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Multiplex ligation-dependent probe amplification (MLPA) long probe preparation method, transgene corn MLPA long probe and detection method

A technology of transgenic corn and detection methods, applied in DNA preparation, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of cumbersome biological preparation methods of MLPA long probes

Inactive Publication Date: 2014-03-12
深圳出入境检验检疫局动植物检验检疫技术中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The purpose of the present invention is to provide a simple, pollution-free and efficient method for preparing MLPA long probes for the cumbersome biological preparation method of the above-mentioned MLPA long probes

Method used

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  • Multiplex ligation-dependent probe amplification (MLPA) long probe preparation method, transgene corn MLPA long probe and detection method
  • Multiplex ligation-dependent probe amplification (MLPA) long probe preparation method, transgene corn MLPA long probe and detection method
  • Multiplex ligation-dependent probe amplification (MLPA) long probe preparation method, transgene corn MLPA long probe and detection method

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Experimental program
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Effect test

Embodiment 1

[0129] The preparation of embodiment 1 transgenic maize MLPA long probe

[0130] 1. Materials and Equipment

[0131] (1) Asymmetric PCR amplification reaction reagents: 10×high-fidelity PCR buffer, 2.5mmol / L dNTP mixture, 50mmol / L MgSO4 solution, 10μmol / L upstream primer, 1μmol / L downstream primer, 5U / μL PlantinumTaq enzyme high-fidelity DNA polymerase, 1ng / μL PUC18 plasmid solution, sterilized distilled water;

[0132](2) Electrophoresis detection reagents: TakaraDL500 molecular weight marker, 6×LoadingBuffer, 10mg / mL ethidium bromide (EB), agarose, 1×TAE buffer;

[0133] (3) PCR product purification kit: QIAquick PCR Purification kit (QIAGEN);

[0134] (4) Gel extraction kit: QIAquickGelExtractionkit (QIAGEN);

[0135] (5) Materials: transgenic maize lines MON810 and MON88017;

[0136] (6) Main equipment: PCR instrument (BiometraT), electrophoresis instrument (BioRad300Xi type), ultraviolet crosslinking instrument (Spectronics, XJ-1000), gel image analyzer (CEE, GAS7001...

Embodiment 2

[0165] Embodiment 2 MLPA long probe detects

[0166] 1. MLPA long probe gel electrophoresis detection

[0167] Using the same method as above, the present invention also prepared long probes for detecting transgenic maize lines NK603, MON863, MON89034, MIR604, GA21, BT11, MON59122, MON3272, CBH351, LY038, and carried out all the prepared MLPA long probes on agarose Gel electrophoresis, to qualitatively detect whether the length of each probe is in line with the expectation. Specifically, the agarose gel electrophoresis is performed on an electrophoresis apparatus for 20-60 min using 3% gel. The result shows that the length of the MLPA single-chain long probe prepared by the present invention is all in line with expectations, and the length of the probe is progressively increasing in a gradient of about 20bp, see image 3 .

[0168] 2. Determination of MLPA long probe concentration

[0169] In order to facilitate the determination of the amount of the prepared long MLPA pro...

Embodiment 3

[0172] Example 3 Transgenic corn MLPA detection

[0173] Select the MLPA long probes prepared above for the detection of transgenic maize lines MON810 and MON88017, Probe810-2 and Probe88017-2, and chemically synthesize the MLPA detection probes Zein-1 and Zein-2 of the endogenous gene Zein at the same time, and synthesize and detect The short probe Probe810-1 of MON810 and the short probe Probe88017-1 of MON88017 (Table 3) were applied to the detection of MLPA in transgenic maize. The MLPA reaction product was analyzed by ABI3100 sequencer, and the results showed that MON810, MON88017 and the endogenous gene Zein all had amplification signals. Among them, MON810 has a 170bp amplification signal, MON88017 has a 252bp amplification signal, and the endogenous gene Zein has a 91bp amplification signal ( Figure 4 ), which is consistent with the theoretical length, which proves that the prepared MLPA long probe meets the design requirements and can be used for MLPA detection.

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Abstract

Disclosed are a method for preparing an MLPA long probe, an MLPA long probe and short probe for transgenic corn, and an MLPA detection method for transgenic corn. The MLPA long probe in the invention is prepared by using technologies such as asymmetric PCR amplification, enzyme digestion, gel electrophoresis and single-strand DNA gel extraction.

Description

technical field [0001] The invention relates to the field of nucleic acid molecular detection, in particular to a method for preparing a long probe for MLPA detection technology, the long probe for MLPA detection of transgenic corn prepared by the method, and the MLPA detection of transgenic corn. Background technique [0002] At present, gene detection is mainly based on PCR technology, which is fast, sensitive, accurate and reliable. However, the PCR method only analyzes one gene in a single tube, and the efficiency is low. At present, it is necessary to screen many transgenic varieties and strains one by one, the workload is too large and the cost is high. Therefore, there is an urgent need for a high-throughput detection method for genetic testing. [0003] Common high-throughput gene detection technologies include gene chip, multi-channel capillary electrophoresis, and DHPLC separation technology. However, these methods generally can only carry out "one-to-many" dete...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11C12N15/10
CPCC12Q1/6844C12Q2600/13C12Q1/6895C12Q2600/16
Inventor 凌杏园陈枝楠章桂明康林向才玉潘广陈菲
Owner 深圳出入境检验检疫局动植物检验检疫技术中心
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