CN101285095B - Process for detecting mononucleotide polymorphism of deoxyribonucleic acid - Google Patents
Process for detecting mononucleotide polymorphism of deoxyribonucleic acid Download PDFInfo
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- CN101285095B CN101285095B CN2008100249076A CN200810024907A CN101285095B CN 101285095 B CN101285095 B CN 101285095B CN 2008100249076 A CN2008100249076 A CN 2008100249076A CN 200810024907 A CN200810024907 A CN 200810024907A CN 101285095 B CN101285095 B CN 101285095B
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Abstract
The invention relates to a method for detecting mononucleotide property of deoxyribonucleic acid. The method is as follows: a target sequence (containing substitution sites) on one single chain of a DNA fragment to be tested is determined; a base quenching probe corresponding to the target sequence is prepared; one end of the probe is combined with a fluorescent group; a pair of primers corresponding to the DNA fragment to be tested is prepared; then a polymerase chain reaction is performed; then the probe is hybridized on the target sequence (containing substitution sites) of the augmented DNA fragment to be tested; the melting temperature is measured; and whether mononucleotide of the substitution sites of the DNA fragment to be tested has mononucleotide polymorphism is judged. The method has the characteristics of low cost, convenient operation, and shorter detection time.
Description
Technical field
The present invention relates to a kind of method that detects the mononucleotide character of thymus nucleic acid.
Background technology
(Single nucleotide polymorphisms SNPs) is meant on genomic level polymorphum by the caused dna sequence dna of variation of single Nucleotide to SNP.The SNPs of gene control region and coding region more possibly cause the variation on the function than the SNPs on other position.These diallele signs of somatotype give to identify Disease-causing gene, set up the drug targeting treatment and individual administration provides very big possibility.Therefore, be academia or business circles to set up a kind of fast, the method for responsive and economic detection SNP all has keen interest.
Many classic methods; Like restriction fragment length polymorphism (Restriction fragment length polymorphism; RFLP) analysis and gel mobility shift assay method etc. are because of the restriction that receives loaded down with trivial details schedule of operation, mutational site and target sequence conformation has limited range of application.These methods are long required detection time, and need empirical technician and analyze net result.Recently, the technology that many new fast typing SNP occurred.As: based on FRET (fluorescence resonance energy transfer; FRET) the quick SNP detection method of the single tube of principle, based on allele-specific primers extend and utilize green and red fluorescence mark mononucleotide gene type method, utilize quenching group extend (Quencher extension, QEXT) in real time stopped pipe detect SNP method, duplex scorpion type primer and analyze SNP technology, solid phase and support the SNP typing, use exonuclease III/ s1 nuclease/the direct forward of PNA system detects SNP method, twin probe method, polymorphic nucleic probe coupling polymer detection method and utilizes omnidistance TaqMan real-time quantitative data automatic gene typing etc.Yet, the reagent that Technology Need mentioned above is expensive, and in China, except TaqMan probe and molecular beacon probe, the probe (comprising the FRET probe) of other type is modified by little company.In the aforesaid method, long like the enzyme cutting method equal time, label range is also less.
Adopt molecular beacon probe to carry out the method that SNP detects in addition: (oligonucleotide is one type of general name of having only the short chain Nucleotide of 30 following bases to the fluorescent mark oligonucleotide probe of employing synthetic; Connect the fluorescence report group or/and the fluorescent quenching group then constitutes probe in the end of oligonucleotide); The two ends of this probe are formed with the irrelevant complementary base of target preface by 5～7; One end mark fluorescent reporter group, the other end mark quenching group.Like this under unbound state; The complementation of fluorescent probe two ends is folded into loop-stem structure; The fluorescence report group of oligonucleotide probe and quenching group are very approaching in the space; The fluorescent energy that this moment, the fluorescence report group was launched after exciting is absorbed by quenching group, does not produce fluorescent signal, shows as fluorescent quenching.And after probe and target DNA hybridization, the loop-stem structure of probe is destroyed, and quenching group leaves the fluorescence report group, and the fluorescent signal that the fluorescence report group is produced after exciting outwards sends.Thereby can judge whether base mutation according to the situation of institute's emitting fluorescence.But this method is when carrying out the design of probe, and the scope of its mark is less.
