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RNAi fragment for interfering with object sequence and RNAi expression vector

A technology of target sequence and interference target, applied in DNA/RNA fragments, introduction of foreign genetic material using vectors, DNA preparation, etc., can solve the problems of high cost, complicated operation process, time-consuming and labor-intensive, etc.

Active Publication Date: 2011-02-23
深圳华大基因农业控股有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method requires multi-step enzyme digestion and connection, the operation process is complicated, time-consuming and labor-intensive
Or use artificial synthesis of the entire forward and reverse fragments and loop loop sequence, but the cost is high, and the effect of direct enzyme digestion is poor

Method used

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  • RNAi fragment for interfering with object sequence and RNAi expression vector
  • RNAi fragment for interfering with object sequence and RNAi expression vector
  • RNAi fragment for interfering with object sequence and RNAi expression vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Embodiment 1: Design of oligo sequence of RNAi interference BGIosR1 gene

[0056] The full sequence of the BGIosR1 gene is as follows:

[0057] ATGGGAGCTAATTGCTGTATAGCTGCAAAAGAGAGGCCTCAGCCATGTGTTACTCCAATTGAAGTGTCAGCATTCAGGAACGTAAGACACTCTCCATCATGGAGCTTCCGGTGGGATAACCGCACACACATAGAAGACATAATGGAAATGCCCGCATTGTTTTCCAACCATAGTAGTGGAAGCATTCGGCCTGAAACAAAGAGTGGCTCAATTGCGCCAACTGATGGCTTTTCTAACGGAGGCAGCCCTTCTGATATGTTCAACAAGCTCAAGTGCCATAAATCTGACAGGAAGAGGGAGTCATCTAAGATTGCGAGATCTGACCTTCGAGCAGGTAGATCCACCACAAGCAATTCTTCCCCTGAGGCAAAGCTTTCCAGAAAATCGCTAGACACAGTAAGTGTGGCTTCAGATTCGAAGATGTCAATCTCTGTTCCTTCAACGCCGCCTGCTATATCCAGAGCAGATCCTTCATCATCCTCCAGGGGGCATTCTCTACCAACAGATGCAGATTCAATGAGGAAAGCACG AGGGCGCCTCTCCTGAAGGAAGGCCATCCTCCTCCATGCTCTCAGTCTGCAGCAACGATTTATCTGCAGTAGGATCACATGGCGAGTCATCTGATGGTTGGTCAATGCGAACATTTTCTGAAATGGTCGCATCATCTCAGAGAGAGAGATGGTCAGTTGATAGTGAGCTCTTAGGTTCTGTTTCGAGTAAAATGACAAGATCAAATGCTTCTAATAATCCTACTACACATTCACCTGACCAAGAGGTGTGCAAGCTATGTTTAAAGCTATTAAAGGAACGGTCCACATGGAATGCTCAGGAACTGGC...

Embodiment 2

[0075] Embodiment 2: RNAi interferes with the preparation of the interference fragment of BGIosR1 gene

[0076] Linker sequence phosphorylation system: 20μl

[0077] wxya 2 O 14 μl

[0078] 10×PNK Buffer A 2μl

[0079] 10×ATP 2μl

[0080] PNK 1μl

[0081] Primer 1μl

[0082] React at 37°C for 30min

[0083] The 5 linker oligo sequences were phosphorylated according to the above steps.

[0084] where PNK is T 4 polynucleotide kinase (T 4 Polynucleotide Kinase) (available from enzymematics company).

[0085] Interference fragment preparation reaction:

[0086] Adapter sequence F1 5μl

[0087] Adapter sequence F2 5μl

[0088] Adapter sequence R1 5μl

[0089] Adapter sequence R2 5μl

[0090] Adapter sequence M1 10μl

[0091] Mix the above phosphorylated 5 oligo sequences according to the above ratio, react in a water bath at 94°C for 45s, and then naturally cool to room temperature, and the obtained interference fragment RNAiF( image 3 ), whose sequence is: 5'GAT...

