CN105755125B - The high-efficiency fluorescence allele-specific polymerase chain reaction method and detection methods of genotyping of primer based on Automated Design - Google Patents
The high-efficiency fluorescence allele-specific polymerase chain reaction method and detection methods of genotyping of primer based on Automated Design Download PDFInfo
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Abstract
The present invention relates to a kind of automatic primer design method of allele-specific polymerase chain reaction, the method for carrying out the method for high-efficiency fluorescence allele-specific polymerase chain extension reaction using the primer of above-mentioned design and detecting Genotyping is additionally provided；For any SNP site, obtain four inner primers of sequence design of 40~60bp of its upstream and downstream, the design of any one inner primer is since 3 ends ', all there are a specific matching in 3 ends ' of any one inner primer on mononucleotide diversity point, -1~-3 in 3 end ' of primer of the matching site of the mononucleotide diversity point；After obtaining inner primer, each inner primer 5 ' end plus one not with the matched short-movie section of template specificity, inner primer and short-movie section collectively constitute outer primer.It can realize the detection specificity of high Genotyping analysis under identical reaction conditions through the invention.
Description
Technical field
The present invention relates to field of biological genes more particularly to a kind of allele-specific polymerase chain reaction (AS-
PCR it) reacts, specifically the high-efficiency fluorescence allele-specific polymerase chain reaction side of the primer based on Automated Design
Method and detection methods of genotyping.
Background technique
With the further investigation to human genome, genetic test increasingly shows important role.It is big absolutely in recent years
Partial detection is both for mononucleotide diversity (SNP).Because it be (accounting for 95% or more) most generally existing in genome,
Form most simple (base changes to another base), research are most clear, and (positions of millions of a SNP sites, frequency are all
Very clear, they have a large amount of research with the relationship etc. of disease).Not only have in scientific research widely to the phenotypic analysis of SNP
Demand, and with a large amount of developments of the association study based on full-length genome SNP, a SNP sites up to ten thousand are shown and various diseases
The definite relation of disease, health, personal characteristics.Precisely medical treatment and accurate health control are carried out with genetic test has become one
Specific novel Feeder Road.
Therefore, the technology detected to SNP is very crucial.The wherein high-throughput Genotyping analysis based on solid-state chip
Technology is quite mature, however its monolithic basic cost of solid-state chip is high, and site sets fixation and cannot be adjusted, and the development time
It is long, it is not suitable for middle primary flux (flux refers to the number of sites that the once can detecte) detection of the fast and flexible of present market demands
System.Existing middle small throughput technology is using Tagman, SNaPshot and MassARRAY as representative, but these technology heavy dependences
Large-scale precision instrument and import reagent, and experimental procedure is complicated, heavy workload, the period is long.Especially primer this.It is most of
The prior art needs the primer marked by specially treated or fluorophor.Since the primer in each site is exclusive not generally applicable
, these modification steps cause testing cost and development cost high.
Allele-specific polymerase chain reaction (AS-PCR) technology occurs as soon as very early, has flexibility big, step
Simply, instrument dependence is low, is suitable for the important advantages such as modularized production.However existing AS-PCR technology has that sensitivity is low, position
The disadvantages of point design failure rate is high.Adaptation primer its optimum reaction condition for being especially different that site designs often difference
It is very big, it is difficult to be detected under unified instrument and reaction condition, so the genetic test based on AS-PCR technology does not have always
There are successfully standardization and industrialization.
The basic principle of ApoE gene is to design special primer according to SNP site.Conventional method is wherein
3 ' ends of one chain (special chain) are matched with the base specific of SNP site, another chain (common chain) according to a conventional method into
Row design.For general SNP there are two different allele, such as A/G, then the first set primer designed is can be special with A type
Property it is matched, second set with G specificity match.Under ideal PCR reaction condition, if the site in sample DNA template
Genotype is AA, then the PCR reaction of the specific primer building of A can generate chain amplification, produces a large amount of DNA double chain and produces
Object；And to the primer of G specificity since there are sequence mismatch between DNA profiling, then normal PCR reaction cannot occur, no
A large amount of amplified production can be generated.It is generally checked whether there is by running gel electrophoresis after the completion of traditional AS-PCR expected
DNA product, if having found DNA band on set molecular weight of product, the allele of specific primer label is
Otherwise the positive is feminine gender.But there are following some disadvantages by tradition AS-PCR:
1) primer specificity is not high.It is also likely to the primer that traditional scheme designs even if having mismatch with template DNA
Non-specific amplification reaction occurs, to generate false positive.
