CN100564542C - Detect the method for the single nucleotide polymorphism of thymus nucleic acid - Google Patents
Detect the method for the single nucleotide polymorphism of thymus nucleic acid Download PDFInfo
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- CN100564542C CN100564542C CNB2006100973815A CN200610097381A CN100564542C CN 100564542 C CN100564542 C CN 100564542C CN B2006100973815 A CNB2006100973815 A CN B2006100973815A CN 200610097381 A CN200610097381 A CN 200610097381A CN 100564542 C CN100564542 C CN 100564542C
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Abstract
The present invention relates to a kind of method that detects the mononucleotide character of thymus nucleic acid.This method is determined first sequence and second sequence on the strand of dna fragmentation to be measured, and first sequence may have variant sites, preparation corresponds respectively to the responsive probe and the grappling probe of first sequence and second sequence, and an end of a responsive probe only end is combined with a kind of in fluorescence report group and the quenching group, one end of a grappling probe only end is combined with another kind in fluorescence report group and the quenching group, preparation and the corresponding a pair of primer of dna fragmentation to be measured, carry out the polymerase chain reaction then, again on the strand that contains first sequence and second sequence with the dna fragmentation to be measured of probe hybridization after increasing, measure melting temp again, can whether have single nucleotide polymorphism to the mononucleotide of the thymus nucleic acid under the dna fragmentation to be measured and judge.The present invention has the advantages that cost is lower, easy to operate, detection time is short.
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 polymorphism by the caused dna sequence dna of variation of single Nucleotide to single nucleotide polymorphism.The SNPs of gene control region and coding region more may cause 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, as restriction fragment length polymorphism (Restriction fragment length polymorphism, RFLP) analysis and gel mobility shift assay method etc. are because of the restriction that is subjected to 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) (fluorescence resonance energy transfer, FRET) the quick SNP detection method of the single tube of principle, extend and utilize the mononucleotide gene type method of green and red fluorescence mark based on allele-specific primers, utilize quenching group to extend (Quencher extension, QEXT) SNP of stopped pipe detection in real time method, duplex scorpion type primer is analyzed the SNP technology, solid phase is supported the SNP typing, use exonuclease III/ s1 nuclease/direct forward of PNA system to detect the SNP method, twin probe method, polymorphic nucleic acid probe coupling polymer detection method and utilize omnidistance TaqMan real-time quantitative data automatic gene typing etc.Yet, the reagent of Technology Need costliness mentioned above, 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 as 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: the fluorescent mark oligonucleotide probe that adopts synthetic, the two ends of this probe are made up of 5～7 complementary bases that have nothing to do with the target preface, 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, do not produce fluorescent signal, show 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.
Chinese patent application 200310119928.3 discloses a kind of method that SNP detects of carrying out with FRET (fluorescence resonance energy transfer), this method is selected two kinds of probes, a kind of probe is combined with the fluorophore donor, another kind of probe is combined with the fluorophore acceptor, under the irradiation of exciting light, the fluorophore donor is excited to provide fluorescence to the fluorophore acceptor, the fluorophore acceptor sends fluorescence, thereby can when being lower than melting temperature(Tm), this temperature value then determine to exist in the purpose nucleic acid single nucleotide polymorphism according to the variable quantity that the fluorophore acceptor the sends fluorescence pairing temperature value of peak value of determining to unwind.Along with the coming off of probe, fluorescence intensity rather than is changed from small to big from large to small in this method.Can certain requirement be arranged with the oligonucleotide chain bonded fluorophore donor or the choosing of fluorophore acceptor of probe, thereby cost is higher.
Summary of the invention
The purpose of this invention is to provide that a kind of cost is lower, easy to operate, detection time is short, alternative label range is big and the method for the single nucleotide polymorphism of success ratio higher detection thymus nucleic acid.
