CN103789436B - A kind of quantitative abrupt climatic change system based on manually modified primer - Google Patents

Abstract

The invention belongs to biological technical field.More specifically, the present invention relates to the detection of transgenation.The invention provides a kind of quantitative abrupt climatic change system based on manually modified primer.Quantitative abrupt climatic change system of the present invention mainly comprises design of primers, PCR reaction and product and detects three steps.By introducing one or more than one artificial mutation in the region that primer 3 ' holds 5 bases, and based on adjustment 3 ' hold the thermodynamic property that is combined with template of primer guarantee archaeal dna polymerase in conjunction with time there is certain stability, but be unlikely to too high, thus reduce false positive, significantly improve selectivity; By adding fluorescence and coupling fluorescent capillary electrophoresis tube detects PCR primer at primer 5 ' end, further increase selectivity, and the semi-quantitative analysis to sample sudden change can be realized.

Description

A kind of quantitative abrupt climatic change system based on manually modified primer

Technical field

The invention belongs to biological technical field.More specifically, the present invention relates to the detection of transgenation.

Background technology

Along with the develop rapidly of sequencing technologies, the mankind and various animals and plants, microbial genome have been sequenced, and the genomics data of magnanimity propose new challenge to the gene functional research in the fields such as medical science, biology, agronomy and forestry.

The change that gene in sudden change phalangeal cell occur.It comprises the sudden change caused by single sequence change, or the insertion of multiple base, lacks and repeat.That studies along with gene and disease association gos deep into, and the abrupt climatic change of Disease-causing gene, medicaments insensitive and tolerance gene has been widely used in the research of disease genesis mechanism.Meanwhile, animals and plants breeding is also made to have striden into the molecular breeding epoch with the molecular genetic marker technique sporting basis.Because abrupt climatic change is in the widespread use in the research of disease genesis mechanism and the field such as animals and plants breeding and genetic improvement, the accurate rapid gene mutation detection methods of highly sensitive, highly selective is made to receive unprecedented concern.

Technique of gene detection is developed so far of a great variety, there is single stranded conformational isomery Polymorphism Analysis technology (Single-strand conformation polymorphism, SSCP), heterogeneous double-stranded conformational polymorphism analysis (Heteroduplex, HTX), denaturing gradient gel electrophoresis (Denaturing Gradient Get Electrophoresis, DGGE), mismatch cleavage method (Dismate cleavage, DC), dhplc analysis (Denaturing high-performance liquid chromatograph, DHPLC), allele specific oligonucleotide hybridization (Allele-Specific Oligonucleotide Hybridization, ASOH), PCR (Polymerase Chain Reaction) sequencing, allele specific amplification (Allelegene-specific Amplification, ASA) etc.But can possess simultaneously highly sensitive, highly selective accurate method for quick also few in number.

At present, maximum gene testers is used to be PCR sequencing, also known as polymerase chain reaction method.PCR sequencing gives tacit consent to " gold standard " at present.But PCR sequencing susceptibility is lower, larger false positive and false negative can be produced to inferior quality or the lower pattern detection of content.PCR primer in PCR sequencing is positioned at both sides, mutational site, normal sequence and mutant nucleotide sequence all can be increased.Due to mutant nucleotide sequence, often proportion is minimum, and in pcr amplification reaction, the thymus nucleic acid (Deoxyribonucleic acid, DNA) of large percentage contained by template has amplification advantage.Therefore, the selectivity of PCR sequencing is lower, is only 10%-30%.Meanwhile, the method can not carry out quantitative analysis to sudden change contained by sample.

Therefore, someone develops one and is called amplification refractory mutation system method (Amplification Refractory Mutation System, ARMS), and it is the one of allele specific amplification method (ASA).ARMS method utilizes 3 ' of the PCR primer principle of holding last bit base could effectively must increase with its template DNA complementation, design ApoE gene amplimer.Research shows, the selectivity of abrupt climatic change can be brought up to 1% by ARMS method, but low for quality or that content is low DNA sample, and such selectivity is still on the low side.In addition, ARMS method relies on conventional gel electrophoresis or probe hybridization to detect PCR primer, for the variants causing amplification efficiency lower because samples contg is lower, electrophoretic band may not show.Meanwhile, the method can not be carried out quantitatively or semi-quantitative analysis sudden change contained by sample.

