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: 2012-04-04
SHENZHEN AUDAQUE DATA TECH
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  • Description
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Problems solved by technology

[0008] The purpose of the present invention is to provide a simple, pollution-free and efficient method for ...

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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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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 reagent: Takara DL 500 molecular weight marker, 6×Loading Buffer, 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: QIAquick Gel Extraction kit (QIAGEN);

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

[0136] (6) Main equipment: PCR instrument (Biometra T), electrophoresis instrument (BioRad 300Xi), UV crosslinking instrument (Spectronics, XJ-1000), gel image analyzer (CEE, GAS7001X), trac...

Embodiment 2

[0165] Example 2 MLPA long probe detection

[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 all the prepared MLPA long probes were subjected to agarose Gel electrophoresis was used to qualitatively detect whether the length of each probe was in line with the expected one. The agarose gel electrophoresis is specifically, using 3% gel, electrophoresis on the electrophoresis apparatus for 20-60min. The results show that the lengths of the MLPA single-stranded long probes prepared by the present invention are all in line with expectations, and the lengths of the probes increase in a gradient of about 20 bp. See image 3 .

[0168] 2. MLPA Long Probe Concentration Determination

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

Embodiment 3

[0172] Example 3 Detection of transgenic corn MLPA

[0173] Select the MLPA long probes, Probe810-2 and Probe88017-2, prepared above for the detection of transgenic maize lines MON810 and MON88017, and chemically synthesize the MLPA detection probes Zein-1 and Zein-2 of the endogenous gene Zein, and synthesize the detection probes. The short probe Probe810-1 for MON810 and the short probe Probe88017-1 for detecting MON88017 (Table 3) were applied to the MLPA detection of transgenic maize. The MLPA reaction products were analyzed by ABI3100 sequencer. The results showed that MON810, MON88017 and the endogenous gene Zein 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

The invention relates to the field of nucleic acid molecule detection and particularly discloses a multiplex ligation-dependent probe amplification (MLPA) long probe preparation method, a transgene corn MLPA long probe, a transgene corn MLPA short probe and a transgene corn MLPA detection method. The MLPA long probe preparation method can be carried out by adopting conventional laboratory technology of asymmetric polymerase chain reaction (PCR) amplification, enzyme restriction, gel electrophoresis, cut gel recovery and the like. Compared with the existing preparation method, the method has the advantages that the operation is simple, the cost is low, and the operation can be realized in ordinary laboratories. The problem of limited application of the MLPA detection method caused by difficult long probe preparation and high cost is solved. The transgene corn MLPA long probe and the transgene corn MLPA short probe can be used for detecting transgene corn, and the basis is provided for the high-flux MLPA detection of the transgene corn. The transgene corn MLPA detection method carried out on the basis has good expansion performance and high specificity, a simple, convenient, effective and reliable high-flux detection method is provided for the transgene corn detection, and the method is particularly suitable for being used for departments such as inspection and quarantine departments and the like.

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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IPC IPC(8): C12Q1/68C12N15/11C12N15/10
CPCC12Q1/6844C12Q2600/13C12Q1/6895C12Q2600/16C12Q1/68C12Q2531/113C12Q2537/143C12Q2561/125C12Q2525/204
Inventor 凌杏园陈枝楠章桂明康林向才玉潘广陈菲
Owner SHENZHEN AUDAQUE DATA TECH
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