The technical scheme that realizes the object of the invention is: the method for the single nucleotide polymorphism of detection thymus nucleic acid of the present invention, have following steps: 1. determine and will certain gene of certain biological thymus nucleic acid be detected, just determine dna fragmentation to be measured, and have first sequence and second sequence on the strand of this dna fragmentation to be measured, described first sequence is meant one section nucleotide sequence that the some specific nucleotides in this sequence may morph, and second sequence is meant the one section nucleotide sequence mutually close with first consecutive nucleotides; 2. preparation can be described as a kind of oligonucleotide probe of responsive probe and the another kind of oligonucleotide probe that preparation can be described as the grappling probe; Described responsive probe is complementary to the corresponding sequence of the corresponding DNA fragments of the corresponding wild type gene of first sequence of dna fragmentation to be measured or is complementary to corresponding sequence with the corresponding DNA fragments of the corresponding mutated genes of first sequence of dna fragmentation to be measured, and an end of a responsive probe only end is combined with fluorescence report group or quenching group; The second sequence complementation of described grappling probe and dna fragmentation to be measured, an end of grappling probe only an end are combined with quenching group, are combined with the fluorescence report group when responsive probe are combined with quenching group when responsive probe is combined with the fluorescence report group; The value of the melting temp of responsive probe is lower than the value of the melting temp of grappling probe; 3. prepare two suitable Oligonucleolide primers, one end complementation of an Oligonucleolide primers in these two Oligonucleolide primers and the strand with first sequence and second sequence of above-mentioned dna fragmentation to be measured, the other end complementation 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 responsive probe and grappling probe, and the quantity of responsive probe and grappling probe is basic identical; 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, 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 are adjacent to one another and quantity is suitable, so if the fluorescence report group on the probe is excited, then the fluorescence that 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 demonstrate 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 single nucleotide polymorphism.
The value of the melting temp of the grappling probe of above-mentioned steps in 2. be lower than step 3. in the value of melting temp of Oligonucleolide primers.The fluorescence report group that step is incorporated into probe in 2. can be the 6-Fluoresceincarboxylic acid, and the quenching group that is incorporated into probe can be a 6-carboxyl tetramethyl-rhodamine.
Thymus nucleic acid to be measured in the above-mentioned steps system 4. is 1.6～3.2ng/ μ l.The concentration of each probe in system that step is added in 4. is 4 * 10
3～8 * 10
4PM.The method that step is carried out the polymerase chain reaction in 4. is, pre-sex change is 1 minute under 90 ℃～95 ℃, then begin circulation, also promptly kept 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 finished a circulation; Move 25～40 circulations altogether.Step 4. in the dna fragmentation to be measured of thymus nucleic acid to be measured can be Tumor Necrosis Factor Receptors II or Apoliprotein M.
It is 90 ℃～95 ℃ that the 5. middle heating systems of above-mentioned steps makes the double-stranded unfolded temperature of dna fragmentation to be measured, and lowering the temperature and making responsive probe and the temperature of grappling probe hybridization on the strand that contains first sequence and second sequence of the dna fragmentation to be measured after the amplification is 30 ℃～40 ℃;
The 6. middle heat-up rate of above-mentioned steps is 0.1 ℃ of a per second, and top temperature is 80 ℃.Step is (6., when the absolute value of the difference of the melting temp that splits away off when the responsive 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 single nucleotide polymorphism.