Conventional PCR method and ARMS method all can not solve false-positive problem.First, when DNA replication dna, the nucleic acid that polymerase covers probably has 10 bases, and 6 bases are double-strand (having primer to combine), and other 4 bases are the template sequences that namely will synthesize.So these 6 double-strand bases are exactly the key that can polymerase effectively combine, the space conformation that also can be understood as the combination of primer and template is only polysaccharase and combines and the key extended.In the experiment of reality, often there is the situation that 3 ' end mispairing still can extend.Particularly, 3 ' end mispairing can not stop extension completely, especially, when mispairing template (as normal sequence) is with the template of mating completely (mutant nucleotide sequence) large percentage, false positive often has the reason of generation to be exactly the space conformation that these primers and template define that polysaccharase can identify.Such as, in some cases, although 3 ' end is mispairing, higher owing to being close to 3 ' the base combination stability held, and then 3 ' end mispairing firmly raw " drawing " that should be distant is together, creates so-called " closing on " effect.Secondly, although some is mispairing, its combination still has quite high stability, such as " G-G " mispairing.For this two classes situation, general PCR method does not consider that some mispairing is thermodynamically still more stable, can trigger the situation of PCR extension yet; And ARMS method, do not consider that 6 double-strand base zones below that primer is combined with template are on the impact of archaeal dna polymerase binding ability, therefore existing method does not all properly settle false positive issue.

Summary of the invention

In order to solve above-mentioned technology Problems existing, invention introduces artificial mutation and consider the stability of archaeal dna polymerase, based on the design of primers theory that this is brand-new, provide and a kind ofly add fluorescence and in conjunction with the retardance round pcr method of capillary electrophoresis, highly sensitive and highly selective can carry out the detection by quantitative of suddenling change.

The present invention relates to the quantitative abrupt climatic change system (Mutation Detection System by Artificial Modified Primers, AMP-MDS) based on manually modified primer, its schematic diagram as shown in Figure 1.

Therefore, the invention provides a kind of method detecting sudden change, described method comprises the steps:

1) for the sudden change that will detect, the combination of design outer primer and inner primer, hold the region of 5 bases at described inner primer 3 ', the direction of from 3 ' to 5 ', introduce artificial mismatches according to thermodynamic stability " height height " or the height phase inter mode of " low height is high " successively;

2) carry out PCR reaction with primer, obtain matching the product increased;

3) semi-quantitative analysis is carried out to described product.

In a specific embodiment, described method comprises the steps:

1) for the sudden change that will detect, design the combination of a pair outer primer and a pair inner primer;

On the intron that described outer primer is positioned at mutated site both sides or across intron;

In opposite directions, 3 ' end is positioned at variant positions in described two inner primer directions, 3 ' end and normal base complementrity of an inner primer, and 3 ' end of another inner primer is complementary with the mutating alkali yl of mutated site;

Described two outer primers respectively with one inner primer can match and carry out pcr amplification, and they self also can carry out pairing amplification;

Fluorescence is added in 5 ' end of outer primer, and uses the fluorescence dye of different colours to mark two inner primers;

The product of described two outer primers pairing amplification and described two outer primers match the length difference of two products of generation respectively with described two inner primers institute must at 5 more than bp, preferably 10 more than bp, more preferably 20 more than bp;

Hold the region of 5 bases reciprocal to introduce mispairing to described inner primer 3 ', according to the pattern that thermodynamic stability height is alternate, detailed principle is as follows:

If 3 ' end mispairing has certain stability, so corresponding mispairing (as shown in Fig. 2 a, b) can be introduced antepenulatimate and the 5th;

If 3 ' end mispairing has lower destructiveness to stability, so then need to judge whether further introduce mispairing (as shown in Fig. 2 c, d) according to the stability of penultimate base pair;

If 3 ' end mispairing has stronger destructiveness (i.e. less stable) for stability, so do not introduce mispairing in antepenulatimate, but when penultimate makes an exception (as shown in Fig. 2 e, f) when having the base pair compared with stiff stability; Such as, for this group primer of AGCGA/TCGCT, its thermodynamic stability is: _---_ (" _ " represents low thermodynamic stability, "-" represents high thermodynamic stability), therefore mispairing can be introduced in antepenulatimate, its thermodynamic stability changes into: _-_-_, mispairing is also introduced as alternative primer in fourth from the last position, its thermodynamic stability is simultaneously: _ _ _-_; Here reflect that the binding ability of primer and template is strong and weak with thermodynamic stability height; Low thermodynamic stability refer to primer and template binding ability weak, built on the sand, such as, AT containing two hydrogen bonds matches, or the mispairing such as AC, AG; High thermodynamic stability refers to primer and template binding ability by force, more firmly, as the CG containing three hydrogen bonds matches.

