CN104513855B - Thermodynamic optimization-based specificity nucleic acid hybridization method - Google Patents

Abstract

The invention relates to a thermodynamic optimization-based high specificity nucleic acid hybridization method which is as follows: the method is mainly based on a nucleic acid reaction thermodynamic nearest neighbor model, DNA and RNA reaction thermodynamic parameters are calculated, the theoretical limit of the probe specificity can be theoretically illuminated, the specific differences and thermodynamic properties of different single base mutation can be calculated simultaneously, different nucleic acid reaction results can be simulated and predicted on a computer; and a theoretically optimized high specificity nucleic acid probe can be designed. The specificity of the probe designed according to thermodynamic calculation theoretically achieves optimal results; stability is good, the probe can be stored for a long time; the detection process is particularly simple; the detection time is short; the detection process is not influenced by ambient temperature and not influenced by solution salt ion concentration, and the thermodynamic optimization-based high specificity nucleic acid hybridization method can meet the clinical demand, and has a good application prospect in medical aspects.

Description

A kind of specific nucleic acid hybridizing method based on thermodynamic optimization

Technical field

The invention belongs to nucleic acid hybridization field, particularly to a kind of high specific nucleic acid hybridization side based on thermodynamic optimization Method.

Background technology

Nucleic acid is the basis of all living things cell, encodes and adjust the expression of hereditary information in vivo.Opposite The great biosiss such as the growth of object, growth, breeding, heredity and variation play an important role.The biological importance of nucleic acid promotees Make nucleic probe and primer in biotechnology such as:Polymerase chain reaction (PCR), biochip (microarray) and fluorescence are former The having a wide range of applications of aspect such as position hybridization.

Nucleic acid can be used in biology, and the determinant attribute of biotechnology and nanometer biotechnology aspect is exactly that it has can Predictability and special Watson Crick hybridization complementary characteristic.But unless near melting temperature, many correct bases The thermodynamics increase of pairing can cover the thermodynamics error of some mispairing, and also can produce non-compared with the hybridization between longer nucleic acid chain Specifically.

High temperature or chemical modification are normally used for improving the specificity of hybridization.Under the conditions of high temperature or chemical modification, standard Hybridization free energy weaker, based on the adhesion between complementary seriess, slightly different sequence just can be identified.However, In many target hybridization system, many different hybridizations must be carried out simultaneously, and this when utilizes near a certain melting temperature Hybridization is clearly infeasible to improve specificity.In addition, melting temperature depends on the concentration of salt, nucleic acid concentration in hybridization solution Deng some factors, the very difficult accuracy predicting hybridization with this.Equally, chemical modification also weakens Watson-Crick base pairing Principle, equally has potential limitation.

In general a preferable nucleic probe should meet three conditions：High-affinity (can be securely and complementary Target combination), (environmental factorss are not easy to affect, different experiments for high selectivity (not being combined with mismatch) and high stability Room testing result has concordance), and this is difficult to meet in actual practice comprehensively.

The side being hybridized in the important function of the aspects such as biotechnology and current high specific according to nucleic acid specificity hybridization Method still suffers from some defects.

Content of the invention

The technical problem to be solved is to provide a kind of high specific nucleic acid hybridization side based on thermodynamic optimization Method, the method is directed to problem and shortage present in traditional high specific hybridization check, is visited using thermodynamic argument and strand displacement The optimization of pin high specific nucleic acid hybridization conditions, proposes a kind of parameter weighing high specific hybridization temperature influence degree and calculating Method, the high specific hybridization for nucleic acid and the specific detection of single base mutation provide effective methodology foundation.

A kind of high specific nucleic acid hybridization based on thermodynamic optimization of the present invention, including：

(1) it is directed to the purpose target X of pre-detection, the chain that the multiple structures of design are had difference and matched with purpose target is put Change probe PC, as reaction 1, product is P and XC to the reaction with purpose target sequence, with the target with single base mutation As reaction 2, product is P and SC for the reaction of S；Calculate the standard of each nucleic acid molecules in reaction 1 and reaction 2 respectively Gibbs free energy Δ G⁰, standard entropy Δ S⁰And Standard Enthalpies Δ H⁰, and the standard Ji cloth of reaction 1 and reaction 2 is calculated according to this This Gibbs free amount Δ_rG⁰, standard entropy variation delta_rS⁰And Standard Enthalpies variation delta_rH⁰；Initially dense in conjunction with each reactant Degree, finally calculates the hybrid product yield of reaction 1 and reaction 2 respectively；

(2) the hybrid product yield according to step (1), selects the probe tending to react 1；It is finally synthesizing the spy selected Pin, realizes the detection of purpose target high specific.