The present invention has positive effect: (1) method of the present invention is simple to operate, the time is short, can be in single tube rapid detection SNP, thereby cost is lower.Method of the present invention only needs two primers and two probes, is one of simple, the most most economical SNP classifying method.In China, a lot of biotech companies all carry out the modification of FAM and TAMRA, and therefore, researchist or reagent development company are easy to obtain this service.Though this law has been used two probes, total expenses synthetic and that modify only is about 1000RMB/5OD.This is than synthetic and modify a TaqMan probe also cheap (1600RMB/5OD).In general, the PCR product is saturated～10 usually
11Individual molecule/μ l, therefore, each reaction uses the 2pmol probe just enough when detecting SNP.It should be noted that because the fluorescence that discharges of hybridization probe can not be by cancellation, thereby the probe of high density can influence the susceptibility of this law because of increasing the fluorescence background under the situation of being excited.Therefore, the probe of 5OD 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 by the variation of responsive probe melting temp.When temperature slowly raise, responsive probe came off from target sequence, and two fluorophors are no longer adjacent, the fluorescence that quenching group is excited to send to the fluorescence report group cancellation effect that do not recur, thus cause 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 fluorescent quenching probe
MValue must be lower than the T of primer
MValue is to avoid the cutting of Taq archaeal dna polymerase.And the T of responsive probe
MValue should be lower than the T of grappling probe
MValue only depends on responsive probe with the generation that guarantees fluorescent signal when melt curve analysis is analyzed.(4) the present invention is in preparation during probe, because alternative general planning just can reach 16 kinds, so bigger for alternative label range of target gene, thus can make success ratio higher.(5) this detection method is applicable to the research of gene type in enormous quantities by the checking of dna sequencing.
Description of drawings
Fig. 1 is the detection schematic diagram of method of the present invention.
Fig. 2 is the conceptual schematic drawing of method middle probe of the present invention.
Fig. 3 is the melt curve analysis analysis chart of Tumor Necrosis Factor Receptors II in the method for the present invention; Wherein curve 1 is a G/G homozygote melt curve analysis analysis chart; Curve 2 is a T/T homozygote melt curve analysis analysis chart; Curve 3 is a T/G heterozygote melt curve analysis analysis chart; Curve 4 negative contrast melt curve analysis analysis charts.
Fig. 4 is the part sequencing result figure of Tumor Necrosis Factor Receptors II for the dna fragmentation to be measured of thymus nucleic acid 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 is the melt curve analysis analysis chart of Apoliprotein M in the method for the present invention; Wherein curve 1 is a T/T homozygote melt curve analysis analysis chart; Curve 2 is a C/C homozygote melt curve analysis analysis chart; Curve 3 is a T/C heterozygote melt curve analysis analysis chart; Curve 4 negative contrast melt curve analysis analysis charts.
Fig. 6 is the part sequencing result figure of Apoliprotein M for the dna fragmentation to be measured of thymus nucleic acid to be measured in the method for the present invention; Wherein Fig. 6 (A) is mutated genes part sequencing result figure, and Fig. 6 (B) is wild type gene part sequencing result figure, and the arrow indication is the mutational site among the figure.
Embodiment
Fig. 1 is the schematic diagram of method of the single nucleotide polymorphism of detection thymus nucleic acid of the present invention.(A)～(D) among Fig. 1 is the synoptic diagram of pcr process, and in this process, probe is not joined to target DNA and gets on.
(A) among Fig. 1 provided the synoptic diagram that mixes of target DNA, sense primer, antisense primer, responsive probe and grappling probe.Target DNA wherein is thymus nucleic acid to be measured, wherein contain dna fragmentation to be measured, have first sequence and second sequence on the strand of this dna fragmentation to be measured, first sequence wherein is meant one section nucleotide sequence that some Nucleotide wherein may morph, and second sequence is meant the one section nucleotide sequence mutually close with first consecutive nucleotides.This moment, target DNA was in normal duplex state.Sense primer is meant one section sequence complementary primer with 5 ' end of the strand that comprises first sequence and second sequence of the dna fragmentation to be measured of thymus nucleic acid to be measured, and antisense primer is meant one section sequence complementary primer with 3 ' end of corresponding another strand of this strand of this dna fragmentation.Responsive probe adopts the oligonucleotide that is combined with quenching group, this oligonucleotide is complementary to the corresponding sequence of the corresponding DNA fragments of the corresponding wild type gene of first sequence of dna fragmentation to be measured or is complementary to corresponding sequence with the corresponding DNA fragments of the corresponding mutated genes of first sequence of dna fragmentation to be measured, and described quenching group is 6-carboxyl tetramethyl-rhodamine (6-carboxy-N, N, N, N-tetrachlorofluorescein, TAMRA); The grappling probe adopts and to be combined with the fluorescence report group and second sequence complementary oligonucleotide dna fragmentation to be measured, and described fluorescence report group be the 6-Fluoresceincarboxylic acid (6-carboxyfluorescein, FAM).