One Chinese patent application 200310119928.3 discloses a kind of method that SNP detects of carrying out with FRET; This method is selected two kinds of probes; A kind of probe is combined with the fluorophore donor, and another kind of probe is combined with the fluorophore acceptor, under the irradiation of exciting light; The fluorophore donor is excited to the fluorophore acceptor fluorescence to be provided; The fluorophore acceptor sends fluorescence, thereby can then confirm to exist in the purpose nucleic acid SNP when this temperature value is lower than melting temperature(Tm) according to the variable quantity that the fluorophore acceptor the sends fluorescence pairing temperature value of peak value of confirming to unwind.Along with the coming off of probe, fluorescence intensity from large to small in this method.To can certain requirement being arranged with the oligonucleotide chain bonded fluorophore donor or the choosing of fluorophore acceptor of probe, thereby cost is higher in this method.
Chinese patent document CN1952178A discloses a kind of method that detects the mononucleotide character of thymus nucleic acid.This method has following steps: confirm that 1. first sequence and second sequence and first sequence on the strand of dna fragmentation to be measured possibly have variant sites, second sequence be meant with first consecutive nucleotides near one section nucleotide sequence; 2. preparation corresponds respectively to the responsive probe of first sequence and corresponding to the second sequence grappling probe; One end of a responsive probe only end is combined with a kind of in fluorescence report group (also can be described as fluorophor) and the quenching group, and an end of a grappling probe only end is combined with the another kind in fluorophor and the quenching group; 3. preparation and the corresponding a pair of primer of dna fragmentation to be measured; 4. by excessive substrate, archaeal dna polymerase, excessive Oligonucleolide primers and the thymus nucleic acid anabolic reaction system that contains dna fragmentation to be measured; Also add base cancellation probe; In above-mentioned reaction system, carry out the polymerase chain reaction then, and make above-mentioned dna fragmentation amplification to be measured; 5. be heated to and make the dna fragmentation sex change to be measured after the amplification and its two strands is untied, lower the temperature then and make responsive probe and grappling probe hybridization on the strand that contains first sequence and second sequence of the dna fragmentation to be measured after the amplification; Because fluorescence report group and quenching group is adjacent to one another and quantity is suitable; So if this moment the fluorescence report group on the probe is excited; The fluorescence that then it was inspired is incorporated into the fluorescent quenching group cancellation of same another probe on the chain, does not externally show fluorescence; 6. the system after heating is hybridized; The responsive probe that is incorporated on first sequence on the strand of dna fragmentation to be measured can split away off earlier, if excite the fluorescence report group on the probe this moment, then externally demonstrates fluorescence because of coming off of responsive probe; Continuation rising along with temperature; The responsive probe increase that comes off, fluorescence intensity be also along with enhancing, and wherein fluorescence intensity increment is changed temperature when maximum as melting temp; If between the segmental corresponding melting temp value of corresponding D NA of wild type gene tangible difference is arranged under this melting temp value and the similarity condition, then the explanation thymus nucleic acid of surveying has SNP.The characteristics that cost is lower, easy to operate though this method has, detection time is short; But need to prepare two kinds of probes and two kinds of fluorescent marks; Thereby when the design of carrying out probe (comprise the definite kernel nucleotide sequence and fluorophor or quenching group are connected on the Nucleotide of an end of (or claim mark) nucleotide sequence), the scope of alternative oligonucleotide is still waiting to increase, corresponding success ratio also awaits increasing.
Summary of the invention
The method of the SNP of the detection thymus nucleic acid of the purpose of this invention is to provide that a kind of cost is lower, more convenient operation, detection time are shorter, and bigger, the corresponding success ratio of the scope of the probe that this method adopted alternative oligonucleotide when design is then higher.
Total technical conceive of the present invention is: method of the present invention comprises the target sequence that comprises variant sites on the strand confirming dna fragmentation to be measured; Preparation is corresponding to the base cancellation probe of target sequence; One end of a probe only end is combined with fluorophor; The polymerase chain reaction is carried out in preparation and the corresponding a pair of primer of dna fragmentation to be measured then, again on the target sequence that contains variant sites with the dna fragmentation to be measured of probe hybridization after increasing; Measure melting temp again, can whether have SNP to the mononucleotide of the variant sites of dna fragmentation to be measured and judge.