2) reaction system is sensitive, and stability is not high.The positive detection judgement of AS-PCR is based on can be in primer and template
PCR amplification is successfully carried out when matching.However traditional PCR reaction is influenced by many factors, including sample quality and reagent it is old
Change degree etc..Since common PCR primers and reaction condition are affected by these factors, it be easy to cause the knot of false negative
Fruit.
3) the condition difference of the AS-PCR of difference SNP is big.The greatest problem of traditional AS-PCR is that different loci designs
Detection reaction, optimum reaction condition is often widely different.This causes experiment to carry out special reality for each SNP site
Condition optimizing is tested, is unfavorable for quantifying detection on a large scale.
4) detection is time-consuming and laborious, and detection flux is low.Since traditional AS-PCR needs to be judged with gel electrophoresis that amplification is anti-
It is answering as a result, and gel electrophoresis production is complicated, the site detected every time is limited, and detection must carry out electrophoresis process extremely every time
It is the process of industrialization and volume production difficult to realize less more than half an hour.
Therefore, critical issue is that have a set of automatic design of primers system, design primer be based on AS-PCR principle,
Detection can be completed under unified experiment condition based on conventional primer synthesis, and for different SNP reaction.Most
Afterwards, testing process needs are simple and quick, stability is high, can realize on routine experiment instrument, and can neatly change
The site of detection and flux.
Summary of the invention
The present invention is to overcome above-mentioned shortcoming, and it is an object of the present invention to provide a kind of allele-specific polymerase chain reaction
The automatic primer design method answered, the automatic primer design method of this allele-specific polymerase chain reaction
The technology that the quick property of allele-specific polymerase chain reaction in the prior art is low, site design failure rate is high is solved to ask
Topic.
The present invention second is designed to provide a kind of high-efficiency fluorescence allele-specific of primer using Automated Design
Polymerase chain extension reaction carries out two step method amplification using primer, and stable reaction, detection is cheap, specificity is high.
Third of the present invention is designed to provide a kind of detecting using real-time fluorescence based on chain type as described above extension reaction
Water is substituted for the fluorescent dye for being used for real-time fluorescence PCR by the method for SNP site Genotyping.
The present invention the 4th is designed to provide a kind of examining using end-point method fluorescence based on chain type as described above extension reaction
The method for surveying SNP site Genotyping extends after the reaction was completed in chain type, fluorescent reagent is added to carry out terminal readings method, detects glimmering
Luminous intensity judges the genotype of SNP.Detection can be realized under the conditions of same PCR.
The present invention is to reach above-mentioned purpose by the following technical programs: a kind of allele-specific polymerase chain reaction
Automatic primer design method, comprise the following steps that
(1) for any SNP site, four inner primers of sequence design of 40~60bp of its upstream and downstream are obtained, any one
Since 3 ends ', 3 ends ' of any one inner primer all have on mononucleotide diversity point specifically for the design of inner primer
Property matching, the matching site meaning -1~-3 of one 3 end ' of inner primer in office of the mononucleotide diversity point, four
The annealing temperature of a inner primer is between 44-60 DEG C；
(2) described each inner primer 5 ' end plus one not with the matched short-movie section of template specificity after obtaining inner primer
The length of short-movie section collectively constitutes outer primer, the annealing temperature of four outer primers in 2~10bp, inner primer and short-movie section
Between 55~75 DEG C.
Preferably, the annealing temperature of four inner primers is preferably 51 DEG C.
Preferably, the length and sequence of short-movie section added by each inner primer can be different.
Preferably, the annealing temperature of four outer primers is preferably 59 DEG C.
Preferably, with 3 ' ends of any one inner primer any company does not occur for 5 ' ends of four outer primers
The matching of continuous 3 bp or more.
A kind of high-efficiency fluorescence allele-specific polymerase chain extension reaction using design primer as described above, is adopted
Two step method amplification, reaction condition are carried out with primer are as follows: 85~98 DEG C, 3min；85~98 DEG C, 10~40s；55~75 DEG C, 2~
40s；4 DEG C of preservations after 30~60 circulations of execution.
