Probe set for detecting small RNA and method for detecting small RNA using same

A technology of DNA probes and probe sets, applied in the direction of DNA/RNA fragments, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problems of increasing the difficulty of preparation and the cost of experiments

Inactive Publication Date: 2009-12-02
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these methods need to use improved probes such as Taqman or LNA to ensure the specificity of detection, which significantly increases the difficulty of preparation and experimental cost

Method used

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  • Probe set for detecting small RNA and method for detecting small RNA using same
  • Probe set for detecting small RNA and method for detecting small RNA using same
  • Probe set for detecting small RNA and method for detecting small RNA using same

Examples

Experimental program
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Effect test

Embodiment 1

[0037] Design and acquisition of linear probes. The following will combine figure 2 The design of the linear probe is described in detail. figure 2 2a in the figure is a schematic diagram of miRNA-122 sequence, which has 22 nucleotides and is not long enough for PCR amplification. Probe 2b has 31 nucleotides, and 2c has 30 nucleotides. When the pair of DNA probes are first hybridized with miRNA-122 and then connected at 2d under the action of T4 DNA ligase, they can A DNA strand of 61 nucleotides is formed, which is long enough for PCR amplification. The DNA probes used in the experiment were purchased from Invitrogen Company in Shanghai, China.

Embodiment 2

[0039] Design of probes with a stem-loop structure. The following will combine image 3 The design of a probe with a stem-loop structure is detailed. image 3 3a in the figure is a schematic diagram of miRNA-122 sequence, which has 22 nucleotides and is not long enough for PCR amplification. Probe 3b has 33 nucleotides, and 3c has 39 nucleotides. When the temperature rises, the stem-loop structure of this pair of probes can be opened to become a straight-chain DNA molecule, and then hybridized with miRNA-122. Then, under the action of T4 DNA ligase, after joining at 3d, a DNA chain with 72 nucleotides can be formed, which is long enough for PCR amplification.

Embodiment 3

[0041] The detection method of miRNA. In this example, a probe with a stem-loop structure similar to that in Example 2 will be used to detect target miRNAs from a system containing 6 miRNAs. The six miRNAs are Mmu-mir-122, syn-aa, aga-mir-210, has-mir-549, has-mir-214 and has-mir-21, and their nucleotide sequences are shown in Figure 4 and shown in Table 1. Mmu-mir-122 is the target miRNA. Among the six miRNAs, except for the syn-aa sequence which was artificially designed to meet the requirements of this experiment, the rest of the miRNA sequences were obtained from the Sanger database. The above six miRNAs used in the experiment were all purchased from Shanghai Gemma Pharmaceutical Technology Co., Ltd. Figure 4 The part of the nucleotide sequence identical to the target miRNA in the five non-target miRNA nucleotide sequences is shown with an emphasis number. It can be seen from the figure that there are 2-8 nucleotide sequences in the non-target miRNA that are identical...

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Abstract

The invention relates to a probe set for detecting a small RNA and a method for detecting the small RNA by using the same and belongs to the field of small RNA detection. The DNA probe set for detecting the small RNA of the invention comprises at least two probes, wherein a segment of nucleotide sequence containing all connections of the connected at least two probes is complementary to the nucleotide sequence of a target small RNA molecular. The method for detecting the small RNA of the invention comprises the following steps: making the probes and the target small RNA first crossed and then connected under the action of a ligase; and finally, detecting the target small RNA by gel electrophoresis, real-time polymerase chain reaction and the like. The method for detecting the small RNA of the invention has the advantages of high sensitivity, large dynamic range, strong anti-interface capacity, low price and the like, and has wide application prospects in various fields related to small RNA detection, such as scientific experiments and clinical diagnosis.

Description

technical field [0001] The invention relates to the detection field of small RNA. Background technique [0002] Small RNA is an important part of the noncoding RNA family (noncoding RNA, referred to as ncRNA), including microRNA (microRNA or miRNA), small interfering RNA (siRNA), small nuclear RNA, small sequence RNA, piwi protein-binding RNA (piRNA) Wait. Studies have found that small RNAs play a vital role in the development, growth, and differentiation of cells and tissues in organisms, and even the occurrence of diseases, as well as the invasion and defense of viruses. There are also various ways in which small RNAs function. They can directly regulate the expression of genes by modifying DNA, and can also regulate the amount of proteins by changing the stability of gene transcription products and so on. Among various types of small RNAs, microRNAs are the most important in function. [0003] MicroRNA is a non-coding RNA molecule with a length of 18-25 nucleotides in ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 席建忠姚波李娟
Owner PEKING UNIV
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