CN103343162A - PCR-DHPLC detection primers and detection method for maize ubiquitin protein promoter gene - Google Patents
PCR-DHPLC detection primers and detection method for maize ubiquitin protein promoter gene Download PDFInfo
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- CN103343162A CN103343162A CN2013102224515A CN201310222451A CN103343162A CN 103343162 A CN103343162 A CN 103343162A CN 2013102224515 A CN2013102224515 A CN 2013102224515A CN 201310222451 A CN201310222451 A CN 201310222451A CN 103343162 A CN103343162 A CN 103343162A
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Abstract
The invention discloses PCR-DHPLC detection primers and a detection method for a maize ubiquitin promoter gene. The primers have stronger specificity, can be used for a PCR, and are specifically amplified to form a DNA fragment of the maize ubiquitin promoter gene; and the amplified product can be suitable for a subsequent DHPLC analysis. The detection method provides the maize ubiquitin promoter gene detection method having convenient operation, good expansibility and strong specificity. A DHPLC is utilized to analyze the PCR amplified product, allows the distinguishing rate of the fragment size of the PCR amplified product to reach a plurality of bases, and has high resolution. The primers and the detection method provide the simple, convenient, effective and reliable detection method for the detection of the maize ubiquitin promoter gene, and are especially applicable to port inspection and quarantine and other departments.
Description
Technical field
The application relates to the detection of transgenic product, and the PCR-DHPLC that particularly relates to a kind of corn ubiquitin protein promoter gene detects primer and detection method.
Background technology
At present, genetically modified crops such as transgene cotton, paddy rice, wheat, corn, soybean are more and more, the detection research of genetically modified crops also become the focus of various countries.Wherein, the promotor of often using in the genetically modified crops, corn ubiquitin protein promotor (being screening-gene ubiquitin) are to detect the important target gene of genetically modified crops.Detection method to corn ubiquitin protein promoter gene mainly adopts conventional PCR, real-time fluorescence PCR, southern blotting technique method, RNA blotting and gene chips etc. at present.Traditional conventional PCR detection method has certain limitation aspect platform extension, along with the increase of target to be checked, need consumption and the ratio of the every cover primer in the system be optimized again, and take into account factors such as amplification efficiency, and workload is bigger; On the other hand, the method for the common gel electrophoresis analysis amplified production that adopts, it is not high to distinguish efficient, and detected result is undesirable.More multiple conventional PCR detection method has superiority though real-time fluorescence PCR is at aspects such as detection sensitivities, but because the restriction of present instrument self and fluorescence dye development, non-interfering 4 fluorescence channels only can be provided simultaneously, also limit this technology and detected the flux expansion, and detected the cost height.Southern blotting technique method and RNA blotting need to change film, hybridization, and complex operation, expense height, detection time are long, and practical application is also less.Based on the gene chip detection methods of overlapping PCR of routine more, need many cover primers to increase, complex operation step, and be not suitable for large-scale high throughput testing.
(Denaturing High-performance Liquid Chromatography is a kind of method for nucleic acid analysis of simple, quick, non-gel DHPLC) to the sex change high-efficient liquid phase chromatogram technology, has advantages such as good resolution, extendability are strong, sensitivity height.This method is at 50 ℃ of condition analysis samples, and the wash-out at sample peak is only determined by the quantity of base pair that the base pair different amts elution order of sample is also different, thereby produces different sample peaks.Concrete, the acetonitrile concentration that has served as post improves, and nucleic acid fragment can be come out by wash-out according to molecular weight order from small to large.Relatively determine the molecular weight size by elution peak and the DNA marker that obtains, thereby judge in the sample whether contain the target detect gene.PCR-DHPLC is the technology that PCR is combined with DHPLC, namely adopts pcr amplification to go out target sequence earlier, and then amplified production is carried out DHPLC analyze, thereby improve sensitivity and the specificity that detects.At present, the detection method that does not still have the PCR-DHPLC of corn ubiquitin protein promoter gene.
Summary of the invention
The application's purpose provides the primer that a kind of new PCR-DHPLC that is used for corn ubiquitin protein promoter gene detects, and based on the PCR-DHPLC detection method of the corn ubiquitin protein promoter gene of this primer.
For achieving the above object, the application has adopted following technical scheme:
The application discloses the primer that a kind of PCR-DHPLC for corn ubiquitin protein promoter gene detects, and the upstream primer of this primer contains sequence shown in the Seq ID No.1, and downstream primer contains sequence shown in the Seq ID No.2;
Seq?ID?No.1：5’-CAGAAATTGCGTGGCGGA-3’
Seq?ID?No.2：5’-GGGCGAGGAAGGGAAAGC-3’。
Need to prove, the application's primer is the Auele Specific Primer that designs at corn ubiquitin protein promoter gene, can carry out specific amplification to corn ubiquitin protein promoter gene, therefore, be appreciated that this primer except the PCR-DHPLC that is used for the application detects, be equally applicable to conventional PCR and detect, and the detection of other PCR-based amplification, as real-time fluorescent PCR, gene chip etc.