Inside and outside primer keeps melting temperature(Tm) as far as possible consistent, such as, within differing 6 degrees Celsius, preferably within 4 degrees Celsius, more preferably within 2 degrees Celsius;

Inside and outside primer GC content is consistent as far as possible, such as, differ within 20%, preferably within 15%, more preferably within 5%;

Inside and outside primer length is consistent as far as possible, such as, within differing 6 nt, preferably within 4 nt;

Inside and outside primer self can not form hairpin structure;

Can not dimer be formed between inside and outside primer, non-specific amplification occurs;

2) carry out PCR reaction with the primer of step 1), obtain the product 1 of described two outer primers pairing amplification and described two outer primers and match the product 2 of generation and product 3 respectively with described two inner primers institute;

3) above-mentioned product is carried out semi-quantitative analysis by fluorescent capillary electrophoresis tube, wherein product 1, product 2 and product 3 meet all have obvious peak value time, representing has mutating alkali yl at mutated site; Further the peak value of each peak value and standard substance is carried out ratio proccessing, sxemiquantitative can be carried out to the content of the content of mutating alkali yl and normal base and compare.

In the present invention, thermodynamic stability height is used to the binding ability power reflecting primer and template; Low thermodynamic stability refer to primer and template binding ability weak, built on the sand, such as, AT containing two hydrogen bonds matches, or the mispairing such as AC, AG; High thermodynamic stability refers to primer and template binding ability by force, more firmly, as the CG containing three hydrogen bonds matches.Such as, for this group primer of AGCGA/TCGCT, its thermodynamic stability is: _---_ (" _ " represents low thermodynamic stability, "-" represents high thermodynamic stability), therefore mispairing can be introduced in antepenulatimate, its thermodynamic stability changes into: _-_-_, mispairing is also introduced as alternative primer in fourth from the last position, its thermodynamic stability is simultaneously: _ _ _-_.

Technical superiority of the present invention is as follows:

1) present method is suddenlyd change by the technology for detection of PCR-based, has very high sensitivity, and being low to moderate 5-10 copy can accurately detect.

2) present method is by holding the region of 5 bases to introduce one or more than one artificial mutation at primer 3 ', and ensure that the stability that archaeal dna polymerase combines on the one hand, another one aspect greatly improves selectivity again.

3) by adding fluorescence at primer 5 ' end and detect PCR primer by fluorescent capillary electrophoresis tube, improve selectivity further, global selectivity is 10 ^-4-10 ^-5between.

4) outer primer of present method design is positioned at intron region, adding fluorescence dye, avoiding introducing extra probe, further increasing specificity, reducing false positive rate by holding at primer 5 '.

5) present method combined with fluorescent capillary electrophoresis technique, can realize the semi-quantitative analysis to sample sudden change.

6) present method process is simple, only comprises two steps, i.e. PCR process and capillary electrophoresis, and therefore detection speed is fast, and whole process only need less than 1 hour.

Accompanying drawing explanation

Fig. 1 illustrates: based on the schematic diagram of the quantitative abrupt climatic change of manually modified primer.

Fig. 2 illustrates: primer 3 ' holds 5 base zones artificially to introduce the design of primers schematic diagram of mispairing.

Except 3 ' holds the mispairing of last base to design according to mutational site, other mispairing is artificial introducing, introduces mispairing Specific Principles and is:

If 3 ' end mispairing has certain stability, so corresponding mispairing (a, b) can be introduced antepenulatimate and the 5th;

If 3 ' end mispairing has lower destructiveness to stability, so then need to judge whether to introduce mispairing (c, d) further according to the stability of penultimate base pair;

If 3 ' end mispairing has stronger destructiveness for stability, so do not advise introducing mispairing in antepenulatimate, but when penultimate is when having the base pair compared with stiff stability, then can consider (e, f).

Arrows thermodynamic stability, long arrow represents high stability base pair, and short arrow then represents low stability base pair, x represents mispairing and destroys lower to stability, X represents mispairing and destroys stability comparatively large, although it is mispairing that x and upward arrow thereof represent, but still has certain stability.

Fig. 3 illustrates: the positive that capillary electrophoresis detects and negative findings comparative example figure.

Fig. 4 illustrates: PCR-sequencing sensitivity experiment results peaks figure.

Fig. 5 illustrates: based on the product electrophorogram example of the quantitative abrupt climatic change of manually modified primer.

Fig. 6 illustrates: utilize the quantitative mutation detection methods based on manually modified primer to detect the capillary electrophoresis fluorescence peak figure result of people's cancerous lung tissue EGFR gene L858R point mutation standard substance of 0%, 0.02%, 0.2%, 2%, 10%, 20%, 50% and 100% respectively.

Fig. 7 illustrates: utilize PCR sequencing, AMRS method, AMP-MDS method to carry out the accuracy rate of the L858R abrupt climatic change of EGFR gene.

