Gene chip for detecting transgenic cattle and preparation method and application thereof

A gene chip and transgene technology, which is applied in the field of gene chip detection of transgenic cattle, can solve the problems of lack of detection and monitoring means for transgenic animals and products, weak foundation of safety evaluation and detection of genetically modified animals and products, etc.

Inactive Publication Date: 2011-09-14
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

my country has a relatively weak foundation in the safety evaluation and testing of transgenic animals and products, and lacks accurate and effective testing and monitoring methods for some transgenic animals and products with great commercialization prospects

Method used

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  • Gene chip for detecting transgenic cattle and preparation method and application thereof
  • Gene chip for detecting transgenic cattle and preparation method and application thereof
  • Gene chip for detecting transgenic cattle and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029]Embodiment 1 transgenic animal

[0030] References for the preparation method of transgenic dairy cows with human lactoferrin "Yang P., Wang J., Gong G., et al. Cattle maternal bioreactor generated by a novel procedure of transgenic cloning for large-scale production of functional human lactoferrin[J] .PLoS One, 2008, 3(10):e3453".

[0031] References for the preparation method of cows with transgenic recombinant human alpha-lactalbumin in the milk of transgenic cloned cows[J].J Dairy Sci, 2008, 91(12): 4466-4476".

[0032] References for the preparation method of transgenic human lysozyme cows "Z Yu, Q Meng, H Yu. Expression and Bioactivity of Recombinant Human Lysozyme in the Milk of Transgenic Mice [J]. Journal of Dairy Science, 2006, 89(18): 2911 ~2918".

[0033] References for preparation method of transgenic pigs with Omega-3 fatty acid desaturase gene "Pan Dengke, Zhang Li, Zhou Yanrong. Somatic cell nuclear transfer to produce sFat-1 cloned pigs with omega-3 f...

Embodiment 2

[0034] Design and synthesis of embodiment 2 primers and probes

[0035] According to the published mitochondrial genes (BOS mtDNA16s rRNA, Ovis mtDNA12s rRNA, Sus NADH5) of cattle, sheep, and pigs in Genbank, the respective codes of the three proteins of human lactoferrin, human lysozyme, and human-α-lactalbumin Genes, as well as marker genes EGFP and NPT II related sequences commonly used in transgenic animals, were screened by DNAstar, primer5.0, DNAman, Mega, etc. and designed 8 pairs of detection primers and 16 specific probes. Synthesized by Shanghai Invitrogen Company. In order to facilitate the reading and judgment of the amplification products by the chip, the downstream primers were modified with TAMRA.

[0036] Table 1: Transgenic Chip Primer Sequences

[0037]

[0038]

[0039] Table 2: Transgenic chip probe sequences

[0040]

[0041]

Embodiment 3

[0042] The preparation of embodiment 3 gene chips

[0043] A 9×8 chip array was designed according to the detection probes (see figure 1 ). The array consists of 4 parts: QC is a spotting positive reference (ctr3) (SEQ ID No.35), which is a fluorescently modified nucleotide; NC is a spotting negative control; PC is a hybridization positive reference (ctr2) (SEQ ID No. .34), that is, TAMRA fluorescent modification is on the single strand of known sequence, which is complementary to the hybridization quality control probe (ctr1) (SEQ ID No.33); other sites are detection probes; each probe has three repeat points .

[0044] After diluting the probes in Table 2 to 12.5 μM, use the BioDotAD1500 chip spotter, according to the chip arrangement diagram ( figure 1 ) arrangement for spotting. After spotting, place the chip under an ultraviolet crosslinker for cross-linking (240mj), then wash with 0.2% SDS for 5-10min, and store in a dry place at room temperature and protected from li...

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Abstract

The invention discloses a gene chip for detecting transgenic cattle and a preparation method and application thereof. The chip provided by the invention contains solid phase carrier and an oligonucleotide probe fixed on the carrier, wherein the oligonucleotide probe contains a detection probe and a quality control probe; and the detection probe is a specific probe of mitochondrial genes of cattle, sheep and pig, human lactoferrin gene, human lysozyme gene, human-alpha-lactoalbumin gene, green fluorescent protein gene and neomycin phosphotransferase gene. By adopting the gene chip and the construction method to detect transgenic cattle, the accuracy, specificity and sensitivity are all higher; and the gene chip is suitable for the high throughout screening of transgenic cattle.

Description

technical field [0001] The invention belongs to the field of animal molecular biology, in particular to a gene chip for detecting transgenic cattle, its preparation method and application. Background technique [0002] Transgenic technology refers to a modification technology that introduces artificially isolated and modified genes into the genome of organisms, introduces gene expression, and causes the traits of organisms to be heritable. Organisms modified by this technology are called genetically modified organisms, commonly known as Genetically modified organisms (GMOs). Transgenic animals are the purposeful, planned, well-founded, and predictable changes in the genetic composition of animals according to human wishes. It is a challenging experimental technology based on modern molecular biology and animal embryo and gamete bioengineering. Its research results have shown broad application prospects in improving production capacity, improving product quality, producing b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 韩雪清王慧煜林祥梅
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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