CN105506101B - AS-PCR primer design method, gene pleiomorphism detecting method and kit - Google Patents
AS-PCR primer design method, gene pleiomorphism detecting method and kit Download PDFInfo
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
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- C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
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Abstract
The present invention relates to a kind of AS-PCR primer design method, gene pleiomorphism detecting method and kit, the method and kit are for detecting the target sequence that may include allelic variation region to determine that allelic variant whether there is.The AS-PCR primer design method is the following steps are included: (i) design AS-PCR primer for the target sequence comprising allelic variation region to be measured；(ii) AS-PCR primer designed by step (i) is modified, holds 1~8 one fluorophor of base internal labeling in primer 5 ', the base in allelic variation region is marked with quenching group；(iii) end of the AS-PCR primer 3 ' designed by step (ii) additionally synthesizes 1~3 base not complementary with testing gene.The detection site ability single compared to traditional AS-PCR, gene pleiomorphism primer design method of the invention and detection method and kit can annex identification mutational site, and probe and primer can be combined into one, primer synthesis cost, design time are saved, design also facilitates understandable, is easily mastered.
Description
Technical field
The invention belongs to diagnostic nucleic acid reagent fields, and in particular to a kind of improved allele-specific polymerase chain
React (AS-PCR) primer design method.The invention further relates to a kind of improved AS-PCR gene pleiomorphism detecting method and reagents
Box, the method and kit are used to detect with true the possible target sequence of (suspecting) comprising allelic variation region
Determining allelic variant whether there is.
Background technique
Allele specific oligonucleotide analytic approach (allele-specific oligonucleotide, ASO) is base
In the mutation detection techniques of hybridization, commonly use to detect known mutations.The oligonucleotide fragment of one section of such as 15~20bp is designed,
In contain occur known mutations position, as probe, when hybridizing with the sample DNA being fixed on film, due to one
The difference of base will lead to Tm value and decline 5~7.5 DEG C, therefore by strict control hybridization conditions, can identify in sample DNA
With the presence or absence of mutation.
It is current a kind of common by the allele-specific polymerase chain reaction (AS-PCR) that ASO is combined with PCR
In analysis gene pleiomorphism, the especially method of single nucleotide polymorphism (SNP).Polymorphism (polymorphism) refers to one
In a biocenose, while and being frequently present of two or more discontinuous anomalies or genotype (genotype) or equipotential base
Because of (allele), also known as genetic polymorphism (genetic polymorphism) or gene pleiomorphism.Intrinsically, polymorphic
Property result from variation on gene level, typically occur in the region for not encoding albumen in gene order and without important adjusting function
The region of energy.Single nucleotide polymorphism (SNP), that is, the difference for the single base being dispersed in, missing including single base, insertion and
Displacement, wherein in the majority with the situation of single base replacement.SNP is a kind of polymorphism to receive much attention at present, since it often exists
Have the function of genetic marker in the research such as genetic disease, disease susceptibility, adverse drug reaction, therefore, is detected as disease
Disease diagnosis and early diagnosis, materia medica research etc. provide huge potentially possible.
In AS-PCR, one of primer of PCR or two primers are designed to anneal at the site of sequence variations, make
Its not iso-allele that can distinguish same gene, these allelic gene types include the replacement, insertion and missing of base.
The fidelity of archaeal dna polymerase is utilized in AS-PCR: for 3 ' hold the last one base correctly to match primer, 3 ' ends are last
The primer of one base mispairing substantially reduces (usually 1/100 to 1/100,000) on extending efficiency.3 ' hold the last one alkali
Thus the primer of base mispairing leads to extend difficult, PCR amplification reduction, therefore be easy to distinguish mutation by detection.