The technical scheme is that：

(1) high specific of nucleotide sequence is detected：It is first depending on the reaction equation of nucleic acid strand displacing probes, for this Nucleotide sequence calculates several comparisons rational probe design.Then the real reaction according to the probe designing and target Result selects the optimum probe design of specificity.Thus reaching the purpose of the high specific detection to nucleotide sequence.

(2) high specific of single base mutation is detected：It is first depending on the reaction equation of nucleic acid strand displacing probes, for This single base mutation calculates several comparisons rational probe design.Then the thermodynamics according to single base mutation itself are joined Number calculates the temperature characterisitic of this mutation, selects the optimal temperature range of detection.Finally according to actual probe and target Mark reaction result, selects optimized probe design and reaction temperature.Thus reaching high specific to detect single base mutation Purpose.

Technical scheme is specially：

(1) high specific of nucleotide sequence is detected：

Nucleic acid strand displacement reaction equation is：

X+PC→P+XC 1

S+PC→P+SC 2

Wherein X represents purpose target, and PC represents strand displacing probes, P and C represents two nucleic acid of strand displacing probes respectively Chain.Through reaction, X displaces the P chain in probe PC to X and PC, thus forming product XC and the P chain being released, Jing Guoyi After fixing time, reaction reaches balance, and this reaction is called reaction 1.Represent the target with single base mutation, then S+PC → SC+ with S P represents the target of mutation and the reaction equation of probe PC, and this reaction is called reaction 2, and course of reaction is similar with aforementioned.

The first step：According to the nucleotide sequence of pre-detection, design matched strand displacing probes.

Calculate the standard Gibbs free energy Δ G of each nucleic acid molecules in participation " reaction 1 " and " reaction 2 "⁰, standard Entropy Δ S⁰And Standard Enthalpies Δ H⁰.And calculated according to this, the standard Gibbs free energy variation delta of this reaction 1 and reaction 2_rG⁰, standard entropy variation delta_rS⁰And Standard Enthalpies variation delta_rH⁰, to reacting 1, its computing formula is as follows：

Δ_rG⁰(1)=Δ G⁰(XC)+ΔG⁰(P)-ΔG⁰(X)-ΔG⁰(PC)

Δ_rH⁰(1)=Δ H⁰(XC)+ΔH⁰(P)-ΔH⁰(X)-ΔH⁰(PC)

Δ_rS⁰(1)=Δ S⁰(XC)+ΔS⁰(P)-ΔS⁰(X)-ΔS⁰(PC)

Wherein Δ_rG⁰(1) the standard free Gibbs energy variable quantity of " reaction 1 ", Δ are represented_rH⁰(1) it is the mark of " reaction 1 " Quasi- enthalpy change amount, Δ_rS⁰(1) it is the standard entropy variable quantity of " reaction 1 ".ΔG⁰(XC)、ΔH⁰And Δ S (XC)⁰(XC) it is respectively and divide The standard Gibbs free energy of sub- XC, Standard Enthalpies and standard entropy.Other standard Gibbs free energies participating in reaction molecular, mark Quasi- enthalpy, standard entropy method for expressing is similar to.

For " reaction 2 ", its standard Gibbs free energy variation delta_rG⁰(2), Standard Enthalpies variation delta_rH⁰(2) and mark Quasi- entropy variation delta_rS⁰(2) computational methods are identical with the computational methods of " reaction 1 ".

