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Primer and method for PCR-DHPLC (polymerase chain reaction-denaturing high-performance liquid chromatography) detection for endogenous genes of wheat

A PCR-DHPLC and endogenous gene technology is applied to the PCR-DHPLC detection primers and detection fields of wheat endogenous genes, and achieves the effects of a reliable detection method, good expansion performance, high sensitivity and resolution

Inactive Publication Date: 2013-03-06
SHENZHEN AUDAQUE DATA TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no PCR-DHPLC detection technology (PCR-DHPLC) for wheat endogenous genes

Method used

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  • Primer and method for PCR-DHPLC (polymerase chain reaction-denaturing high-performance liquid chromatography) detection for endogenous genes of wheat
  • Primer and method for PCR-DHPLC (polymerase chain reaction-denaturing high-performance liquid chromatography) detection for endogenous genes of wheat
  • Primer and method for PCR-DHPLC (polymerase chain reaction-denaturing high-performance liquid chromatography) detection for endogenous genes of wheat

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 DNA Extraction

[0031] Sample DNA was extracted by CTAB method, as follows:

[0032] a) Weigh 5 g of the sample, add liquid nitrogen to the mortar and grind until the sample is a powder with a size of about 0.5 mm;

[0033] b) Weigh 300 mg of the ground sample, quickly transfer it to a 2 mL centrifuge tube, add 700 μL of CTAB extract solution preheated at 65 °C, mix well, and put it in a water bath at 65 °C for 30 min;

[0034] c) Add 5 μL RNase (10 mg / mL), and bathe in water at 37°C for 30 minutes;

[0035] d) Add an equal volume of Tris saturated phenol, mix thoroughly, and centrifuge at 12000r / min for 15min;

[0036] e) Take the supernatant, add an equal volume of chloroform / isoamyl alcohol (24:1) to mix, and centrifuge at 12000r / min for 15min;

[0037] f) Take the supernatant, add an equal volume of chloroform / isoamyl alcohol (24:1) to mix, and centrifuge at 12000r / min for 15min;

[0038] g) Add an equal volume of pre-cooled isopropanol, shake gently,...

Embodiment 2

[0041] Example 2 DNA concentration determination

[0042] The concentration and purity of the extracted sample DNA were measured; the absorbance values ​​at 260nm and 280nm were measured by an ultraviolet spectrophotometer, and the purity and concentration of nucleic acid were calculated respectively. The calculation formula is as follows:

[0043] DNA purity = OD260 / OD280

[0044] DNA concentration=50×OD260mg / mL

[0045] The purity ratio of DNA was between 1.7 and 1.9, and the concentration was greater than 10ng / μL.

Embodiment 3

[0046] Embodiment 3 PCR amplification

[0047] Design the binding site of the specific detection primer according to the wheat endogenous gene acc1, and add the regulatory sequence at the 5' end of the specific detection primer binding site, and synthesize the detection primer containing the regulatory sequence (Table 1). PCR amplification with upstream / downstream primers, sample settings include: DNA from 3 wheat samples and DNA from non-GM rapeseed, DNA from potato line EH92-527-1, DNA from non-GM rice, DNA from non-GM cotton, DNA from non-GM DNA from corn, DNA from papaya and water blank.

[0048] Table 1 The detection primer binding sites and primers of wheat endogenous gene acc1

[0049]

[0050]

[0051] The total volume of the PCR reaction system is 50 μL, and the components are: multiplex PCR reaction mixture Multiplex PCR Mix (TaKaRa) 25 μL, 10 μmol / L primers 1 μL, DNA 2 μL, 5U / μL Taq enzyme 0.25 μL, with sterilized double distilled water Make up to 50 μL.

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Abstract

The invention discloses a primer and a method for PCR-DHPLC (polymerase chain reaction-denaturing high-performance liquid chromatography) detection for endogenous genes of wheat. The primer is high in specificity and can be used for PCR amplification and DHPLC analysis. The method for detection for the endogenous genes of the wheat is simple in operation, good in extensibility and high in sensitivity. PCR amplification products are analyzed by means of DHPLC, the segment size resolution of the PCR amplification products can reach multiple basic groups, and the resolution ratio is high. The primer and the method have the advantages that the method is a simple, convenient, effective and reliable method for detection for the endogenous genes of the wheat, and the primer and the method are particularly suitable for port inspection and quarantine departments and the like.

Description

technical field [0001] The invention relates to a gene detection primer and a method, in particular to a PCR-DHPLC detection primer and a detection method of wheat endogenous genes. Background technique [0002] At present, the detection methods of wheat genes mainly adopt conventional PCR, real-time fluorescent PCR, PCR-gene chip and other detection methods. The traditional conventional PCR detection method has certain limitations in terms of platform expansion. With the increase of targets to be detected, it is necessary to re-optimize the amount and ratio of each set of primers in the system, and take into account factors such as amplification efficiency and workload. On the other hand, the commonly used gel electrophoresis method to analyze the amplification products has low discrimination efficiency and unsatisfactory detection results. Although multiple real-time fluorescent PCR has advantages over multiple conventional PCR detection methods in terms of detection sens...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 章桂明向才玉凌杏园潘广程颖慧康林李鹤遥
Owner SHENZHEN AUDAQUE DATA TECH
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