The primer 3 ' of traditional AS-PCR holds the last one base particularly critical, it is necessary to be gene polymorphism sites.With detection
For SNP, principle is as shown in Figure 1.In Figure 1A, allele-specific primers are designed for the target sequence comprising SNP site
(calling AS primer in the following text) 1-1,1-2, that is, on the basis of Standard PCR design of primers, SNP site to be measured is made to be located exactly at AS primer 1-
1, only the base of 3 ' ends is different (respectively corresponding wild type and possible saltant type) from 1-2 for the 3 ' ends of 1-2, i.e. primer 1-1,
Remaining base sequence is consistent, wherein 5 ' end X represent hydroxyl or phosphate in base, 3 ' end Y represent the hydroxyl in base.Figure 1B
In, 2-1 and 2-2 are respectively the wild type and mutant DNA double-strand where allele, and wherein 2-1b and 2-2b is respectively and AS
The template strand of primer pairing.As shown in Figure 1 C, the principle for holding the last one base strictly complementary to match according to 3 ' realizes equipotential base
The specific amplification of cause.That is, forming Fig. 1 C if the AS primer in Figure 1A is matched with the template complete complementary in Figure 1B
Duplex structure shown in middle 3-1,3-2, PCR reaction are gone on smoothly, if forming situation shown in 3-3,3-4,3 ' terminal bases in Fig. 1 C
Mispairing, then PCR reaction are not available for.By analysis PCR amplification as a result, determining SNP situation, if primer 1-2 is expanded and primer 1-1
Without obvious amplification, then show that there are the mutation.
Requirement of traditional AS-PCR to archaeal dna polymerase is relatively high.Common DNA is poly- in PCR (non-AS-PCR) reaction system
Synthase has A type and two kinds of Type B, and Aform DNA polymerase has DNA polymerase activity of the 5 ' ends to 3 ' ends, such as Taq archaeal dna polymerase
Deng；B-form DNA polymerase not only has DNA polymerase activity of the 5 ' ends to 3 ' ends, also with 3 ' ends to the DNA excision enzyme at 5 ' ends
Activity can identify immediately and cut off the base mismatch (because mispairing causes to tilt) at 3 ' ends, so that the base of remaining non-mispairing obtains
To continue to expand, such as pfu archaeal dna polymerase, KOD archaeal dna polymerase, etc..Since traditional AS-PCR is just being needed using polymorphic
Property site mismatched primers, so be only capable of in amplified reaction using Aform DNA polymerase, cannot use can cut off base mismatch
B-form DNA polymerase.
Fig. 2A and 2B schematically shows the operative condition of A type and B-form DNA polymerase in normal PCR system.Its
In, Fig. 2A is operative condition of the Aform DNA polymerase in normal PCR system, when primer (3 ' ends are hydroxyl) and template complete
Timing, in the case where Aform DNA polymerize enzyme effect, primer is able to amplification (left side), and when the end of primer 3 ' is with template mispairing, under annealing temperature
Mispairing part tilts, cannot be in conjunction with template, and Aform DNA polymerase does not work, and primer cannot expand (right side), using this characteristic,
Traditional AS-PCR is achieved the detection of polymorphic allele；Fig. 2 B is work of the B-form DNA polymerase in normal PCR system
With situation, when primer and template exact matching, in the case where B-form DNA polymerize enzyme effect, primer is able to amplification (left side), and works as primer 3 '
When end is with template mispairing, mispairing part is tilted, and B-form DNA polymerase first plays the circumscribed enzyme effect of DNA that 3 ' ends are held to 5 ', will be wrong
It is cut off with part, the primer of remaining non-mispairing is able to continue to expand (right side) under archaeal dna polymerase effect, is obviously not used in this way
Identify polymorphic allele in traditional AS-PCR.
Traditional AS-PCR technology has the disadvantage in that
The first, since AS primer will be designed for saltant type to be measured, so tradition AS-PCR technology is only capable of to known
Polymorphic site detected, unknown mutation type cannot be detected.
The second, for tradition AS-PCR technology in terms of detecting amplification, one is use electrophoresis analytical method, this mode
It is complicated for operation time-consuming, it is at high cost, and technology is more difficult universal；Another kind is using real-time fluorescence quantitative PCR, this is also required to additionally close
At the fluorescence probe for being marked with fluorophor and fluorescent quenching group.
Therefore, there are the needs of improvement tradition AS-PCR technology in industry.In the Chinese invention patent for belonging to same obligee
In ZL201410106312.0, a kind of new AS-PCR technology is proposed, wherein modifying AS-PCR primer, is made described
The 3 ' of AS-PCR primer, which hold the last one base to have, can shield the group that the AS-PCR primer directly extends, and use
The PCR reaction that B-form DNA polymerase carries out, so that the modified AS-PCR primer extends when with template strand mispairing, and with
The template strand mutual added time does not extend.