Obtaining the standard Gibbs free energy variation delta of each reaction equation_rG⁰, Standard Enthalpies variation delta_rH⁰With Standard entropy variation delta_rS⁰Afterwards." reaction 1 " and the hybridization yield of " reaction 2 " can be calculated further：

Wherein, 1 and 2 represent the hybridization yield of " reaction 1 " and " reaction 2 " respectively.By comparing calculate gained two instead The hybridization yield answered and respective Standard Enthalpies variation delta rH⁰With standard entropy variation delta rS⁰Relation.Determine and ensure that " instead Answer 1 " yield 1 considerable simultaneously " reaction 2 " yield 2 level off to 0 optimum Δ rH⁰With Δ rS⁰Value.

Determine " reaction 1 " and the Δ rH of " reaction 2 "⁰With Δ rS⁰Concrete numerical value after, by Amending design probe PC The base number of some parts and species, the actual reaction of adjustment and the Δ rH calculating gained⁰With Δ rS⁰Concrete numerical value phase Unanimously.In specific experiment, design multiple structures and there is difference probe, the real reaction result of Binding experiment, select best performance Probe.It is finally reached, strand displacing probes PC is very considerable with the reaction yield of purpose target X.But strand displacing probes PC is almost Do not react with single base mutation target S, thus realizing the high specific detecting.

Second step：Detection process.

It is the preparation of probe first, after strand displacing probes design and synthesize, two chains of probe are passed through hybridization etc. Process is allowed to form duplex structure.Reaction solution system is arbitrarily to can ensure that the solution body of nucleic acid normal hybridisation stable existence System.According to the concentration range of purpose target to be detected, determine concentration and probe concentration substantially, mix homogeneously with purpose target, room temperature Standing.The sign of end reaction result can be with nucleic acid gel electrophoresis or the fluorescence letter modifying fluorescence and quenching group on probe Number or other all can apply the means of this probe.

(2) high specific of single base mutation is detected：

Reaction equation is：

X+PC→P+XC 1

S+PC→P+SC 2

Wherein X represents purpose target, and PC represents strand displacing probes, P and C represents two nucleic acid of strand displacing probes respectively Chain.Through reaction, X displaces the P chain in probe PC to X and PC, thus forming product XC and the P chain being released, Jing Guoyi After fixing time, reaction reaches balance, referred to as " reaction 1 ".Represent the target with single base mutation, then S+PC → SC+P generation with S The target of table mutation and the reaction equation of probe PC, referred to as " reaction 2 ", course of reaction is similar with aforementioned.

The first step：According to the nucleotide sequence of pre-detection, design matched strand displacing probes.

Nucleic acid strand displacing probes are used for the process of single base mutation detection, and its probe designs the first step of part and to nucleic acid The first step of the high specific detection of sequence is identical, i.e. the first step in (1).

Second step：Detection process

In the detection process to single base mutation, probe all can keep high specific at 0 DEG C to 50 DEG C.Treat when not knowing During the concrete species of single base mutation of detection, detection process is also identical with second step in (1).

When knowing the concrete species of single base mutation to be detected, dashed forward with further this kind of single base being calculated by formula The temperature characterisitic becoming, so that it is determined that go out to detect the optimum temperature range of specificity, thus improving the specificity of detection further.Tool Body method is as follows：

Calculate the standard enthalpy difference Δ Δ H and standard entropy difference Δ Δ S of this kind of single base mutation, specific formula for calculation first For：

Δ Δ H=Δ_rH⁰(2)-Δ_rH⁰(1)

Δ Δ S=Δ_rS⁰(2)-Δ_rS⁰(1)

Then the reaction yield that reacts of strand displacing probes of this kind of single base mutation sequence and design is calculated according to formula, Computing formula is as follows：

Wherein, 2 represent the yield of reaction 2, and T is the ambient temperature of reaction.By studying under different temperatures 2 value, can To find, single base mutation can be divided three classes according to its temperature characterisitic difference

, when temperature is relatively low, 2 is less for first kind single base mutation.With the rising of temperature, it can significantly increase, for this Class single base mutation, the specificity Optimal Temperature of its detection should be relatively low, such as 0 DEG C to 25 DEG C.

For Equations of The Second Kind single base mutation, temperature change is inconspicuous with the impact of probe reaction yield to it, and its detection is special The opposite sex will not by temperature change and occur substantially to change, for such abrupt climatic change temperature range in the range of 0 DEG C to 50 DEG C ?.

