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PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) detection primer and detection method for genetically modified maize strain BT11

A technology of PCR-DHPLC and transgenic corn, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., to achieve reliable detection methods, good scalability, and easy operation

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

Currently, there is no PCR-DHPLC detection technique (PCR-DHPLC) for transgenic maize line BT11

Method used

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  • PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) detection primer and detection method for genetically modified maize strain BT11
  • PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) detection primer and detection method for genetically modified maize strain BT11
  • PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) detection primer and detection method for genetically modified maize strain BT11

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 65 °C water bath 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, pla...

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 transgenic maize BT11 strain, and add the regulatory sequence at the 5' end of the specific detection primer binding site to synthesize the detection primer containing the regulatory sequence (Table 1). / Downstream primers for PCR amplification, sample settings include: non-transgenic corn DNA, DNA of corn line MON88017, DNA of corn line MIR604, DNA of corn line Bt176, DNA of corn line Bt11, DNA of corn line MON810, and corn line GA21 DNA of corn line NK603, DNA of corn line MON863, DNA of corn line MON89034, DNA of corn line T25, DNA of soybean line A2704-12, DNA of soybean line A5547-12, DNA of non-transgenic canola, LLcotton25 DNA of the line, non-transgenic cotton DNA, DNA of the Bt63 line, DNA of the potato line EH92-527-1.

[0048] Table 1 Detection primer binding sites and primers of transgenic maize line BT11

[0049]

[0050] The total volume o...

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Abstract

The invention discloses a PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) detection primer and a detection method for genetically modified maize strain BT11. The primer has strong specificity and can be used for PCR amplification and DHPLC analysis. The detection method is simple and convenient to operate, good in expansion performance and high in sensitivity. The DHPLC is used to analyze PCR amplified products, fragment size resolution can reach multiple bases, and resolution ratio is high. The PCR-DHPLC detection primer and the detection method for genetically modified maize strain BT11 have the advantages that the detection method is simple, convenient, effective, reliable and especially suitable for departments of port inspection and quarantine and the like.

Description

technical field [0001] The invention relates to a detection of a transgenic product, in particular to a PCR-DHPLC detection primer and a detection method of a transgenic corn strain. Background technique [0002] At present, the detection methods of transgenic corn 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 real-time fluorescent PCR has advantages over conventional PCR detection methods in terms of detection sensitivity, due ...

Claims

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Application Information

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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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