The application's another side also discloses a kind of PCR-DHPLC detection method of corn ubiquitin protein promoter gene, this detection method comprises the primer that adopts the application's design, DNA with detected sample is that template is carried out pcr amplification, and the product of pcr amplification is carried out dhplc analysis.
Need to prove, the primer of the application's design is the Auele Specific Primer at corn ubiquitin protein promoter gene, therefore, be appreciated that, if contain the corn ubiquitin protein promoter gene composition of capacity in the detected sample, can amplify corresponding target fragment, and when DHPLC analyzes, produce corresponding elution peak.In addition, the application's primer also is simultaneously to design according to the specific demand that DHPLC analyzes, and the product that this primer amplification comes out can be good at being applicable to that DHPLC analyzes.Also need to prove, thereby though can detecting corn ubiquitin protein promoter gene from various samples, the application's primer judges whether contain transgene component in the test sample, but, be appreciated that, this also is not suitable for the sample that contains corn, because itself just contains this promotor the corn kind, have no way of judging that this promotor is that corn itself has or genetically modified crops have.Therefore, the application's primer and method are used for not containing the transgenosis sample detection of corn composition only.
Further, it is that reference standard is carried out dhplc analysis that the application's detection method also comprises with DNA marker, and the dhplc analysis result of pcr amplification product and the dhplc analysis result of DNA marker are compared.
Preferred the application's detection method comprises the steps:
(A) adopting the application's primer, is template with the DNA of detected sample, carries out pcr amplification;
(B) pcr amplification product with step (A) is that sample carries out the DHPLC analysis, is that reference standard is carried out the DHPLC analysis with DNA marker simultaneously;
(C) the DHPLC analytical results of pcr amplification product in the step (B) and the DHPLC analytical results of DNA marker are compared, determine the molecular weight size of pcr amplification product, thereby judge whether to contain the target sequence of detection.
Need to prove, in theory, the application's primer is the Auele Specific Primer according to the design of corn ubiquitin protein promoter gene, can amplify the fragment of about 165bp to the corn ubiquitin protein promoter gene composition in the detected sample, therefore, can analyze the elution peak of 165bp by DHPLC; And other composition is not had amplification, namely do not have other elution peak and occur.Among the application, judge whether to contain corn ubiquitin protein promoter gene target sequence foundation namely, whether the elution peak of 165bp is arranged.
Because adopt above technical scheme, the application's beneficial effect is:
The application's corn ubiquitin protein promoter gene detects primer and has stronger specificity, can be used in follow-up multiplex PCR amplification and DHPLC and analyzes.The application combines PCR at traditional undesirable problem of electrophoretic analysis pcr amplification result with DHPLC, a kind of easy and simple to handle, scalability good, resolving power is high corn ubiquitin protein promoter gene detection method is provided.Utilize the pcr amplification product of DHPLC to analyze, can reach several bases to different big or small PCR product fragment differentiation rates, even can distinguish the fragment of 1 base difference in size.The application's primer and detection method are particularly suitable for department's uses such as Check and Examination of Port quarantine for the detection of corn ubiquitin protein promoter gene provides a kind of simple, convenient, effective, reliable detection method.
Description of drawings
Fig. 1: the partial results figure that is the DHPLC analysis of pcr amplification product in the embodiment of the present application;
Wherein, top-down curve is respectively, and M representation DNA marke, 1 represents transgenic corns MON88017,2 and represents transgenic corns T25,3 and represent transgenic wheat, 4 and represent non-transgenic soybean, 5 and represent non-transgenic rape, 6 and represent potato EH92-527-1,7 and represent non-transgenic paddy rice, 8 and represent non-transgenic corn, 9 and represent pawpaw, the contrast of the blank water of 10 representatives.Each peak value of DNA marker is followed successively by 80bp, 102bp, 174bp, 257bp, 267bp, 296bp, 434bp, 458bp, 587bp from left to right.
Embodiment
The application provides the primer of the PCR-DHPLC detection that is used for corn ubiquitin protein promoter gene, and this primer designs at the distinguished sequence of corn ubiquitin protein promoter gene, and the specific amplification that can be used in corn ubiquitin protein promoter gene detects.The application also provides the PCR-DHPLC detection method based on the corn ubiquitin protein promoter gene of the application's primer on this basis.