A method of SNP site Genotyping is detected using real-time fluorescence based on chain type as described above extension reaction,
In chain type extension reaction system, water is substituted for the fluorescent dye for being used for real-time fluorescence PCR, two allele are done respectively
One real-time PCR reactions carries out 30 or more PCR cycles, the fluorescence in the low temperature period detection architecture of each PCR cycle
Value, for the power of fluorescence signal with the double-stranded DNA total amount in linearly or non-linearly reflection system, reading from real-time curve should
The genotype of SNP.
A kind of side using end-point method fluorescence detection SNP site Genotyping based on chain type as described above extension reaction
Method, chain type extension after the reaction was completed, add the fluorescent reagent to carry out terminal readings method, fluorescence intensity judges the gene of SNP
Type.
The beneficial effects of the present invention are: (1) of the invention primer, which be not required to modification, can be carried out PCR, and react steady
Determine, detection is cheap, specificity is high；(2) present invention can realize detection for arbitrary SNP under the conditions of same PCR；(3) originally
The PCT reaction system of invention only needs 3ul, micro to survey；Meanwhile the present invention does not need not depending on large-scale instrument, only
PCR instrument is needed, fluorescence detection equipment can be carried out detecting, and at low cost, effect is good；(4) it designs by means of the present invention
Primer can realize under identical reaction conditions high Genotyping analysis detection specificity.
Detailed description of the invention
Fig. 1 is the schematic diagram for the Automated Design primer that the embodiment of the present invention 1 describes；
Fig. 2 is the 384 orifice plates detection sample-adding table schematic diagram in the embodiment of the present invention 2；
Fig. 3 is that the same SNP site in the embodiment of the present invention 3 uses real-time fluorescence quantitative PCR instrument in three different samples
The fluorescence curve figure of detection；
Fig. 4 is to show same SNP site high-volume pattern detection result scatter plot in the embodiment of the present invention 4.
Specific embodiment
The present invention is described further combined with specific embodiments below, but protection scope of the present invention is not limited in
This:
Embodiment 1: it as shown in Figure 1, carrying out Automated Design for a SNP site, first has to take the site upstream and downstream
The sequence of 50bp or so, the design of any one inner primer is since 3 ends '.The present invention is different from tradition AS-PCR, is design
All there are specificity matching in 3 ends ' of two primers of upstream and downstream in SNP site, and (the matching site of SNP is -the 1 of 3 ends '
~-3).Therefore also at 3 ends ' overlapping (1-5bp's is overlapping) occurs for positive negative strand primer.Then from 3 ends ' to 5 ends ' into
Row extends, and determines a sequence with template matching (reversion is complementary) respectively in positive minus strand.The annealing temperature of primer is (fixed herein
Justice is inner primer temperature) it needs within the specified range (44-60 degree, 51 degree of ideal temperature).With the molecule thermokinetics mould of primer
Type calculates.Its formula is as follows:
Annealing temperature=dH-273.15+16.6 (logA) dS+dS0+R(ln(C/4))；
dS0=-10.8；
R=1.987；
A=50；
C=250；
DH and dS refers respectively to the enthalpy and entropy generated in two continuous base-pair match reactions, design parameter such as following table
Shown in 1:
| Interaction | dH°kcal/mol | dS°cal/mol per°K |
| AA/TT | -9.1 | -24.0 |
| AT/TA | -8.6 | -23.9 |
| TA/AT | -6.0 | -16.9 |
| CA/GT | -5.8 | -12.9 |
| GT/CA | -6.5 | -17.3 |
| CT/GA | -7.8 | -20.8 |
| GA/CT | -5.6 | -13.5 |
| CG/GC | -11.9 | -27.8 |
| GC/CG | -11.1 | -26.7 |
| GG/CC | -11.0 | -26.6 |
| XX/XX | -6.0 | -16.9 |
Table 1
We are inner primer the matched primer of this section and DNA template sequence specificity designed.Four inner primers point
It Yong not Pinf1 (No.1 inner primer normal chain), Pinr1 (No.1 inner primer minus strand), Pinf2 (No. two inner primer normal chain), Pinr2
(No. two inner primer minus strands).Pinf1 and Pinr1 is directed to first allele, and Pinf2 and Pinr2 are directed to second equipotential base
Cause.By traversing all possibilities, make the annealing temperature of this four inner primers closest to above-mentioned ideal inner primer annealing temperature
Degree, and the mutual temperature difference is minimum.
