CN101638683A - Dual-PCR method for detecting tomato TY-1 gene and Mi gene at the same time - Google Patents
Dual-PCR method for detecting tomato TY-1 gene and Mi gene at the same time Download PDFInfo
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- CN101638683A CN101638683A CN200810041081A CN200810041081A CN101638683A CN 101638683 A CN101638683 A CN 101638683A CN 200810041081 A CN200810041081 A CN 200810041081A CN 200810041081 A CN200810041081 A CN 200810041081A CN 101638683 A CN101638683 A CN 101638683A
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Abstract
The invention provides a dual-PCR method for detecting a tomato TY-1 gene and an Mi gene at the same time, which comprises the following steps: (1) performing PCR reaction on a PCR system comprising the sequences of a Ty-1 primer and an Mi primer to obtain a PCR product; (2) keeping an enzyme cutting system at a temperature of 65 DEG C for 1.5 hours to obtain an enzyme cutting product; and (3) detecting the PRC product and the enzyme cutting product, using EB for dyeing the final result, adopting a Bio-RAD gel imaging system for taking pictures, and analyzing the result. The dual-PCR techniquehas the advantages that: 1, two genes are detected at the same time in the PCR, and the used time is just one half of that for detecting the two genes respectively so that the identification time isgreatly saved; and 2, the medicines used by the PCR are greatly saved, and the experimental cost is reduced.
Description
Technical field
The present invention relates to detect simultaneously the detection method of tomato TY-1 gene and Mi gene.
Background technology
Tomato yellow leaf curl virus (Tomato Yellow Leaf Curl virus) disease and root knot nematode (Root-knotnematode) disease are two kinds of worldwide diseases.TY-1 gene and Mi gene are respectively the important gene of tomato resisting etiolation curve leaf disease virus and root knot nematode disease.
Sick and root knot nematode disease the increasing the weight of year by year at home along with tomato yellow leaf curl virus in recent years, the tomato variety that seed selection contains TY-1 gene and Mi gene becomes urgent further.
At present, document: Zamir, D., Ekstein-Michelson, I.., Zakay, Y., Navot, N., Zeidan, M., Sarfatti, M., Eshed, Y., Harel, E., Pleben, T., Van-Oss, H., Jedar, N., Rabinowitch, H.D.and Czosnek, H.., 1994, Mapping and introgression of aTomato yellow leaf curl virus tolerance gene, Ty-1.Theoretical and AppliedGenetics, 88:141～146 reports: found the CAPS mark of TYLCV Ty-1 gene, document: Williamson, V.M., Ho, J.Y., Wu, F.F., Miller, N., and Kaloshian, I., 1994 A PCR-based marker tightly linked to the nematode resistance gene, Mi, in tomato.Theoretical and Applied Genetics, 87,757-763 reports, has obtained the CAPS mark of tomato root-knot nematode resistant Mi gene.
At present, the conventional detection TY-1 gene and the method for Mi gene, as Zamir, D document disclosed method, but it is to detect TY-1 gene and Mi gene simultaneously that there is an obvious defects in this method, therefore, qualification time is long, appraisal cost is high, can not satisfy the needs of the parties concerned.
Summary of the invention
The objective of the invention is to set up a kind of dual-PCR method that detects tomato TY-1 gene and Mi gene simultaneously, be intended to save qualification time, reduce experimental cost, accelerate the pyramiding breeding process of tomato Ty-1 gene and Mi gene.
Method of the present invention comprises the steps:
(1) the PCR system is carried out the PCR reaction according to following program:
95 ℃ of sex change 5min, then 94 ℃ of sex change 1min, 61 ℃ of annealing 1min, 72 ℃ extend 2min, 35 circulations, last 72 ℃ are extended 10min, obtain the PCR product, 4 ℃ of preservations;
Said PCR system is the aqueous solution, and component and content are:
Template DNA 20ng/L
Ty-1 primer 0.2umol/L
Mi primer 0.2umol/L
10×buffer 2ul/L
Mg
2+ 3.5mmol/L
dNTPs 250umol/L
Taq enzyme 0.2uL/L
Said template DNA refers to tomato DNA, and the preparation method is as follows:
1, gets the tomato tender leaf and put into mortar, add liquid nitrogen, be ground into powder, get 40mg and pack in the round end centrifuge tube of 2mL.
2, add 700ul CTAB extracting solution, acutely rock, make CTAB extracting solution and sample thorough mixing even.
(100mmol/L Tris-HCl (pH 8.0), 20mmol/L EDTA, 1.4mol/L NaCl, 3%CTAB, 2%PVP, 2% beta-mercaptoethanol)
3,65 ℃ of water-bath 1h.
