CN105132528A - Double-stranded displacing probe high-specificity detection single-base change method based on thermodynamics optimization - Google Patents

Double-stranded displacing probe high-specificity detection single-base change method based on thermodynamics optimization Download PDF

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CN105132528A
CN105132528A CN201510423384.2A CN201510423384A CN105132528A CN 105132528 A CN105132528 A CN 105132528A CN 201510423384 A CN201510423384 A CN 201510423384A CN 105132528 A CN105132528 A CN 105132528A
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毛红菊
武振华
张宏莲
金庆辉
赵建龙
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Shanghai Institute of Microsystem and Information Technology of CAS
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Abstract

The invention relates to a double-stranded displacing probe high-specificity detection single-base change method based on thermodynamics optimization, wherein the method comprises the following steps: (1) in accordance with a pre-detected target sequence, designing a double-stranded displacing probe and designing a corresponding PCR amplification primer, and amplifying a target sequence through the primer, wherein by designing the probe and the primer, change in free energy of reaction 1 is close to 0 and theoretically optimum specificity is achieved; and (2) according to the design of the step (1), synthesizing actual probe and primer, so as to achieve high-specificity detection of the target sequence. The method disclosed by the invention, by virtue of the designed double-stranded displacing probe, further improves the specificity of single-base change detection; the method is free from influence of the length of a detection sequence, good in stability, long in preservation duration, quite simple in detection process and short in detection time, and the detection process is not affected by ambient temperature or the concentration of salt ions in a solution.

