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Method for detecting microRNA (microribonucleic acid) on basis of rolling circle amplification and upconversion material

A technology for converting materials and detecting probes, applied in the fields of molecular biology and nucleic acid chemistry, can solve the problem of high DNA dye background, and achieve the effects of overcoming light scattering, good sequence selectivity, and long fidelity time

Inactive Publication Date: 2016-04-20
武汉顺可达生物科技有限公司 +1
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Problems solved by technology

However, due to the presence of double-stranded parts in the pre-amplification system, DNA dyes have the problem of high background

Method used

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  • Method for detecting microRNA (microribonucleic acid) on basis of rolling circle amplification and upconversion material
  • Method for detecting microRNA (microribonucleic acid) on basis of rolling circle amplification and upconversion material

Examples

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Embodiment 1

[0028] 1. Design of microRNAs detection system

[0029] (1) if figure 1 As shown, the microRNAs detection probes used in the examples were synthesized by Shanghai Sangong Co., Ltd., and the probes were synthesized against the microRNAslet-7a sequence. The sequence of the microRNAs is: UGAGGUAGUAGGUUGUAUAGUU; the 3' end segment of the microRNA detection probe has 11 bases are complementary paired with the 5' end of the target microRNA, 11 bases are complementary paired with the 3' end of the target microRNA in the 5' end segment, and the middle segment is single-stranded DNA. The probe sequence from 5' end to 3' end is: phospho-CTACTACCTCATTTGCATTTCAGTTTACGGTTTAGCATTTCGCAATTTTAACTATACAAC. The added primer is a DNA sequence modified with biotin at the 5' end, which is exactly the same as a sequence in the probe. The primer sequence is: biotin-CAACCTACTACCTCATTTGC. In addition, the DNA modified on the upconversion material is characterized by the DNA sequence modified by the am...

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Abstract

The invention relates to a method and kit for detecting microRNA (microribonucleic acid). The method comprises the following steps: adding a microRNA detection probe into a microRNA-containing system so that the terminal region of the probe is combined with the target microRNA in a base pairing mode, and adding a T4 DNA (deoxyribonucleic acid) ligase so that the 3' end of the probe is connected with the 5' end to form a cyclic structure; adding a DNA primer of which the 5' end is modified with biotin, adding a Phi 29 DNA polymerase, and carrying out rolling circle amplification; after the amplification finishes, adding streptavidin magnetic beads into a system to be detected, taking with a ferromagnet, and eluting; adding a DNA-modified upconversion material into the system to be detected so that the single-chain DNA on the upconversion material is combined with the primer DNA taken by the magnetic beads in a base pairing mode; incubating, adsorbing the magnetic beads with the ferromagnet, eluting to remove the unpaired upconversion material, and outputting the left magnetic-bead-adsorbed upconversion material part as a signal; and finally, exciting by using 980nm exciting light on a fluorescence spectrophotometer, and detecting the microRNA content by using the obtained fluorescence signal of the upconversion material.

Description

technical field [0001] The invention belongs to the fields of molecular biology and nucleic acid chemistry, and relates to a method for detecting microRNA based on rolling circle amplification and up-conversion materials. Background technique [0002] Mature microRNAs (microRNAs; miRNA, also translated as small molecule RNA) are a type of single-stranded non-coding RNA found in eukaryotic cells that can regulate gene expression. Generally speaking, microRNA consists of about 22 nucleotides (the number of nucleotides varies from 18 to 25), and it widely regulates gene expression through transcription in animals, plants and viruses. microRNA inhibits post-transcriptional gene expression by specifically binding to target messenger ribonucleic acid (mRNA). Studies have shown that microRNA regulates more than half of human genes, and one microRNA can have hundreds of target genes. [0003] Due to the importance of microRNA in disease prediction, microRNA has become a new genera...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6844C12Q1/6876C12Q2600/178C12Q2521/501C12Q2521/101C12Q2531/125C12Q2563/107C12Q2563/143C12Q2563/149
Inventor 周翔肖珩王少儒杨雨青李娓
Owner 武汉顺可达生物科技有限公司
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