
Two, the preparation of sample to be tested
The synthetic DNA sequence is as follows:
Sequence 12 (no base mutant DNA sequence Tc):
5’-GATTGCGGAAAGAAGGTATGAGATAATGTCAC-3’；
Sequence 13 (single base mutation dna sequence dna T 1NC):
5’-GATTGCGGAAAGAAGGTATGAGATATTGTCAC-3’。
Three, detect the single base mutation of sequence 12 and sequence 13
1) design D1 and D2 use fluorescein-labelled D2, and the sequence of D1 and D2 is as follows:
D1：5’-GTGACATTATCTCATA?CCTTCTTTCCGCAATCGGA-3’；
D2：5’-FAM-CTGGCCTCCGATTGCGGAAAGAAGGTATGAGATCGGAGGCCAG-3’。
2) reduce to room temperature behind the 80 ℃ of hydridization 20min of D2 (10 μ M) with the D1 (10 μ M) of 7.5 μ L and 5.0 μ L and obtain duplex structure.
3) preparation feedback liquid
Reaction solution A: with the restriction enzyme damping fluid of 2.5 μ L, 1.5 the restriction enzyme restriction endonuclease HaeIII of μ L (10unit/ μ L) (NEB company), 7.5 the Millipore water of DNA of μ L sequence 12 (10 μ M) and 1.0 μ L together is incorporated in 37 ℃ of reaction 50min, is the control systems first.
Reaction solution B: with the restriction enzyme damping fluid of 2.5 μ L, the restriction enzyme restriction endonuclease HaeIII of 1.5 μ L (10unit/ μ L) (NEB company), the Millipore water of DNA of 7.5 μ L sequences 13 (10 μ M) and 1.0 μ L together are incorporated in 37 ℃ of reaction 50min.
Reaction liquid C: with the restriction enzyme damping fluid of 2.5 μ L, the Millipore water of the restriction enzyme restriction endonuclease HaeIII of 1.5 μ L (10unit/ μ L) (NEB company) and 1.0 μ L together is incorporated in 37 ℃ of reactions 50min, second in contrast.
4) mensuration of fluorescence spectrum
Get three kinds of reaction solutions that 10 μ L steps 1 obtain respectively, join respectively 2.0mL the HEPEs damping fluid (contain HEPEs 25mM, pH=8.0) in, add the PFP (2 * 10 of 1.5 μ L respectively -3M) after, under 380nm, excite, detect fluorescence spectrum.
Carry out revision test three times, data results is the mean value of 3 tests.
The I of reaction liquid C 424nm/ I 527nmBe about 0.45 ± 0.04; The I of reaction solution A 424nm/ I 527nmBe about 1.65 ± 0.08; The I of reaction solution B 424nm/ I 527nmBe about 1.12 ± 0.045, reduced by 32% than reaction solution A.The result shows that the transfer efficiency reduction of the energy transfer efficiency comparison photograph of reaction solution A reaches 3 times.Increase I along with the mutating alkali yl number 424nm/ I 527nmReduce gradually, energy transfer efficiency increases gradually.DNA for single base mutation can observe very obvious variation.
Embodiment 5, utilize DNAzyme and PFP to detect base mutation among the DNA
One, PFP1's is synthetic
Step 1 with embodiment 1.
Two, the preparation of sample to be tested
The synthetic DNA sequence is as follows:
Sequence 14 (no base mutant DNA sequence Mc): 5 '-CGGTGAAGCTACAACGAGAGC-3 ';
Sequence 15 (single base mutation dna sequence dna M 1NC): 5 '-CGTTGAAGCTACAACGAGAGC-3 ';
Sequence 16 (2 base mutation dna sequence dna M 2NC): 5 '-CGTTGAAGCAACAACGAGAGC-3 ';
Sequence 17 (3 base mutation dna sequence dna M 3NC): 5 '-CGGAGAAGCTTCAACGTGAGC-3 ';
Sequence 18 (5 base mutation dna sequence dna M 5NC): 5 '-CGGAGAAGCTTGAGCGAGTGC-3 '.