The group that primer directly extends can be shielded since the end of AS-PCR primer 3 ' of above-mentioned design has, when with template
When DNA is combined, if primer and template complete complementary, 3 ' due to primer have carried out de-hydroxylated modification, can not occur with dNTP
Polymerization reaction, therefore primer does not extend.Conversely, if primer and template mispairing, i.e., not fully complementary, then mispairing part is stuck up when annealing
It rises not in conjunction with template, 3 ' ends of B-form DNA polymerase are worked to 5 ' end 5 prime excision enzyme activities at this time, and the end of primer 3 ' is tilted
The not part excision of complementary pairing, (phosphodiester bond in DNA chain between dNTP exists for 3 ' the terminal hydroxy groups exposing of remaining primer portion
After B-form DNA polymerization enzyme effect is lauched solution, the end of primer 3 ' generates hydroxyl, and the 5 ' ends of the dNTP or DNA chain under hydrolyzing form phosphoric acid
Base), in this way, the primer after digestion can extend under archaeal dna polymerase effect.
Fig. 3 A and 3B schematically show action principle of the B-form DNA polymerase in above-mentioned AS-PCR system.In Fig. 3 A
Primer 3 ' holds the end of primer 5 ' in group Y ', Fig. 3 B for being connected with shielding primer extend to be connected with fluorophor X0, 3 ' ends are connected with
Quenching group Y0.When primer and template exactly match, since 3 ' terminal hydroxy groups are shielded, primer can not be expanded on (left side), and worked as and drawn
When object 3 ' holds base and template mispairing, mispairing part is tilted under annealing temperature, and B-form DNA polymerase first plays 3 ' ends to 5 ' ends
The circumscribed enzyme effect of DNA, mispairing part is cut off, and the primer of remaining non-mispairing is able to continue to expand under archaeal dna polymerase effect
(right side).
Therefore, and complementary with template strand with traditional AS-PCR on the contrary, above-mentioned AS-PCR primer only just extends in mispairing
Shi Ze does not extend, and can not thus distinguish mutation type and detecting polymorphic allele whether there is.Specifically, if
There are a variety of possible mutation, such as the insertion of single or multiple bases, missing or replacement for a certain allele, and people only need
When test sample is with the presence or absence of mutation type specific without determination is mutated, then AS-PCR can be designed only for wild type and drawn
Object if amplification shows that sample has mutation, otherwise does not expand and shows that sample is wild type.Compared with traditional AS-PCR primer,
Extend when complementary with template strand and mispairing occurs and does not extend then, therefore to be mutated design primer for each, this new method
Advantage is apparent.
With the progress of kilobase marker technology, at present in any position mark fluorophor of primer sequence or quenching group
It is all easy to accomplish, such as raw work biology, Thermoscientific have such service.So the present inventor is at upper one
Further research and development have been carried out on the basis of patent, have obtained a kind of new AS-PCR primer design method, and this method is directed to target sequence
Allelic variation region (including SNP and the insertion of one or more base or missing) carry out design primer, should to determine
The concrete type in allelic variation region.This method not only provides a kind of replacing different from ZL201410106312.0 technology
For scheme, and B-form DNA polymerase be 3 ' hydroxyl dependent form in the case where still can normally play a role (due to
The 3 ' of primer have carried out de-hydroxylated modification in ZL201410106312.0 technology, and 3 ' hydroxyl dependent form B-form DNA polymerases may
It does not play a role).Thus for the scope of application of archaeal dna polymerase, method ratio ZL201410106312.0 technology of the invention
More there is application advantage.
Summary of the invention
Objects and advantages of the present invention a part will be listed in the following description, another part skill common for this field
It is easy description based on following for art personnel and expects.