After (B) among Fig. 1 illustrated elevated temperature, the target DNA sex change made the template that its double-stranded DNA is untied becomes strand.
(C) among Fig. 1 illustrates in annealing process, and a pair of primer in the reaction system also is that sense primer and antisense primer match renaturation with two strands of the target dna fragment that has unwind respectively.
(D) among Fig. 1 illustrates that on the annealed basis DNA polymerase infiltrates mononucleotide from 3 ' end under suitable temperature, and extended to 3 ' extreme direction by 5 ' end along template, synthetic new dna fragmentation.Circulate on this basis.
When (E) expression polymerase chain reaction among Fig. 1 finished, responsive probe and grappling probe and dna fragmentation to be measured be (as 42 ℃ or 30 ℃) hybridization under lower temperature conditions.Because the fluorescence report group FAM of grappling probe and the quenching group TAMRA of responsive probe are close mutually, the fluorescence that fluorescence report group FAM is excited to be sent is by quenching group TAMRA cancellation.Arrow indication position is the SNP site among the figure.
(F) among Fig. 1 expression is when temperature slowly raises, and responsive probe is melted the disengaging target DNA earlier, and make responsive probe with quenching group no longer with the grappling probe with the fluorescence report group mutually close, and make fluorescence intensity sharply increase suddenly.If the dna fragmentation to be measured of thymus nucleic acid to be measured has single nucleotide variations, then the segmental corresponding melting temp value of the corresponding D NA of its melting temp value and wild type gene has evident difference.
Fig. 2 is the conceptual schematic drawing of responsive probe of the present invention and grappling probe.The sequence of the oligonucleotide of responsive probe wherein is complementary to the corresponding sequence of the corresponding DNA fragments of the corresponding wild type gene of first sequence of dna fragmentation to be measured or is complementary to corresponding sequence with the corresponding DNA fragments of the corresponding mutated genes of first sequence of dna fragmentation to be measured; The previous case is meant: the first sequence complementation of the sequence of the oligonucleotide of responsive probe and the dna fragmentation to be measured that does not morph; Latter event is meant: the first sequence complementation of the sequence of the oligonucleotide of responsive probe and the dna fragmentation to be measured that morphs.The length of the sequence of the oligonucleotide of responsive probe can be determined with the length of first sequence in addition, in other words, whether first sequence can be by having oligonucleotide corresponding with it to exist and determining, also promptly this oligonucleotide is except that the corresponding Nucleotide of its possibility change point, rest part all with the first sequence complementary oligonucleotide, and as long as selected oligonucleotide sequence meets the primary condition of probe; And the corresponding Nucleotide of the possible change point of this oligonucleotide is meant the Nucleotide of the corresponding position of Nucleotide at this oligonucleotide and possible change point place first sequence; And the corresponding Nucleotide of this possibility change point can with this of first sequence may variation place unmanifest Nucleotide complementation, perhaps with variation after the Nucleotide complementation.
Still see Fig. 2, in the design of responsive probe, after the sequence of the oligonucleotide of having determined responsive probe, can select a group that it is arranged on 3 of this oligonucleotide ' end or 5 ' end in fluorescence report group and quenching group (these two groups can be commonly referred to as fluorophor), responsive probe just has 4 kinds of forms like this.First kind of form wherein is that 3 of this oligonucleotide ' end is provided with the fluorescence report group, second kind of form is that 5 of this oligonucleotide ' end is provided with the fluorescence report group, the third form is that 3 of this oligonucleotide ' end is provided with quenching group, and the 4th kind of form is that 5 of this oligonucleotide ' end is provided with quenching group.