Embodiment

The present invention relates to a kind of quantitative abrupt climatic change system based on manually modified primer (Mutation Detection System by Artificial Modified Primers, AMP-MDS).In order to reasonably estimate that primer is combined with template 3 ' holds the impact combined archaeal dna polymerase, take into account stability and specificity, our emphasis is considered to introduce mispairing in the region of 3 ' end, 5 bases, ensure that on the one hand necessary stability is beneficial to archaeal dna polymerase and combines, what be combined with material impact to DNA is 3 ' end, 6 bases.Here adopt conservative way, get 5 bases and carry out manual intervention; On the other hand, according to pairing particular case, suitably introduce mispairing, avoid the caused false positive results of 3 ' end causing stability too high.Compare with traditional method, the innovation of method of the present invention holds the region of 5 bases to introduce the design of primers theory of or more than one artificial mutation according to the alternate pattern of thermodynamic stability height at primer 3 ', both consider the fact that some mispairing is also stable, have also contemplated that the impact on archaeal dna polymerase; Last and combine capillary electrophoresis technique, make it highly sensitive and highly selective to carry out the detection by quantitative of suddenling change.

Goal of the invention relates generally to following 6 points:

1) present method detects the design of primers of sudden change.

For sudden change, present method designs a pair inner primer and a pair outer primer, and wherein 3 ' end of inner primer is positioned at variant positions, 3 ' end and normal base complementrity of an inner primer, and 3 ' end of another inner primer is complementary with mutating alkali yl; Outer primer is positioned at catastrophe point both sides, and respectively with one inner primer can match and carry out pcr amplification, and they self also can carry out pairing amplification.For, the amplified production of sudden change, only needs the length difference of guarantee three products namely to analyze by capillary electrophoresis more than 5 bases.

2) present method is by holding the region of 5 bases to introduce one at primer 3 ' or more than one artificial mutation improves selectivity.

For the inner primer of sudden change, present method adds artificial mismatches in the region of its 3 ' end 5 bases, and compare with the mispairing in ARMS technology, mispairing is incorporated into except penultimate by present method, is also incorporated into antepenulatimate, the 4th, even the 5th; Further, base mismatch number is not confined to 2, even can reach three or four.The principle introducing base mismatch is according to the alternate pattern of thermodynamic stability height, the region of 3 ' end, 5 bases is avoided to occur stronger thermodynamic stability, but make it to remain on lower more uniform thermodynamics level, thus prevent 3 ' stronger end of combination stability, avoid non-specific amplification (i.e. false positive).

3) present method is by adding fluorescence at primer 5 ' end and detect PCR primer by fluorescent capillary electrophoresis tube, improves selectivity further.

For sudden change, fluorescence is added in 5 ' end of outer primer, and uses the fluorescence dye of display different colours to mark two outer primers.Because fluorescent capillary electrophoresis tube is by detecting the accumulation of fluorescent signal, even if the mutant DNA content therefore in sample is less, in pcr amplification, be also in competitive disadvantages, and the content of PCR primer is also relatively low, fluorescent capillary electrophoresis tube still can accurately detect.

4) present method being added fluorescence dye by holding at primer 5 ', being avoided introducing extra probe, reduce further false positive rate.

Compare with common fluorescent quantitative PCR technique, because the fluorescence in present method is added in 5 ' end of primer, there is no extra probe, therefore compare the step that it also avoid hybridization with traditional method, reduce false-positive risk.

5) present method combined with fluorescent capillary electrophoresis technique, can realize the semi-quantitative analysis to sample sudden change.

By comparing target fluorescence intensity in the fluorescence intensity of mutant DNA and fluorescent capillary electrophoresis tube, calculate a uncalibrated visual servo, can the scale parameter of lateral comparison, for carrying out sxemiquantitative description to the sudden change content in sample.

6) present method is by the position of design outer primer, and transcript can be prevented the pollution of pcr amplification product.

In present method, outer primer design on intron or across an intron, thus avoids the interference of transcription product for PCR primer, reduce further false-positive risk.

The schematic diagram of AMP-MDS as shown in Figure 1.

The invention provides a kind of method detecting sudden change, described method comprises step:

1) for the sudden change that will detect, design the combination of a pair outer primer and a pair inner primer;

On the intron that described outer primer is positioned at mutated site both sides or across intron;

In opposite directions, 3 ' end is positioned at variant positions in described two inner primer directions, 3 ' end and normal base complementrity of an inner primer, and 3 ' end of another inner primer is complementary with mutating alkali yl;

Described 2 outer primers respectively with one inner primer can match and carry out pcr amplification, and they self also can carry out pairing amplification;

Fluorescence is added in 5 ' end of outer primer, and two outer primers use the fluorescence dye of different Show Color;

The product of described two outer primers pairing amplification and described two outer primers match the length difference of two products of generation respectively with described two inner primers institute must at more than 5bp;

Hold the region of 5 bases reciprocal to introduce mispairing to described inner primer 3 ', according to the pattern that thermodynamic stability height is alternate, detailed principle is as follows:

If 3 ' end mispairing has certain stability, so corresponding mispairing (as shown in Fig. 2 a, b) can be introduced antepenulatimate and the 5th;

