CN109825558A - A kind of base mutation sequence amplification detection method based on primer activation - Google Patents
A kind of base mutation sequence amplification detection method based on primer activation Download PDFInfo
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Abstract
The present invention relates to a kind of base mutation sequence amplification detection methods based on primer activation, comprising: (1) offer includes the reaction mixture of genomic DNA, first kind primer, the second class primer, third class primer, nucleotide monomer mixture, endoribonuclease and nucleic acid polymerase；(2) in the reaction mixture, when the first kind primer is mutated in conjunction with base site with genomic DNA in target alkali, endoribonuclease can shear 3 ' ends of the first kind primer, the primer being activated；(3) reaction mixture that step (2) obtains is placed in steady temperature, constant temperature strand displacement amplification reaction occurs to obtain widened genome amplification product；(4) amplified production is analyzed to identify targeting base type.Base mutation sequence amplification detection method high sensitivity of the present invention based on primer activation, high specificity, and constant temperature stopped pipe single step reaction, it is possible to prevente effectively from cross contamination.
Description
Technical field
The invention belongs to molecular biology fields, more particularly, to a kind of base mutation sequence amplification based on primer activation
Detection method.
Background technique
Base mutation is generally existing in living nature, be primarily referred to as genomic level occur base-pair replacement, increase
And missing, caused by gene structure change.It includes lacking for point mutation caused by single sequence change or multiple bases
It loses, repeat and is inserted into.Gene mutation not only may be from the heredity of family, but also may originate from the random mutation generated by changes such as environment.
Because base mutation would generally change the character of gene expression albumen where it, or cause its correlation RNA and protein expression level
Change, to generate harm to organism.
In recent years, the detection method of base mutation emerged one after another, while classical method is also modified constantly.At present
Relatively conventional method mainly includes traditional PCR- high throughput sequencing technologies and PCR- pyrosequencing techniques and emerging
ARMS-PCR technology (amplification refractory mutation system), HRM technology (high-
Resolution melting analysis), PNA- sonde method and streaming fluorescent hybridization etc..First two detection mode is all first
Target gene is first carried out to PCR amplification to be sequenced again.ARMS-PCR technology is obtained just by a pair of special primer amplification
The PCR product of Chang Xulie and mutant nucleotide sequence distinguish the mutation type of detection site further according to the difference of solubility curve.HRM is
According to the difference of the length of gene order, G/C content and base complementrity, sample is divided using high-resolution solubility curve
Analysis.PNA- sonde method and streaming fluorescent hybridization are sensitivity and the high gene tester of accuracy.However, the above method exists
Usually have that analytical cycle is long, sensitivity is low, limited specificity, at high cost or cumbersome multiple in the detection of base mutation
It is miscellaneous, high to operator technical requirements, easily cause sample pollution, the problems such as instrument platform is complicated.
Therefore, it is badly in need of a kind of one for capable of overcoming mainstream base mutation detection method, multiple or whole defects at present
Improved base mutation detection method.
Summary of the invention
The present invention provides a kind of base mutation sequence amplification detection methods based on primer activation, to solve existing method
Specificity it is not high, sensitivity is low, cross contamination, complicated for operation, inefficiency, it is at high cost the problems such as.The described method includes:
(1) reaction mixture is provided, wherein the reaction mixture includes that genomic DNA, first kind primer, the second class are drawn
Object, third class primer, nucleotide monomer mixture, endoribonuclease and nucleic acid polymerase reaction mixture, wherein institute
First kind primer, the second class primer and the third class primer stated are respectively and before and after targeting base site in the genomic DNA
One section or two sections of sequences it is consistent or complementary, condition is the 3 ' terminal sequence of first kind primer and genomic DNA in targeting base
Site is complementary, and base complementary with targeting base in the first kind primer is ribonucleotide, while described the
A kind of primer 3 ' it is terminal modified have nucleic acid polymerase extension blocking group, the second class primer and third class primer are only
It is made of DNA and 3 ' ends can be extended by nucleic acid polymerase；
(2) reaction mixture is placed in reaction temperature, the first kind primer and genomic DNA are in targeting base
Locations complementary enables endoribonuclease to hydrolyze the di-phosphate ester of ribonucleotide upstream in the first kind primer
Key removes the terminal modified nucleic acid polymerase extension blocking group of the first kind primer 3 ', and obtaining 3 ' ends can be gathered by nucleic acid
The primer product for the activation that synthase extends；
(3) primer product of activation described in step (2) can with the second class primer described in the reaction mixture and
Third class primer hybridizes before and after targeting base with the genomic DNA respectively, and further under nucleic acid polymerization enzyme effect into
Row constant temperature strand displacement nucleic acid amplification is to obtain the special sequence amplification product of targeting base；
(4) amplified production is analyzed to identify targeting base type, including real-time fluorescent analysis, real-time nephelometric analysis,
The reaction mixture is wherein provided before the step (2), the step (3) and the step (4).