The technical scheme that realizes the method for a kind of SNP that detects thymus nucleic acid of providing of the object of the invention is: have following steps:
1. confirm and will certain gene of certain biological thymus nucleic acid be detected, just confirm dna fragmentation to be measured, and the some specific nucleotides in one section nucleotide sequence on the strand of this dna fragmentation to be measured possibly morph; The nucleotide sequence that this section comprises the specific nucleotide that possibly morph can be described as target sequence; And the residing position of the specific nucleotide that possibly morph wherein can be described as variant sites;
2. prepare a kind of base cancellation probe of forming by oligonucleotide and fluorophor; The oligonucleotide of base cancellation probe is complementary to the corresponding sequence of the corresponding DNA fragments of the corresponding wild type gene of target sequence of dna fragmentation to be measured or is complementary to the corresponding sequence with the corresponding DNA fragments of the corresponding mutated genes of target sequence of dna fragmentation to be measured; One end of the oligonucleotide of a base cancellation probe only end is combined with fluorophor; Fluorophor can be marked at 5 ' or 3 ' end of the oligonucleotide of base cancellation probe; When fluorescent mark in 5 ' when end, 3 ' end of the oligonucleotide of probe must adopt phosphate group to seal;
3. prepare two suitable Oligonucleolide primers; One section sequence complementation of one end of an Oligonucleolide primers in these two Oligonucleolide primers and a strand of above-mentioned dna fragmentation to be measured, one section sequence complementation of the other end of another Oligonucleolide primers in these two Oligonucleolide primers and another strand of above-mentioned dna fragmentation to be measured;
4. by excessive substrate, archaeal dna polymerase, excessive Oligonucleolide primers and the thymus nucleic acid anabolic reaction system that contains dna fragmentation to be measured; Also add base cancellation probe; In above-mentioned reaction system, carry out the polymerase chain reaction, and make above-mentioned dna fragmentation amplification to be measured;
5. to the above-mentioned system heating of carrying out behind the polymerase chain reaction; Dna fragmentation sex change to be measured after making amplification and its two strands is untied; The oligonucleotide hybridization of lowering the temperature then and making base cancellation probe is on the target sequence of the strand of the dna fragmentation to be measured after the amplification, and the oligonucleotide of the base cancellation probe of this moment is in the state with target complement sequence; If the fluorophor to base cancellation probe excites when the oligonucleotide of base cancellation probe is in the strand state; A little less than the fluorescigenic cancellation effect of base pair fluorophor institute of then adjoining with fluorophor; If the fluorophor to base cancellation probe under the complementary state after the oligonucleotide of base cancellation probe and target sequence hybridization generate two strands excites, the fluorescigenic cancellation effect reinforcement of base pair fluorophor institute of then adjoining with fluorophor;
6. when the fluorophor to probe excites; System after the heating hybridization, the base cancellation probe that is incorporated on the target sequence of strand of dna fragmentation to be measured can split away off, and the fluorescence that makes it return to the strand state and externally demonstrate that comes off of base cancellation probe strengthens; Continuation rising along with temperature; The increase that comes off of base cancellation probe, fluorescence intensity be also along with enhancing, and wherein fluorescence intensity increment is changed temperature when maximum as melting temp; If between the segmental corresponding melting temp value of corresponding D NA of wild type gene tangible difference is arranged under this melting temp value and the similarity condition, then the explanation thymus nucleic acid of surveying has SNP.
Thymus nucleic acid to be measured in the above-mentioned steps system 4. is preferably 1.6～3.2ng/ μ l.
The 4. middle concentration of institute's adding probe in system of above-mentioned steps is preferably 4 * 10
3～8 * 10
4PM.
The value of the melting temp of the base cancellation probe of above-mentioned steps in 2. be lower than step 3. in the value of melting temp of Oligonucleolide primers.
The method that above-mentioned steps is carried out the polymerase chain reaction in 4. is; Preparatory sex change is 1 minute under 90 ℃～95 ℃, then begins circulation, also promptly keeps 0 second under 90 ℃～95 ℃ the temperature; With the temperature transition rate is that the speed of 20 ℃ of per seconds is cooled to 55 ℃～61 ℃, kept 10 seconds; Being that the speed of 20 ℃ of per seconds is warming up to 70 ℃～75 ℃, kept 10 seconds with the temperature transition rate again, is that the speed of 20 ℃ of per seconds is warming up to 90 ℃～95 ℃ with the temperature transition rate again, has so just accomplished a circulation; Move 25～40 circulations altogether.
Above-mentioned steps 5. in heating systems make the double-stranded unfolded temperature of dna fragmentation to be measured be preferably 90 ℃～95 ℃, cooling and make the temperature of base cancellation probe hybridization on the strand that contains target sequence of dna fragmentation to be measured after the amplification be preferably 20 ℃～65 ℃;
The 6. middle heat-up rate of above-mentioned steps is preferably 0.1～0.3 ℃ of per second, and top temperature is 95 ℃.