Three, detect the base mutation of sequence
1) design K1 and K2, the sequence of K1 and K2 is as follows:
K1：5’-GCTCTCGTTGTAGCTTCACCG-3’：
K2：5’-TACAGCTAATAGGCTAGCTACAACGAGAGC-3’。
Design substrate S uses fluorescein-labelled S, and the sequence of S is as follows:
5’-FAM-GCTCrArUATTAGCTGTA-3’。
2) K2 (10 μ M) of the K1 of 2.0 μ L (10 μ M) and 2.0 μ L reduces to room temperature and obtains duplex structure behind 80 ℃ of hydridization 20min.
3) preparation feedback liquid
Reaction solution A: the HEPEs buffered soln that double-stranded DNA is joined 1mL (contains 25mM HEPEs, 50mMMgCl 2, pH=7.5) in, add the substrate S (10 μ M) of 0.5 μ L and the DNA (10 μ M) of 2.0 μ L sequences 14 then, be the control systems first.
Reaction solution B: the HEPEs buffered soln that double-stranded DNA is joined 1mL (contains 25mM HEPEs, 50mMMgCl 2, pH=7.5) in, add the substrate S (10 μ M) of 0.5 μ L and the DNA (10 μ M) of 2.0 μ L sequences 15 then.
Reaction liquid C: the HEPEs buffered soln that double-stranded DNA is joined 1mL (contains 25mM HEPEs, 50mMMgCl 2, pH=7.5) in, add the substrate S (10 μ M) of 0.5 μ L and the DNA (10 μ M) of 2.0 μ L sequences 16 then.
Reaction solution D: the HEPEs buffered soln that double-stranded DNA is joined 1mL (contains 25mM HEPEs, 50mMMgCl 2, pH=7.5) in, add the substrate S (10 μ M) of 0.5 μ L and the DNA (10 μ M) of 2.0 μ L sequences 17 then.
Reaction solution E: the HEPEs buffered soln that double-stranded DNA is joined 1mL (contains 25mM HEPEs, 50mMMgCl 2, pH=7.5) in, add the substrate S (10 μ M) of 0.5 μ L and the DNA (10 μ M) of 2.0 μ L sequences 18 then.
Reaction solution F: the HEPEs buffered soln that double-stranded DNA is joined 1mL (contains 25mM HEPEs, 50mMMgCl 2, pH=7.5) in, add the substrate S (10 μ M) of 0.5 μ L then, in contrast second.
4) mensuration of fluorescence spectrum
The solution of previous step is induced 1h at 37 ℃, add back under 380nm, the exciting of the PFP (0.1mM) of 10 μ L and the K1 of 3.0 μ L (10 μ M are in order to remove non-specific interaction) and obtain fluorescence spectrum.
Carry out revision test three times, data among the figure and result are the mean value of 3 tests.
Fig. 2 is for using the figure as a result of DNAzyme DNA detection base mutation; 3a is the fluorescence spectrum of reaction solution A, B and F; 3b is the FRET efficient histogram of reaction solution A, B, C, D and E, [M C]=[M 1NC]=[M 2NC]]=[M 3NC]]=[M 5NC]]=20nM.
The result shows, the I of reaction solution F 424nm/ I 527nm=0.6 ± 0.10, the I of reaction solution A 424nm/ I 527nm=1.847 ± 0.121; The I of reaction solution B 424nm/ I 527nm=0.957 ± 0.035; The I of reaction liquid C 424nm/ I 527nm=0.78 ± 0.07; The I of reaction solution D 424nm/ I 527nm=0.71 ± 0.04; The I of reaction solution E 424nm/ I 527nm=0.67 ± 0.04, can observe very obvious variation.Increase I along with the mutating alkali yl number 424nm/ I 527nmReduce gradually, energy transfer efficiency increases gradually.
Embodiment 6, utilize DNAzyme and PFP to detect single base mutation
One, PFP2's is synthetic
Step 1 with embodiment 2.