It is an object of the present invention to provide a kind of design methods of AS-PCR primer, the described method comprises the following steps:
(i) AS-PCR primer is designed for the target sequence comprising allelic variation region to be measured；(ii) to designed by step (i)
AS-PCR primer is modified, and 1~8 one fluorophor of base internal labeling is held in primer 5 ', to allelic variation region
Base is marked with quenching group；(iii) the additional synthesis in the end of the AS-PCR primer 3 ' designed by step (ii) 1~3 with it is to be measured
The not complementary base of gene.Using the above method of the present invention design primer can be used for determining allele to be measured whether with it is known
The different AS-PCR reaction of gene type, such as distinguishing wild type and saltant type, and with traditional AS-PCR design of primers
Difference considers that the specific mutation type of allele to be measured is no longer necessary.
Compared with the AS-PCR technology for using fluorescent marker in ZL201410106312.0 patent of invention, of the invention draws
There is the base not complementary with template in the allelic variation region rear of object, while fluorescent quenching group label is in allele
In the base of variable region rather than in terminal bases, in this way, the terminal bases of primer still possess hydroxyl group, so as to avoid
Hydroxyl dependent form B-form DNA polymerase generates the testing result of mistake.When carrying out PCR reaction using B-form DNA polymerase, due to
There is the base not complementary with template in allelic variation region rear, annealing temperature lower end tilts, and B-form DNA polymerase is first
3 ' ends are played to the circumscribed enzyme effect of DNA at 5 ' ends, mispairing part is cut off, the primer for being left non-mispairing is acted in archaeal dna polymerase
Under be able to continue to expand.Therefore, no matter the allelic variation region of primer whether with template matching, primer all by cause chain prolong
It stretches, referring to fig. 4 B, only when primer and template mispairing, the allelic variation region with fluorescent quenching group is by together
Excision, enables the fluorescent label signal of amplified production to be detected；And when primer and template matching, have fluorescent quenching
The allelic variation region of group is retained, and only the not complementary base at its rear is removed, thus the fluorescence of amplified production
Signal will not be detected.Therefore, the allelic variation region for being assured that template by detecting specific fluorescent signal, example
Such as distinguishing wild type and saltant type.
Fig. 4 A schematically shows the AS- with 3 ' hydroxyl dependent form B-form DNA polymerases in ZL201410106312.0
Action principle in PCR system.At this time since primer 3 ' is held without hydroxyl, 3 ' hydroxyl dependent form B-form DNA polymerases do not play work
With no matter whether primer matches with template, cannot all cause chain extension, so that wild type and saltant type cannot be distinguished.
Another object of the present invention is to provide a kind of AS-PCR gene pleiomorphism detecting method, and the method is used for possible
Target sequence comprising allelic variation region is detected to determine that allelic variant whether there is, it is characterised in that described
Method is the following steps are included: (i) design AS-PCR primer for the target sequence comprising allelic variation region to be measured；(ii) right
AS-PCR primer designed by step (i) is modified, and 1~8 one fluorophor of base internal labeling is held in primer 5 ', to equipotential
The base in genetic mutation region is marked with quenching group；(iii) end of the AS-PCR primer 3 ' designed by step (ii) is additional closes
At 1~3 base not complementary with testing gene；(iv) target sequence described in the AS-PCR primer pair for using step (iii) to be modified
Column carry out PCR amplification, and the PCR amplification uses B-form DNA polymerase；(v) amplification of analytical procedure (iv) determines described etc.
Position genetic mutation whether there is.
In AS-PCR primer design method and/or AS-PCR gene pleiomorphism detecting method of the invention, it is preferable that step
Suddenly the length of primer described in (i) is 18bp~30bp.Make in this way B-form DNA polymerase digestion identification region as close to
3 ' end of primer, and still there is enough chain lengths after being digested, it is taken off from template strand in order to avoid the primer after digestion is too short and easy
It falls, reduces amplification efficiency.
Preferably, the 5 ' of the AS-PCR primer in the amplification reaction is made to hold the Tm value for arriving the allelic variation region
At 48 DEG C~52 DEG C.As Tm increases, primer and template mispairing probability increase, and as Tm is reduced, primer falls off several from template
Rate increases.
Preferably, the fluorophor includes but is not limited to: FAM, VIC, HEX, ROX, Taxas Red or CY5, etc.,
The fluorescent quenching group includes but is not limited to: BHQ1, BHQ2, BHQ3, Dabcyle, Temra, Eclipse, etc..It is above-mentioned glimmering
Light group and fluorescent quenching group and its labeling method are known in the art.