Embodiment 3

[0093] Embodiment 3: RNAi interferes with the oligo sequence design of BGIosR2 gene

[0094] The full sequence of the BGIosR2 gene is as follows:

[0095] ATGACGTGTCACTCTGCCATGGCGGCTCTCACCGTTGGGCACGCGGCGATCGTCCATGCCACCACGCGTCTCGAGGACGCCCGGTCCACCGGCCGTCGGCGGCGGCGGCGGGGGATGATCACGGTGCGCGCGGCGGCGGCGGCGACGAGCGGGTGGGAGCCGGGCAGCTGGAGGGCGCGGCCGGCGGCGAGGCAATCCCGGCGTGG GGGGACGCCGCCATGGGGCGGGCCTTCCTCCTGCAGGGCGGCGACTGCGCCGAGAGCTTCAAAGAGTTCGCCGCCAACAACATCCGCGACACCTTTCGCCTCATGCTCCAGATGGCCGTCGTCCTCACCTTTGGCGGCCAGATGCCCACCATCAAGGTTGGAAGGATGGCTGGCCAATTTGCAAAGCCAAGATCAAACCCAACTGAGACTATAGATGGAGTGACACTTCCTTCCTATCGAGGTGATATTATTAACAGCGATGGTTTTGATGAGAAGTCGCGTGCACCAGATCCTGAAAGGTTAATTAGAGCCTACAGCCAGTCCGCGAGCACCCTGAATCTTCTGAGAGGATTTGCTCATGGAGGGTATGCAGATCTGCAGAGAGTCACCCAGTGGAATCTTGACTTCTTGAGGGACAGCACGCAAGGGGACAGGTATATGGAGCTGTCCGAGAGGGTTCACGATGCCATCGGATTTATGGTTGCTGCTGGTCTGACTCCTCAGCATCCCATCATGACGACGGCTGAATTCTGGACATCTCATGAGTGCCTTCATTTGCCATATGAGCAAGCATTGACTAGGGTGGACTCCATTTCTGGGCTTTACTACGATTGCTCTGCTCATATG...

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Abstract

The invention belongs to molecular biology, and relates to an RNAi fragment for interfering with an object sequence and a preparation method thereof. The method comprises the following steps of: obtaining a theoretical sequence capable of forming a neck ring structure according to the object sequence, designing and piecewise synthesizing the sequence and a reverse complementary sequence of the sequence according to the theoretical sequence, phosphorylating the piecewise synthesized sequences, then mixing, denaturizing and annealing the phosphorylated sequences, and connecting the annealed sequences and an enzyme-cut RNAi expression vector to obtain an RNAi expression vector for interfering with the object sequence. The invention also relates to a recombinant vector containing the RNAi fragment for interfering with the object sequence and a preparation method thereof, and application of the RNAi fragment in constructing the RNAi expression vector. The method for constructing the RNAi expression vector can simplify the experiment steps, save the experiment time and reduce the experiment cost.

Description

technical field [0001] The present invention relates to molecular biology, in particular to an RNAi fragment for interfering with a target sequence, an RNAi expression vector, and a preparation and construction method thereof. Background technique [0002] RNA interference (RNA interference, RNAi) is an efficient, specific, and easy-to-operate gene silencing technology developed in recent years. Compared with antisense and co-suppression technology, it can obtain higher gene silencing efficiency. It is a phenomenon of degradation of homologous RNA mediated by double-stranded RNA (double-stranded RNA, dsRNA) in eukaryotes. In cells, the long dsRNA is cleaved into small interfering RNA (small interfering RNA or short interfering RNA, siRNA) of 21-26 nucleotides (nucleotide, nt) by Dicer enzyme similar to RNase III. Subsequently, the siRNA binds to the protein complex to form the RNA-induced silencing complex (RISC), and melts. The active RISC binds to its complementary trans...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/113C12N15/10C12N15/66
Inventor 张耕耘全志武夏秋菊倪雪梅
Owner 深圳华大基因农业控股有限公司