Two, the preparation of sample to be tested
The synthetic DNA sequence is as follows:
Sequence 19 (no base mutant DNA sequence Mc): 5 '-CGGTGAAGCTACAACGAGAGC-3 ';
Sequence 20 (single base mutation dna sequence dna M 1NC): 5 '-CGGTGTAGCTACAACGAGAGC-3 '.
Three, detect the single base mutation of sequence
1) design K1 and K2, the sequence of K1 and K2 is as follows:
K1：5’-GCTCTCGTTGTAGCTTCACCG-3’；
K2：5’-TACAGCTAATAGGCTAGCTACAACGAGAGC-3’。
Design substrate S uses fluorescein-labelled S, and the sequence of S is as follows:
5’-FAM-GCTCrArUATTAGCTGTA-3’。
2) K2 (10 μ M) of the K1 of 2.0 μ L (10 μ M) and 2.0 μ L reduces to room temperature and obtains duplex structure behind 80 ℃ of hydridization 20min.
3) preparation feedback liquid
Reaction solution A: the HEPEs buffered soln that double-stranded DNA is joined 1mL (contains 25mM HEPEs, 50mMMgCl 2, pH=7.5) in, add the substrate S (10 μ M) of 0.5 μ L and the DNA (10 μ M) of 2.0 μ L sequences 19 then, be the control systems first.
Reaction solution B: the HEPEs buffered soln that double-stranded DNA is joined 1mL (contains 25mM HEPEs, 50mMMgCl 2, pH=7.5) in, add the substrate S (10 μ M) of 0.5 μ L and the DNA (10 μ M) of 2.0 μ L sequences 20 then.
Reaction liquid C: the HEPEs buffered soln that double-stranded DNA is joined 1mL (contains 25mM HEPEs, 50mMMgCl 2, pH=7.5) in, add the substrate S (10 μ M) of 0.5 μ L then, in contrast second.
4) mensuration of fluorescence spectrum
The solution of previous step is induced 1h at 37 ℃, add back under 380nm, the exciting of the PFP (0.1mM) of 10 μ L and the K1 of 3.0 μ L (10 μ M are in order to remove non-specific interaction) and obtain fluorescence spectrum.
Carry out revision test three times, data results is the mean value of 3 tests.
The result shows, the I of contrast second 424nm/ I 527nm=0.58 ± 0.08; The I of reaction solution A 424nm/ I 527nm=1.95 ± 0.12; The I of reaction solution B 424nm/ I 527nm=1.02 ± 0.08, reduced by 48% than reaction solution A.Can observe very obvious variation.Increase I along with the mutating alkali yl number 424nm/ I 527nmReduce gradually, energy transfer efficiency increases gradually.
Sequence table
＜110〉Institute of Chemistry, Academia Sinica
＜120〉a kind of method that detects the DNA base mutation
<130>CGGNARY81218
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＜213〉artificial sequence
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＜213〉artificial sequence
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ccctaaccct?aacactaacc?c 21
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＜213〉artificial sequence
<400>3
cccaaaccca?aacccaaacc?c 21
<210>4
<211>21
<212>DNA
＜213〉artificial sequence
<400>4
cccaatccca?atcccaatcc?c 21
<210>5
<211>15
<212>DNA
＜213〉artificial sequence
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ccaaccacac?caacc 15
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＜213〉artificial sequence
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gattgcggaa?agaaggtatg?agataatatc?ac 32
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＜213〉artificial sequence
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gattgcgtaa?agaaggtatg?agataatatc?ac 32
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＜213〉artificial sequence
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gattgcgtaa?agaaggtatg?agacaatatc?ac 32
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＜213〉artificial sequence
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gattacggaa?agtaggtctg?agatcatgcc?ac 32
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＜213〉artificial sequence
<400>12
gattgcggaa?agaaggtatg?agataatgtc?ac 32
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＜213〉artificial sequence
<400>13
gattgcggaa?agaaggtatg?agatattgtc?ac 32
<210>14
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<400>14
cggtgaagct?acaacgagag?c 21
<210>15
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<212>DNA