After obtaining inner primer, the present invention design each inner primer 5 ' end plus one not with the matched short-movie of template specificity
Section, and length is in 2~10bp.This short-movie section be in order to after inner primer starts the specific amplification with template, when
Increase the annealing temperature of reaction when doing template with amplified production to increase stability, avoids reaction to DNA sample quality and its
The sensibility of his factor and there is the result of false positive.The length and sequence of short-movie section added by each inner primer can be different,
Inner primer and short-movie section collectively constitute outer primer, corresponding to name following Pouf1, Pour1, Pouf2, Pour2.Same traversal calculates
Make the annealing temperature of this four outer primers closest to ideal outer primer annealing region (55~75 degree, best 59
Degree), and the mutual temperature difference is minimum, and the 5 ' ends of four outer primers is required arbitrary continuation not to occur with any 3 ' end
The matching of 3 bp or more.
Embodiment 2: the extension of high-efficiency fluorescence allele-specific polymerase chain is carried out using the primer that embodiment 1 designs
(UFAS-PCR) it reacts:
(1) reaction system is in ratio shown in following table 2, by taking the 3ul system for adapting to 384 orifice plates as an example:
| Distilled water | 0.568μl |
| 10x buffer | 0.3μl |
| dNTP | 0.06μl |
| Mg2+ | 0.06μl |
| 0.6 μm of primer | 1μl |
| Taq archaeal dna polymerase | 0.012μl |
| Template DNA (1.5ng/ μ l) | 1μl |
| Total system | 3μl |
Table 2
(2) 384 orifice plates detection sample-adding format is as shown in Fig. 2, odd-numbered line (A, C, E, G, I, K, M) plus the first allele
Primer and reaction system (being identified with a1)；The primer and reactant of even number line (B, D, F, H, J, L, N) plus the second allele
It is (being identified with a2).The same sample is added in every device to hole.
(3) PCR reaction condition is as follows:
Using two step method PCR, only two temperature conditions of height, low temperature is for annealing and extending, and high temperature is for being denaturalized.
Embodiment 3: real-time fluorescence detection is carried out, 0.15ul water is substituted for evagreen or class in the system of embodiment 2
As be used for the fluorescent dye of real-time fluorescence PCR, and the fluorescent value in the low temperature period detection architecture of each PCR cycle.
Evagreen class fluorescence refers to not severe jamming PCR reaction, can be independent of fluorescence of the sequence-specific in conjunction with DNA double chain
Dyestuff, the strong and weak double-stranded DNA total amount that can linearly or non-linearly in reflection system of fluorescence signal.
The method of real-time fluorescence detection can carry out 30 or more (optimal conditions 60) by the reaction condition of embodiment 2
PCR cycle does a real-time PCR reactions to two allele, to read the gene of the SNP from real-time curve respectively
Type.The time point that the curve occurrence index of positive reaction increases is much earlier than the curve of negative reaction.For each SNP,
There is a wide in range region that can distinguish negative and positive.Many algorithms can be set to determine that curve belongs to positive or yin
Property.Such as simplest threshold method, in a threshold cycle number NtestOn, if current curves fluorescent value is more than Stest, then it is judged to
Otherwise the positive is feminine gender.
As shown in figure 3, carrying out fluorescence detection to SNP site shown in embodiment 1 in three different DNA samples.The SNP
Genotype in these three individuals is respectively AA, GG and AG.Pitch black curve shows that the signal of A, light gray curve show the letter of G
Number.The N of vertical line instruction setting in figuretestValue is 43, and horizontal dotted line shows signal threshold value StestIt is 0.8.It is deep in most left figure
Black curve is in NtestBefore be obviously improved show A for the positive, grey curve do not increase indicate G for feminine gender, synthesis result read gene
Type is " AA ", and is actually consistent.In the figure in the middle, grey curve is in NtestBefore be obviously improved and show G as the positive, pitch black curve do not have
There is raising to indicate A as feminine gender, it is " GG " that synthesis result, which reads genotype, and is actually consistent；In most right figure, grey black curve all exists
NtestBefore be obviously improved and show A and G is the positive, it is " AG " that synthesis result, which reads genotype, and is actually consistent.
