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Method for detecting insertion flanking sequence and copying quantity of Tgf2 transposon in goldfish genome

A technology of flanking sequences and transposons, which is applied to the detection of Tgf2 transposon insertion flanking sequences and copy numbers in the goldfish genome, achieving high-efficiency and low-cost effects

Inactive Publication Date: 2012-08-08
SHANGHAI OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on a method for detecting the flanking sequence and copy number of Tgf2 transposon insertion in the goldfish genome

Method used

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  • Method for detecting insertion flanking sequence and copying quantity of Tgf2 transposon in goldfish genome
  • Method for detecting insertion flanking sequence and copying quantity of Tgf2 transposon in goldfish genome
  • Method for detecting insertion flanking sequence and copying quantity of Tgf2 transposon in goldfish genome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Extraction of fish genomic DNA with Tgf2 transposon insertion

[0041] Due to the insertion of Tgf2 transposon in the goldfish genome, take about 100-200 mg of caudal fin of 95% alcohol or live goldfish, cut it into pieces, and dry at 55°C for 5-6 minutes; add 410 μL of STE lysate (pH=8.10), 80 μL SDS (10%, pH=8.14) and 10 μL of proteinase K (20 μg / ml); turn over and shake well, and keep warm in a 56°C water bath for 14-15 hours; add 340 μL to 400 μL of saturated sodium chloride solution and mix well, shake gently 3min; add 340μL to 400μL of chloroform, shake well; centrifuge at 12000rpm for 20min at 4°C, transfer the supernatant to another clean centrifuge tube; add 450μL to 500μL of isopropanol (-20°C), and centrifuge at 12000rpm for 20min (4 ℃); pour off the supernatant, save the precipitate, add 800μL of 75% ethanol to wash; centrifuge at 12000rpm for 5min to 15min (4℃); to remove ethanol, dry the precipitate, add 100μL to 200μL of double distilled water...

Embodiment 2

[0043] Synthesis of embodiment 2 Splinkerette linker

[0044] Entrusted Shanghai Shenggong Company to synthesize a long oligonucleotide sequence (SEQ ID No: 1) and a short oligonucleotide sequence (SEQ ID No: 2) required for the splinkerette adapter; The short sequence was dissolved in sterile double-distilled water to 50 μm / μl, and 50 μl of oligonucleotide long sequence and short sequence were mixed in equal volumes, incubated at 95°C for 5 minutes, and then gradually lowered to 4°C at 1°C / 15s on the PCR instrument , The long sequence and the short sequence can form a splinkerette joint during the annealing process, and store at -20°C. The synthetic splinkerette linker has the "GATC" cohesive end of restriction endonuclease Sau3A1 on one side, and a 7-bp hairpin structure formed by complementary AT base pairing in the short sequence to form a mismatch region on the other side figure 2 ).

Embodiment 3

[0045] Example 3 Digestion of Goldfish Genomic DNA and Ligation of Splinkerette Adapters

[0046] The goldfish genomic DNA containing the Tgf2 transposon extracted in Example 1 was digested with the restriction endonuclease Sau3A1 in a water bath at 37°C for 12 hours. The digestion reaction was carried out in a 30 μl system containing 10 μl Genomic DNA (2.5 μg), 3μl 10×Sau3A1 Restriction buffer, 4μl Sau3A1 enzyme (10U / μl), 0.3μl Acetylated BSA (10mg / ml), 12.7μl ddH 2 O. The digested products were subjected to 1.2% agarose gel electrophoresis, using TBE as a buffer, and electrophoresis at a voltage of 5V / cm to detect the effect of enzyme digestion ( image 3 ). The Sau3A1 digested DNA product of the complete goldfish genome was placed in a water bath at 65°C for 20 minutes to terminate the activity of the restriction endonuclease, and stored at -20°C for future use. Ligate 4.5 μl of the Sau3A1-digested DNA product and 1 μl of the splinkerette adapter with 4 μl of T4 DNA li...

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Abstract

The invention relates to a method for detecting the insertion flanking sequence and copying quantity of a Tgf2 transposon in a goldfish genome. The method comprises the following steps of: extracting the DNA (Deoxyribose Nucleic Acid) of a goldfish genome with a Tgf2 transposon; performing restrictive incision enzyme digestion on the DNA of the genome; connecting an enzyme-digested DNA fragment with a splinkerette joint; and performing primary PCR (Polymerase Chain Reaction) and secondary PCR respectively to amplify flanking sequences of transposon insertion loci 5' and 3' respectively; and performing cloning and sequence measurement on a product of the secondary PCR to detect the flanking sequence of the transposon in a fish genome and presume an inserted copying quantity. The method has the advantages of easiness, convenience, high efficiency and low cost, and a rapid and accurate method can be provided for the detection of the copying quantities and flanking sequences of other fish genome insertion mutation and transgenic descendants mediated by the Tgf2 transposon.

Description

technical field [0001] The present invention relates to the field of fish genetic engineering, specifically, a method for detecting the flanking sequence and copy number of Tgf2 transposon insertion in the goldfish genome, and its application in the detection of goldfish Tgf2 transposon insertion site, suitable for Rapid and accurate identification of genomic insertion copy number and flanking sequences in goldfish and farmed fish Tgf2 transposon-mediated transgenic or insertional mutagenesis offspring. Background technique [0002] DNA transposon (transposon) is a kind of mobile genetic element that can change the insertion position in the host genome, and the process of changing the insertion position is called transposition. Genomic insertional mutagenesis mediated by many different transposons (such as P factor and Mos1) has been used as a tool for large-scale genetic screens, not only in unicellular organisms such as bacteria and yeast, but also in multicellular organis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 邹曙明田玉梅蒋霞云
Owner SHANGHAI OCEAN UNIV
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