In AS-PCR gene pleiomorphism detecting method of the invention, the B-form DNA polymerase of step (iv) include but
It is not limited to pfu archaeal dna polymerase, KOD archaeal dna polymerase, etc..The B-form DNA polymerase is known in the art, and can
To be obtained from open channel, for example, pfu archaeal dna polymerase can be purchased from TAKARA；KOD archaeal dna polymerase can be adopted from TOYOBO
Purchase.
Another object of the present invention is to provide a kind of AS-PCR genetic polymorphism detection kit, wherein at least comprising following
Ingredient: AS-PCR primer designed by (i)-(iii) the step of a. above-mentioned AS-PCR gene pleiomorphism detecting method；B. above-mentioned
B-form DNA polymerase used in the step of AS-PCR gene pleiomorphism detecting method (iv).
The detection site ability single compared to traditional AS-PCR, mutation identification primer design method of the invention and detection
Method and kit remain all advantages of ZL201410106312.0 patent of invention technology, and most notable one is, can
To annex identification mutational site in Candidate Mutant Type Range, such as use wild type as abrupt climatic change sequence, without examining
Consider specific mutation type, this is in complicated human tumour genetic mutation detection, such as Human epidermal growth factor receptor (EGF-R ELISA) gene
40 kinds or more of the mutation or people C-KIT (one of subclass of tyrosine kinase) gene the 11st that 19th exon occurs are aobvious outside
The mutation of 25 kinds of son or more, it is only necessary to design one or several wild type marker primer corresponding with mutantional hotspot regional sequence i.e.
It can.Moreover, because the end of primer is not carried out de-hydroxylated modification by method of the invention, so as to avoid hydroxyl dependent form
B-form DNA polymerase generates the testing result of mistake.
Detailed description of the invention
For above and other purpose, feature, advantage and embodiment of the invention can be clearer and more comprehensible, to saying for institute's attached drawing
It is bright as follows:
Fig. 1 schematically shows the principle of traditional AS-PCR detection SNP；
Fig. 2A -2B schematically shows the operative condition of A type and B-form DNA polymerase in PCR system, wherein Fig. 2A
It is operative condition of the Aform DNA polymerase in PCR system, Fig. 2 B is operative condition of the B-form DNA polymerase in PCR system；
Fig. 3 A and 3B schematically show action principle of the B-form DNA polymerase in AS-PCR system of the invention；
Fig. 4 A schematically shows the AS- with 3 ' hydroxyl dependent form B-form DNA polymerases in ZL201410106312.0
Action principle in PCR system；
Fig. 4 B is schematically shown with 3 ' hydroxyl dependent form B-form DNA polymerases in AS-PCR system of the invention
Action principle；
Fig. 5 A and 5B are the typical amplification figure of the heterozygous and homozygous sample that are detected according to the method for the present invention.
Specific embodiment
In order to keep the objectives, technical solutions, and advantages of the present invention clearer, below in conjunction with the drawings and the specific embodiments,
The present invention will be described in further detail.It should be appreciated that the specific embodiments described herein are only used to explain this hair
It is bright, it is not intended to limit the present invention.
1. people's methylenetetrahydrofolate reductase (MTHFR) gene pleiomorphism AS-PCR of embodiment detection:
The specific steps condition of PCR can refer to " Molecular Cloning:A Laboratory guide (third edition) " (the 8th chapter " polymerase chain reaction
Answer amplification in vitro DNA ", 597-701 pages, Science Press, in August, 2002) record.
1.1. people's mthfr gene polymorphism AS-PCR design of primers:
People MTHFR-01 is for detecting SNP (GTCTGCGGGAGCGATTTCAT) site T allele, people
MTHFR-02 is for detecting SNP (GTCTGCGGGAGCGATTTCAT) site C allele, people MTHFR-03 are downstream
Primer.
