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

A technology of PCR-DHPLC and transgenic corn, applied in biochemical equipment and methods, recombinant DNA technology, measuring devices, etc., to achieve high-throughput detection methods, good scalability, 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

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 DNA Extraction

[0028] Sample DNA was extracted by CTAB method, as follows:

[0029] 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;

[0030] 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;

[0031] c) Add 5 μL RNase (10 mg / mL), and bathe in water at 37°C for 30 minutes;

[0032] d) Add an equal volume of Tris saturated phenol, mix thoroughly, and centrifuge at 12000r / min for 15min;

[0033] e) Take the supernatant, add an equal volume of chloroform / isoamyl alcohol (24:1) to mix, and centrifuge at 12000r / min for 15min;

[0034] f) Take the supernatant, add an equal volume of chloroform / isoamyl alcohol (24:1) to mix, and centrifuge at 12000r / min for 15min;

[0035] g) Add an equal volume of pre-cooled isopropanol, shake gently,...

Embodiment 2

[0038] Example 2 DNA concentration determination

[0039] 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:

[0040] DNA purity = OD260 / OD280

[0041] DNA concentration=50×OD260mg / mL

[0042] The purity ratio of DNA was between 1.7 and 1.9, and the concentration was greater than 10ng / μL.

Embodiment 3

[0043] Embodiment 3 PCR amplification

[0044] Design specific detection primer binding sites according to transgenic maize lines MON863, NK603, MON810, T25, MON88017, MIR604, 59122, and add at the 5' end of the specific detection primer binding sites of MON810, T25, MON88017, MIR604 lines Regulatory sequence, synthetic detection primers containing regulatory sequences (Table 1), synthetic primer pair 1 upstream / downstream primer specific binding site, primer pair 2 upstream / downstream primer specific binding site, primer pair 7 upstream / downstream primer specificity The binding site and the primer pair 3 up / downstream primers adding regulatory sequences, the primer pair 4 upper / downstream primers adding regulatory sequences, the primer pair 5 upper / downstream primers adding regulatory sequences, the primer pair 6 upper / downstream primers adding regulatory sequences The downstream primers were used as primers for PCR amplification, and the PCR sample settings included the mixe...

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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 maize. 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 the genetically modified maize 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 maize 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 and the like.

Description

technical field [0001] The invention relates to the detection of a transgenic product, in particular to a multiple PCR-DHPLC detection primer and a detection method of a transgenic corn. Background technique [0002] Currently, detection methods for transgenic corn mainly use multiplex PCR, multiplex real-time fluorescent PCR, PCR-gene chip and other detection methods. The traditional multiplex conventional PCR detection method has certain limitations in the platform expansion method. 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. On the other hand, the commonly used gel electrophoresis method to analyze the amplified 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...

Claims

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Application Information

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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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