The fluorophor that above-mentioned steps is incorporated into the oligonucleotide of probe in 2. is preferably the 6-Fluoresceincarboxylic acid.
The to be measured dna fragmentation of above-mentioned steps in 1. is the fragment of Apoliprotein M gene.
Above-mentioned steps 6. in; When the absolute value of the difference of the melting temp that splits away off when the base cancellation probe on the strand of dna fragmentation to be measured and the segmental corresponding melting temp value of corresponding D NA of wild type gene is 5 ℃～10 ℃, judge that then the thymus nucleic acid of surveying has SNP.
The present invention has positive effect: the operation of (1) method of the present invention is simpler, the time is shorter, can be in single tube rapid detection SNP, thereby cost is lower.Method of the present invention only needs two kinds of primers and a kind of probe, and fluorescent mark also needs only a kind of, is so far one of simple, the most most economical SNP classifying method.In China, a lot of biotech companies all carry out fluorescence modifies, and therefore, researchist or reagent development company are easy to obtain this service.This law has been used a probe, and total expenses synthetic and that modify only is about 300 RMB/5 OD.This is than synthetic and modify a TaqMan probe also cheap (1600 RMB/5 OD).In general, the PCR product is saturated～10 usually
11Individual molecule/μ l, therefore, each reaction uses 2 pmol probes just enough when detecting SNP.The probe of 5 OD can detect 10000 parts of samples approximately.In this sense, this law is very economical and practical.(2) melt in the process at the PCR product, the difference of gene order can detect through the variation of probe melting temp.When temperature slowly raise, base cancellation probe came off from target sequence, and probe recovers the strand state, and the cancellation effect of the base pair fluorophor that adjoins with fluorophor of the oligonucleotide of probe weakens, thereby causes fluorescence to roll up suddenly.Temperature when the fluorescence increment changes maximum is called melting temp.The melting temp of mutated genes and the melting temp of wild type gene have evident difference.Typical wild-type homozygote sample can present the single paddy of melting, and for heterozygote, then visible two melt paddy.The paddy of melting with wild-type homozygote differing temps then can appear in the mutant homozygote.The temperature drift that is caused by a base mispairing and is easy to identification usually between 5 ℃～10 ℃.(3) T of base cancellation probe
MValue must be lower than the T of primer
MValue is to avoid the cutting of Taq archaeal dna polymerase.(4) during probe, probe can carry out the mark of fluorophor at 5 ' or 3 ' end of oligonucleotide in preparation in the present invention, and the scope of the alternative oligonucleotide that is used for probe that therefore is directed to target gene is bigger, has higher success rate thereby make.(5) this detection method is applicable to the research of gene type in enormous quantities through the checking of dna sequencing.
Description of drawings
Fig. 1 is the schematic diagram of method of the SNP of detection thymus nucleic acid of the present invention; Fig. 1 wherein (a) is after the expression polymerase chain reaction finishes, the synoptic diagram after base cancellation probe and dna fragmentation to be measured are hybridized under lower temperature conditions.The base cancellation probe of this moment becomes double-stranded state (or claiming complementary state) by the strand state; If the fluorophor (FAM) of probe is excited, the cancellation effect of the fluorescence that the base pair fluorophor of the oligonucleotide that then adjoins with fluorophor is sent is stronger; Arrow indication position is mutational site (SNP site just) among the figure.Fig. 1 (b) is for representing when temperature slowly raises; Base cancellation probe comes off from target sequence; Probe recovers the strand state, and the cancellation effect of the fluorescence that fluorophor sent of the base pair probe that adjoins with fluorophor weakens, thus the synoptic diagram that causes fluorescence to roll up suddenly.
Probe and a pair of primer design conceptual scheme of Fig. 2 for being adopted in the method for the present invention.
Fig. 3 A is to be dna fragmentation to be measured with the Apoliprotein M gene in the method for the present invention; After on the strand that contains target sequence of base cancellation probe (A cancellation probe) being hybridized the dna fragmentation to be measured after increasing, the graphic representation that the fluorescence signal intensity of the three kinds of genotype (A/A, A/G, G/G) along with the rising of temperature in the system changes thereupon;
Fig. 3 B is the genotypic melt curve analysis figure of three kinds among Fig. 3 A.
Fig. 4 is the part sequencing result figure of Apoliprotein M gene for the dna fragmentation to be measured in the method for the present invention; Wherein Fig. 4 (A) is mutated genes part sequencing result figure, and Fig. 4 (B) is wild type gene part sequencing result figure, and the arrow indication is the mutational site among the figure.