3rd class single base mutation, its temperature characterisitic is determined by its position, with above-mentioned formula relation less, such single base The edge of the closer sequence in position of mutation, when the temperature increases, because the conformation of its concrete product is different, it will make class The Gibbs free amount Δ rG of reaction⁰(2) significantly raised when high temperature, thus reducing reaction yield 2.So such mutation Will have when temperature is higher and preferably detect specificity, such as 25 DEG C to 50 DEG C.

In the other parts and (1) of its course of reaction after detection temperature scope has been selected according to the species of detected mutation Second step is identical.

(3) nucleic acid strand displacement probe specificity is not by managing that the change of solution salt ionic concentration and variation of ambient temperature are affected By basis.

It is this kind of probe theoretical basiss independent of temperature variations first, the formula being obtained by the first step in (1)：

In this formula, T represents temperature.Designed by accurate probe, by the standard entropy variation delta of reaction_rH⁰(1) adjust For approximating 0, because the coefficient before temperature T in formula is Δ_rH⁰(1), so when this coefficient approximates 0, no matter temperature T How to change, its product is 0.In this way, temperature change can be made not affect reaction yield, thus it is special to reach detection The purpose of different in nature not temperature influence.

Next to that the theoretical basiss that this kind of probe is not affected by salt ionic concentration change in solution, by formula：

ΔS⁰=Δ S⁰[1MNaCl]+0.368×N/2×ln[Na⁺]

Wherein Δ S⁰For the standard entropy of nucleic acid molecules, [1MNaCl] and [Na⁺] respectively represent both materials concentration, by The impact that formula can be seen that the change response parameter of salt ionic concentration in solution is to be divided by changing the nucleic acid of residual reaction The standard entropy of son is realized.I passes through accurately probe design, by the standard entropy variation delta of reaction_rS⁰Numerical value be adjusted to about Equal to 0 it is possible to eliminate the impact to reaction result for the salt ionic concentration change in solution.

The present invention passes through accurately thermodynamic argument and calculates, and calculates and visits close to optimized nucleic acid strand displacement in theory Pin so as to reaction standard entropy Δ_rS⁰With Standard Enthalpies Δ_rH⁰Close to 0.According to result of calculation, design several structures and have carefully The other probe of elementary errors.The probe designing is completed through the preparation of the processes such as bulk crossing.When probe is used for detecting, by probe Mix according to certain concentration phase with target.And according to actual reaction result, select specificity and the best probe of sensitivity. Realize the high specific detection to specific nucleic acid sequence.

The present invention passes through accurately thermodynamic argument and calculates, and for different single base mutation species, calculates single base The temperature characterisitic of mutation.The optimum environment temperature of the specificity of this temperature characterisitic suitable is determined for detected targeted mutagenesis Degree.And calculate in theory close to optimized nucleic acid strand displacing probes so as to reaction standard entropy Δ_rS⁰With Standard Enthalpies Δ_rH⁰ Close to 0.According to result of calculation, design several structures have nuance probe.According to actual reaction result, select Specificity and the best probe of sensitivity.Realize the high specific detection to single base mutation.

The present invention is calculated by accurate thermodynamic argument and probe design, designs and has the nucleic acid chains of high specific and put Change probe.The standard entropy Δ that will be reacted by probe design_rS⁰With Standard Enthalpies Δ_rH⁰Adjust to close to 0.Eliminate environment temperature The impact to detection process of degree and the change of solution salt ionic concentration.It is achieved thereby that all protecting within the temperature range of 0 DEG C to 50 DEG C Hold high specific, all keep under any salt ionic concentration that ensure that nucleic acid normal hybridisation the nucleotide sequence of high specific with Single base mutation detects.Achieve under a wide range of ambient can steady operation high specific nucleic acid probe design. Be simultaneously achieved in the solution environmental under any salt ionic concentration can steady operation high specific nucleic acid probe design.

The strand displacing probes that the present invention designs are applied to the method that any one can be detected using this spy, finally The sign of reaction result can with nucleic acid gel electrophoresis or the fluorescence signal that fluorescence and quenching group are modified on probe or other All can apply the means of this probe.