People MTHFR-01:5'-AAGGAGAAGGTGTCTGCGGGAGC a c -3'
People MTHFR-02:5'-AAGGAGAAGGTGTCTGCGGGAGT a c -3'
People MTHFR-03:5'-gaaagatcccggggacgatg-3'
1.2. Modify to primer:
People MTHFR-01:5'(FAM)-AAGGAGAAGGTGTCTGCGGGAGC (BHQ1) a c -3'
People MTHFR-02:5'(VIC)-AAGGAGAAGGTGTCTGCGGGAGT (BHQ1) ac -3'
1.3. templated synthesis:
People MTHFR sequence 1 is synthesized as template DNA (italic thickened portion is primer binding sequence):
CTTCATCCCTCGCCTTGAACAGGTGGAGGCCAGCCTCTCCTGACTGTCATCCCTATTGGCAGGTTACC
CCAAAGGCCACCCCGAAGCAGGGAGCTTTGAGGCTGACCTGAAGCACTTGCGATTTCATCATCACGCAGCTTTTCTTTGAGGCTGACAC
ATTCTTCCGCTTTGTGAAGGCATGCACCGACATGGGCATCACTTGCCC CCCATCCAGGTGAGGGGCCCAGGAGAGCCCATAAGCTCCCTCCACCCCACTCTCACCGCACCGTCCTCGCACAG
The sequence 2 for synthesizing people MTHFR is used as template DNA:
CTTCATCCCTCGCCTTGAACAGGTGGAGGCCAGCCTCTCCTGACTGTCATCCCTATTGGCAGGTTACC
CCAAAGGCCACCCCGAAGCAGGGAGCTTTGAGGCTGACCTGAAGCACTTGCGATTTCATCATCACGCAGCTTTTCTTTGAGGCTGACAC
ATTCTTCCGCTTTGTGAAGGCATGCACCGACATGGGCATCACTTGCCC CCATCCAGGTGAGGGGCCCAGGAGAGCCCATAAGCTCCCTCCACCCCACTCTCACCGCACCGTCCTCGCACAG
1.4. match liquid: preparing reaction solution: 2mM dNTP, 2.5mM Mg according to following concentration2+, 1U/ reaction pfu DNA polymerization
Enzyme, 10mM pH8.3Tris, 50mM KCl, 0.1pmol/ul primer people MTHFR-01,0.1pmol/ul primer people MTHFR-02,
0.1pmol/ul primer people MTHFR-03, template DNA, adding water to volume is 20ul.
The template DNA of sequence 1 and sequence 2 carried out gradient dilution respectively after (1pg/ul, 10pg/ul, 100pg/ul,
1000pg/ul), it is expanded for fluorescent PCR.
Template consumption: ddH2O (i.e. 0), 2pg/ reaction, 20pg/ reaction, 200pg/ reaction, 2000pg/ reaction.
1.5. the loop parameter used: 95 degrees Celsius 5 minutes
(95 degrees Celsius 10 seconds 58 degrees Celsius 45 seconds) 10 circulations
(95 degrees Celsius 10 seconds 60 degrees Celsius 45 seconds) 35 circulations
1.6. fluorescence quantitative PCR detection: Bio-rad CFX96 fluorescent PCR instrument is used.
1.7. result: concentration is respectively the Ct of the sample of 0pg/ul, 1pg/ul, 10pg/ul, 100pg/ul, 1000pg/ul
Value see the table below 1.
Table 1
"/" indicates negative sample, without this SNP site；" N " indicates that testing result is feminine gender；Template additional amount (sequence 1+ sequence
Column 2) refer to the amount that MTHFR sequence 1 and MTHFR sequence 2 are put into PCR reaction.
1.8. result judgement method: if Ct (VIC, C) and Ct (FAM, T) are both greater than 35, then illustrate that sample concentration is low or has
Inhibit, need to reform.If having a Ct value less than 32 in Ct (VIC, C) or Ct (FAM, T), then following result judgement is pressed:
I), Ct (VIC, C)-Ct (FAM, T) < -1 then judges that SNP site is homozygous for CC；
Ii), Ct (VIC, C)-Ct (FAM, T) > 1 then judges that SNP site is homozygous for TT；
Iii), -1 < Ct (VIC, C)-Ct (FAM, T) < 1 then judges SNP site for CT heterozygous.