4, place 4 ℃ of refrigerators or room temperature to be cooled to below 15 ℃.
5, add 24: 1 chloroform/primary isoamyl alcohol of 700 μ l, slowly put upside down mixing up and down, extracting 15min is the milkiness shape to solution, assurance sample and chloroform thorough mixing.
6, the centrifugal 10min of 12000rpm.
7, get supernatant liquor in the 1.5ml centrifuge tube, add the Virahol of isopyknic precooling-20 ℃, mixing gently turns upside down.
8 ,-20 a ℃ refrigerator leaves standstill more than the 3h.
9, the centrifugal 10min of 12000rpm abandons supernatant liquor.Precipitate 2 times with 70% washing with alcohol.
10, dry up DNA, make the ethanol volatilization clean.
11, add 65 ℃ of water-bath dissolving DNAs of 200 μ L TE.
(TE:10mmol/L Tris-Hcl and 1mmol/LEDTA, PH=8)
The Ty-1 primer sequence:
CAPS1F：5’-TAATCCGTCGTTACCTCTCCTT-3’
CAPS1R：5’-CGGATGACTTCAATAGCAATGA-3’
The Mi primer sequence:
CAPS2F：5’-TCGGAGCCTTGGTCTGAATT-3’
CAPS2R：5’-GCCAGAGATGATTCGTGAGA-3’；
10 * buffer is a kind of PCR reaction buffer, is 10～50mmol/LTris-Hcl (PH8.3～8.8), can adopt Shanghai to give birth to the product of worker company;
Mg
2+Derive from the compound of magnesium chloride;
DNTPs is the necessary thing of a kind of PCR reaction, and its chemical name is a triphosphate deoxy-nucleotide, can adopt Shanghai to give birth to the product of worker company;
Taq zymochemistry name is called hot resistant DNA polymerase.Can adopt Shanghai to give birth to the worker ' product of company;
(2) enzyme is cut the preparation of product:
Enzyme is cut system at 65 ℃ of insulation 1.5h, and acquisition and enzyme are cut product;
The said enzyme system of cutting is the aqueous solution, and component and content are:
The enzyme system of cutting is:
PCR product 0.35uL/L,
Taq I enzyme 0.05uL/L,
Buffer0.1uL/L，
(3) sepharose of configuration 2.5% adds 1 μ L EB (0.5 μ g/ml) and pours in the glue groove, inserts comb, treats that gelling detects the back admittedly.Get 10uL PCR product and 10uL enzyme respectively and cut product and detect, the termination fruit adopts the Bio-RAD gel imaging system to take pictures with EB dyeing;
The chemical name of said EB is an ethidium bromide, can adopt the commercially available prod, and as the product of the living worker in Shanghai company, dyeing process is prior art, as Williamson, and the method for V.M. bibliographical information;
The Bio-RAD gel imaging system is a kind of general system, can adopt the product of Bio-RAD company;
(4) interpretation of result: the special segment that all becomes 398bp and 750bp before anti-sense genotype enzyme is cut.After the TaqI enzyme is cut, isozygoty and contain Ty-1 becomes 570bp, 303bp, 180bp, 95bp with the material of Mi gene specific fragment, heterozygosis contains Ty-1 becomes 750bp, 570bp, 398bp, 303bp, 180bp, 95bp with the material of Mi gene specific fragment, heterozygosis contains the isozygoty material that contains the Mi gene of Ty-1 and becomes the specific fragment of 570bp, 398bp, 303bp, 180bp, 95bp, heterozygosis contains the isozygoty material that contains the Ty-1 gene of Mi and becomes the specific fragment of 750bp, 570bp, 303bp, 180bp, 95bp, and the material that do not contain Ty-1 and Mi gene of isozygotying is not digested.
Description of drawings
Fig. 1 is among the embodiment 1, the photo before enzyme is cut.
Fig. 2 is among the embodiment 1 photo after enzyme is cut.
Fig. 3 is among the embodiment 2, the photo before enzyme is cut.
Fig. 4 is among the embodiment 2 photo after enzyme is cut.
This double PCR technology has following advantage: 1, in a PCR, detect simultaneously two genes, The used time is just detected respectively half of two used times of gene, has greatly saved qualification time; 2, greatly save the used medicine of PCR, reduced experimental cost.
Embodiment
Get the tender leaf of tomato 2300 kinds, adopt Williamson, V.M document disclosed method is extracted DNA respectively, is template DNA.
Tomato 2300 is that Shanghai academy of agricultural sciences horticulture institute tomato is organized the height of seed selection for self-mating system.