Beneficial effect

The present invention has reached optimal result in theory based on the probe specificity that calculation of thermodynamics is designed；Good stability, Holding time is long；Detection process and its simple；And detection time is short；Detection process is not influenced by ambient temperature, is not subject to molten simultaneously Salt ionic concentration impact in liquid；The method disclosure satisfy that clinical demand, has good application prospect in terms of medical science；The present invention It is expected to develop into the new method of multiple nucleic acids Sequence Detection and single base mutation detection.

Brief description

Fig. 1 is purpose target nucleic acid sequence (figure a) and the testing process of single base mutation target nucleic acid sequence (figure b)；Its Middle figure a is reacted with purpose target X for strand displacing probes, and release chain P chain is released, and purpose target X and complementary strand C hybridizes；Scheming b is Strand displacing probes are reacted with single base mutation target S, and this reaction is hardly carried out, and the yield of its product is minimum；

Fig. 2 is the real reaction in embodiment 1；

Fig. 3 is the testing result of embodiment 1.

Specific embodiment

With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention Rather than restriction the scope of the present invention.In addition, it is to be understood that after having read the content of present invention instruction, people in the art Member can make various changes or modifications to the present invention, and these equivalent form of values equally fall within the application appended claims and limited Scope.

Embodiment 1

(1) design of probe and preparation

First, according to the purpose target microRNA-125 to be detected, carry out strand displacing probes design.Specific design method For designing the single nucleic acid strands with purpose target complete complementary, then adding some bases in one end of this single nucleic acid strands, claim Make complementary strand.When another chain of design probe, should be identical with target sequence partial sequence to be detected, referred to as discharge Chain.In adjustment probe, the base sequence of some positions makes standard enthalpy of reaction Δ_rH⁰With reaction normal entropy Δ_rS⁰Numerical value about etc. In 0.The probe designing is transferred to company to synthesize.Probe sequence after synthetic, by complementary strand and release chain according to a definite proportion Example bulk crossing is simultaneously diluted to suitable concentration.The concentration of probe is 50nM in the present embodiment.

In the present embodiment, for the ease of observation reaction result, by two chains of probe modified respectively fluorescence molecule and Fluorescent quenching agent molecule.Fluorescence molecule is FAM, and quencher is Dabcyl.When two chains of probe hybridize mutually, FAM and quenching Agent of going out contacts.Fluorescence is not then now had to send.When target is hybridized with probe, in probe a chain will be put by chain Change reaction and be released.Fluorescence molecule separates with quencher, then fluorescence signal strengthens.Fluorescence intensity directly reflects reaction and produces Amount.As shown in Fig. 2 two chains of wherein strand displacing probes have modified quencher and fluorescence molecule, respectively if target occurs therewith Reaction, then the release chain P being modified with quencher is released, the product of the fluorescence recovery, fluorescence intensity and product of fluorescence molecule Amount is directly proportional.

Three kinds of different probes, respectively 7/6 probe, 7/5 probe and 7/4 probe is devised near result of calculation.With As a example 7/6 probe, the strand displacement initiation sequence that wherein numeral 7 represents probe is 7 bases longs, that is, in one section of interpolation of probe 7 bases.The sequence that 6 other ends representing probe add is six bases, as the equilibrium shape models of strand displacement.Other probes In numeral equivalent in meaning.

Nucleic acid target used and probe are by the synthesis of the precious biology of big chain (TAKARA) company limited.Sequence used is shown in Table 1.

Table 1 strand displacing probes sequence and microRNA-125 sequence and its various mutant nucleotide sequence

In the present embodiment, four kinds of mutation standard enthalpy difference Δ Δ H of targets and standard entropy difference Δ Δ S and when 0 DEG C Standard free energy difference Δ Δ G^0℃It is listed in table 2.

