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Sugarcane genome endogenous reference gene isopentenyl diphosphate delta-isomerase gene PCR (polymerase chain reaction) primer sequences and amplification method

A technology of prenyl diphosphate and internal standard gene, which is applied in recombinant DNA technology, DNA/RNA fragments, DNA preparation, etc., can solve the problem that there is no research report on copy number, and achieve easy determination, strong specificity, The effect of wide applicability

Inactive Publication Date: 2015-02-04
FUJIAN AGRI & FORESTRY UNIV
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Problems solved by technology

There have been many studies on the prenyl diphosphate δ-isomerase gene that produces this enzyme, but no studies have been reported on the copy number of this gene in the genome of sugarcane species

Method used

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  • Sugarcane genome endogenous reference gene isopentenyl diphosphate delta-isomerase gene PCR (polymerase chain reaction) primer sequences and amplification method
  • Sugarcane genome endogenous reference gene isopentenyl diphosphate delta-isomerase gene PCR (polymerase chain reaction) primer sequences and amplification method
  • Sugarcane genome endogenous reference gene isopentenyl diphosphate delta-isomerase gene PCR (polymerase chain reaction) primer sequences and amplification method

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Embodiment 1

[0021] The sequence of PCR primers for prenyl diphosphate delta-isomerase gene is: ScIDI2-F : GCATCATAGCGTCCAGGTCTA; ScIDI2-R : CCATCAGCATCAGTTTTTGTG.

[0022] PCR reaction system: 1.0 U Taq DNA polymerase, 1×PCR buffer solution, 0.25 mM dNTP, 3.0 mM MgCl2, primers ScIDI2-F and ScIDI2-R Each 0.25 mM, DNA template 25 ng, the total volume of the reaction system is 25 μl;

[0023] PCR amplification conditions: pre-denaturation at 95°C for 6 min; denaturation at 94°C for 30 s; annealing at 56°C for 30 s, extension at 72°C for 40 s, a total of 35 cycles, and a final extension at 72°C for 7 min.

[0024] (2) Electrophoresis detection of PCR product of sugarcane prenyl diphosphate δ-isomerase gene and analysis of product specificity and generality

[0025] Using a pair of primers for the prenyl diphosphate δ-isomerase gene, 12 sugarcane varieties were detected by PCR. It was found that the primers for the prenyl diphosphate δ-isomerase gene only One band was amplified and t...

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Abstract

The invention discloses sugarcane genome endogenous reference gene isopentenyl diphosphate delta-isomerase gene PCR (polymerase chain reaction) primer sequences and an amplification method. The primer sequences are composed of ScIDI2-F and ScIDI2-R. The PCR amplification method comprises a reaction system and amplification conditions. The invention provides a genome endogenous reference gene for PCR detection and species specific identification of transgenic sugarcane, and meanwhile, provides insurance for detection precision and result accuracy of the transgenic sugarcane.

Description

technical field [0001] The invention belongs to the field of biological technology, and in particular relates to a PCR primer sequence and an amplification method of an internal standard gene prenyl diphosphate delta-isomerase gene of sugarcane genome. Background technique [0002] Transgenic technology is becoming more and more mature, and the types of genetically modified sugarcane are becoming more and more diverse. At present, herbicide-resistant, insect-resistant, and disease-resistant genetically modified sugarcane has been field tested in 14 countries including the United States, Australia, India, and Brazil, creating conditions for the commercial cultivation of genetically modified sugarcane. In my country, sugarcane gene cloning and transgenic research only started in the mid-1990s. Although it started late, it has made rapid progress. The transgenic sugarcane that has been obtained includes insect-resistant genetically modified sugarcane, drought-resistant genetica...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12N15/10
Inventor 陈平华林冰陈忠伟陈利平施桂姣张卓项慰刘迪夏峰王恒波高三基许莉萍陈如凯
Owner FUJIAN AGRI & FORESTRY UNIV
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