Still see Fig. 2, in the design of grappling probe, the second sequence complementation of the sequence of its oligonucleotide and dna fragmentation to be measured, and the primary condition that meets probe, and second sequence of dna fragmentation to be measured can be the one section nucleotide sequence (can be described as right side second sequence) that is positioned at the right side of first sequence, also can be another section nucleotide sequence (can be described as left side second sequence) that is positioned at the left side of first sequence.Therefore, its sequence and the right side second sequence complementary oligonucleotide can be called the right side oligonucleotide of grappling probe, and its sequence and the left side second sequence complementary oligonucleotide be called the left side oligonucleotide of grappling probe.
The grappling probe matches aspect two with responsive probe in use.The cooperation of first aspect is position cooperation between the two, when just the oligonucleotide of grappling probe is the right side oligonucleotide, when in use hybridizing with dna fragmentation to be measured, be positioned at the responsive probe right side, when the oligonucleotide of grappling probe is the left side oligonucleotide, when in use hybridizing, be positioned at the responsive probe left side with dna fragmentation to be measured.Second aspect is the cooperation of fluorophor, just when the fluorophor on the responsive probe is the fluorescence report group, fluorophor on the grappling probe then is a quenching group, and when the fluorophor on the responsive probe was quenching group, the fluorophor on the grappling probe then was the fluorescence report group.So,,, 4 kinds of fit forms are arranged all corresponding to 4 kinds of forms of responsive probe each for the grappling probe; First kind of fit form is: oligonucleotide is the right side oligonucleotide, and fluorophor is positioned at 3 of this oligonucleotide ' end; Second kind of fit form is: oligonucleotide is the right side oligonucleotide, and fluorophor is positioned at 5 of this oligonucleotide ' end; The third fit form is: oligonucleotide is the left side oligonucleotide, and fluorophor is positioned at 3 of this oligonucleotide ' end; The 4th kind of fit form is: oligonucleotide is the left side oligonucleotide, and fluorophor is positioned at 5 of this oligonucleotide ' end.
By above explanation as can be known, of the present invention when carrying out probe design, alternative design has 16 kinds.
Describe the present invention below in conjunction with embodiment, but the present invention is not limited to these embodiment.
The method of present embodiment has following steps:
1. determine dna fragmentation to be measured.This dna fragmentation to be measured is Tumor Necrosis Factor Receptors II (Tumornecrosis factor receptor II, the TNFRII) gene in the human genome.TNFRII is 1 type transmembrane protein, its assignment of genes gene mapping is at 1p36.2, total length 43 kbp, contain 10 exons and 9 introns, 96 diallele SNP signs of TNFRII gene the 6th exons 1 produce 3 kinds of genotype, be respectively T/T homozygote, G/G homozygote and T/G heterozygote, wherein the T/T homozygote belongs to wild type gene.There are two kinds of different allelotrope of 96 ATG → AGG of the 6th exons 1 in the TNFR2 gene, can cause the change of methionine(Met) (M) → arginine (R).This polymorphism and autoimmune disorder, relevant as systemic lupus erythematous and familial rheumatoid arthritis.The segmental length of TNFRII gene amplification product is 529bp, first sequence wherein is from this segmental the 91st bit base, finish to the 107th bit base, the 101st bit base wherein then is the some position that possible morph, and putting in order of first consecutive nucleotides is: 5 '-GACTGCATCCCTGCTTG-3 ' (possible variant sites is wherein represented with mutant).Second sequence is from this segmental the 59th bit base, and to the end of the 74th bit base, putting in order of second sequence is: 5 '-GCCATACTCCGGGTGG-3 '.