＜213〉artificial sequence
<400>15
cgttgaagct?acaacgagag?c 21
<210>16
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＜213〉artificial sequence
<400>16
cgttgaagca?acaacgagag?c 21
<210>17
<211>21
<212>DNA
＜213〉artificial sequence
<400>17
cggagaagct?tcaacgtgag?c 21
<210>18
<211>21
<212>DNA
＜213〉artificial sequence
<400>18
cggagaagct?tgagcgagtg?c 21
<210>19
<211>21
<212>DNA
＜213〉artificial sequence
<400>19
cggtgaagct?acaacgagag?c 21
<210>20
<211>21
<212>DNA
＜213〉artificial sequence
<400>20
cggtgtagct?acaacgagag?c 21

Claims (9)

1. one kind is detected the method that whether has base mutation among the DNA, may further comprise the steps successively:
1) with fluorescein-labelled strand polynucleotide M; The normal sequence of described M and DNA to be measured is complementary fully and can form the G-tetramer structure;
2) make fluorescein-labeled M form the G-tetramer structure;
3) M and the ethidium bromide that will form the G-tetramer structure mixes, the compound that adds DNA to be measured and following formula (I), the control systems of the compound of dna fragmentation that adding is made up of the normal sequence of DNA to be measured and following formula (I) is set simultaneously, determines as follows whether DNA to be measured exists sudden change: if the I of DNA reaction system to be measured 600nm/ I 422nmSmaller or equal to 2 ± 0.07 of control systems, there is base mutation in DNA to be measured; If the I of DNA reaction system to be measured 600nm/ I 422nmEqual 1.5 ± 0.08 of control systems, there is not base mutation in DNA to be measured; Described I 600nmFor emission wavelength is the fluorescence intensity of the emission peak of 600nm; Described I 422nmFor emission wavelength is the fluorescence intensity of the emission peak of 422nm;
Figure S2008101034584C00011
In the formula (I), m=1～10, n=2～100; R 1, R 2, R 3, R 4=C XH 2X+1Or-O (C XH 2X+1), x=1～10; R 5=C XH 2X+1, x=1～3; R 6=Br, Cl, I, CF 3COO or CH 3COO.
2. the method for claim 1 is characterized in that: in the described formula (I), and m=6, n=6, R 1=H, R 2=H, R 3=H,, R 4=H, R 5=CH 3, R 6=Br.
3. the method for claim 1 is characterized in that: in the described formula (I), and m=6, n=6, R 1=H, R 2=H, R 3=H,, R 4=H, R 5=CH 2CH 3, R 6=I.
4. one kind is detected the method that whether has base mutation among the DNA, may further comprise the steps successively:
1) preparation strand polynucleotide D1, the normal sequence of described D1 and DNA to be measured is complementary fully;
2) preparation strand polynucleotide D2 is with fluorescein-labelled sequence D 2; Described D2 can form hairpin dna, and stem ring and D1 are complementary fully, and stem and D1 have at least 6nt not complementary, and the restriction enzyme site of restriction enzyme is contained in the stem district;
3) D1 and D2 are mixed, add DNA to be measured again, add the corresponding restriction enzyme of restriction enzyme site with D2 stem district then, add the compound of following formula (I) again; System that the dna fragmentation of forming with the normal sequence of DNA to be measured replaces DNA to be measured is set simultaneously in contrast;
Determine as follows whether DNA to be measured exists sudden change: if the I of DNA reaction system to be measured 424nm/ I 527nmSmaller or equal to 2.57 ± 0.05 of control systems, there is base mutation in DNA to be measured; If the I of DNA reaction system to be measured 424nm/ I 527nmEqual 3.8 ± 0.11 of control systems, there is not base mutation in DNA to be measured; Described I 424nmFor emission wavelength is the fluorescence intensity of the emission peak of 424nm; Described I 527nmFor emission wavelength is the fluorescence intensity of the emission peak of 527nm;
Figure S2008101034584C00021
In the formula (I), m=1～10, n=2～100; R 1, R 2, R 3, R 4=C XH 2X+1Or-O (C XH 2X+1), x=1～10; R 5=C XH 2X+1, x=1～3; R 6=Br, Cl, I, CF 3COO or CH 3COO.