In some embodiments, the first kind primer from 5 ' end to 3 ' end comprising with position in the genomic DNA with
Hold consistent sequence, the sequence complementary with targeting base, 3 ' terminal modified nucleic acid polymerase extensions in targeting base upstream 5 '
Blocking group, and in the base complementary with targeting base be the ribonucleotide that can be matched with natural acid.Some
In embodiment, in the first kind primer with targeting base before and after the base of complementary sequence at complementary with targeting base be
Ribonucleotide, condition are that ribonucleotide is located at 2-10 base of 3 ' end upstreams.In some embodiments, described 3 '
Terminal modified nucleic acid polymerase extension blocking group can be selected from: the nucleotide that not can be extended can block nucleic acid polymerase
The other groups extended.In some embodiments, the second class primer includes and the genomic DNA from 5 ' ends to 3 ' ends
The complementary sequence in the end of targeting base site upstream 5 ' and more consistent sequence far upstream, condition be the second class primer with it is described
The complementary section of genomic DNA is different from the first kind primer in the covering section of the genomic DNA, and is located at described
5 ' end upstreams of the first kind primer in the covering section of the genomic DNA.In some embodiments, the third class primer
Comprising the sequence complementary with the consistent sequence in the genomic DNA targeting base site upstream 5 ' end or the end of downstream 3 ', condition is
The sequence context that the third class primer is covered in the genome is located at the first kind primer and second class is drawn
The outside of object sequence coverage range, and coverage area is not overlapped.
In some embodiments, the endoribonuclease has thermostabilization and endonuclease activity.In some implementations
In mode, the endoribonuclease is selected from: RNase H, RNase A and any combination thereof.
In some embodiments, the nucleic acid polymerase has thermostabilization and/or strand-displacement activity.In some embodiment party
In formula, the nucleic acid polymerase is selected from: Phi29DNA polymerase, Bst archaeal dna polymerase, Pyrophage 3137, Vent polymerization
Enzyme (such as the Vent polymerase of Thermococcus litoralis, Deep Vent polymerase, Vent (- exo) polymerase,
Deep Vent (- exo) polymerase), TOPOTaq archaeal dna polymerase, 9 ° of Nm polymerases, Klenow Fragment archaeal dna polymerase
I, MMLV reverse transcriptase, AMV reverse transcriptase, HIV reverse transcriptase, the mutation of T7phase archaeal dna polymerase (lack 3 ' -5 ' excision enzymes
Activity), super fidelity dna polymerase, Taq polymerase, Bst archaeal dna polymerase (overall length), E.coli archaeal dna polymerase,
LongAmpTaq archaeal dna polymerase, OneTaq archaeal dna polymerase and any combination thereof.
In some embodiments, the reaction mixture further includes pH adjusting agent, so that the reaction mixes
The pH value of object maintains between 7.5-9.5.
In some embodiments, the reaction mixture further includes the ingredient of multiple choices the following group: including Mg2+、K+、(NH4)+、H+、Cl-、SO2-, Tris-Cl and cell surface activating agent.
In some embodiments, any temperature that the reaction temperature is 55 DEG C~95 DEG C, condition are that (1) will be described anti-
Mixture is answered to be placed in the temperature that can open the double-strand of the genomic DNA；(2) institute can be made by being placed in the reaction mixture
State first kind primer, the second class primer, temperature of the third class primer in conjunction with DNA single-stranded template；It (3) will be described anti-
Answering mixture to be placed in can make the first kind primer in conjunction with DNA single-stranded template, and in the endoribonuclease
The temperature of lower 3 ' the end blocking group of removal of effect, to generate the primer product for the activation that can be extended by nucleic acid polymerase；(4) by institute
Stating reaction mixture and being placed in can be such that the first kind primer in conjunction with DNA single-stranded template prolongs under the action of the nucleic acid polymerase
The temperature of elongation, to generate amplified production；(5) being placed in the reaction mixture can make the amplified production fall off into list
The temperature of chain.
