Method for detecting miRNA by ultra-branch rolling circle amplification triggered by connection reaction

A technology of rolling circle amplification and ligation reaction, applied in the fields of molecular biology and nucleic acid chemistry, which can solve problems such as unsatisfactory ligation efficiency and impact on detection sensitivity, so as to avoid the interference of background fluorescent signals, improve ligation efficiency, and improve sensitivity Effect

Inactive Publication Date: 2017-06-13
WUHAN UNIV
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  • Claims
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Problems solved by technology

But before this, it is necessary to connect the padlock probe to form a circle as the template for amplification, so the efficiency of the ligation reaction is also an important factor affecting the detect

Method used

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  • Method for detecting miRNA by ultra-branch rolling circle amplification triggered by connection reaction
  • Method for detecting miRNA by ultra-branch rolling circle amplification triggered by connection reaction
  • Method for detecting miRNA by ultra-branch rolling circle amplification triggered by connection reaction

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Embodiment 1

[0026] In this example, miRNA21 (sequence 5'-uagcuuaucagacugauguuga-3') was used as the detection target.

[0027] 1. Design and synthesis of 5' phosphorylated padlock probe P-21 and secondary primer S-1

[0028] (1) The sequence of the phosphorylated padlock probe P-21 at the 5' end is:

[0029] 5'- CTGATAAGCTA TTTGCATTTCAGTTTACGGTTTAGCATTTCGCAATTTT TCAACATCAGT -3'.

[0030] Among them, the underlined sequences at both ends can be complementary paired with miRNA21 (miR-21), respectively, and there is a sequence in the middle with a length of 38 bases.

[0031] (2) The sequence of the secondary primer S-1 is: 5'-AGTCTGATAAGCTA TTTGCA-3', which is identical to the partial sequence of the phosphorylated lock probe, and can be complementary to the linear rolling circle amplification product, thereby triggering hyperbranched rolling circle amplification. increase.

[0032] 2. Detection of miRNA21

[0033] (1) Ligation reaction

[0034] In 10μL ligation reaction system, ad...

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Abstract

The invention discloses a method for detecting miRNA by ultra-branch rolling circle amplification triggered by a connection reaction, and belongs to the field of molecular biology and nucleic acid chemistry. The method is mainly composed of three parts, wherein the first part is to mutually supplement and pair two terminals of a lock type probe of 5'end phosphorylation and a target marker minRNA, and then connect them by a SplintR ligase to be a ring; the second part is an ultra-branch roll ring amplification; after the lock type probe is used as a template after forming to be a ring, the target market miRNA is used as a primer; under the function of phi 29 DNA polymerase, the linear roll ring amplification is generated; through introducing a second-grade primer, the linear roll ring amplification is expanded along a branch chain direction, so as to further amplify the signal; the third part is to add SYBR GREEN I dye in the roll ring amplification product to detect. The method is simple in operation, low in cost, single in system, high in sensitivity, free from the background fluorescence signal disturbance, and suitable for detecting miRNA in a complex biotic environment.

Description

technical field [0001] The invention belongs to the fields of molecular biology and nucleic acid chemistry, and in particular relates to a method for detecting miRNA by hyperbranched rolling circle amplification triggered by a ligation reaction. Background technique [0002] miRNA (microRNA) is a class of non-coding single-stranded ribonucleic acid molecules encoded by endogenous genes with a length of about 18-25 nucleotides, which participate in post-transcriptional gene expression regulation in animals and plants. miRNA and siRNA have similar lengths, and both are cut by Dicer enzyme. But the difference is that the mature miRNA is processed once by the original pri-miRNA to form pre-miRNA (microRNA precursor), and then processed to form mature miRNA. These processing processes require Dorsha enzyme and Dicer enzyme. In terms of biological function, the complete complementary pairing of miRNA and target messenger RNA will lead to the degradation of the messenger RNA of th...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/682C12Q2525/207C12Q2531/125C12Q2563/107C12Q2533/107
Inventor 周翔刘奕侬王少儒田沺张小娥黄俊捷杨伊文
Owner WUHAN UNIV
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