5. method as claimed in claim 4 is characterized in that: in the described formula (I), and m=6, n=6, R 1=H, R 2=H, R 3=H,, R 4=H, R 5=CH 3, R 6=Br.
6. method as claimed in claim 4 is characterized in that: in the described formula (I), and m=6, n=6, R 1=H, R 2=H, R 3=H,, R 4=H, R 5=CH 2CH 3, R 6=I.
7. one kind is detected the method that whether has base mutation among the DNA, may further comprise the steps successively:
1) preparation strand polynucleotide K1, the normal sequence of described K1 and DNA to be measured is complementary fully;
2) preparation strand polynucleotide K2, the middle portion of described K2 has the DNAzyme activity, and K2 and K1 have at least 15nt complementary fully, and include the DNAzyme sequence of 10nt at least, and K2 and K1 at least respectively have 6nt not complementary end to end;
3) preparation DNAzyme substrate S uses fluorescein-labelled S; The DNA part of substrate S is complementary fully with K2, and the restriction enzyme site that comprises two special RNA bases is arranged in the middle of the substrate S;
4) K1 and K2 are mixed, add substrate S then, the compound that adds DNA to be measured and following formula (I), the control systems of the compound of dna fragmentation that adding is made up of the normal sequence of DNA to be measured and following formula (I) is set simultaneously, determines as follows whether DNA to be measured exists sudden change: if the I of DNA reaction system to be measured 424nm/ I 527nmSmaller or equal to 1.595 ± 0.06 of control systems, there is base mutation in DNA to be measured; If the I of DNA reaction system to be measured 424nm/ I 527nmEqual 3.08 ± 0.1 of control systems, there is not base mutation in DNA to be measured; Described I 424nmFor emission wavelength is the fluorescence intensity of the emission peak of 424nm; Described I 527nmFor emission wavelength is the fluorescence intensity of the emission peak of 527nm.
Figure S2008101034584C00031
In the formula (I), m=1～10, n=2～100; R 1, R 2, R 3, R 4=C XH 2X+1Or-O (C XH 2X+1), x=1～10; R 5=C XH 2X+1, x=1～3; R 6=Br, Cl, I, CF 3COO or CH 3COO.
8. method as claimed in claim 7 is characterized in that: in the described formula (I), and m=6, n=6, R 1=H, R 2=H, R 3=H,, R 4=H, R 5=CH 3, R 6=Br.
9. method as claimed in claim 7 is characterized in that: in the described formula (I), and m=6, n=6, R 1=H, R 2=H, R 3=H,, R 4=H, R 5=CH 2CH 3, R 6=I.
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CN107091834A (en) * 2017-05-24 2017-08-25 青岛科技大学 It is a kind of that the method that amplifying technique detects DNA is circulated based on hair fastener mispairing

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CN102558511A (en) * 2011-12-20 2012-07-11 中国科学院化学研究所 Method for evaluating antimicrobial susceptibility of drugs
CN102558511B (en) * 2011-12-20 2014-07-30 中国科学院化学研究所 Method for evaluating antimicrobial susceptibility of drugs
CN102731405A (en) * 2012-07-06 2012-10-17 中国科学院化学研究所 Photodynamic treatment medicament, medical composition and preparation method thereof
CN102731405B (en) * 2012-07-06 2014-06-18 中国科学院化学研究所 Photodynamic treatment medicament, medical composition and preparation method thereof
CN107091834A (en) * 2017-05-24 2017-08-25 青岛科技大学 It is a kind of that the method that amplifying technique detects DNA is circulated based on hair fastener mispairing
CN107091834B (en) * 2017-05-24 2019-07-02 青岛科技大学 A method of DNA is detected based on hair fastener mispairing circulation amplifying technique

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