Test confirms that each sample testing result is consistent with sequencing background；Wherein 1 result of sample is feminine gender, and sample 2 is
SNP site is the homozygous sample of C, and sample 3 is the homozygous sample that SNP site is T, and sample 4 and 5 is heterozygous sample.Ginseng
See that Fig. 5, Fig. 5 A are the typical amplification curve of homozygous sample, one of two primers show amplification curve, but have amplification to produce
Object；Fig. 5 B is the typical amplification curve of heterozygous sample, and two primers have amplification.
2. Human epidermal growth factor receptor genetic mutation design of primers of embodiment and its application:
The various mutations that the present embodiment may occur for the mutantional hotspot area of the 19th exon of Human epidermal growth factor receptor gene carry out not
The abrupt climatic change of mutation type is distinguished, wherein having chosen 64 kinds of mutation types, synthesis template sequence is verified.
2.1. Human epidermal growth factor receptor design of primers:
Human epidermal growth factor receptor -01 is for detecting SNP site mutated gene, Human epidermal growth factor receptor -02 for detecting total EGFR gene quantity, people
EGFR-03 is downstream primer.(it is EGFR wild-type DNA-sequence that SNP type, which is shown in Table 4, E-1, and E-2~E-65 is to mutate
Gene order, wherein leukorrhagia is marked as the region of gene delection, italic be after gene mutation with the distinguishing sequence area of wild type
Domain)
The mutation type of table 2:EGFR
Human epidermal growth factor receptor -01:5'-AAATTCCCGTCGCTATCAAGGAATTAca -3'
Human epidermal growth factor receptor -02:5'-GTGAGAAAGTTAAAATTCCCGTAca -3'
Human epidermal growth factor receptor -03:5'-ATCGAGGATTTCCTTGTTG-3'
2.2. Modify to primer:
Human epidermal growth factor receptor -01:5'(FAM)-AAATTCCCGTCGCTATCAAGGAATTA (BHQ1) ca -3'
Human epidermal growth factor receptor -02:5'(VIC)-GTGAGAAAGTTAAAATTCCCGTA (BHQ1) ca -3'
2.3. templated synthesis:
Human epidermal growth factor receptor sequence E-1~E-65 is synthesized as template DNA:
2.4. match liquid: preparing reaction solution: 2mM dNTP, 2.5mM Mg2+, 1U/ reaction B-form DNA polymerization according to following concentration
Enzyme (pfu archaeal dna polymerase), 10mM pH8.3Tris, 50mM KCl, 0.1pmol/ul primer Human epidermal growth factor receptor -01,0.1pmol/ul
Primer Human epidermal growth factor receptor -02,0.1pmol/ul primer Human epidermal growth factor receptor -03, template DNA, adding water to volume is 20ul.
Wherein template DNA has been carried out respectively after gradient dilution (1pg/ul, 10pg/ul, 100pg/ul, 1000pg/ul), is used
It is expanded in fluorescent PCR.
Template consumption: ddH2O, 2pg/ reaction, 20pg/ reaction, 200pg/ reaction, 2000pg/ reaction.
2.5. the loop parameter used: 95 degrees Celsius 5 minutes
(95 degrees Celsius 10 seconds 58 degrees Celsius 45 seconds) 10 circulations
(95 degrees Celsius 10 seconds 60 degrees Celsius 45 seconds) 35 circulations
2.6. fluorescence quantitative PCR instrument instrument: Bio-rad CFX96 fluorescent PCR instrument.
2.7. result: the synthetic DNA of 50pg/ul is taken to do template, the Ct value of detection see the table below 4.
2.8. result judgement method: if Ct (VIC, W) and Ct (FAM, M) are both greater than 35, then illustrate that sample concentration is low or has
Inhibit, need to reform.If having a Ct value less than 32 in Ct (VIC, W) or Ct (FAM, M), then following result judgement is pressed.
I), Ct (FAM, M)-Ct (VIC, W) < 6 then judges that EGFR mutates；
Ii), Ct (FAM, M)-Ct (VIC, W) > 6 then judges EGFR to be wild.