Description

A kind of displacement of the double-strand based on thermodynamic optimization nucleic acid probe high specific detects the method for single sequence change
Technical field
The invention belongs to single sequence change detection field, particularly a kind of displacement of the double-strand based on thermodynamic optimization nucleic acid probe high specific detects the method for single sequence change
Background technology
Nucleic acid is the basic molecule in all living things cell, encodes in vivo and regulates the expression of genetic information.The growth of organism, growth, breeding, the great biological phenomena such as heredity and variation are played an important role.The biological importance of nucleic acid impels nucleic acid probe and primer in biotechnology as polymerase chain reaction (PCR), having a wide range of applications of the aspects such as biochip (microarray) and nanoassemble material.
And the single sequence change in nucleotide sequence, be form bacterium, virus and the drug-fast genetic base of tumour, also have substantial connection with various diseases simultaneously.Detect the single sequence change in nucleotide sequence fast and accurately, all will have important effect to biological study and medical diagnosis on disease.
In traditional method, high temperature or chemical modification are normally used for the specificity improving hybridization.Under high temperature or chemical modification condition, the target of single sequence change and the combination rate of probe will be starkly lower than the sequence of complete complementary.But these class methods often have limitation.First they need harsh reaction conditions to control, and can not be used for many targets simultaneously and detect simultaneously.In addition, when detected sequence is longer, its specificity will be subject to single secondary structure impact practicing nucleic acid molecule, thus loses the separating capacity to single sequence change.
General, based on the probe of thermodynamic principles design, highly sensitive (with mating target mortise completely) can be met simultaneously, highly selective (being combined with single sequence change target hardly), the requirements such as high stability (not affecting by solution salt ionic concn while of detecting not influenced by ambient temperature).Such as molecular beacon and strand displacement probe.
Existing molecular beacon and strand displacement probe in detecting process can be subject to secondary structure impact, cannot carry out reliable single sequence change detect long sequence.In addition, its specificity still has much room for improvement, and be can further improve the specificity of probe by more excellent design.
Summary of the invention
Technical problem to be solved by this invention is to provide the method that a kind of double-strand based on thermodynamic optimization displacement nucleic acid probe high specific detects single sequence change, and the double-stranded displacing probe that the method is designed further increases single sequence change detection specificity; Not examined sequence length impact; Good stability, the shelf time is long; Testing process and simple; And detection time is short; Testing process is not influenced by ambient temperature, does not affect by salt ionic concentration in solution simultaneously.
A kind of displacement of the double-strand based on thermodynamic optimization nucleic acid probe high specific of the present invention detects the method for single sequence change, comprising:
(1) for the aim sequence of pre-detection, design double-stranded displacing probe, and design corresponding pcr amplification primer; Go out object target sequence by primer amplification, the reaction of the target sequence of probe and complete complementary is as reaction 1, and gained reaction product is product 1 and product 2; Probe with there is the reaction of target sequence of single sequence change as reaction 2, gained reaction product is product 3 and product 4; Calculate the standard Gibbs free energy Δ G of each nucleic acid molecule in reaction 1 and reaction 2 respectively 0, and the standard Gibbs free energy variation delta of reaction 1 and reaction 2 is calculated according to this rg 0; By probe and design of primers, make the free energy change amount of reaction 1 close to 0, reach theoretical optimum specificity;
(2) according to the design of step (1), synthesize actual probe and primer, the high specific realizing object target sequence detects.
Amplification procedure in described step (1) is asymmetric pcr.
The standard Gibbs free energy Δ G of the reaction 1 in described step (1) 0calculation formula be:
Δ rg 0(1)=Δ G 0(product 1)+Δ G 0(product 2)-Δ G 0(probe)-Δ G 0(target sequence).
By probe and design of primers in described step (1), make the standard entropy change of reaction 1 close to 0.Salt ionic concentration change calculations formula is: Δ S 0=Δ S 0[1MNaCl]+0.368 × N/2 × ln [Na +], the standard entropy change of reaction 1 is Δ rs 0(1) ≈ 0, thus ensure that this testing process does not affect by salt ionic concentration.
Technical scheme of the present invention is:
The high specific of single sequence change is detected: first according to the reaction equation of nucleic acid double chain displacement probe, calculate the best probe design of this double-strand replacement(metathesis)reaction specificity for complete complementary target sequence to be detected.Then according to design result, the probe that synthesis is actual and corresponding asymmetric pcr primer, go out target sequence to be detected by asymmetric pcr primer amplification, probe and target sequence react.Thus reach the object that high specific detects single base mutation.
Technical scheme is specially:
Nucleic acid double chain replacement(metathesis)reaction equation is:
X+ double-chain probe → product 1+ product 21
S+ double-chain probe → product 3+ product 42
Wherein X represents the target sequence of complete complementary, and S represents the target sequence of single sequence change.X and probe are through reacting, and formation reaction product 1 and product 2, after certain hour, reaction reaches balance, claims this reaction to be reaction 1.Have the target sequence of single sequence change with S representative, then S+ double-chain probe → product 3+ product 4 represents the target sequence of single sequence change and the reaction equation of probe, claims this reaction to be reaction 2, reaction process and aforementioned similar.
The first step: according to the nucleotide sequence of pre-detection, designs the double-stranded displacing probe and asymmetric pcr primer that match with it.
Calculate the standard Gibbs free energy Δ G of each nucleic acid molecule in participation " reaction 1 " and " reaction 2 " 0.And the standard Gibbs free energy variation delta of reaction 1 and reaction 2 is calculated according to this rg 0, to reaction 1, its calculation formula is as follows:
Δ rg 0(1)=Δ G 0(product 1)+Δ G 0(product 2)-Δ G 0(X)-Δ G 0(double-chain probe)
Wherein Δ rg 0(1) the free Gibbs energy variable quantity of standard of " reaction 1 " is represented.Δ G 0the standard Gibbs free energy that (product 1) is product 1.The standard Gibbs free energy method for expressing of other participation reaction molecular is similar.
By base number and the kind of some part of Amending design double-stranded displacing probe, the standard Gibbs free energy variation delta of adjustment reaction 1 rg 0be approximately equal to 0, thus design the probe of specificity optimum.According to the probe designed, design corresponding asymmetric pcr primer.Target sequence to be detected is gone out by primer amplification.The probe that final design goes out and primer, can reach, considerable with the hybridization output of the target sequence X of complete complementary, but double-stranded displacing probe changes target sequence S with single alkali hardly to react, thus realize the high specific of detection.
Second step: testing process.
First be the preparation of probe, after double-stranded displacing probe designs and synthesizes, four of probe chains made it to form duplex structure by processes such as hybridization.Then, target sequence to be detected is gone out with the asymmetric pcr primer amplification designed.Reaction soln system is the solution system that can ensure arbitrarily nucleic acid normal hybridisation stable existence.According to the concentration range of object target sequence to be detected, determine concentration and probe concentration roughly, mix with object target, room temperature leaves standstill.The sign of end reaction result can modify on probe the fluorescent signal of fluorescence and quenching group or other all can apply the means of this probe.