(1) the PCR system is carried out the PCR reaction according to following program:
95 ℃ of sex change 5min, then 94 ℃ of sex change 1min, 61 ℃ of annealing 1min, 72 ℃ extend 2min, 35 circulations, last 72 ℃ are extended 10min, obtain the PCR product, 4 ℃ of preservations;
Said PCR system is the aqueous solution, and component and content are:
Template DNA 20ng/L
Ty-1 primer 0.2umo1/L
Mi primer 0.2umol/L
10×buffer 2ul/L
Mg
2+ 3.5mmol/L
dNTPs 250umol/L
Taq enzyme 0.2uL/L
The Ty-1 primer sequence:
CAPS1F：5’-TAATCCGTCGTTACCTCTCCTT-3’
CAPS1R：5’-CGGATGACTTCAATAGCAATGA-3’
The Mi primer sequence:
CAPS2F：5’-TCGGAGCCTTGGTCTGAATT-3’
CAPS2R：5’-GCCAGAGATGATTCGTGAGA-3’；
(2) enzyme is cut the preparation of product:
Enzyme is cut system at 65 ℃ of insulation 1.5h, and acquisition and enzyme are cut product;
The said enzyme system of cutting is the aqueous solution, and component and content are:
PCR product 0.35uL/L,
TaqI enzyme 0.05uL/L,
Buffer0.1uL/L，
(3) sepharose of configuration 2.5% adds 1 μ L EB (0.5 μ g/ml) and pours in the glue groove, inserts comb, treats that gelling detects the back admittedly.Get 10uL PCR product and 10uL enzyme respectively and cut product and detect, the termination fruit adopts the Bio-RAD gel imaging system to take pictures with EB dyeing;
The chemical name of said EB is an ethidium bromide, can adopt the commercially available prod, and as the product of the living worker in Shanghai company, dyeing process is prior art, as Williamson, and the method for V.M. bibliographical information;
The Bio-RAD gel imaging system is a kind of general system, can adopt the product of Bio-RAD company;
Analytical results such as Fig. 1 and Fig. 2, result show that tomato 2300 does not contain TY-1 gene and Mi gene.(because tomato 2300 enzymes all become 750 before and after cutting, the band of 398bp, so tomato 2300 does not contain TY-1 gene and Mi gene).
Get the tender leaf of tomato 2583 or 2697 kinds, adopt Williamson, V.M document disclosed method is extracted DNA respectively, is template DNA.
Tomato 2583 or 2697 is the kind of Shanghai academy of agricultural sciences horticulture institute tomato group seed selection.
(1) the PCR system is carried out the PCR reaction according to following program:
95 ℃ of sex change 5min, then 94 ℃ of sex change 1min, 61 ℃ of annealing 1min, 72 ℃ extend 2min, 35 circulations, last 72 ℃ are extended 10min, obtain the PCR product, 4 ℃ of preservations;
Said PCR system is the aqueous solution, and component and content are:
Template DNA 20ng/L
Ty-1 primer 0.2umol/L
Mi primer 0.2umol/L
10×buffer 2ul/L
Mg
2+ 3.5mmol/L
dNTPs 250umol/L
Taq enzyme 0.2uL/L
The Ty-1 primer sequence:
CAPS1F：5’-TAATCCGTCGTTACCTCTCCTT-3’
CAPS1R：5’-CGGATGACTTCAATAGCAATGA-3’
The Mi primer sequence:
CAPS2F：5’-TCGGAGCCTTGGTCTGAATT-3’
CAPS2R：5’-GCCAGAGATGATTCGTGAGA-3’；
(2) enzyme is cut the preparation of product:
Enzyme is cut system at 65 ℃ of insulation 1.5h, and acquisition and enzyme are cut product;
The said enzyme system of cutting is the aqueous solution, and component and content are:
PCR product 0.35uL/L,
TaqI enzyme 0.05uL/L,
Buffer0.1uL/L，
(3) sepharose of configuration 2.5% adds 1 μ L EB (0.5 μ g/ml) and pours in the glue groove, inserts comb, treats that gelling detects the back admittedly.Get 10uL PCR product and 10uL enzyme respectively and cut product and detect, the termination fruit adopts the Bio-RAD gel imaging system to take pictures with EB dyeing;
The chemical name of said EB is an ethidium bromide, can adopt the commercially available prod, and as the product of the living worker in Shanghai company, dyeing process is prior art, as Williamson, and the method for V.M. bibliographical information;
The Bio-RAD gel imaging system is a kind of general system, can adopt the product of Bio-RAD company;
Photographic analysis result such as Fig. 3 and Fig. 4, result show that tomato 2583 does not contain TY-1 gene and Mi gene, and 2697 heterozygosis contain TY-1 gene and heterozygosis Mi gene.