Embodiment 4: end-point method fluorescence detection, as shown in figure 4, with the PCR specification of embodiment 2 to SNP shown in embodiment 1
Site is detected, and the negative quality inspection sample of 188 different individual DNA and 4 (replacing DNA sample with deionized water) is a same
One 384 orifice plate completes detection.Embodiment 2 PCR after the reaction was completed, add sybrgreen class fluorescent reagent carry out terminal
Readings method, fluorescence intensity judge the genotype of SNP.One is completed in regular-PCR instrument with system shown in embodiment 2
Set reaction cycle number (30-60, it is optimal 42) to then take out system, a certain amount of Sybrgreen class final value method fluorescence is added
Then dyestuff reads the fluorescence signal of the special wavelength of the fluorescent dye with fluorescence microplate reader.The fluorescence signal is more than one solid
Determine threshold value and be then judged to the positive, is otherwise feminine gender.Judged each DNA sample in the base in the site according to two allele signals
Because of type.Fig. 4 X-axis shows the signal of A allele, and Y-axis shows the signal of G allele.Threshold value 1 and threshold value 2 are jointly two dimension
Signal is divided into 4 regions.The signal of lower right corner A is the positive, and the signal of G is feminine gender, base of the sample in the region on the SNP
Because type is AA；The signal of the upper right corner A and G is not positive, and genotype of the sample in the region on the SNP is AG；Upper left corner A's
Signal is feminine gender, and the signal of G is the positive, and genotype of the sample in the region on the SNP is GG；The signal of the lower left corner A and G are equal
Unnegative, the sample in the region is judged to negative Quality Control.
It is specific embodiments of the present invention and the technical principle used described in above, if conception under this invention institute
The change of work when the spirit that generated function is still covered without departing from specification and attached drawing, should belong to of the invention
Protection scope.
Claims (9)
1. a kind of automatic primer design method of allele-specific polymerase chain reaction, which is characterized in that including step
It is as follows:
(1) for any SNP site, four inner primers of sequence design of 40~60bp of its upstream and downstream are obtained, are drawn in any one
Since 3 ends ', all there are specificity in 3 ends ' of any one inner primer on mononucleotide diversity point for the design of object
Match, in -1~-3, four of matching site one 3 end ' of inner primer of meaning in office of the mononucleotide diversity point
The annealing temperature of primer is between 44-60 DEG C；
(2) after obtaining inner primer, each inner primer 5 ' end plus one not with the matched short-movie section of template specificity, the short-movie
The length of section collectively constitutes outer primer in 2~10bp, inner primer and short-movie section, and the annealing temperature of four outer primers is 55
Between~75 DEG C.
2. a kind of automatic primer design method of allele-specific polymerase chain reaction according to claim 1,
It is characterized by: the annealing temperature of four inner primers is preferably 51 DEG C.
3. a kind of automatic primer design method of allele-specific polymerase chain reaction according to claim 1,
It is characterized by: the length of short-movie section added by each inner primer and sequence difference.
4. a kind of automatic primer design method of allele-specific polymerase chain reaction according to claim 1,
It is characterized by: the annealing temperature of four outer primers is preferably 59 DEG C.
5. a kind of automatic primer design method of allele-specific polymerase chain reaction according to claim 1,
It is characterized by: 5 ' ends of four outer primers do not occur arbitrary continuation 3 with 3 ' ends of any one inner primer
The matching of bp or more.
6. a kind of high-efficiency fluorescence allele-specific polymerase chain using design primer as described in claim 1 extends anti-
It answers, it is characterised in that: two step method amplification, reaction condition are carried out using primer are as follows: 85~98 DEG C, 3min；85~98 DEG C, 10~
40s；55~75 DEG C, 2~40s；4 DEG C of preservations after 30~60 circulations of execution.
7. a kind of detect SNP site Genotyping using real-time fluorescence based on chain type as claimed in claim 6 extension reaction
Method, it is characterised in that: in chain type extension reaction system, water is substituted for the fluorescent dye for being used for real-time fluorescence PCR, to two
A allele does a real-time PCR reactions respectively, 30 or more PCR cycles is carried out, in the low temperature period of each PCR cycle
Fluorescent value in detection architecture, fluorescence signal it is strong and weak with the double-stranded DNA total amount in linearly or non-linearly reflection system, from
The genotype of the SNP is read in real-time curve.
8. a kind of use end-point method fluorescence detection SNP site Genotyping based on chain type as claimed in claim 6 extension reaction
Method, it is characterised in that: chain type extension after the reaction was completed, add fluorescent reagent carry out terminal readings method, fluorescence intensity
To judge the genotype of SNP.
9. a kind of automatic primer design method of allele-specific polymerase chain reaction according to claim 1,
It is characterized by: the length of short-movie section added by each inner primer is identical with sequence.
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