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A small RNA detection kit and quantitative method based on unbiased recognition and constant temperature amplification

A detection kit and isothermal amplification technology, applied in the field of small RNA detection, can solve problems such as affecting enzyme recognition ability and inaccurate detection results, achieve rapid enzyme-coordinated cascade isothermal amplification reaction, improve sensitivity, and sample demand. less effect

Active Publication Date: 2018-04-17
XI AN JIAOTONG UNIV
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Problems solved by technology

Although the 3' end methylation of these small RNAs does not change the nucleotide sequence, it brings great challenges to the existing detection technologies based on enzyme excision, enzyme polymerization, and enzyme ligation. These enzyme reactions need to be combined with The 3' end of the small molecule RNA acts, and the methylation of the 3' end will affect the recognition ability of the enzyme, inhibit the efficiency of the enzyme reaction, and eventually lead to inaccurate detection results

Method used

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  • A small RNA detection kit and quantitative method based on unbiased recognition and constant temperature amplification
  • A small RNA detection kit and quantitative method based on unbiased recognition and constant temperature amplification
  • A small RNA detection kit and quantitative method based on unbiased recognition and constant temperature amplification

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Embodiment 1

[0047] A small RNA detection kit based on unbiased recognition and constant temperature amplification, including:

[0048] A composition that forms a ternary hybrid structure with target small RNA, including 10nM 3-WJ primer and 10nM3-WJ template:

[0049] The 3-WJ primer consists of two parts of the sequence, the 5'end is complementary to the 3'end of the target small RNA, and the 3'end is complementary to the middle section of the 3-WJ template;

[0050] The 3-WJ template consists of three parts of the sequence, the 3'end is complementary to the 5'end of the target small RNA, the middle segment is complementary to the 3'end of the 3-WJ primer, and the 5'end contains two nucleic acid nickase recognition sites The SDA template region of dots; one of the restriction sites has been modified by sulfo;

[0051] The nucleotide sequence of the 3-WJ primer is (5’to 3’):

[0052] GTG CTC ACT CAT CCA AAA(SEQ.ID.NO.1)

[0053] The nucleotide sequence of the thio-modified 3-WJ template containing ...

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Abstract

The invention discloses a small RNA (ribonucleic acid) detection kit and quantitative method based on unbiased recognition and isothermal amplification. Through nucleic acid complementary hybridization, target small RNA specifically identifies a 3-WJ primer and a 3-WJ template, a stable three-way cross structure is formed, and the 3-WJ primer starts an SDA (strand displacement amplification) reaction along with the phosphorothioate-modified 3-WJ template and produces a large number of single-chain SDA products with restriction enzyme cutting sites. The single-chain DNA opens stem-loop structures of molecular beacons to enable fluorescence to recover. A double-chain complementary structure formed by the molecular beacons and the single-chain SDA products contains nucleic acid nickase recognition sites, the sites are recognized by nickase after the double-chain structure is formed, the cut molecular beacons fall off from the double-chain structure to produce fluorescence signals, and the released SDA products and new molecular beacons can form hybrid double chains and produce more fluorescence signals. The quantitative method and the kit are high in sensitivity, and cascade amplification of the SDA reaction is realized skillfully by the aid of the phosphorothioate-modified template.

Description

Technical field [0001] The invention belongs to the technical field of small RNA detection, and specifically relates to a small RNA detection kit and a quantitative method based on unbiased recognition and constant temperature amplification. Background technique [0002] Small RNA is a type of cell-endogenous non-protein-coding RNA molecule that is widely present in eukaryotes. Its length is about 20-30 bases. It mainly includes small interfering RNAs (siRNAs), microRNAs (miRNA) and piwi interacting RNA (piRNA). Small RNA binds to a specific Argonaute family protein (AGO protein) to guide AGO to approach its target molecule (DNA or RNA), and specifically reduces the expression of target genes through RNA-induced silencing complexes. Small RNA participates in the regulation of cell growth, development, differentiation, proliferation and apoptosis and other life processes, affects almost all signal pathways, participates in various physiological and pathological processes, and pla...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6851C12Q1/6844
Inventor 赵永席陈锋赵越
Owner XI AN JIAOTONG UNIV
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