If 3 ' end mispairing has lower destructiveness to stability, so then need to judge whether further introduce mispairing (as shown in Fig. 2 c, d) according to the stability of penultimate base pair;

If 3 ' end mispairing has stronger destructiveness for stability, so do not introduce mispairing in antepenulatimate, but when penultimate makes an exception (as shown in Fig. 2 e, f) when having the base pair compared with stiff stability;

Such as, AGCGA/TCGCT, this group primer, its thermodynamic stability is: _---_ (" _ " represents low thermodynamic stability, "-" represents high thermodynamic stability), therefore can introduce mispairing in antepenulatimate, its thermodynamic stability changes into: _-_-_, mispairing is also introduced as alternative primer in fourth from the last position, its thermodynamic stability is simultaneously: _ _ _-_;

Inside and outside primer keeps melting temperature(Tm) as far as possible consistent, such as, within differing 6 degrees Celsius;

Inside and outside primer GC content is consistent as far as possible, such as, differ within 20%;

Inside and outside primer length is consistent as far as possible, such as, within differing 6 nt;

Inside and outside primer self can not form hairpin structure;

Can not dimer be formed between inside and outside primer, non-specific amplification occurs;

2) PCR reaction

Carry out PCR reaction with the primer of step 1, obtain the product of described two outer primers pairing amplification and described two outer primers and match the product of generation respectively with described two inner primers institute;

Such as, PCR reaction reaction system and reaction conditions as follows:

Reaction system

Reaction system	Reaction conditions
		RTaq enzyme	2.5 μl
10 × damping fluid	5 μl
		dNTP-MIX	200 μmol/L
MgCl 2	1.5-2.0 mmol/L
		Outer primer	Each 1 μ l
Methane amide	1 μl
		Inner primer	Each 1 μ l
Water	Supply system
		DMSO	1 μl
Template	1 μl
		Total system	50 μl

Reaction conditions:

PCR reaction conditions is:

95 DEG C of denaturations 5 minutes;

95 DEG C of sex change 30 seconds, 60 DEG C of annealing 30 seconds, 72 DEG C extend 30 seconds, 10 circulations;

95 DEG C of sex change 15 seconds, 69 DEG C of annealing 15 seconds, 72 DEG C extend 15 seconds, 25 circulations;

Finally extend 7 minutes.

3) product detects

Above-mentioned product is carried out semi-quantitative analysis by fluorescent capillary electrophoresis tube.

In one embodiment, described semi-quantitative analysis is specially STR and detects, i.e. STR (Short Tandem Repeat, STR) analysis platform, for a kind of technique means of genetic material difference between working sample (STR difference).STR (STR) detects STR also known as microsatellite DNA (micro-satellite DNA).

Concrete steps are as follows:

(1) mix with the ratio of methane amide in 1:13130 with ABI GS500LIZ.

(2), after mixing liquid being taken out 9 μ l, testing sample 0.5 μ l is added.

(3) on ABI3730 sequenator, carry out capillary electrophoresis detection, obtain corresponding glue figure and peak figure.

Below in conjunction with the embodiment of the present invention and accompanying drawing, the present invention is further detailed.

Embodiment one, detects the single mutation in the paraffin-embedded tissue pathological slice tissue of nonsmall-cell lung cancer.With technical project series primer of the present invention in the present embodiment, and detect EGFR gene, the i.e. transgenation in 21 exons the 858th mutational site of EGF-R ELISA (Epidermal Growth Factor Receptor) in sample in conjunction with capillary electrophoresis.

Experiment material is as follows:

Reagent	Manufacturer
		RTaq enzyme	TAKALA (precious biological) company
10 × damping fluid	TAKALA (precious biological) company
		dNTP-MIX	TAKALA (precious biological) company
MgCl 2	TAKALA (precious biological) company
		Primer synthesizes	Beijing Qing Kexin industry Bioisystech Co., Ltd
Methane amide	SIGMA company of the U.S.
		DMSO	SIGMA company of the U.S.
Proteinase K	QIAGEN company
		QIAamp MinElute post	QIAGEN company
AW1, AW2, ATL etc.	QIAGEN company

Sample and reference standards preparation process as follows:

1. detect sample

Be derived from hospital pathology department through paraffin-embedded pathological section, tissue slice is put in dye sheet cylinder, adds dimethylbenzene and fully soak 2 hours, with alcohol rinsing 2 times, add 100% alcohol immersion 10 minutes, dry.The tissue slice dried is kept flat on experiment table fixing, with careful tissue is peeled off from slide of flat scraper, rapidly tissue sample is put into 1.5 ml EP pipes and build lid.Add 180 μ l ATL damping fluids and 20 μ l Proteinase Ks, fully mix with oscillator, mixture is put into water-bath 56 DEG C digestion 60 minutes.Postdigestive mixture is put into water-bath 90 DEG C digestion 60 minutes.Add 200 μ l AL damping fluids fully to mix; add 200 μ l 100% ethanol mixings again; biased sample is put into QIAamp MinElute column; centrifugal 2 min of 8000 rpm; then respectively with 500 μ l Buffer AW1 and 500 μ l Buffer AW2,6000 × g (8000 rpm) centrifugal 1 min wash-out respectively, last 20000 × g; 14000 rpm are centrifugal, and 3 min dry, and add 100 μ l ATE20000 × g; Collect eluting liquid after the centrifugal 1 min wash-out of 14000 rpm and carry out DNA quality evalution.

