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

A technology of PCR-DHPLC and detection method, which is applied in the field of multiple PCR-DHPLC detection primers and detection of transgenic cotton, and achieves the effects of good expansion performance, high sensitivity and resolution, 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 technology for transgenic cotton (PCR-DHPLC)

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1D

[0043] Example 1DNA Extraction

[0044] Sample DNA was extracted by CTAB method, as follows:

[0045] 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;

[0046] 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;

[0047] c) Add 5 μL RNase (10 mg / mL), and bathe in water at 37°C for 30 minutes;

[0048] d) Add an equal volume of Tris saturated phenol, mix thoroughly, and centrifuge at 12000r / min for 15min;

[0049] e) Take the supernatant, add an equal volume of chloroform / isoamyl alcohol (24:1) to mix, and centrifuge at 12000r / min for 15min;

[0050] f) Take the supernatant, add an equal volume of chloroform / isoamyl alcohol (24:1) to mix, and centrifuge at 12000r / min for 15min;

[0051] g) Add an equal volume of pre-cooled isopropanol, shake gently, ...

Embodiment 2D

[0054] Example 2 DNA concentration determination

[0055] 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:

[0056] DNA purity = OD260 / OD280

[0057] DNA concentration=50×OD260mg / mL

[0058] The purity ratio of DNA was between 1.7 and 1.9, and the concentration was greater than 10ng / μL.

Embodiment 3

[0059] Embodiment 3PCR amplification

[0060] According to the sad I gene, transgenic lines MON531 and MON15985 design the binding site of the specific detection primer, design the specific primer binding site of the strain according to the transgenic line MON1445, and add the regulatory sequence at the 5' end of the above primer binding site, Synthesize detection primer pairs containing regulatory sequences (Table 1).

[0061] Table 1 Transgenic cotton detection primer binding sites and primers

[0062]

[0063] 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.

[0064] PCR reaction conditions: denaturation at 94°C for 1min; then 35 cycles of 94°C for 30s, 57°C for 1min, and 72°C for 1min; after the cycle, extend at 72°C for 10min.

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Abstract

The invention discloses a multiplex PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) detection primer and a detection method for genetically modified cotton. 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, and multi-target detection of genetically modified cotton is realized. The DHPLC is used to analyze PCR amplified products, fragment size resolution can reach multiple bases, and resolution ratio is high. The multiplex PCR-DHPLC detection primer and the detection method for genetically modified cotton have the advantages that the detection method is simple, convenient, effective, reliable, high in throughput and especially suitable for departments of port inspection and quarantine, agricultural production, plant protection and the like.

Description

technical field [0001] The invention relates to the detection of a transgenic product, in particular to a multiplex PCR-DHPLC detection primer and detection method for transgenic cotton. Background technique [0002] The current detection methods for transgenic cotton mainly include conventional PCR, real-time fluorescent PCR, PCR-gene chip and so on. The scalability of traditional conventional PCR detection methods has certain limitations. 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 the workload is relatively large. ; On the other hand, the commonly used gel electrophoresis method for analyzing 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 to...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11G01N30/02
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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