The present invention, by accurately thermodynamic argument calculating, calculates in theory close to specificity optimized nucleic acid double chain displacement probe, the standard free energy change amount Δ making it react rg 0close to 0.According to calculation result designing probe.The probe designed has been prepared through processes such as bulk crossings.Design the asymmetric pcr primer corresponding to probe simultaneously, when probe is for detecting, utilizes asymmetric pcr primer amplification to go out target sequence to be detected, probe is mixed according to certain concentration mutually with target sequence.Realize detecting the high specific of specific single sequence change.
The present invention is calculated and probe design by accurate thermodynamic argument, designs the nucleic acid double chain displacement probe with high specific.By the standard entropy Δ of probe design by reaction rs 0with Standard Enthalpies Δ rh 0be adjusted to close to 0.Eliminate envrionment temperature and the impact of solution salt ionic concn change on testing process.Thus achieve and in the temperature range of room temperature, all keep high specific at 0 DEG C, any can ensure the salt ionic concentration of nucleic acid normal hybridisation under all keep single sequence change of high specific to detect.Achieve under a wide range of ambient can steady operation high specific double-strand displacement nucleic acid probe design.Achieve in the solution environmental under any salt ionic concentration can the high specific duplex displacement property nucleic acid probe design of steady operation simultaneously.
The double-stranded displacing probe that the present invention designs is applicable to any one and this spy can be used to carry out the method detected, the sign of end reaction result can be used in the fluorescent signal of probe being modified fluorescence and quenching group or other all can apply the means of this probe.
beneficial effect
The present invention is based on the double-stranded displacing probe that calculation of thermodynamics designs and further increase single sequence change detection specificity; Not examined sequence length impact; Good stability, the shelf time is long; Testing process and simple; And detection time is short; Testing process is not influenced by ambient temperature, does not affect by salt ionic concentration in solution simultaneously; The method can meet clinical demand, has good application prospect in medical science; Be expected to the novel method developing into the detection of multiple nucleic acids sequence list sequence change.
Accompanying drawing explanation
Fig. 1 is the testing process of complementary nucleic acid target sequence (reaction 1) and single sequence change nucleic acid target sequence (reaction 2); Wherein react 1 middle probe and complementary target sequence exchanges a nucleic acid chains, reaction yield is considerable; Reaction 2 is probe and the reaction of single sequence change target sequence, and this reaction process does not occur substantially, and reaction yield can be ignored;
Fig. 2 is the real reaction in embodiment 1;
Fig. 3 is the detected result of embodiment 1.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.In addition should be understood that those skilled in the art can make various changes or modifications the present invention, and these equivalent form of values fall within the application's appended claims limited range equally after the content of having read the present invention's instruction.
Embodiment 1
(1) Design & preparation of probe
First, according to the object K-ras sequence for detecting, and the ultimate principle of double-stranded displacing probe is designed and can be ensured that the free energy change amount of reaction equation 1 is the probe of 0.Specific design method is, design four with the single nucleic acid strands of two chain complete complementaries of aim sequence, then add some bases respectively in one end of these wherein four single nucleic acid strands.In adjustment probe, the base sequence of some position makes reaction equation 1 standard free energy change amount Δ rg 0approximate 0.Company is transferred to synthesize the probe designed.Probe sequence after synthetic, by each bar chain of probe bulk crossing be diluted to suitable concentration according to a certain percentage.
In the present embodiment, for the ease of the result of observation reaction, the chain of two in probe fluorescence molecule and fluorescent quenching agent molecule are modified respectively.Fluorescence molecule is FAM, and quencher is Dabcyl.When four chains of probe are hybridized mutually, FAM and quench reagent.Fluorescence is not then now had to send.When target sequence and probe hybridization time, two chains of probe will be separated by double-strand replacement(metathesis)reaction.Fluorescence molecule is separated with quencher, then fluorescent signal strengthens.Fluorescence intensity directly reflects reaction yield.
Nucleic acid probe used and asymmetric pcr primer are by the synthesis of large chain precious biological (TAKARA) company limited.Sequence used is in table 1.
Table 1 double-stranded displacing probe sequence and PCR primer sequence
Reagent and instrument: TEbuffer (SangonBiotech), RNase-freewater (SangonBiotech), primer and probe (Takarabiotechnology (Dalian)), Taq enzyme (Takara), DNTPs, BX51 fluorescent microscope (OLYMPUS), fluorescence spectrophotometer FluoroMax-4spectrofluorometer (HORIBA).
(2) amplification of target sequence
The first step pcr amplification: all primer RNAse-freewater are diluted to 10uM stand-by.The first step increases, and the primer is outer primer F and outer primer R, and amplification system is 20uL, and primer respectively adds 0.5uL, final concentration 250nM.Sample form DNA adds 2uL.Taq enzyme adds 0.5 to 2 unit.MgCl 2final concentration 2.5mM.10 × taqbuffer adds 2uL.DNTPs final concentration 0.25mM.The first step amplified production is obtained through amplification.
Second step pcr amplification: the first step amplified production RNAse-freewater dilutes 100 times as second step amplification template.Second amplification the primer is three kinds of F1, R1, R2, and amplification system is divided into two pipes, and numbering is respectively No. 1 and No. 2.
No. 1 pipe: sample form DNA adds 0.5 to 2uL.Taq enzyme adds 2 units.MgCl2 final concentration 2.5mM.10 × taqbuffer adds 2uL.DNTPs final concentration 0.25mM.F1 final concentration 500nM, R1 final concentration 5nM.
No. 2 pipes: sample form DNA adds 0.5 to 2uL.Taq enzyme adds 2 units.MgCl2 final concentration 2.5mM.10 × taqbuffer adds 2uL.DNTPs final concentration 0.25mM.F1 final concentration 5nM, R2 final concentration 500nM.
Amplification procedure is through the amplification of number wheel, and obtaining asymmetric pcr product two, is second step amplified production.
3rd step: two of second step articles of amplified productions are mixed in hybridization solution, obtains target sequence to be detected through processes such as hybridization.
(3) probe preparation: probe four sections is diluted to 1uM with TEbuffer respectively.Balanced mix, and add some NaCl aqueous solution, eventually pass through the processes such as hybridization and obtain probe solution.
(4) the high specific testing process of single sequence change
Testing process: get probe and mix with sample product, mixed solution is 20uL, leaves standstill.Probe is divided into two parts after mixing with each target, reacts 3 hours respectively under 4 DEG C and room temperature.Control group is that probe mixes with the buffer without any target sequence.Then reaction centrifuge tube used is placed in fluorescence microscopy Microscopic observation fluorescent signal.If there is bright fluorescence to produce, then illustrate that tool sequence has sudden change to be detected.
Single sequence change detected result judges:
In above-mentioned testing process, the excitation wavelength of FAM fluorescence molecule is 492nm, emission wavelength is 518nm, select the spectral filter of respective wavelength on fluorescent microscope, each reacted centrifuge tube is placed in basis of microscopic observation, and take pictures, utilize image processing software Image-ProPlus to analyze its fluorescence intensity.In this experiment, designing probe becomes sequence complete complementary with the single sequence change for detecting, then having fluorescent signal to produce explanation has this single base to change existence.And wild type sequence and control group are all without obvious fluorescent signal.Signal ratio is up to 100 times.4 DEG C of groups all observe the above results with room temperature group.