The standard enthalpy difference Δ Δ H of 2 four kinds of mutation targets of table and standard entropy difference Δ Δ S and the standard free energy when 0 DEG C Difference Δ Δ G^0℃

The data of each mutation target is substituted into formula respectively：

Can get the function of relation between each the mutation distinctive reaction yield of target and temperature.Through analysis, it is mutated target Mark 1 reaction yield not vary with temperature and change, mutation target 2 reaction yield raises with temperature and reduces, mutation target 3 reacts Yield raises with temperature and raises, and mutation target 3 reaction yield does not vary with temperature.

(2) the high specific detection of microRNA-125 sequence and the high specific detection process of specific single base mutation

At this, probe dilution preparing is 50nM, each of synthesis target is diluted to corresponding concentration, will visit Pin and each target are mixed in centrifuge tube respectively according to corresponding proportion.Separately take one group of probe simple and without used by any target Solution environmental be TE buffer (10mM Tris-HCl, pH=8 and 1mM EDTA), NaCl concentration be 1M.

Probe is divided into two parts after mixing with each target, react 3 hours respectively at 4 DEG C and 50 DEG C.Then institute will be reacted Centrifuge tube is placed in fluorescence microscopy Microscopic observation fluorescence signal.

1. the testing result of MicroRNA-125 sequence judges：

In above-mentioned detection process, a length of 492nm of excitation light wave of FAM fluorescence molecule, launch wavelength is 518nm, selects glimmering The optical filter of respective wavelength on light microscope, each reacted centrifuge tube is placed in basis of microscopic observation, and takes pictures, using figure Analyze its fluorescence intensity as processing software I mage-Pro Plus.Probe and purpose target sequence MicroRNA-125 reaction result There is the strong fluorescence signal of trial of strength, probe does not all have fluorescence signal or only faint with the reaction that other contain single base mutation sequence Fluorescence signal.4 DEG C of groups observe the above results with 50 DEG C.

2. specific single base mutation testing result is judged：

In the detection process to single base mutation, probe all shows good single base in 4 DEG C of groups with 50 DEG C of groups and dashes forward Become separating capacity.When be intended to detect sport known when, the temperature characterisitic of this single base mutation can be precalculated first, thus logical Toning full employment temperature range, improves the specificity of detection further.In the present embodiment, four kinds of single base mutation sequences of design Arrange, result of calculation is：As shown in figure 3, the mutation target 1,2,3,4 with single base mutation all only has extremely faint fluorescence letter Number, at 4 DEG C and 50 DEG C, reaction result is all such, shows very high specificity, and is not affected by variation of ambient temperature；Separately Outward, when probe is used for detecting certain specific single base mutation, the fluorescence signal of mutation target is lower, represents that its detection is special Property is better.The temperature characterisitic that the experimental result of four kinds of single base mutations shows is consistent with result of calculation, and mutation target 1 is special Property not temperature influence, mutation target 2 specificity raises with temperature and increases, and mutation target 3 specificity raises with temperature and subtracts Little.Mutation target 4 specificity not temperature influence.Reaction result is consistent with above-mentioned result of calculation.

Claims (4)

1. a kind of specific nucleic acid hybridizing method based on thermodynamic optimization, including：

(1) it is directed to the purpose target X of pre-detection, design multiple structures and be there is difference the strand displacement spy being matched with purpose target Pin PC, as reaction 1, product is P and XC to the reaction with purpose target sequence, with the target S's with single base mutation As reaction 2, product is P and SC for reaction；Calculate the standard Ji cloth of each nucleic acid molecules in reaction 1 and reaction 2 respectively This free energy Δ G⁰, standard entropy Δ S⁰And Standard Enthalpies Δ H⁰, and according to this calculate reaction 1 and reaction 2 standard Gibbs from By energy variation delta_rG⁰, standard entropy variation delta_rS⁰And Standard Enthalpies variation delta_rH⁰；In conjunction with the initial concentration of each reactant, The final hybrid product yield calculating reaction 1 and reaction 2 respectively；