Fig. 5 A is 4 kinds of different bases (A, T, C, G) among Fig. 2 when the cancellation probe is used to measure the SNP of Apoliprotein M gene, the homozygous melt curve analysis figure of A/A wherein;
When Fig. 5 B is used to measure the SNP of Apoliprotein M gene for 4 kinds of different base cancellation probes among Fig. 2, the melt curve analysis figure of A/G heterozygote wherein;
When Fig. 5 C is used to measure the SNP of Apoliprotein M gene for 4 kinds of different base cancellation probes among Fig. 2, the homozygous melt curve analysis figure of G/G wherein.
Embodiment
Describe the present invention below in conjunction with embodiment, but the present invention is not limited to these embodiment.
(embodiment 1)
The method of present embodiment has following steps:
1. confirm dna fragmentation to be measured.Some specific nucleotides in one section nucleotide sequence on the strand of dna fragmentation to be measured possibly morph, and the nucleotide sequence that this section comprises the specific nucleotide that possibly morph can be described as target sequence; And the residing position of the specific nucleotide that possibly morph wherein can be described as variant sites.The dna fragmentation to be measured of present embodiment is Apoliprotein M (apolipoprotein M, apoM) fragment of gene in the human genome.ApoM is the lipophorin of finding recently, and its assignment of genes gene mapping is at 6p21.31.At the nearly promoter region of apoM one T-778C (rs805296) SNP is arranged.Produce 3 kinds of genotype at this SNP sign, be respectively T/T homozygote, C/C homozygote and T/C heterozygote, T/T homozygote wherein belongs to wild type gene.This SNP is relevant with SUV and fasting plasma glucose, and also increases the susceptibility of diabetes B among the crowd of Han nationality.The length of the fragment of apoM gene amplification product (being dna fragmentation to be measured) is 410bp, and the 180th bit base wherein then is the site (the arrow indication site among Fig. 4 (B)) that possibly morph.
2. prepare base cancellation probe.Base cancellation probe is made up of oligonucleotide and fluorophor.The oligonucleotide of base cancellation probe is complementary to the corresponding sequence of the corresponding DNA fragments of the corresponding wild type gene of target sequence of dna fragmentation to be measured or is complementary to the corresponding sequence with the corresponding DNA fragments of the corresponding mutated genes of target sequence of dna fragmentation to be measured.End in 5 ' or the 3 ' end of the oligonucleotide of a base cancellation probe only end is combined with fluorophor; That is to say that fluorophor has only one, and this fluorophor is marked on of Nucleotide that two of oligonucleotide of base cancellation probe are arranged in the termination; When 5 ' when end of fluorescent mark at oligonucleotide, 3 ' end of the oligonucleotide of probe must connect phosphate group and seal.We have designed 4 kinds of base cancellation probes by sequence shown in Figure 2, and the oligonucleotide of these 4 kinds of probes is apoM T-778C oligonucleotide, and the Nucleotide that is marked with fluorophor of the oligonucleotide of these 4 kinds of base cancellation probes has corresponding base.Each base cancellation probe is complementary to the segmental corresponding sequence of corresponding D NA with the target sequence corresponding mutant of step in 1., and the fluorophor that is incorporated into the oligonucleotide of probe is the 6-Fluoresceincarboxylic acid.The all synthetic and modifications of Ying Jun company of apoM T-778C oligonucleotide and corresponding base cancellation probe in Shanghai.Because the oligonucleotide of above-mentioned 4 kinds of base cancellation probes is with reference to coding strand design and synthetic (being the part of this coding strand sequence); Therefore; If carry out somatotype according to template strand, when detecting if detected be A/A homozygote or G/G homozygote, then its somatotype is T/T homozygote or C/C homozygote; And if detected be the A/G heterozygote, then its somatotype is the T/C heterozygote.The base cancellation probe that present embodiment adopted is an A cancellation probe, among other embodiment, also can adopt T cancellation probe shown in Figure 2, C cancellation probe or G cancellation probe.
3. prepare two suitable Oligonucleolide primers, they are sense primer and antisense primer.The sense primer of present embodiment is the one section sequence complementary oligonucleotide that comprises 3 ' end with a strand of above-mentioned dna fragmentation to be measured, and antisense primer is the one section sequence complementary oligonucleotide that comprises 3 ' end with another strand of above-mentioned dna fragmentation to be measured.The oligonucleotide of the sense primer of present embodiment has 30, and its sequence is 5 '-ACTGACACATTCACTCAACATTTATTACTA-3 '; The oligonucleotide of the antisense primer of present embodiment has 21, and its sequence is 5 '-AGGGGTTGGTGGTGTTTTGTT-3 '.These two kinds of primers are given birth to the synthetic and modification of worker in Shanghai.The value of the melting temp of two Oligonucleolide primers is higher than the value of the melting temp of the base cancellation probe of step in 2..