Claims (5)
1. detect the dual-PCR method of tomato TY-1 gene and Mi gene simultaneously, it is characterized in that, comprise the steps:
(1) the PCR system is carried out the PCR reaction, obtain the PCR product;
The Ty-1 primer sequence:
CAPS1?F：5’-TAATCCGTCGTTACCTCTCCTT-3’
CAPS1?R：5’-CGGATGACTTCAATAGCAATGA-3’
The Mi primer sequence:
CAPS2?F：5’-TCGGAGCCTTGGTCTGAATT-3’
CAPS2?R：5’-GCCAGAGATGATTCGTGAGA-3’；
(2) enzyme is cut the preparation of product:
Enzyme is cut system at 65 ℃ of insulation 1.5h, and acquisition and enzyme are cut product;
(3) get PCR product and enzyme respectively and cut product and detect, the termination fruit adopts the Bio-RAD gel imaging system to take pictures analytical results with EB dyeing.
2. method according to claim 1 is characterized in that, the PCR system is carried out PCR reaction according to following program: 95 ℃ of sex change 5min, then 94 ℃ of sex change 1min, 61 ℃ of annealing 1min, 72 ℃ extend 2min, 35 circulations, last 72 ℃ are extended 10min.
3. method according to claim 1 is characterized in that, said PCR system is the aqueous solution, and component and content are:
Template DNA 20ng/L
Ty-1 primer 0.2umol/L
Mi primer 0.2umol/L
10×buffer 2ul/L
Mg
2+ 3.5mmol/L
dNTPs 250umol/L
Taq enzyme 0.2uL/L
Said template DNA refers to tomato DNA.
4. method according to claim 1 is characterized in that, the said enzyme system of cutting is the aqueous solution, and component and content are:
PCR product 0.35uL/L,
Taq I enzyme 0.05uL/L,
Buffer0.1uL/L。
5. method according to claim 1 is characterized in that, in the step (3), gets 10uL PCR product and 10uL enzyme respectively and cuts product and detect.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104894285A (en) * | 2015-06-29 | 2015-09-09 | 天津市农业生物技术研究中心 | Method for detecting genes Cf-9 and I-2 according to dual-PCR (polymerase chain reaction) technology |
| CN105087567A (en) * | 2015-08-28 | 2015-11-25 | 青岛农业大学 | Duplex PCR (polymerase chain reaction) primer and method for identifying tomato chlorosis viruses and tomato yellow leaf curl viruses |
| CN105463082A (en) * | 2015-12-10 | 2016-04-06 | 江苏省农业科学院 | Multiple PCR (polymerase chain reaction) detection method of tomato Mi-1 gene and ty-5 gene |
| CN105463081B (en) * | 2015-12-10 | 2018-08-24 | 江苏省农业科学院 | A kind of multi-PCR detection method of tomato Ty-1 genes and ty-5 genes |
| CN105463079B (en) * | 2015-12-10 | 2018-08-24 | 江苏省农业科学院 | A kind of multi-PCR detection method of tomato Ty-3 genes and ty-5 genes |
-
2008
- 2008-07-28 CN CN200810041081A patent/CN101638683A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104894285A (en) * | 2015-06-29 | 2015-09-09 | 天津市农业生物技术研究中心 | Method for detecting genes Cf-9 and I-2 according to dual-PCR (polymerase chain reaction) technology |
| CN105087567A (en) * | 2015-08-28 | 2015-11-25 | 青岛农业大学 | Duplex PCR (polymerase chain reaction) primer and method for identifying tomato chlorosis viruses and tomato yellow leaf curl viruses |
| CN105463082A (en) * | 2015-12-10 | 2016-04-06 | 江苏省农业科学院 | Multiple PCR (polymerase chain reaction) detection method of tomato Mi-1 gene and ty-5 gene |
| CN105463081B (en) * | 2015-12-10 | 2018-08-24 | 江苏省农业科学院 | A kind of multi-PCR detection method of tomato Ty-1 genes and ty-5 genes |
| CN105463079B (en) * | 2015-12-10 | 2018-08-24 | 江苏省农业科学院 | A kind of multi-PCR detection method of tomato Ty-3 genes and ty-5 genes |
| CN105463082B (en) * | 2015-12-10 | 2018-10-30 | 江苏省农业科学院 | A kind of multi-PCR detection method of tomato Mi-1 genes and ty-5 genes |
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Open date: 20100203 |