Claims (4)

1. the double-strand based on thermodynamic optimization replaces the method that nucleic acid probe high specific detects single sequence change, comprising:
(1) for the aim sequence of pre-detection, design double-stranded displacing probe, and design corresponding pcr amplification primer; Go out object target sequence by primer amplification, the reaction of the target sequence of probe and complete complementary is as reaction 1, and gained reaction product is product 1 and product 2; Probe with there is the reaction of target sequence of single sequence change as reaction 2, gained reaction product is product 3 and product 4; Calculate the standard Gibbs free energy Δ G of each nucleic acid molecule in reaction 1 and reaction 2 respectively 0, and the standard Gibbs free energy variation delta of reaction 1 and reaction 2 is calculated according to this rg 0; By probe and design of primers, make the free energy change amount of reaction 1 close to 0, reach theoretical optimum specificity;
(2) according to the design of step (1), synthesize actual probe and primer, the high specific realizing object target sequence detects.
2. a kind of displacement of the double-strand based on thermodynamic optimization nucleic acid probe high specific according to claim 1 detects the method for single sequence change, it is characterized in that: the amplification procedure in described step (1) is asymmetric pcr.
3. a kind of displacement of the double-strand based on thermodynamic optimization nucleic acid probe high specific according to claim 1 detects the method for single sequence change, it is characterized in that: the standard Gibbs free energy Δ G of the reaction 1 in described step (1) 0calculation formula be: Δ rg 0(1)=Δ G 0(product 1)+Δ G 0(product 2)-Δ G 0(probe)-Δ G 0(target sequence).
4. a kind of displacement of the double-strand based on thermodynamic optimization nucleic acid probe high specific according to claim 1 detects the method for single sequence change, it is characterized in that: by probe and design of primers in described step (1), make the standard entropy change of reaction 1 close to 0.
CN201510423384.2A 2015-07-17 2015-07-17 Double-stranded displacing probe high-specificity detection single-base change method based on thermodynamics optimization Pending CN105132528A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115011672A (en) * 2022-06-30 2022-09-06 重庆邮电大学 Ultralow frequency gene mutation detection method

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