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Preparing method for tr-gene products detecting oligonucleotides chip and use thereof

A production method and technology of oligonucleotide probes, which are applied in the production of oligonucleotide chips and the detection of genetically modified products, and can solve the problems of inability to apply, detection probe sequences and target gene amplification primers to be detected, etc.

Inactive Publication Date: 2005-02-23
国家质量监督检验检疫总局动植物检疫实验所 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the instructions do not provide any information about the detection probe sequence and the amplification primers of the target gene to be detected, so it cannot be applied to actual work at all.

Method used

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  • Preparing method for tr-gene products detecting oligonucleotides chip and use thereof
  • Preparing method for tr-gene products detecting oligonucleotides chip and use thereof
  • Preparing method for tr-gene products detecting oligonucleotides chip and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0182] Modification of embodiment 1 slide

[0183] Soak clean glass slides in 95% acetone solution containing 1% 3-aminopropyltrimethylsilane for 2 minutes, soak in acetone for 5 minutes, repeat 6 times, bake at 110°C for 45 minutes, and then immerse the slides in 0.2% 1,4-Phenyl diisocyanate in 10% pyridine / DMF solution for 2 hours, washed once with acetone solution, dried at room temperature, and stored at 4°C for later use.

Embodiment 2

[0184] The design of embodiment 2 probe

[0185] heterologous insertion gene

probe

length

T m

CaMV35S promoter

TGCTCCACCATGTTGACGAAG1

21

61.6

FMV35S promoter

AAAACCAAGAAGGAACTCCCATC4

23

60.7

CAMV35 terminator

GAAACCCTTAGTATGTATTTGTATTTGTAA7

30

60.0

Nos promoter

GACCTTAGGCGACTTTTGAACG10

22

60.9

Nos terminator

AATCCTGTTGCCGGTCTTGC13

22

62.3

NptII

GCTCCTGCCGAGAAAGTATCC16

21

60.6

bar

CTGTGCCTCCAGGGACTTCA19

20

60.5

PAT

GCCACAACACCCTCAACCTCA22

21

62.8

Barnase

GTCTGAAGATAATCCGCAACCC25

22

60.4

Barstar

ACCTGGACGCTTTATGGGATT28

21

60.3

EPSPS (soybean)

GGAAAGGCCAGAGGATTTGC31

20

61.1

EPSPS (rape)

GGGTCTTGTTGGTGTTTACGATT34

23

60.1

Cry I A(b)

CAGCACGGGGTTGGTGTAGAT37

21 ...

Embodiment 3

[0190] The immobilization of embodiment 3 probe

[0191] Synthesize the above-mentioned probe (synthesized by TaKaRa Company), add TE (PH7.5) according to the given nmol number to make a 100uM stock solution, take 10ul and dilute it 5 times with TE (PH7.5) to make a 20uM concentration of 50ul ( It is best to use GeneQuant to quantify and adjust the concentration to be the same), from which 20ul and 20ul 0.1M PH 9.0 Na 2 CO 3 / NaHCO 3 The buffer solution was double-diluted to 40ul of the spotting solution with a final concentration of 10uM. At the same time, the spotting solution without oligonucleotide fragments was set up as a blank control for monitoring the background after hybridization.

[0192] Take 8 ul of each of the above spotting solutions in a 384-well plate, and spot the desired matrix with a spotting instrument. Each probe was repeatedly spotted 5 times, and the spot spacing was 500um. Place at 37°C for 1-2h, rinse once with 1% ammonia water, and dry for late...

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Abstract

A method for preparing oligonucleotide chip for transgene product detection and use are disclosed. It includes: designing specific oligonucleotide probe and related primer by heterogeneric inserting gene, species internal standard gene, specific boundary sequence in transgene product gene set, fixing probe on glass slide to form transgene product detecting chip, amplifying DNA of plant to be tested by related primmer, marking by fluorescence, and hybridizing with chip. It can be use to acquire variable information.

Description

technical field [0001] The invention relates to a nucleic acid detection technology, in particular to a method for making an oligonucleotide chip for detection of transgenic products and its application. Background technique [0002] With the rapid development of biotechnology, by introducing heterologous genes into the genome of natural plants, the transformation of their original genes can be realized, in order to obtain new plant varieties with optimized traits such as high nutrition and high stress resistance, and further improve the quality of plants. The quality of food and / or feed, which is a raw material, is currently being extensively studied and applied. [0003] According to the statistics of the International Agricultural Biotechnology Application Consulting Service Center (ISAAA), in 2001, the planting area of ​​genetically modified products in the world was 52.6 million hectares, and more than ten countries in the world have planted genetically modified product...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 缪海珍朱水芳李瑶黄文盛张谦黄新华杨喆毛裕民谢毅
Owner 国家质量监督检验检疫总局动植物检疫实验所
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