4. carry out the polymerase chain reaction (Polymerase chain reaction, PCR).With the dna profiling of 40～80ng, 10 * PCR damping fluid of 2.5 μ l, the MgCl of 2.5mM
2, excessive substrate 0.5 μ l 4 * dNTPs, available from the Shen, Shanghai can betting office the Taq archaeal dna polymerase, sense primer 10 pmol and the antisense primer 10 pmol anabolic reaction systems that 3. step obtains of 1.25U, also add A cancellation probe 2 pmol that 2. step obtains.The volume of above-mentioned whole system is 25 μ l, and the concentration of dna profiling in system is 1.6～3.2ng/ μ l, and the concentration of probe in system is 8 * 10
4PM.Dna profiling also promptly contains the thymus nucleic acid of dna fragmentation to be measured; This dna profiling is to give birth to UIIQ-10 pillar poba gene group DNA extraction agent box extraction respectively from 200 human coronary artery disease patients' blood that the worker makes with Shanghai, therefrom selects arbitrarily then to obtain.。In above-mentioned reaction system, carry out the polymerase chain reaction, and make above-mentioned dna fragmentation amplification to be measured; The method of carrying out the polymerase chain reaction is; Preparatory sex change is 1 minute under 90 ℃～95 ℃, then begins circulation, also promptly keeps 0 second under 90 ℃～95 ℃ the temperature; With the temperature transition rate is that the speed of 20 ℃ of per seconds is cooled to 55 ℃～61 ℃, kept 10 seconds; Being that the speed of 20 ℃ of per seconds is warming up to 70 ℃～75 ℃, kept 10 seconds with the temperature transition rate again, is that the speed of 20 ℃ of per seconds is warming up to 90 ℃～95 ℃ with the temperature transition rate again, has so just accomplished a circulation; Move 25～40 circulations altogether, thereby obtain the PCR product.
5. A cancellation probe and PCR product are carried out molecular hybridization.Still on the LightCycler of Roche Holding Ag gene amplification detector, carry out; Heat above-mentioned carry out behind the polymerase chain reaction system (wherein mainly being the PCR product) to 95 ℃, kept 30 seconds; Make the dna fragmentation sex change to be measured after the amplification and its two strands is untied; Be cooled to 30 ℃ then, kept 4 minutes; (Fig. 1 a), thereby the oligonucleotide of the base cancellation probe of this moment is in the state with target complement sequence to the oligonucleotide hybridization that makes base cancellation probe on the target sequence of the strand of the dna fragmentation to be measured after the amplification.If this moment, the fluorophor to base cancellation probe excited; Because it is stronger with the fluorescigenic cancellation effect of base pair fluorophor institute that fluorophor adjoins; So fluorophor fluoresce is because of by this base cancellation, therefore externally the fluorescence intensity of demonstration weakens greatly.
6. measure the melting temp of dna fragmentation to be measured.Still on the LightCycler of Roche Holding Ag gene amplification detector, carry out, the fluorescence channel of opening on the detector 1 (F1) detects, and the fluorophor of probe is excited.System after the heating hybridization; Speed with 0.1 ℃ of per second makes system be warming up to 80 ℃ from 30 ℃; In temperature-rise period, the base probe splits away off (Fig. 1 b) from the strand of dna fragmentation to be measured, because the cancellation effect of base (being connected with the base of the Nucleotide of fluorophor) to fluorescence that fluorophor sends under the strand state with fluorophor adjoins mutually of the oligonucleotide of probe weakens; Thereby the fluorescence intensity in the system increases; Along with the continuation of temperature raises, increases that come off of base cancellation probe, fluorescence intensity is also along with enhancing (Fig. 3 A); Wherein fluorescence intensity increment is changed temperature when maximum as melting temp (Melting temperature, T
M).Melting temp T
MCorresponding with one of following three kinds of situation (Fig. 3 B): first kind of situation is because G/G homozygote and base cancellation probe mate fully, in the homozygous melt curve analysis of G/G, produces a higher melting temp, greatly about about 48.6 ℃; Second kind of situation is that A/A homozygote and base cancellation probe have a base to be unworthy of, T in the homozygous melt curve analysis of A/A
MValue then floats to about 41.3 ℃; The third situation is to have occurred two in the genotypic melt curve analysis of heterozygote A/G to melt paddy, T
MValue is respectively 48.6 ℃ and 41.3 ℃.Two temperature heads (△ T) of melting between the paddy are about 7.3 ℃.After measuring the melting temp of dna fragmentation, just can whether have SNP and judge this dna fragmentation.Because what second kind of situation represented is the segmental corresponding melt curve analysis of corresponding D NA of wild type gene, its melting temp is about 41.3 ℃.