2. the preparation of reference standards

Analysis is compared, direct labor's synthetic standards sequence SEQ ID NO.1 according to EGFR gene 21 exon the 858th mutational site gene wild-type of Cosmic warehouse publication and mutant gene sequence:

5-GCAGCGGGTTACATCTTCTTTCATGCGCCTttccattctttggatcagtagtca ctaacgttcgccagccataagtcctcgacgtggagaggctcagagcctGGCATGAA CTACTTGGAGGACCGTCGCTTGGTGCACCGCGACCTGGCAGCCAGGAACGTACTGG TGAAAACACCGCAGCATGTCAAGATCACAGATTTTGGGC kgGCCAAACTGCTGGGTGCGGAAGAGAAAGA ATA CCATGCAGAAGGAGGCAAAGTAAGGAGGTGGCT-3, wherein K is t(wild-type) or G(sudden change)

3. design of primers

Inner Fp1 CGCAGCATGTCAAGATCACAGATTTTGGGC T（SEQ ID NO.2 ）

Inner Rp1 CTTTCTCTTCCGCACCCAGCAGTTTGGCC C（SEQ ID NO.3 ）

Inner Fp2 CGCAGCATGTCAAGATCACAGATTTTGGG TT（SEQ ID NO.4 ）

Inner Rp2 CTTTCTCTTCCGCACCCAGCAGTTTGGC TC（SEQ ID NO.5 ）

Inner Fp3 CGCAGCATGTCAAGATCACAGATTTTGG ACT（SEQ ID NO.6 ）

Inner Rp3 CTTTCTCTTCCGCACCCAGCAGTTTGG TCC（SEQ ID NO.7 ）

Inner4 Fp CGCAGCATGTCAAGATCACAGATTTTG GATT（SEQ ID NO.8 ）

Inner4 Rp CTTTCTCTTCCGCACCCAGCAGTTTG GTTC（SEQ ID NO.9 ）

Inner5 Fp CATGTCAAGATCACAGATTTT GGATT（SEQ ID NO.10 ）

Inner5 Rp TCTTCCGCACCCAGCAGTTT GGTTC（SEQ ID NO.11 ）

Outer Fp GCAGCGGGTTACATCTTCTTTCATGCGCCT （SEQ ID NO.12 ）

Outer Rp AGCCACCTCCTTACTTTGCCTCCTTCTGCA(fluorescent mark) (sEQ ID NO.13 )

4. pcr amplification

PCR reaction system is as follows:

Reaction system	Reaction conditions
		RTaq enzyme	2.5 μl
10 × damping fluid	5 μl
		dNTP-MIX	200 μmol/L
MgCl 2	1.5-2.0 mmol/L
		Outer primer	Each 1 μ l
Methane amide	1 μl
		Inner primer is each	1 μl
Water	Supply system
		DMSO	1 μl
Template	1 μl
		Total system	50 μl

PCR reaction conditions is: 95 DEG C of denaturations 5 minutes;

95 DEG C of sex change 30 seconds, 60 DEG C of annealing 30 seconds, 72 DEG C extend 30 seconds, 10 circulations;

95 DEG C of sex change 15 seconds, 69 DEG C of annealing 15 seconds, 72 DEG C extend 15 seconds, 25 circulations;

Finally extend 7 minutes.

5. detect

Utilize the goal gene with fluorescence in gs500 detection PCR result.

6. specificity analyses

Using the positive of synthesis, negative DNA as template detection target fragment contrast experiment, result as shown in Figure 3.

7. sensitivity experiment

Sample Positive and negative respectively according to 1:0,0:1,1:1,1:4,1:50,1:500 and 1:5000(and 1:X shown below) amount ratio mix, and after making mixing, cumulative volume is 100 μ l.Calculate volume required according to concentration, process is as follows:

C ₁V ₁:C ₂V ₂=1:X；

V ₁+V ₂=100；

Comprehensive above two equations draw V ₁=200C ₂/ (XC ₁+ C ₂), V ₂=100-V ₁.

Having recorded each sample concentration is C ₁=33 ng/ μ l, C ₂=41.95 ng/ μ l.

