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Simple and new method for identifying copy numbers of transgenic plants

A technology of transgenic plants and copy number, applied in the field of molecular detection of transgenic plants, can solve the problems of expensive experimental equipment and consumables, and achieve the effects of increasing the difficulty of the experiment, shortening time-consuming and low-cost.

Inactive Publication Date: 2009-06-03
新疆农业科学院核技术生物技术研究所
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Problems solved by technology

However, this method is based on real-time quantitative PCR, and its experimental equipment, reagents, and consumables are relatively expensive.

Method used

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  • Simple and new method for identifying copy numbers of transgenic plants
  • Simple and new method for identifying copy numbers of transgenic plants
  • Simple and new method for identifying copy numbers of transgenic plants

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0040] Example 1. Determination of the copy number of the offspring of transgenic Bt gene insect-resistant cotton

[0041] 1 Routine PCR molecular detection.

[0042] Extract the total DNA of the transgenic Bt gene insect-resistant cotton sample, adjust the DNA concentration of the sample to 100ng / μl, and do PCR molecular detection. figure 1 It is the agarose gel electrophoresis picture of the PCR products of 10 transgenic materials. Among the positive reactions, the PCR product band of the No. 2 individual strain is the brightest, and the DNA of the No. 2 individual strain is used to determine the cycle number.

[0043] 2 Detection of target gene primers of transgenic Bt cotton and primers of internal standard single-copy genes

[0044] Primer sequences of Bt gene:

[0045] cry1A-F: 5'GAAGGATTGAGCAATCTCTAC 3'

[0046] cry1A-R: 5'CAATCAGCCTAGTAAGGTCGT 3'

[0047] The expected amplified fragment size is 340bp.

[0048] Internal standard single copy gene SAD1 primer sequenc...

example 2

[0062] Example 2. Determination of the copy number of the offspring of transgenic Susy cotton

[0063] 1. According to the experimental process of Example 1, the optimal number of PCR cycles to determine the transgenic Susy cotton is 32 cycles.

[0064] 2. Target gene primers

[0065] Susy-F: 5'CATCATTGCGATAAAGGAAAGG 3'

[0066] Susy-R: 5'GGCTCAAAATCCAATTCCAAAAA 3'

[0067] The expected amplified fragment size is a fragment of 720bp.

[0068] 3. Determination of copy number

[0069] Divide the PCR reaction mixture of the sample to be tested (20μl PCR system, 2.0μl 10×Taq Buffer, 1.6μl dNTP (2.5mM), 0.2μl Taq enzyme, 14.6μl water, 1.0μl DNA template) into 2 equal parts One part was added to the target gene Susy primer, and one part was added to the internal standard gene SAD1 primer. The target Susy gene cycling conditions were 94°C preheating for 2 minutes, 94°C for 30 s, 58°C for 30 s, 72°C for 1 min, and 32 cycles of 72°C extension for 10 min. The internal standard SAD...

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Abstract

The invention provides a simple and new method of using a common PCR cycler and a specific single copy gene as interior labels to realize the identification of copy numbers of transgenic plants. The invention is characterized by extracting the total DNA of young leave samples to be detected, adjusting the concentration of the DNA of all the samples to be consistent, designing specific primers of target genes and interior label genes, further determining the best PCR cycle number on the basis of PCR molecular detection, then making parallel PCR and agarose electrophoresis of the target genes and the interior label genes under the same cycle number and finally determining the copy numbers of the target genes according to the ratio of optical density values of the target genes and the interior genes in an electrophoresis image. The invention has the advantage that the invention is beneficial to screening the transgenic plants with stable expression in transgenic plant breeding; the invention has the advantages of simple operation, short time consumption, low cost, safety and accuracy; and the invention combines the PCR molecular detection of the transgenic plants and causes a laboratory for performing the PCR molecular detection of the transgenic plants to realize batch detection without adding experimental equipment and increasing the difficulty.

Description

technical field [0001] The invention relates to a simple and new method for identifying the copy number of transgenic plants, which belongs to the field of molecular detection of transgenic plants. Background technique [0002] With the development of plant genetic engineering technology, it is possible to transfer exogenous genes into plants to improve plant yield, quality, stress resistance or bioreactors for certain secondary metabolites. Transgenic plants are playing an increasingly important role in modern agricultural technology. The effect of transgenics depends on the stable expression of exogenous genes in transgenic plants. However, transgenic plants with multiple copies of repeated transgenic sequences often make it impossible for foreign genes to be stably expressed in transgenic plants, or even not expressed at all. This is a phenomenon that easily occurs in transgenic plants. Especially the pollen tube transgenic method is more prone to this phenomenon due t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 王冬梅范玲李建平陈勋基胡文冉
Owner 新疆农业科学院核技术生物技术研究所
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