If the melting temp of the dna fragmentation of surveying has only one and be in about 48.6 ℃ then belong to first kind of situation, sudden change has been described.If the melting temp of the dna fragmentation of surveying has only one and be in about 41.3 ℃ then belong to second kind of situation, explain and do not undergo mutation.If the melting temp of the dna fragmentation of surveying has two and one to be in about 48.6 ℃ another and to be in about 41.3 ℃ then to belong to the third situation, sudden change has been described.
T between the different experiments
MValue possibly fluctuate between ± 0.8 ℃, and the △ T that difference is melted between the paddy possibly fluctuate between ± 0.6 ℃.This law is why with melting paddy, rather than representes with melting the peak, is because there is not the mapping analysis function of dF/dT in LightCycler 3.5 versions.We hope that this function adds in new version, so that the user selects corresponding function according to demand separately.
The accuracy of this detection method is through the dna sequencing checking.Dna sequencing work is accomplished by Shanghai Ying Jun company, and Fig. 4 has shown sequencing result.
In addition, we also compare the effect of different base pair fluorescent quenchings.For the relatively influence of four kinds of base pair fluorescent quenchings; On each corresponding oligonucleotide of 4 kinds of base cancellation probes shown in Figure 2 that we designed respectively mark different bases; Fig. 5 has shown four kinds of base (A; T, C and G) the cancellation probe in the process of three kinds of genotypings to the cancellation effect of fluorescence.Comparative result shows; Four kinds of base cancellation probes can both be used for the gene type of A/A (Fig. 5 A), A/G (Fig. 5 B), G/G (Fig. 5 C) accurately and effectively, and A cancellation probe, T cancellation probe, the C cancellation probe maximum rate that fluorescence increases when melting will be apparently higher than G cancellation probe.
Claims (10)
1. method that detects the SNP of thymus nucleic acid has following steps:
1. confirm and will certain gene of certain biological thymus nucleic acid be detected, just confirm dna fragmentation to be measured, and the some specific nucleotides in one section nucleotide sequence on the strand of this dna fragmentation to be measured possibly morph; The nucleotide sequence that this section comprises the specific nucleotide that possibly morph is called target sequence; And the residing position of the specific nucleotide that possibly morph wherein is called variant sites;
2. prepare a kind of base cancellation probe of forming by oligonucleotide and fluorophor; The oligonucleotide of base cancellation probe is complementary to the corresponding sequence of the corresponding DNA fragments of the corresponding wild type gene of target sequence of dna fragmentation to be measured or is complementary to the corresponding sequence with the corresponding DNA fragments of the corresponding mutated genes of target sequence of dna fragmentation to be measured; The oligonucleotide of base cancellation probe has only an end to be combined with fluorophor; Fluorophor is marked at 5 ' or 3 ' end of the oligonucleotide of base cancellation probe; When fluorescent mark in 5 ' when end, 3 ' end of the oligonucleotide of probe must adopt phosphate group to seal;
3. prepare two suitable Oligonucleolide primers; One section sequence complementation of one end of an Oligonucleolide primers in these two Oligonucleolide primers and a strand of above-mentioned dna fragmentation to be measured, one section sequence complementation of the other end of another Oligonucleolide primers in these two Oligonucleolide primers and another strand of above-mentioned dna fragmentation to be measured;
4. by excessive substrate, archaeal dna polymerase, excessive Oligonucleolide primers and the thymus nucleic acid anabolic reaction system that contains dna fragmentation to be measured; Also add base cancellation probe; In above-mentioned reaction system, carry out the polymerase chain reaction, and make above-mentioned dna fragmentation amplification to be measured;
5. to the above-mentioned system heating of carrying out behind the polymerase chain reaction; Dna fragmentation sex change to be measured after making amplification and its two strands is untied; The oligonucleotide hybridization of lowering the temperature then and making base cancellation probe is on the target sequence of the strand of the dna fragmentation to be measured after the amplification, and the oligonucleotide of the base cancellation probe of this moment is in the state with target complement sequence; If the fluorophor to base cancellation probe under the complementary state after the oligonucleotide of base cancellation probe and the target sequence hybridization generation two strands excites; Then fluorophor fluoresce is by the base cancellation of adjoining with fluorophor; If the fluorophor to base cancellation probe excites when the oligonucleotide of base cancellation probe is in the strand state, then intensity that fluorophor fluoresces increases;
6. when the fluorophor to probe excites; System after the heating hybridization, the base cancellation probe that is incorporated on the target sequence of strand of dna fragmentation to be measured can split away off, and the fluorescence that makes it return to the strand state and externally demonstrate that comes off of base cancellation probe strengthens; Continuation rising along with temperature; The increase that comes off of base cancellation probe, fluorescence intensity be also along with enhancing, and wherein fluorescence intensity increment is changed temperature when maximum as melting temp; When if the absolute value of the difference of the segmental corresponding melting temp value of corresponding D NA of wild type gene is 5 ℃～10 ℃ under this melting temp value and the similarity condition, then the explanation thymus nucleic acid of surveying has SNP.