It is described anti-when in some embodiments, using the special amplified production of targeting base described in real-time fluorescent analysis
Answering mixture further comprises the dyestuff and Metal ion indicator that can generate fluorescence signal during the reaction.In some realities
It applies in mode, the dyestuff and Metal ion indicator are selected from: PicoGreen, SYBR Green I, calcein, hydroxyl naphthalene
Phenol indigo plant and any combination thereof.
In some embodiments, the method further includes analyzing the targeting base specific amplified product to identify
Sequence signature relevant to disease or phenotype.In some embodiments, the sequence signature packet relevant to disease or phenotype
Include point mutation caused by single sequence change, the missing of multiple bases, the repetition of multiple bases, the insertion of multiple bases.
Compared with the existing technology, a kind of base mutation sequence amplification detection method based on primer activation of the present invention
It has the advantage that
(1) a kind of base mutation sequence amplification detection method based on primer activation of the present invention, in conjunction with ribose core
Identification of the sour restriction endonuclease to targeting base complementation ribonucleotide bases activates primer sequence mediated constant temperature strand displacement nucleic acid amplification
Reaction, amplification efficiency is high, can complete amplified reaction in 1 hour, high sensitivity can detecte the object of 22aM, the party
Method specificity is good, there is the single base mismatch differentiation rate close to 10000 times；
(2) it is of the present invention it is a kind of based on primer activation base mutation sequence amplification detection method, reaction constant temperature into
Row does not depend on complicated, accurate temperature control instrument, while the easy interpretation of result, can directly estimate or only need to be by simple luminoscope
Or transmissometer observed result.
(3) a kind of base mutation sequence amplification detection method based on primer activation of the present invention, one step of reaction are complete
It is reacted at and for stopped pipe, avoids cross contamination, simultaneous reactions required time is short, is not necessarily to fluorescent marker, can directly detect base
Because of a group sample, it is not necessarily to amplification procedure and other processing, experimental procedure is simplified, saves time and human cost.
Detailed description of the invention
The attached drawing for constituting a part of the invention is used to provide further understanding of the present invention, schematic reality of the invention
It applies example and its explanation is used to explain the present invention, do not constitute improper limitations of the present invention.In the accompanying drawings:
Fig. 1 is basic schematic diagram of the invention；
It is that the method for the present invention expands and detect the real-time of various concentration Kras rs121913529 mutant nucleotide sequence that A is schemed in Fig. 2
Fluorescence curve.
Scheming B is POI value described in embodiment of the present invention method 1 and Kras rs121913529 mutant nucleotide sequence log concentration
Relationship.
Fig. 3 be described in embodiment of the present invention method 1 under the rs121913529 wild type DNA background of the site Kras to prominent
The real-time fluorescence curves of modification sequence amplification and detection, mutant DNA Mutant Target is in wild type DNA from left to right
Ratio in Wild Target is followed successively by 100%, 10%, 1%, 0.1%, 0.01% and 0%, and the total concentration of DNA is 2.2pM.
Fig. 4 is the gel electrophoresis figure of Kras rs121913529 amplified production described in the embodiment of the present invention 1.Wherein M is
Molecular weight marker, 1 is blank control (be free of target Kras sequence), and 2 be positive control (2.2pM Mutant Target, but mixed
Close free nucleic acid polymerase in liquid), 3 be positive control (2.2pM Mutant Target, but without ribonucleic acid inscribe in mixed liquor
Enzyme), 4 be positive control (2.2pM Mutant Target), and 5 be negative control (2.2pM Wild Target).
Fig. 5 is HT29 cell described in the embodiment of the present invention 2 (rs121913529 mutation is negative) and SW480 cell
The amplification in the mutational site rs121913529 of (rs121913529 mutation is positive) genomic DNA and detection real-time fluorescence curves.