Need to prove, the application's primer is to detect and design at the PCR-DHPLC of corn ubiquitin protein promoter gene, but, be appreciated that, itself also uses this primer as pcr amplification, the pcr amplification of therefore, can be used for conventional pcr amplification, real-time fluorescence PCR amplification equally, being combined with gene chip etc.
Also by reference to the accompanying drawings the application is described in further detail below by concrete experimental example.Following experimental example only is further detailed the application, should not be construed as the restriction to the application.
Embodiment 1 DNA extraction
(1) DNA extraction: with reference to plant genome DNA extract test kit (QIAGEN, the method that Cat.No.69104) provides makes improvements slightly extracts DNA, the concrete operations step is as follows:
1. the AP1 solution of 65 ℃ of preheatings and Buffer AE;
2. the liquid nitrogen grinding sample is got an amount of sample and is gone in the centrifuge tube of 1.5mL after Powdered;
3. add the AP1 solution of 400 μ L preheatings and the RNA enzyme of 4 μ L100mg/mL in the centrifuge tube, fully behind the mixing, 65 ℃ of water-bath 10min-15min, during vibration mixing 2-3 time;
4. after water-bath is finished, directly add 130 μ LAP2 solution, can not shake during ice bath 5min ice bath behind the mixing that fully vibrates, then the centrifugal 5min of 14000r/min;
5. draw supernatant and join in the QIA shredder Mini spin column pillar, be i.e. purple pillar in the test kit, the centrifugal 2min of 14000rpm/min, the volume number of the supernatant liquor that record is drawn;
6. abandon top pillar, add the Buffer AP3/E solution of 1.5 times of volumes in the solution after filter, with liquid-transfering gun mixing gently;
7. get the solution of 650 μ L steps mixing 6. at every turn and transfer in the DNeasy Mini spin column pillar, i.e. the pillar of white in the test kit, the centrifugal 1min of 14000rpm/min abandons filtrate, has filtered fully until the solution of step mixing 6.; Need to prove, the volume that DNeasy Mini spin column post can hold when filtering is about 650 μ L, and therefore step solution 6. is generally all greater than 650 μ L,, 650 μ L solution can only be got at every turn, rest solution could be added again after filtering, abandon filtrate;
8. abandon following collection tube, spin column post is put into a new 2mL collection tube, add 500 μ L Buffer AW solution, the centrifugal 1min wash-out of 14000rpm/min impurity, repeat the operation of wash-out impurity once, abandon filtrate, the centrifugal 1min of 12000rpm/min blank pipe;
9. abandon following collection tube, change a new 1.5mL centrifuge tube, add the Buffer AE dissolving DNA after the 100 μ L preheatings, room temperature leaves standstill 5min deposit D NA, the centrifugal 1min of 12000rpm/min, the solution of collection are that dna solution need to prove that the Buffer AE after the 100 μ L preheatings can add at twice or repeatedly;
10. abandon top pillar, preserve dna solution for-20 ℃.
(2) DNA purifying: when the amount that contains impurity such as protein among the DNA was higher, its purity can not satisfy requirement of experiment, need carry out purifying to it, and concrete purification step is as follows:
1. after the dna solution that extracts being diluted to 500 μ L-700 μ L, add isopyknic phenol and chloroform, fully mixing;
2. the centrifugal 10min of 14000rpm/min gets supernatant liquor;
3. add the Virahol of 0.6 times of volume in supernatant liquor ,-20 ° of C leave standstill more than the 30min;
4. the centrifugal 10min of 14000rpm/min under 4 ° of C conditions abandons supernatant;
5. to 70% the ethanol that adds lmL4 ° of C precooling during step is abandoned the precipitation of supernatant liquor in 4., the centrifugal 10min of 14000rpm/min abandons supernatant, and repetitive operation once;
6. will be air-dry through being deposited under the aseptic condition of step washing with alcohol 5., add an amount of AE Buffer dissolving DNA, namely obtain the higher dna solution of purity.Need to prove, the add-on of AE Buffer is decided according to concrete requirement of experiment, if wish that the concentration of acquisition dna solution is higher, then can add more a spot of AE Buffer, as 10 μ L-50 μ L, also can directly add 100 μ L Buffer AE again, then before DNA concentration and the purifying quite, perhaps add the Buffer AE of volume more to obtain the dna solution of low concentration.
Embodiment 2 DNA concentration determinations
The sample DNA that extracts is carried out concentration and purity testing; Adopt ultraviolet spectrophotometer to measure 260nm and 280nm place absorption value, calculate purity and the concentration of nucleic acid respectively, calculation formula is as follows:
DNA purity=OD260/OD280
DNA concentration=50 * OD260mg/mL
The purity ratio of DNA is between 1.7～1.9, and concentration is greater than 10ng/ μ L.