2. determine responsive probe and grappling probe.The oligonucleotide of responsive probe is made up of 17 bit bases, the first sequence complementation with the employing of step in 1., also promptly with the segmental corresponding sequence complementation of the corresponding D NA of mutant, its 3 ' end is combined with quenching group, the sequence of responsive probe is: 5 '-CAAGCAGGGATGCAGTC-TAMRA-3 ', wherein the 7th site that may undergo mutation with first sequence is corresponding.The oligonucleotide of grappling probe has 16, and its 5 ' end is combined with the fluorescence report group, and the sequence of grappling probe is: 5 '-FAM-CCACCCGGAGTATGGC-3 '.These two kinds of probes are given birth to the synthetic and modification of worker in Shanghai, and the value of the melting temp of responsive probe is lower than the value of the melting temp of grappling probe.
3. determine two suitable Oligonucleolide primers.The oligonucleotide of sense primer has 24, and its sequence is 5 '-CGGGGACGTTCTCCAACACGACTT-3 '; The oligonucleotide of antisense primer has 20, and its sequence is 5 '-TGGCTGCGTGTGTTGGGATC-3 '.These two kinds of primers are given birth to the worker in Shanghai synthetic and modify, and the value of their melting temp is greater than the value of the melting temp of the grappling 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 1.5mM
2, excessive substrate 0.5 μ l 4 * dNTPs, available from the Shen, Shanghai can betting office 1.25U Taq archaeal dna polymerase, sense primer 10pmol that 3. step obtains and antisense primer 10pmol anabolic reaction system and carry out the polymerase chain reaction, also add responsive probe 2pmol that 2. step obtain and grappling probe 2pmol and constitute whole system.Wherein, PCR total reaction system is 25 μ l, and the concentration of dna profiling in system is 1.6～3.2ng/ μ l, and the concentration of each probe in system is 8 * 10
4PM.Dna profiling also promptly contains the thymus nucleic acid of dna fragmentation to be measured, also claims target DNA, and this dna profiling extracts from human coronary artery disease patient's blood for the UIIQ-10 pillar poba gene group DNA extraction agent box of giving birth to worker's manufacturing with Shanghai.Patient's number has 200, and therefore, the dna profiling of present embodiment has 200, and each dna profiling has been carried out the polymerase chain reaction respectively and carried out follow-up detection step respectively.
The polymerase chain reaction is in the LightCycler of Roche Holding Ag gene amplification detector (version number: carry out 3.5).Process is as follows: pre-sex change is 1 minute under 95 ℃, then begin circulation, also promptly kept 0 second under 95 ℃ the temperature, with the temperature transition rate is that the speed of 20 ℃ of per seconds is cooled to 61 ℃, kept 10 seconds, be that the speed of 20 ℃ of per seconds is warming up to 72 ℃, kept 10 seconds with the temperature transition rate again, be that the speed of 20 ℃ of per seconds is warming up to 95 ℃ with the temperature transition rate again, so just finished a circulation; Move 40 circulations altogether.Last circulates in and is warming up to 72 ℃, kept 10 seconds and finishes.
5. probe hybridization is on same strand of dna fragmentation to be measured.Still on the LightCycler of Roche Holding Ag gene amplification detector, carry out, heating systems to 95 ℃, kept 30 seconds, the two strands of the dna fragmentation to be measured after the amplification is untied, be cooled to 42 ℃ then, kept 4 minutes, thereby responsive probe is hybridized on first sequence of the strand that contains first sequence and second sequence of the dna fragmentation to be measured after the amplification, make the grappling probe hybridization on second sequence of the strand that contains first sequence and second sequence of the dna fragmentation to be measured after the amplification.Because the quantity of responsive probe that is added and grappling probe is basic identical, and the probe of hybridizing on the strand of corresponding dna fragmentation to be measured is adjacent to one another, if excite the fluorescence report group on the probe this moment, then the fluorescence that it was inspired is positioned at the quenching group cancellation on another probe on the same strand, does not therefore externally show fluorescence.