Test confirms that each sample testing result is consistent with actual conditions.The method according to the invention is demonstrated, for people
The mutantional hotspot region of EGFR gene can be detected out the gene only with the AS-PCR primer for wild type site design
Mutantional hotspot region various mutations type.
Therefore, the foregoing is merely the preferred embodiment of the present invention, are not intended to limit the invention, and the present invention claims guarantors
The range of shield is limited by claims and its equivalent replacement mode, done within the spirit and principles of the present invention
Any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention.
Claims (7)
1. a kind of design method of AS-PCR primer, the described method comprises the following steps:
(i) AS-PCR primer is designed for the target sequence comprising allelic variation region to be measured；
(ii) AS-PCR primer designed by step (i) is modified, holds 1~8 one fluorescence of base internal labeling in primer 5 '
Group marks the base in allelic variation region with quenching group；
(iii) end of the AS-PCR primer 3 ' designed by step (ii) additionally synthesizes 1~3 alkali not complementary with testing gene
Base.
2. a kind of AS-PCR gene pleiomorphism detecting method, the method is used for the target that may include allelic variation region
Sequence is detected to determine that allelic variant whether there is, it is characterised in that the described method comprises the following steps:
(i) AS-PCR primer is designed for the target sequence comprising allelic variation region to be measured；
(ii) AS-PCR primer designed by step (i) is modified, holds 1~8 one fluorescence of base internal labeling in primer 5 '
Group marks the base in allelic variation region with quenching group；
(iii) end of the AS-PCR primer 3 ' designed by step (ii) additionally synthesizes 1~3 alkali not complementary with testing gene
Base；
(iv) target sequence described in the AS-PCR primer pair modified using step (iii) carries out PCR amplification, and the PCR amplification makes
With B-form DNA polymerase；
(v) amplification of analytical procedure (iv) determines that the allelic variant whether there is,
Wherein, the B-form DNA polymerase refers to the not only DNA polymerase activity with 5 ' ends to 3 ' ends, also has 3 ' ends to 5 '
The archaeal dna polymerase of the DNA 5 prime excision enzyme activity at end.
3. method according to claim 1 or 2, wherein allelic variation region described in step (i) is located at the AS-
In 1st~6 base at 3 ' ends of PCR primer, the length of the primer is 18bp~30bp.
4. method according to claim 1 or 2, wherein making the 5 ' of the AS-PCR primer in the amplification reaction to hold to described
The Tm value in allelic variation region is 48 DEG C~52 DEG C.
5. method according to claim 1 or 2, wherein the group that the fluorophor is constituted selected from following substance: FAM,
VIC, HEX, ROX, Taxas Red or CY5, the quenching group are selected from the group that is constituted of following substance: BHQ1, BHQ2, BHQ3,
Dabcyle, Temra or Eclipse.
6. method according to claim 1 or 2, wherein the B-form DNA polymerase is pfu archaeal dna polymerase or KOD DNA
Polymerase.
7. a kind of AS-PCR genetic polymorphism detection kit wherein at least includes following component:
A. AS-PCR primer designed by the method and step (i)-according to any one of claim 2 to 6 (iii)；
B. B-form DNA polymerase used in the method and step according to any one of claim 2 to 6 (iv),
Wherein, the B-form DNA polymerase refers to the not only DNA polymerase activity with 5 ' ends to 3 ' ends, also has 3 ' ends to 5 '
The archaeal dna polymerase of the DNA 5 prime excision enzyme activity at end.
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| CN103911445A (en) * | 2014-03-20 | 2014-07-09 | 刘小芳 | AS-PCR primer design method, gene polymorphism detection method and kit |
| CN104611427A (en) * | 2015-01-16 | 2015-05-13 | 江苏宏泰格尔生物医学工程有限公司 | AS-PCR (allele-specific polymerase chain reaction) primer design method, gene mutation detection method and kit |
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| CN103911445A (en) * | 2014-03-20 | 2014-07-09 | 刘小芳 | AS-PCR primer design method, gene polymorphism detection method and kit |
| CN104611427A (en) * | 2015-01-16 | 2015-05-13 | 江苏宏泰格尔生物医学工程有限公司 | AS-PCR (allele-specific polymerase chain reaction) primer design method, gene mutation detection method and kit |
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