Automatically the V under each X correspondence is calculated with Excel software ₁and V ₂.Obtain 1 to No. 7 fresh sample.

Mixed ratio	X	V 1	V 2	Send and survey numbering
					1 former state		100	0	1
2 former states		0	100	2
					50.00%	1	55.97	44.03	3
20.00%	4	24.12	75.88	4
					1.96%	50	2.4794	97.521	5
0.20%	500	0.2536	99.746	6
					0.02%	5000	0.02542	99.975	7

Order-checking qualification biased sample, result as shown in Figure 4.

Different inner primer design fluorescent capillary electrophoresis tube detection sensitivity experimental result is as following table.

Not suddenly change segment

Sudden change segment

50% sudden change

20% sudden change

10% sudden change

2% sudden change

0.2% sudden change

0.02% sudden change

Inner primer design 1

+

+

+

+

+

+

+

+

Inner primer design 2

--

+

+

+

+

+

+

+

Inner primer design 3

--

+

+

+

+

+

+ -

+

8. selectivity experiment

1) electrophoresis observed result as shown in Figure 5.

2) capillary detection result as shown in Figure 6.

By to interpretation of result: it is very good that Sample Positive and feminine gender detect selectivity according to 1:0,0:1,1:1,1:4,1:50,1:500 and 1:5000 ratio mixing application the inventive method respectively.

9. repeatability and accuracy rate

1) standard substance carry out 10 experiments, repeatability and rate of accuracy reached 100%.

2) choose the routine traditional sequencing of clinical pathology 100 and the inventive method is compared, this experimental technique detection sensitivity, accuracy rate are obviously better than traditional sequencing, greatly reduce the false negative rate of traditional sequencing.

10. detect odds ratio comparatively

We have chosen the clinical case of 10 routine Patients with Non-small-cell Lungs, and utilize PCR sequencing, AMRS method and our AMP-MDS method carry out the L858R abrupt climatic change of EGFR gene and compare respectively, comparative result sees table.As shown in Figure 7, the accuracy rate of AMP-MDS method is the highest, reaches 100%, and false positive rate is better than ARMS method greatly, and false negative rate is better than PCR sequencing greatly.

Note: CR: complete incidence graph, PR: partial rcsponse, SD: stable, PD: progress.

<110> thinks rich AudioCodes bioinformation science and technology (Beijing) company limited

 

<120> mono-kind is based on the quantitative abrupt climatic change system of manually modified primer

 

<130> 20140120

 

<160> 13

 

<170> PatentIn version 3.3

 

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<211> 31

<212> DNA

<213> artificial sequence

 

<400> 4

cgcagcatgt caagatcaca gattttgggt t 31

 

 

<210> 5

<211> 30

<212> DNA

<213> artificial sequence

 

<400> 5

ctttctcttc cgcacccagc agtttggctc 30

 

 

<210> 6

<211> 31

<212> DNA

<213> artificial sequence

 

<400> 6

cgcagcatgt caagatcaca gattttggac t 31

 

 

<210> 7

<211> 30

<212> DNA

<213> artificial sequence

 

<400> 7

ctttctcttc cgcacccagc agtttggtcc 30

 

 

<210> 8

<211> 31

<212> DNA

<213> artificial sequence

 

<400> 8

cgcagcatgt caagatcaca gattttggat t 31

 

 

<210> 9

<211> 30

<212> DNA

<213> artificial sequence

 

<400> 9

ctttctcttc cgcacccagc agtttggttc 30

 

 

<210> 10

<211> 26

<212> DNA

<213> artificial sequence

 

<400> 10

catgtcaaga tcacagattt tggatt 26

 

 

<210> 11

<211> 25

<212> DNA

<213> artificial sequence

 

<400> 11

tcttccgcac ccagcagttt ggttc 25

 

 

<210> 12

<211> 30

<212> DNA

<213> artificial sequence

 

<400> 12

gcagcgggtt acatcttctt tcatgcgcct 30

 

 

<210> 13

<211> 30

<212> DNA

<213> artificial sequence

 

<400> 13

agccacctcc ttactttgcc tccttctgca 30

 

Claims (5)

1., for the method that the detection of non-diagnostic object suddenlys change, described method comprises the steps:

1) for the sudden change that will detect, design the combination of a pair outer primer and a pair inner primer;

On the intron that described outer primer is positioned at mutated site both sides or across intron;

In opposite directions, 3 ' end is positioned at variant positions in described two inner primer directions, 3 ' end and normal base complementrity of an inner primer, and 3 ' end of another inner primer is complementary with mutating alkali yl;

Described 2 outer primers respectively with one inner primer can match and carry out pcr amplification, and they self also can carry out pairing amplification;

Fluorescence is added in 5 ' end of outer primer, and two outer primers use the fluorescence dye of display different colours to mark;