Wherein a, b are respectively to take 106The analysis of a cell extract of SW480 cell 1% and 0.1% is as a result, c, d are respectively to take 106It is a
The analysis result of the cell extract of HT29 cell 1% and 0.1%.
Specific embodiment
Below with reference to examples and drawings, the present invention will be described in detail.
Embodiment 1: the amplification and detection of gene Kras single base mutation site rs121913529
Using the saltant type Kras Plasmid DNA Mutant Target containing the site rs121913529 and contain
The wild type Kras Plasmid DNA Wild Target in the site rs121913529 is respectively as detection target, for verifying the present invention
Sensitivity and specificity.Kras plasmid dna sequence is as shown in table 1 (shown DNA sequence dna and its complementary strand).
1 plasmid sequence of table
Detection primer, including first kind primer DP1, the second class primer are designed according to the mutational site Kras rs121913529
P2, third class primer P3 and P4, wherein the rU in first kind primer DP1 is targeted mutagenesis in ribonucleotide, with Kras sequence
Site is complementary, 3 ' it is terminal modified have C3Spacer, nucleic acid polymerase can be blocked to the extension of the first primer DP1, accordingly
Sequence and it is end modified as shown in table 2.Primer used in the present embodiment is designed according to following principle:
1. first kind primer is from 5 ' ends to 3 ' ends comprising holding consistent sequence and target prominent with targeted mutagenesis alkali yl upstream 5 '
Become sequence complementary before and after base, 3 ' terminal modified extension blocking groups, and with targeted mutagenesis base complementrity at alkali
Base is the ribonucleotide that can be matched with natural acid, and ribonucleotide is located at 2-10 base of 3 ' end upstreams
Place.
2. the second class primer from 5 ' ends to 3 ' ends comprising the sequence complementary with the end of targeted mutagenesis alkali yl upstream 5 ' and it is farther on
Consistent sequence is swum, involved sequence of interval is different from first kind primer and is related to section overlapping, and is located at first kind primer and relates to
And 5 ' the end upstreams in section.
Hold consistent sequence and the end of downstream 3 ' complementary with targeted mutagenesis alkali yl upstream 5 ' 3. third class primer is separately included
Sequence, involved sequence of interval is located at first kind primer and the second class primer is related to the outside of sequence of interval, and sequence area
Between range be not overlapped.
The mutation of table 2 amplification and detection primer sequence table
It configures the base mutation sequence amplification activated based on primer and detects mixed liquor, wherein containing Mg2+、K+、(NH4)+、H+、
Cl-、SO2-、Tris-Cl、X-100, dNTP, RNase H2, Bst archaeal dna polymerase, SYBR Green I, SEQ ID
Primer shown in NO:1, shown in primer and SEQ ID NO:4 shown in primer, SEQ ID NO:3 shown in SEQ ID NO:2
Primer.
Reaction temperature is 63 DEG C.
In reaction process, the first primer DP1 and DNA Complementary hybridization at target single base, when targeting base is prominent in DNA
When modification Mutant Target, ribalgilase RNase H2 identify and shear in the first primer DP1 with target single base mutation
The phosphodiester bond of the upstream ribonucleotide rU of site complementation, force DP1 with blocking group C3Spacer 3 ' end from
It is fallen at RNase H2 shearing, while leaving the OH that can be extended by Bst archaeal dna polymerase at 3 ' ends, first activated is drawn
Object, and stretch nucleic acid polymerization enzyme effect is downward, this extension products can be replaced to obtain single-stranded by the extension products of third primer P4
DNA, 5 ' ends of single stranded DNA, which further fold, forms a hairpin structure；Meanwhile second primer P2 and third primer P3 can be with this
Single stranded DNA extends in nearly 3 ' end different zones in conjunction with and by polymerase, and the extension product of the second primer P2 can be by third primer
The extension product of P3 replaces and becomes single-stranded, so that obtaining both ends all has the amplification template of hairpin structure, in template, first
Primer DP1 is verified and is activated repeatedly, and with the second primer P2 collective effect so that template constantly be extended and generate new expansion
Increase template, obtains high specific relevant to targeting base and highly sensitive DNA cloning, help the accurate detection of targeting base.