Wherein, the computing formula of hybrid product yield is：

${\mathrm{\λ}}_1=\frac{3e^{\frac{1}{R}\left({\mathrm{\Δ}}_r{S}^0\right(1)-\frac{1}{T}{\mathrm{\Δ}}_r{H}^0(1\left)\right)}-\sqrt{e^{\frac{2}{R}\left({\mathrm{\Δ}}_r{S}^0\right(1)-\frac{1}{T}{\mathrm{\Δ}}_r{H}^0(1\left)\right)}+14e^{\frac{1}{R}\left({\mathrm{\Δ}}_r{S}^0\right(1)-\frac{1}{T}{\mathrm{\Δ}}_r{H}^0(1\left)\right)}+1}+1}{2e^{\frac{1}{R}\left({\mathrm{\Δ}}_r{S}^0\right(1)-\frac{1}{T}{\mathrm{\Δ}}_r{H}^0(1\left)\right)}-2};$

${\mathrm{\λ}}_2=\frac{3e^{\frac{1}{R}\left({\mathrm{\Δ}}_r{S}^0\right(2)-\frac{1}{T}{\mathrm{\Δ}}_r{H}^0(2\left)\right)}-\sqrt{e^{\frac{2}{R}\left({\mathrm{\Δ}}_r{S}^0\right(2)-\frac{1}{T}{\mathrm{\Δ}}_r{H}^0(2\left)\right)}+14e^{\frac{1}{R}\left({\mathrm{\Δ}}_r{S}^0\right(2)-\frac{1}{T}{\mathrm{\Δ}}_r{H}^0(2\left)\right)}+1}+1}{2e^{\frac{1}{R}\left({\mathrm{\Δ}}_r{S}^0\right(2)-\frac{1}{T}{\mathrm{\Δ}}_r{H}^0(2\left)\right)}-2}$

Wherein R is ideal gas constant, and T is reaction temperature；

(2) the hybrid product yield according to step (1), selects the probe tending to react 1；It is finally synthesizing the probe selected, real Existing purpose target specificity detection；Described tend to react 1 probe criterion be：

The reaction yield of strand displacing probes PC and purpose target X is more than the reaction of strand displacing probes PC and single base mutation target S Yield.

2. a kind of specific nucleic acid hybridizing method based on thermodynamic optimization according to claim 1 it is characterised in that：Institute State the standard Gibbs free energy Δ G of the reaction 1 in step (1)⁰, standard entropy Δ S⁰And Standard Enthalpies Δ H⁰Computing formula be：

Δ_rG⁰(1)=Δ G⁰(XC)+ΔG⁰(P)-ΔG⁰(X)-ΔG⁰(PC)；

Δ_rH⁰(1)=Δ H⁰(XC)+ΔH⁰(P)-ΔH⁰(X)-ΔH⁰(PC)；

Δ_rS⁰(1)=Δ S⁰(XC)+ΔS⁰(P)-ΔS⁰(X)-ΔS⁰(PC).

3. a kind of specific nucleic acid hybridizing method based on thermodynamic optimization according to claim 1 it is characterised in that：Institute The purpose target stated in step (1) is known single base mutation, calculates the temperature characterisitic of single base mutation by formula, determines The optimum temperature range of detection specificity；By salt ionic concentration change impact formula in solution, select and become by salt ionic concentration Change the minimum strand displacing probes of impact.

4. a kind of specific nucleic acid hybridizing method based on thermodynamic optimization according to claim 3 it is characterised in that：Institute Stating temperature characterisitic computing formula is：Δ Δ H=Δ_rH⁰(2)-Δ_rH⁰(1)；Δ Δ S=Δ_rS⁰(2)-Δ_rS⁰(1)；

${\mathrm{\λ}}_2=\frac{3e^{\frac{1}{R}(\mathrm{\Δ}\mathrm{\Δ}S-\frac{1}{T}\mathrm{\Δ}\mathrm{\Δ}H)}-\sqrt{e^{\frac{2}{R}(\mathrm{\Δ}\mathrm{\Δ}S-\frac{1}{T}\mathrm{\Δ}\mathrm{\Δ}H)}+14e^{\frac{1}{R}(\mathrm{\Δ}\mathrm{\Δ}S-\frac{1}{T}\mathrm{\Δ}\mathrm{\Δ}H)}+1}+1}{2e^{\frac{1}{R}(\mathrm{\Δ}\mathrm{\Δ}S-\frac{1}{T}\mathrm{\Δ}\mathrm{\Δ}H)}-2}.$