The product of described two outer primers pairing amplification and described two outer primers match the length difference of two products of generation respectively with described two inner primers institute must at 5 more than bp;

Hold the region of 5 bases reciprocal to introduce mispairing to described inner primer 3 ', according to the pattern that thermodynamic stability height is alternate, detailed principle is as follows:

If 3 ' end mispairing has certain stability, so introduce corresponding mispairing antepenulatimate and the 5th;

If 3 ' end mispairing has lower destructiveness to stability, so then need to judge whether further introduce mispairing according to the stability of penultimate base pair;

If 3 ' end mispairing has stronger destructiveness for stability, so do not introduce mispairing in antepenulatimate, but make an exception when penultimate is and has the base pair compared with stiff stability;

Inside and outside primer melting temperature(Tm) differs within 6 degrees Celsius;

Within inside and outside primer GC content difference 20%;

Within inside and outside primer length differs 6 nt;

Inside and outside primer self can not form hairpin structure;

Can not dimer be formed between inside and outside primer, non-specific amplification occurs,

Described inner primer is CGCAGCATGTCAAGATCACAGATTTTGGGTT and CTTTCTCTTCCGCACCCAGCAGTTTGGCTC respectively; Or be CGCAGCATGTCAAGATCACAGATTTTGGACT and CTTTCTCTTCCGCACCCAGCAGTTTGGTCC respectively;

Described outer primer is GCAGCGGGTTACATCTTCTTTCATGCGCCT and AGCCACCTCCTTACTTTGCCTCCTTCTGCA respectively;

2) carry out PCR reaction with the primer of step 1), obtain the product 1 of described two outer primers pairing amplification and described two outer primers and match the product 2 of generation and product 3 respectively with described two inner primers institute;

3) above-mentioned product is carried out semi-quantitative analysis by fluorescent capillary electrophoresis tube, wherein product 1, product 2 and product 3 meet all have obvious peak value time, representing has mutating alkali yl at mutated site; Further the peak value of each peak value and standard substance is carried out ratio proccessing, sxemiquantitative can be carried out to the content of the content of mutating alkali yl and normal base and compare,

Wherein said sudden change is EGFR gene 21 exon the 858th mutational site.

2. be added in outer primer described in the process of claim 1 wherein the 5 ' fluorescence dye held is HEX and FAM.

3. the process of claim 1 wherein that described semi-quantitative analysis is specially the STR analysis platform based on ABI3730xl system.

4. detect the outer primer in EGFR gene 21 exon the 858th mutational site and the combination of inner primer, comprise the combination of a pair outer primer and a pair inner primer:

On the intron that described outer primer is positioned at mutated site both sides or across intron;

In opposite directions, 3 ' end is positioned at variant positions in described two inner primer directions, 3 ' end and normal base complementrity of an inner primer, and 3 ' end of another inner primer is complementary with mutating alkali yl;

Described 2 outer primers respectively with one inner primer can match and carry out pcr amplification, and they self also can carry out pairing amplification;

Fluorescence is added in 5 ' end of outer primer, and two outer primers are shown the fluorochrome label of different colours;

The product of described two outer primers pairing amplification and described two outer primers match the length difference of two products of generation respectively with described two inner primers institute must at 5 more than bp;

Hold the region of 5 bases reciprocal to introduce mispairing to described inner primer 3 ', according to the pattern that thermodynamic stability height is alternate, detailed principle is as follows:

If 3 ' end mispairing has certain stability, so introduce corresponding mispairing antepenulatimate and the 5th;

If 3 ' end mispairing has lower destructiveness to stability, so then need to judge whether further introduce mispairing according to the stability of penultimate base pair;

If 3 ' end mispairing has stronger destructiveness for stability, so do not introduce mispairing in antepenulatimate, but make an exception when penultimate is and has the base pair compared with stiff stability;

Inside and outside primer melting temperature(Tm) differs within 6 degrees Celsius;

Within inside and outside primer GC content difference 20%;

Within inside and outside primer length differs 6 nt;

Inside and outside primer self can not form hairpin structure;

Can not dimer be formed between inside and outside primer, non-specific amplification occurs,

Described inner primer is CGCAGCATGTCAAGATCACAGATTTTGGGTT or CTTTCTCTTCCGCACCCAGCAGTTTGGCTC respectively; Or be CGCAGCATGTCAAGATCACAGATTTTGGACT and CTTTCTCTTCCGCACCCAGCAGTTTGGTCC respectively;

Described outer primer is GCAGCGGGTTACATCTTCTTTCATGCGCCT and AGCCACCTCCTTACTTTGCCTCCTTCTGCA respectively.

5. the outer primer of claim 4 and the combination of inner primer, the fluorescence dye that 5 ' of wherein said outer primer is held is HEX and FAM.