2. the method for the SNP of detection thymus nucleic acid according to claim 1 is characterized in that: the thymus nucleic acid to be measured in the step system 4. is 1.6～3.2ng/ μ l.
3. the method for the SNP of detection thymus nucleic acid according to claim 1 is characterized in that: the 4. middle concentration of institute's adding probe in system of step is 4 * 10
3～8 * 10
4PM.
4. the method for the SNP of detection thymus nucleic acid according to claim 1 is characterized in that: the value of the melting temp of the base cancellation probe of step in 2. be lower than step 3. in the value of melting temp of Oligonucleolide primers.
5. the method for the SNP of detection thymus nucleic acid according to claim 1; It is characterized in that: the method that step is carried out the polymerase chain reaction in 4. is; Preparatory sex change is 1 minute under 90 ℃～95 ℃, then begins circulation, also promptly keeps 0 second under 90 ℃～95 ℃ the temperature; With the temperature transition rate is that the speed of 20 ℃ of per seconds is cooled to 55 ℃～61 ℃, kept 10 seconds; Being that the speed of 20 ℃ of per seconds is warming up to 70 ℃～75 ℃, kept 10 seconds with the temperature transition rate again, is that the speed of 20 ℃ of per seconds is warming up to 90 ℃～95 ℃ with the temperature transition rate again, has so just accomplished a circulation; Move 25～40 circulations altogether.
6. the method for the SNP of detection thymus nucleic acid according to claim 1; It is characterized in that: it is 90 ℃～95 ℃ that the 5. middle heating systems of step makes the double-stranded unfolded temperature of dna fragmentation to be measured, and lowering the temperature and making the temperature of base cancellation probe hybridization on the strand that contains target sequence of the dna fragmentation to be measured after the amplification is 20 ℃～65 ℃;
7. the method for the SNP of detection thymus nucleic acid according to claim 1 is characterized in that: the 6. middle heat-up rate of step is 0.1～0.3 ℃ of a per second, and top temperature is 95 ℃.
8. the method for the SNP of detection thymus nucleic acid according to claim 1 is characterized in that: the fluorophor that step is incorporated into the oligonucleotide of probe in 2. is the 6-Fluoresceincarboxylic acid.
9. according to the method for the SNP of the described detection thymus nucleic acid of one of claim 1 to 8, it is characterized in that: the to be measured dna fragmentation of step in 1. is the fragment of Apoliprotein M gene.
10. the method for the SNP of detection thymus nucleic acid according to claim 9; It is characterized in that: step 6. in; When the absolute value of the difference of the melting temp that splits away off when the base cancellation probe on the strand of the dna fragmentation to be measured of Apoliprotein M gene and the segmental corresponding melting temp value of corresponding D NA of wild type gene is 7.3 ± 0.6 ℃, judge that then the thymus nucleic acid of surveying has SNP.
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| CN1570143A (en) * | 2003-03-21 | 2005-01-26 | 普生股份有限公司 | Identification of single nucleotide polymorphisms |
| CN1952178A (en) * | 2006-11-03 | 2007-04-25 | 罗光华 | Method for detecting polymorphism of mononucleotide of DNA |
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| CN1570143A (en) * | 2003-03-21 | 2005-01-26 | 普生股份有限公司 | Identification of single nucleotide polymorphisms |
| CN1952178A (en) * | 2006-11-03 | 2007-04-25 | 罗光华 | Method for detecting polymorphism of mononucleotide of DNA |
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