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Omega structure oligonucleotide primer for detecting short chain ribonucleic acid (RNA) and application thereof

An oligonucleotide, omega technology, applied in DNA/RNA fragments, recombinant DNA technology, biochemical equipment and methods, etc., can solve problems such as intractability

Active Publication Date: 2012-08-01
CHENGDU NUOEN BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method is obviously intractable when the detection involves an increasing miRNA expression profile

Method used

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  • Omega structure oligonucleotide primer for detecting short chain ribonucleic acid (RNA) and application thereof
  • Omega structure oligonucleotide primer for detecting short chain ribonucleic acid (RNA) and application thereof
  • Omega structure oligonucleotide primer for detecting short chain ribonucleic acid (RNA) and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Embodiment 1: Comparison experiment of RNA oligomeric chain and DNA oligomeric chain stability

[0073] Such as image 3 As shown, RNA with a 6-paired base stem and DNA with a 9-paired base stem are the most economical structures. The backbone chains of RNA and DNA are different, and the energy contribution to maintain the secondary structure is also different. As the paired nucleotides in the stem region increase, the Gibbs free energy decreases. However, the contribution of each added base pair to structural stability varies according to its position. For example, adding one base pair to a 4-base convex-stem RNA reduces the free energy by -0.9 kcal / mole (kcal / mole mole), a 5-base convex-stem RNA adds one base pair, and the free energy drops by -3.6 kcal / mole. The 6-base-pair convex-stem RNA is the most economical structure, which yields the greatest free energy gain with the least number of nucleotides. The 6-base pair convex stem structure is the most common RN...

Embodiment 2

[0074] Embodiment 2: the functional experiment of the probe spacer of omega primer of the present invention

[0075] A sequence in the chain of TPL6 designed and synthesized is the same as its 3' end sequence as a template.

[0076]

[0077] TPL6 sequence (complementary strand): SEQ ID NO:2

[0078]

[0079] RT reactions were simulated with omega primers with 8 base pair stem lengths, and nucleotides in italics were used as probe spacers.

[0080]

[0081] Dissolve RT primers with 1x TE buffer at a concentration of 50 μM. Add 1 μl of primers to 500 μl H2O dilution, and use 1 μl of diluted primers per RT reaction. Perform 10-fold serial dilutions of TPL6 with sterilized water, dilution 10 7 – 10 12 as a template. The 5 μl RT reaction system consisted of 1x PCR buffer, 0.1xTE, 50 nM omega primer, 0.05 u / μl Taq DNA polymerase and different dilutions of TPL6 template. The conditions of the RT reaction were: 37°C for 10 minutes, 20°C for 1 minute and 37°C for 10 sec...

Embodiment 3

[0086] Example 3 Contrastive experiment of the influence of probe region length on reverse transcription efficiency and in-chain priming

[0087] The probe types used are:

[0088] A: Stem-loop primers + probes indicated for each plate below

[0089] B: Linear primers + probes as indicated for each plate below

[0090] C: Omega primers + probes indicated for each plate below

[0091] The TLP5 template sequences used for plates 1-5 were (complementary strand):

[0092]

[0093] The primer probe sequences are as follows:

[0094] Board 1. AGT

[0095] Board 2. AGTC

[0096] Board 3. AGTCA

[0097] Plate 4. AGTCAG

[0098] Plate 5. AGTCAGT

[0099] Template TLP1 used for plate 6 - no intrastrand site, the sequence is (complementary strand):

[0100]

[0101] The probe sequences are:

[0102] Plate 6. AGTCAGT

[0103] Primers were dissolved in 1xTE at a concentration of 50 μM, denatured at 95°C 10”, kept at 65°C for 5’, and then cooled to room temperature. Add 1...

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Abstract

The invention discloses an omega structure oligonucleotide primer for detecting short chain RNA. The oligonucleotide primer sequentially comprises a polymerase chain reaction (PCR) primer target area containing 20-30 basic groups, a variable coding area containing 0-50 basic groups, an omega stem-loop, a probe spacer region containing at least one basic group, and a probe region containing 4-11 basic groups from a 5' end to a 3' end. The length of stems of the omega stem-loop is that of 4-12 paired basic groups, and the length of loops of the omega stem-loop is that of 3-20 unpaired basic groups. According to the omega structure oligonucleotide primer for detecting the short chain RNA and an application thereof, starting inside chains and primer dimmer can be avoided, target hybridization accuracy of a probe at the 3' tail end can be increased, transcription specificity and sensitivity can be improved, high reverse transcriptase (RT) efficiency and good specificity can be achieved with few primers, a plurality of primers capable of detecting different targets can be added into a reaction system simultaneously, and PCR resultants obtained from different detected targets can be distinguished simultaneously, therefore multi-channel and high-flux detection is achieved, and detection efficiency is greatly improved.

Description

technical field [0001] The invention relates to a primer, in particular to a primer for detecting short-chain RNA and its application. Background technique [0002] Short-chain RNA plays an important role in gene regulation in many biological, physiological and pathological processes. MicroRNA (miRNA) is the most abundant type of short-chain RNA. Because it is very short compared with its congeners DNA and RNA, it is difficult to find, so its existence has only been discovered in recent years. In 1993, lin-4 was the first 22nt short-chain RNA discovered. It inhibits the initiation of developmental timetable and cell differentiation in nematodes in experiments by regulating the target gene lin-4 messenger RNA (mRNA). It was not until 7 years later that short-chain RNAs with similar structures and functions were discovered in human cells that it was determined that lin-4 was not isolated, and it was classified as a class of molecules of gene regulators, namely microRNAs (miR...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6816C12Q1/68C12Q2525/207C12Q2525/301C12Q1/6876
Inventor 张松柏蒋国彪唐放张菲菲
Owner CHENGDU NUOEN BIOLOGICAL TECH
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