Real-time fluorescent analysis
It is detected with real-time fluorescence spectrometer, corresponding dyestuff SYBR Green I selective exitation wavelength 497nm and transmitting
Wavelength 520nm reads first order fluorescence value at interval of 30s, real-time fluorescence curves figure is as a result recorded as, from sensitivity, quantitative analysis
The property for the base mutation sequence amplification detection that performance and the anisotropic three aspects verifying of saltation zone dtex are activated the present invention is based on primer
Energy.
1. sensitivity is verified
It is detected with real-time fluorescence spectrometer, corresponding dyestuff SYBR Green I selective exitation wavelength 497nm and transmitting
Wavelength 520nm reads first order fluorescence value at interval of 30s, and concrete outcome real-time fluorescence curves figure refers to attached drawing 2A.Real-time fluorescence
Curve graph is shown: for mutant DNA Mutant Target, POI (the point of of the real-time fluorescence curves measured
Inflection) value is reduced with the raising of target concentration, is shown that the method for the present invention is highly sensitive to targeted mutagenesis base and is moved
State response；Meanwhile the concentration that mutant DNA Mutant Target can be detected in the present embodiment can show down to 22aM
The high sensitivity of the method for the present invention and extremely low Monitoring lower-cut.
2. quantitative analysis performance verification
Using the POI value of real-time fluorescence curves each in Fig. 2A to pair of the concentration of mutant DNA target Mutant Target
Number mapping, obtains the paced work curve of the present embodiment, as shown in figure 2b.Paced work curve is shown: as target Mutant
When Target concentration is between 220aM to 22pM, POI value and target concentration are linearly related, show that the method for the present invention can be used for
The quantitative analysis of target.
3. specificity verification
In the case where fixed target total concentration is 2.2pM, Mutant Target is mixed with Wild Target, so that
Mutant Target occupation ratio is followed successively by 100%, 10%, 1%, 0.1%, 0.01% and 0%, and uses the method for the present invention
The amplification and real-time fluorescence detection, resulting real-time fluorescence curves for carrying out mutant sequences refer to attached drawing 3.Real-time fluorescence curves
Display: when Mutant Target target ratio is down to 0.01%, the method for the present invention still can be from ratio a height of 99.99%
0.01% Mutant Target target is distinguished in Wild Target target, resulting real-time fluorescence curves POI value is lower than
The signal (Mutant Target ratio is 0%) of 100%Wild Target target, to saltant type target and wildtype target target
Discrimination is about 10,000 times, shows the superior single base identification specificity of the method for the present invention.
Gel electrophoresis analysis
Glue and electrophoresis process carry out at room temperature, agarose and 0.5 × tbe buffer liquid of the electrophoretic analysis using 3%
(45mM Tris, 45mM Boric Acid, 10mM EDTA, pH 8.0), is dyed with 10 μ L GelRed, in well
Be added 10 μ L amplified production mixed liquor (63 DEG C of reaction solution reaction 35min after product), the electrophoresis 90min under 101V voltage,
After electrophoresis, band is observed with Tanon 4200SF gel imaging system and is taken pictures.
Electrophoretogram refers to attached drawing 4, and wherein M is molecular weight marker, and 1 is blank control (being free of target Kras sequence), and 2 are
Positive control (2.2pM Mutant Target, but free nucleic acid polymerase in mixed liquor), 3 be positive control (2.2pM Mutant
Target, but without endoribonuclease in mixed liquor), 4 be positive control (2.2pM Mutant Target), and 5 be negative right
According to (2.2pM Wild Target).Electrophoretogram is shown: amplified production is only occurred in using Mutant Target as the experiment of target
In system, show that primer distinguishes saltant type target and wildtype target target high specific.In addition, lacking nucleic acid polymerase or core
When ribosomal ribonucleic acid restriction endonuclease, even if saltant type target exists, amplified production is not also obtained, shows that the specific amplified of saltant type target is
The result that nucleic acid polymerase is cooperateed with endoribonuclease.