6. see Fig. 3, measure the melting temp of dna fragmentation to be measured.Still carry out on the LightCycler of Roche Holding Ag gene amplification detector, the fluorescence channel of opening on the detector 1 (F1) detects, and the fluorescence report group of probe is excited.System after the heating hybridization, speed with 0.1 ℃ of per second makes system be warming up to 80 ℃ from 42 ℃, in temperature-rise period, responsive probe at first splits away off from the strand of dna fragmentation to be measured, thereby its quenching group can not carry out cancellation to the fluorescence that the fluorescence report group that is incorporated on the grappling probe on this strand is excited to send, thereby the fluorescence intensity in the system increases, continuation rising along with temperature, the responsive probe increase that comes off, fluorescence intensity is also along with enhancing, wherein fluorescence intensity increment is changed temperature when maximum as melting temp (Melting temperature, T
M).This melting temp T
MCorresponding with one of following three kinds of situations: first kind of situation is because G/G homozygote and responsive probe are mated fully, produces a higher melting temp, greatly about about 58.5 ℃; Second kind of situation is that T/T homozygote and responsive probe have a base to be unworthy of T
MValue then floats to 52.5 ℃; The third situation is that heterozygote T/G genotype then occurs two and melts paddy, T
MValue is respectively 52.5 ℃ and 58.5 ℃.Two temperature heads (Δ T) of melting between the paddy are 6 ℃.After the melting temp of dna fragmentation is measured at the place, just can whether have single nucleotide polymorphism and judge this dna fragmentation.With the third situation explanation sudden change is arranged if belong to first kind of situation, do not undergo mutation if belong to second kind of situation then illustrate.
T between the different experiments
MValue may fluctuate between ± 0.8 ℃, and the Δ T that difference is melted between the paddy may fluctuate between ± 0.6 ℃.This law is why with melting paddy, rather than represents with melting the peak, is because there is not the mapping analysis function of dF/dT in LightCycler 3.5 versions.We wish that this function adds in new version, so that the user selects corresponding function according to demand separately.
See Fig. 4, the accuracy of this detection method is verified by dna sequencing.Dna sequencing work is finished by Shanghai Ying Jun company, and Fig. 4 has shown sequencing result.
Embodiment 2
All the other are identical with embodiment 1, and difference is:
Step in 2. the sequence of definite responsive probe be 5 '-CTACAAAAAGTACAAAAATT-FAM-3 ', the sequence of determined grappling probe is 5 '-GTGGTGGCACACGC-TAMRA-3 '.ApoM T-778C oligonucleotide is synthetic and modification in Shanghai Ying Jun company.The probe of apoM T-778C is with reference to coding strand design and synthetic, and therefore, T/T homozygote and C/C homozygote should be represented with A/A homozygote and G/G homozygote respectively.
4. step carries out in the polymerase chain reaction, and the responsive probe that is added, grappling probe, sense primer and antisense primer are corresponding responsive probe, grappling probe, sense primer and the antisense primer of present embodiment.
The hybridization temperature of step in 5. is 30 ℃.
The melting temp T of step in 6.
MCorresponding with one of following three kinds of situations: first kind of situation is because C/C homozygote and responsive probe are mated fully, produces a higher melting temp, greatly about about 50 ℃; Second kind of situation is that T/T homozygote and responsive probe have a base to be unworthy of T
MValue then floats to 43 ℃; The third situation is that heterozygote T/C genotype then occurs two and melts paddy, T
MValue is respectively 50 ℃ and 43 ℃.Two temperature heads (Δ T) of melting between the paddy are 7 ℃.After the melting temp of dna fragmentation is measured at the place, just can whether have single nucleotide polymorphism and judge this dna fragmentation.With the third situation explanation sudden change is arranged if belong to first kind of situation, do not undergo mutation if belong to second kind of situation then illustrate.(Fig. 5).
The accuracy of this detection method is verified by dna sequencing.Dna sequencing work is finished by Shanghai Ying Jun company, and Fig. 6 has shown sequencing result.