Embodiment 3 pcr amplifications
According to the dna sequence dna design primer (table 1) of corn ubiquitin protein promoter gene, and the DNA of the following sample of extraction carries out specific detection: transgenic corns MON88017, transgenic corns T25, transgenic wheat, non-transgenic soybean, non-transgenic rape, potato EH92-527-1, non-transgenic paddy rice, non-transgenic corn, pawpaw etc.
The detection primer of table 1 corn ubiquitin protein promoter gene
| Title | Sequence 5 '-3 ' | Seq?ID?No. |
| ub-F | CAGAAATTGCGTGGCGGA | 1 |
| ub-R | GGGCGAGGAAGGGAAAGC | 2 |
PCR reaction system cumulative volume is 50 μ L, each composition is respectively: PCR reaction solution 10 * Buffer5 μ L, 2.5mmol/L dNTP 5 μ L, each 1 μ L of the upstream primer of 10 μ mol/L and downstream primer, 5U/ μ L Taq polymerase0.3 μ L, dna solution 1 μ L, supplying with the sterilization distilled water then is 50 μ L.
PCR reaction conditions: 94 ℃ of sex change 1min; Enter 35 circulations then: 94 ℃ of 30s, 58 ℃ of 90s, 72 ℃ of 90s; 72 ℃ are extended 10min after the loop ends.
Embodiment 4 DHPLC detect
The PCR product is placed on the automatic sampling platform of DHPLC; Open DHPLC control software Navigator, in detection method, select Multiplex, i.e. multi-disc piecewise analysis, setting upper limit of detection simultaneously is 600bp, is limited to 70bp down; Move two blank, i.e. empty needle, balance chromatographic column; Move a marker, be used for reference to contrast, the nucleic acid fragment size of test sample; Successively sample to be tested is analyzed, partial results is seen Fig. 1.The DNA marker that the application adopts is SSR (pUC18/MspI) DNA Marker, can obtain by commercially available purchase.
From top to bottom curve among Fig. 1, article one, the DHPLC analytic curve that carries out for DNA marke of curve, second curve to second from the bottom curve is that the DNA of transgenic corns MON88017, transgenic corns T25, transgenic wheat, non-transgenic soybean, non-transgenic rape, potato EH92-527-1, non-transgenic paddy rice, non-transgenic corn and pawpaw carries out respectively carrying out the curve that DHPLC analyzes behind the pcr amplification in regular turn, and the last item curve is to adopt water to carry out the curve that DHPLC analyzes as blank.
The result judges: observe the elution peak that DHPLC obtains, by analyzing the dna fragmentation size of determining the elution peak position automatically with marker comparison and WAVE4500, judge accordingly.
Be that the pcr amplification product of template is that the DHPLC of sample analyzes to the DNA with transgenosis and non-transgenic corn, its analytical results shows, all contains the elution peak of 165bp in these samples; Not that the DNA of the sample of corn is that the pcr amplification product of template is that the DHPLC of sample analyzes and all do not have elution peak with other.As seen, the primer that this example provides has good specificity, can be from plurality of samples the specific corn ubiquitin protein promoter gene that detects.
Above content be in conjunction with concrete embodiment to further describing that the application does, can not assert that the application's concrete enforcement is confined to these explanations.For the application person of an ordinary skill in the technical field, under the prerequisite that does not break away from the application's design, can also make some simple deduction or replace, all should be considered as belonging to the application's protection domain.
Claims (3)
1. primer that the PCR-DHPLC that is used for corn ubiquitin protein promoter gene detects, it is characterized in that: described primer comprises upstream primer and downstream primer, described upstream primer contains sequence shown in the Seq ID No.1, and described downstream primer contains sequence shown in the Seq ID No.2;
Seq?ID?No.1：5’-CAGAAATTGCGTGGCGGA-3’
Seq?ID?No.2：5’-GGGCGAGGAAGGGAAAGC-3’。
2. the PCR-DHPLC detection method of a corn ubiquitin protein promoter gene, it is characterized in that: described detection method comprises the described primer of employing claim 1, DNA with detected sample is that template is carried out pcr amplification, and the product of pcr amplification is carried out dhplc analysis.
3. detection method according to claim 2, it is characterized in that: it is that reference standard is carried out dhplc analysis that described detection method also comprises with DNA marker, and the dhplc analysis result of pcr amplification product and the dhplc analysis result of DNA marker are compared.
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Application publication date: 20131009 |