Embodiment 2: the amplification and detection in the mutational site rs121913529 of cell genomic dna
Respectively by 106A HT29 cell (rs121913529 mutation is negative) and SW480 cell (rs121913529 mutation sun
Property) extracted respectively with ONE-4-ALL Genomic DNA Mini-Preps Kit, then 1% or 0.1% cell is respectively taken to mention
Liquid is taken, and the method for the present invention carries out the amplification and detection in the mutational site rs121913529, augmentation detection mixed liquor used and implementation
It is consistent in example 1.Analysis result real-time fluorescence curves refer to attached drawing 5.Real-time fluorescence curves are shown: two various concentrations
Rs121913529 is mutated positive cell SW480 and gives the real-time fluorescence curves with different POI values in 40min or so, different
The rs121913529 mutation negative cells HT29 of concentration does not provide real-time fluorescence curves then in 90min, shows present invention side
High specific of the method to targeted mutagenesis base amplification.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (17)
1. a kind of base mutation sequence amplification detection method based on primer activation, which comprises
(1) provide reaction mixture, wherein the reaction mixture include genomic DNA, first kind primer, the second class primer,
Third class primer, nucleotide monomer mixture, endoribonuclease and nucleic acid polymerase reaction mixture, wherein described
First kind primer, the second class primer and third class primer respectively with one section before and after targeting base site in the genomic DNA
Or two sections of sequences are consistent or complementary, condition is the 3 ' terminal sequence of first kind primer and genomic DNA in targeting base site
Complementation, and base complementary with targeting base in the first kind primer is ribonucleotide, while the first kind
Primer 3 ' it is terminal modified have nucleic acid polymerase extension blocking group, the second class primer and third class primer are only by taking off
Oxygen ribonucleic acid forms and 3 ' ends can be extended by nucleic acid polymerase；
(2) reaction mixture is placed in reaction temperature, the first kind primer and genomic DNA are in targeting base site
In conjunction with, enable endoribonuclease to hydrolyze the phosphodiester bond of ribonucleotide upstream in the first kind primer,
The terminal modified nucleic acid polymerase extension blocking group of the first kind primer 3 ' is removed, obtaining 3 ' ends can be by nucleic acid polymerase
The primer product of the activation of extension；
(3) primer product of activation described in step (2) can be with the second class primer and third described in the reaction mixture
Class primer hybridizes before and after targeting base site with the genomic DNA respectively, and further under nucleic acid polymerization enzyme effect into
Row constant temperature strand displacement nucleic acid amplification is to obtain the special sequence amplification product of targeting base；
(4) amplified production is analyzed to identify targeting base type, including real-time fluorescent analysis, real-time nephelometric analysis,
The reaction mixture is wherein provided before the step (2), the step (3) and the step (4).
2. method according to claim 1, wherein the first kind primer includes and the gene from 5 ' ends to 3 ' ends
Consistent sequence, the sequence complementary with targeting base, 3 ' terminal modified nucleic acid polymerizations are held with targeting base upstream 5 ' in position in group DNA
Enzyme extension blocking group, and base at complementary with targeting base is the ribose core that can be matched with natural acid
Thuja acid.
3. method described in any one of -2 according to claim 1, wherein in the first kind primer mutually with targeting base front and back
Base of the sequence of benefit at complementary with targeting base is ribonucleotide, and condition is the 2- that ribonucleotide is located at 3 ' end upstreams
At 10 bases.
4. method according to any one of claim 1-3, wherein described 3 ' terminal modified nucleic acid polymerase extensions
Blocking group can be selected from: the nucleotide that not can be extended or other groups that nucleic acid polymerase can be blocked to extend.
5. method according to any of claims 1-4, wherein the second class primer from 5 ' ends to 3 ' ends comprising with
The complementary sequence in the genomic DNA targeting base site upstream 5 ' end and more consistent sequence far upstream, condition are described the
Two class primers are different from the first kind primer in the area of coverage of the genomic DNA with the complementary section of the genomic DNA
Between, and it is located at the first kind primer in 5 ' the end upstreams in the covering section of the genomic DNA.
6. method according to any one of claims 1-5, wherein the third class primer includes and the genomic DNA
Targeting base site upstream 5 ' holds the sequence of consistent sequence or the complementation of the end of downstream 3 ', and condition is the third class primer described
The sequence context covered in genome is located at the outside of the first kind primer and the second class primer sequence coverage range,
And coverage area is not overlapped.
