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High-efficiency separation method for lower molecular multiplex PCR-amplified fragments

A separation method and a technique for amplifying fragments, which are applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve the requirements of limited resolution and the inability to meet the detection requirements of multiple PCR small molecule amplified fragments, Complexity and other issues to achieve the effect of improving purity

Inactive Publication Date: 2010-05-12
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the complexity of the multiplex PCR reaction system, the requirements for subsequent separation methods are more stringent. Traditional agarose gel electrophoresis for separation and analysis has limited resolution and cannot meet the requirements for the detection of multiplex PCR small molecule amplified fragments.

Method used

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  • High-efficiency separation method for lower molecular multiplex PCR-amplified fragments
  • High-efficiency separation method for lower molecular multiplex PCR-amplified fragments
  • High-efficiency separation method for lower molecular multiplex PCR-amplified fragments

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1 Genomic DNA extracted from crispy rice by CTAB method

[0070] 1) The rice cracker sample containing corn components was ground into powder in liquid nitrogen.

[0071] 2) Weigh 200mg and add it into a 1.5mL Eppendof centrifuge tube, and set up a negative control at the same time.

[0072] 3) Add 1000 μL of CTAB solution, oscillate evenly, incubate at 65° C. for 40 minutes, mix at least 5 times, and centrifuge at 12,000 rpm for 10 minutes at 4° C.

[0073] 4) Carefully pipette the supernatant into another new tube, add an equal volume of Tris-saturated phenol:chloroform:isoamyl alcohol (25:24:1) to the supernatant, shake well, and centrifuge at 12,000 rpm for 15 min at 4°C.

[0074] 5) Repeat step (4) once.

[0075] 6) Carefully pipette the supernatant into another new tube, add an equal volume of chloroform: isoamyl alcohol (24:1) to the supernatant, shake evenly, and centrifuge at 12000 rpm for 15 min at 4°C.

[0076] 7) Carefully pipette the supernatant ...

Embodiment 2

[0082] Embodiment 2 is done PCR detection to the DNA that embodiment 1 extracts

[0083] The DNA extracted in Example 1 was used as a template to amplify the rice crispy genome with the designed primers (as shown in Table 2). The PCR reaction system is as follows (30μL): 10×PCR buffer: 3μL (10×PCR buffer: 100mmol / L Tris-HCl, 500mmol / L KCl, 15mmol / L MgCl 2 ); 2.5mmol / L dNTP: 2.4μL; the primer concentration is 10μM, a total of three pairs of primers, each pair of primers upstream and downstream primers were added 1μL; Taq DNA polymerase: 0.4μL, the extracted DNA was added 1μl as a template, and used wxya 2 O water to make up to 30 μl. The PCR amplification conditions were as follows: pre-denaturation: 94°C for 5 min; denaturation: 94°C for 30 s; annealing: 60°C for 30 s; extension: 72°C for 30 s; final extension: 72°C for 7 min; a total of 35 cycles were set.

[0084] Table 2 Primer design

[0085]

[0086] Such as figure 2 As shown, multiple PCR products were detected ...

Embodiment 3

[0087] Example 3 Purification of Multiplex PCR Small Molecule Amplified Fragments Using Magnetic Bead Purification

[0088] 1) Take the multiplex PCR products in a 1.5ml Eppendorf centrifuge tube. Add an equal volume of magnetic bead binding solution (add 2.0mol / L NaCl, 1.0mol / L MgCl2, 15% PEG 6000 and 15% PEG 8000 to 100mmol / L Tris-Cl, 10mmol / L EDTA solution, the pH value 6.5) and 30uL 1mol / L DTT solution;

[0089] 2) Add 200uL magnetic beads. Before aspirating the magnetic beads, the reagent bottle containing the magnetic beads must be vortexed for 30 seconds to ensure that the magnetic beads are evenly suspended. Pipette repeatedly with a pipette or mix well with a vortex mixer to keep the magnetic beads in a suspended state for 5 minutes.

[0090] 3) Place the centrifuge tube on the magnetic rack and let it stand for 30 seconds. The liquid can only be absorbed when the magnetic beads are completely adsorbed on the side of the centrifuge tube near the magnetic rack. The ...

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Abstract

The invention provides a high-efficiency separation method for lower molecular multiplex PCR-amplified fragments, which comprises: firstly, purifying lower molecular multiplex PCR-amplified fragments by using a magnetic bead purification method; and secondly, separating the lower molecular multiplex PCR-amplified fragments by using a capillary electrophoresis method. The method combining a multiplex PCR method and capillary electrophoresis can quickly screen transgenic plants. The multiplex PCR has high template-saving, reagent-saving and time-saving performance; when used to process the products of the multiplex PCR, the magnetic bead method can effectively remove the impurities from a PCR reaction system to make the PCR reaction system more suitable for performing capillary electrophoresis; and the capillary electrophoresis has the characteristics of low detection limit, high sensitivity and short detection time. The method can be safely used, successfully separate and identify the lower molecular multiplex PCR-amplified fragments, keep the detection time within 10.6 minutes and be widely used in fields such as food sanitation quarantine and clinical examination.

Description

technical field [0001] The present invention relates to a method, in particular to a method. The invention can successfully efficiently separate the amplified fragments of multiple PCR small molecules, and after the multiple PCR products are purified by magnetic beads, they can be better separated by capillary electrophoresis. Background technique [0002] The safety of genetically modified food has become one of the hot issues of global concern. In order to identify genetically modified crops, it is usually necessary not only to screen and detect the CaMV35S promoter and NOS terminator of the genetically modified crops, but also to identify the strains of the genetically modified crops and quantify the genetically modified components. [0003] The research on PCR molecular detection technology of deep-processed products has always been a difficulty and hot spot in the research of detection technology, because the genomic DNA fragments have been degraded into very small frag...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/68
Inventor 黄昆仑许文涛张春娇王龑罗云波戴蕴青
Owner CHINA AGRI UNIV
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