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Method of two-way extension detection of miRNA of two nonoverlapped amplification primers based on miRNA bridging
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A technology for amplification primers and detection methods, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problem of low detection sensitivity, high cost of primer design, and inability to distinguish mature miRNA and precursor miRNA and other problems, to achieve the effect of strong specificity and high sensitivity
Active Publication Date: 2018-06-19
斯格特生物
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These methods have some problems in primer design and detection effect, such as high cost of primer design, low detection sensitivity, inability to distinguish mature miRNA from precursor miRNA (pre-miRNA), etc.
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[0052] Amplification buffer (including oligo1 and its stable sequence,
[0053] oligo2 and its stable sequence): 17...
Embodiment 2
[0071] [Example 2] Detection of miRNA specificity
[0072] Four members of the miRNA let7 family (sequences are shown in Table 2) were selected for related experiments, oligos were designed for each miRNA, and four miRNAs were detected simultaneously to verify the specificity of the method for detecting miRNAs.
[0073] Table 2 Sequences of four members of miRNA let7 family
[0074] miR-let-7b-5p
uggagguaguag guuguguguu
miR-let-7c-5p
ugagguaguag guuguauguu
miR-let-7e-5p
ugagguaggag guuguauaguu
miR-let-7d-5p
agagguaguag guugcauaguu
[0075] Table 3 Detection system for four members of the miRNA let7 family
[0076]
[0077]
[0078] A. Amplification of miRNA
[0079] The amplification system was prepared with probes for each miRNA, and the four miRNAs were simultaneously amplified. The specific amplification system is as follows:
[0080] miRNA (10fM): 2μL
[0081] Amplification buffer (including oligo1 and its stable...
Embodiment 3
[0089] [Example 3] Detection of miRNA sensitivity
[0090] Using miR-let-7b-5p, a member of the miRNA let7 family, to conduct related experiments, the synthesized miRNA was dissolved to 100 μM, and then 10-fold gradient dilution was performed to detect miRNAs with different dilutions. The detection results are as follows:
[0091]
[0092]
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Abstract
The invention discloses a method of two-way extension detection of miRNA of two nonoverlapped amplification primers based on miRNA bridging and belongs to the technical field of medical biology. The method adopts two primers: an amplification primer 1 and an amplification primer 2, during the detection of miRNA; the amplification primer 1 complements a half sequence at a 3' terminal of miRNA and comprises a T7 promoter and a tag sequence; the amplification primer 2 is the same as a half sequence of a 5' terminal of miRNA and comprises a tag sequence. Two-way extension of the two amplificationprimers can be achieved by the miRNA bridging by the action of reverse transcriptase, and a dsDNA (double-stranded deoxyribonucleic acid) molecule comprising the tag sequence, miRNA and the T7 promoter is formed. An RNA molecule comprising the tag sequence and miRNA is transcribed from the molecule by the action of T7 polymerase, and qualitative analysis can be performed on the RNA molecule according to a tag molecule. The method has the characteristics of high specificity, high sensitivity and high throughput and provides technical possibility for disease diagnosis taking miRNA as a biomarker.
Description
technical field [0001] The invention relates to the field of nucleic acid molecular detection, in particular to a method and a kit for bidirectionally extending two non-overlapping nucleotide fragments based on miRNA bridging to detect miRNA. Background technique [0002] miRNAs are very important in the regulation of gene expression, and up to 30% of mammalian genetic genes are regulated by miRNAs. So far, more than 1000 miRNAs have been found in the human genome. Mature miRNAs are a class of naturally occurring, small non-coding RNA molecules, many of which differ by only one or a few nucleotides. The expression of miRNAs is tissue-specific and varies in abundance, with differences of several orders of magnitude. More importantly, miRNAs are considered to be the most potential biomarkers in the diagnosis of human diseases. [0003] Compared with ordinary RNA, miRNA has the following three characteristics: (1) miRNAs are very small molecules, about 21-25 nucleotides long...
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