7. method according to claim 1 to 6, wherein the endoribonuclease has thermostabilization and interior
Enzyme cutting activity.
8. method according to any one of claims 1-7, wherein the endoribonuclease is selected from: RNase H,
RNase A and any combination thereof.
9. method according to claim 1 to 8, wherein there is the nucleic acid polymerase thermostabilization and/or chain to set
Change activity.
10. method according to claim 1 to 9, wherein the nucleic acid polymerase is selected from: Phi29DNA polymerization
(such as the Vent of Thermococcus litoralis is poly- for enzyme, Bst archaeal dna polymerase, Pyrophage 3137, Vent polymerase
Synthase, Deep Vent polymerase, Vent (- exo) polymerase, Deep Vent (- exo) polymerase), TOPOTaq DNA polymerization
Enzyme, 9 ° of Nm polymerases, Klenow Fragment DNA polymerase i, MMLV reverse transcriptase, AMV reverse transcriptase, HIV reverse transcription
Enzyme, T7phase archaeal dna polymerase mutation (lacking 3 ' -5 ' 5 prime excision enzyme activities), super fidelity dna polymerase, Taq polymerase, Bst
Archaeal dna polymerase (overall length), E.coli archaeal dna polymerase, LongAmpTaq archaeal dna polymerase, OneTaq archaeal dna polymerase, and its appoint
Meaning combination.
11. method according to claim 1 to 10, wherein the reaction mixture further includes pH value tune
Agent is saved, so that the pH value of the reaction mixture maintains between 7.5-9.5.
12. method described in any one of -11 according to claim 1, wherein the reaction mixture further includes a variety of choosings
Select the ingredient of the following group: comprising Mg2+、K+、(NH4)+、H+、Cl-、SO2-, Tris-Cl and cell surface activating agent.
13. any temperature that -12 reaction temperatures are 55 DEG C~95 DEG C according to claim 1, condition is:
(1) reaction mixture is placed in the temperature that can open the double-strand of the genomic DNA；
(2) first kind primer, the second class primer, the third class primer can be made by being placed in the reaction mixture
Temperature in conjunction with DNA single-stranded template；
(3) being placed in the reaction mixture can make the first kind primer in conjunction with DNA single-stranded template, and in the core
The temperature that 3 ' end blocking groups are removed under the action of ribosomal ribonucleic acid restriction endonuclease, can drawing by the activation that nucleic acid polymerase extends with generation
Object product；
(4) being placed in the reaction mixture can make first kind primer in conjunction with DNA single-stranded template in the nucleic acid polymerization
The temperature of development length under the action of enzyme, to generate amplified production；
(5) being placed in the reaction mixture can make the amplified production fall off into single-stranded temperature.
14. method according to claim 1 to 13, when special using targeting base described in real-time fluorescent analysis
Amplified production when, the reaction mixture further comprises the dyestuff or metal that can generate fluorescence signal during the reaction
Ion indicator.
15. method described in any one of -14 according to claim 1, wherein the dyestuff and Metal ion indicator are selected from:
PicoGreen, SYBR Green I, calcein, hydroxynaphthol blue and any combination thereof.
16. method described in any one of -15 according to claim 1 further comprises analyzing the targeting base specific amplified
Product is to identify sequence signature relevant to disease or phenotype.
17. method described in any one of -16 according to claim 1, the sequence signature relevant to disease or phenotype include
Point mutation, the missing of multiple bases, the repetition of multiple bases, the insertion of multiple bases caused by single sequence change.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111321201A (en) * | 2019-12-27 | 2020-06-23 | 安诺优达基因科技(北京)有限公司 | Method for preparing nucleic acid for sequencing and application thereof |
| CN111705150A (en) * | 2020-08-19 | 2020-09-25 | 中南大学湘雅医院 | Method, primer and kit for detecting clostridium difficile ribosome 027 type virulence regulatory gene tcdC |
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| CN111321201A (en) * | 2019-12-27 | 2020-06-23 | 安诺优达基因科技(北京)有限公司 | Method for preparing nucleic acid for sequencing and application thereof |
| CN111705150A (en) * | 2020-08-19 | 2020-09-25 | 中南大学湘雅医院 | Method, primer and kit for detecting clostridium difficile ribosome 027 type virulence regulatory gene tcdC |
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