Claims (10)
1, a kind of method that detects the single nucleotide polymorphism of thymus nucleic acid has following steps:
1. determine and will certain gene of certain biological thymus nucleic acid be detected, just determine dna fragmentation to be measured, and have first sequence and second sequence on the strand of this dna fragmentation to be measured, described first sequence is meant one section nucleotide sequence that the some specific nucleotides in this sequence may morph, and second sequence is meant the one section nucleotide sequence mutually close with first consecutive nucleotides;
2. preparation is called a kind of oligonucleotide probe of responsive probe and the another kind of oligonucleotide probe that preparation is called the grappling probe; Described responsive probe is complementary to the corresponding sequence of the corresponding DNA fragments of the corresponding wild type gene of first sequence of dna fragmentation to be measured or is complementary to corresponding sequence with the corresponding DNA fragments of the corresponding mutated genes of first sequence of dna fragmentation to be measured, and an end of responsive probe is combined with fluorescence report group or quenching group; The second sequence complementation of described grappling probe and dna fragmentation to be measured, an end of grappling probe are combined with quenching group, are combined with the fluorescence report group when responsive probe are combined with quenching group when responsive probe is combined with the fluorescence report group; The value of the melting temp of responsive probe is lower than the value of the melting temp of grappling probe;
3. prepare two suitable Oligonucleolide primers, one end complementation of an Oligonucleolide primers in these two Oligonucleolide primers and the strand with first sequence and second sequence of above-mentioned dna fragmentation to be measured, the other end complementation 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 responsive probe and grappling probe, and the quantity of responsive probe and grappling probe is basic identical; 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, 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 are adjacent to one another and quantity is suitable, so if the fluorescence report group on the probe is excited, then the fluorescence that 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 demonstrate 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 single nucleotide polymorphism.
2, the method for the single nucleotide polymorphism 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 single nucleotide polymorphism of detection thymus nucleic acid according to claim 1 is characterized in that: the concentration of each probe in system that step is added in 4. is 4 * 10
3～8 * 10
4PM.
4, the method for the single nucleotide polymorphism of detection thymus nucleic acid according to claim 1 is characterized in that: the value of the melting temp of the grappling probe of step in 2. be lower than step 3. in the value of melting temp of Oligonucleolide primers.
5, the method of the single nucleotide polymorphism 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, pre-sex change is 1 minute under 90 ℃～95 ℃, then begin circulation, also promptly kept 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, be that the speed of 20 ℃ of per seconds is warming up to 70 ℃～75 ℃ with the temperature transition rate again, kept 10 seconds, be that the speed of 20 ℃ of per seconds is warming up to 90 ℃～95 ℃ with the temperature transition rate again, so just finished a circulation; Move 25～40 circulations altogether.
6, the method for the single nucleotide polymorphism 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 responsive probe and the temperature of grappling probe hybridization on the strand that contains first sequence and second sequence of the dna fragmentation to be measured after the amplification is 30 ℃～40 ℃;
7, the method for the single nucleotide polymorphism of detection thymus nucleic acid according to claim 1 is characterized in that: the 6. middle heat-up rate of step is 0.1 ℃ of a per second, and top temperature is 80 ℃.
8, the method for the single nucleotide polymorphism of detection thymus nucleic acid according to claim 1, it is characterized in that: the fluorescence report group that step is incorporated into probe in 2. is the 6-Fluoresceincarboxylic acid, and the quenching group that is incorporated into probe is a 6-carboxyl tetramethyl-rhodamine.
9, according to the method for the single nucleotide polymorphism of the described detection thymus nucleic acid of one of claim 1 to 8, it is characterized in that: step 4. in the dna fragmentation to be measured of thymus nucleic acid to be measured be Tumor Necrosis Factor Receptors II or Apoliprotein M.
10, according to the method for the single nucleotide polymorphism of the described detection thymus nucleic acid of one of claim 1 to 8, 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 responsive 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 single nucleotide polymorphism.
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