Structure and application of double-stranded oligonucleotide nucleic acid probe

A nucleotide nucleic acid and oligonucleotide technology, applied in the field of gene detection, can solve the problems of high background, difficult quenching, and uncertainty of differences in enzyme activity, so as to improve the detection sensitivity and greatly promote the application value. , the effect of reducing the fluorescent background

Pending Publication Date: 2019-04-19
ACADEMY OF MILITARY MEDICAL SCI
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Problems solved by technology

Although the TaqMan probe technology is widely used, there are still the following main disadvantages. First, the length of the linear probe limits the energy transfer efficiency, and the quenching is difficult to complete, resulting in a high background; second, the hydrolysis of the probe depends on the outer surface of the Taq enzyme. Dicerase activity is greatly affected by the performance and quality of the enzyme during quantification, and the difference in enzyme activity brings greater uncertainty to the quantification; the third is that the TaqMan probe only labels one fluorescent molecule, and the sensitivity needs to be improved

Method used

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  • Structure and application of double-stranded oligonucleotide nucleic acid probe
  • Structure and application of double-stranded oligonucleotide nucleic acid probe

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Embodiment 1 hepatitis B virus double-stranded oligonucleotide nucleic acid probe detection

[0062] 1. HBV Detection Primer Probe Design

[0063] According to the principle of qualitative and quantitative analysis of double-stranded oligonucleotide nucleic acid probes, design and synthesize primers F, R, long-chain oligonucleotide probe P1, and short-chain oligonucleotide probe according to the DNA sequence of the target molecule HBV to be tested P2, Taqman probe P3, oligonucleotide probe P4 containing mutant bases, the sequences of primers and probes are shown in Table 1.

[0064] Table 1 Primer and probe sequences

[0065]

[0066] (1) Primer

[0067] The upstream primer F has a total of 21 nucleotides, 17 nucleotides away from the long-chain oligonucleotide probe, and 22 nucleotides away from the short-chain oligonucleotide probe.

[0068]The downstream primer R has a total of 21 nucleotides, 27 nucleotides away from the long-chain oligonucleotide probe, and 3...

Embodiment 2

[0110] Example 2 Genotype detection of double-stranded oligonucleotide probe 2C19*2(681G>A) single nucleotide polymorphism site

[0111](1) Prepare the PCR reaction system, including: 2.5 μL of 10× Buffer, 1.2 μL (10 μM) of upstream and downstream primers, 0.6 μL (10 μM) of double-stranded oligonucleotide probe, Mg2+2.5 μL, 50× ST enzyme 0.5 μL (BIORI, China), nucleic acid extraction kit to extract human DNA template 3 μL, the total reaction volume is 25 μL;

[0112] Reaction conditions: 50°C, 2min; 95°C, 5min; 95°C, 20s, 60°C, 45s, a total of 40 cycles, collecting fluorescence during annealing;

[0113] (2) Template: 12 cases each of 2C19*2 gene mutant A / A, wild type G / G, and heterozygous G / A, ddH2O was used as a negative control, and PCR detection was performed according to the operation in step (1);

[0114] (3) The experimental results are shown in Table 7 and Figure 10-12 Shown:

[0115] The double-stranded probe can detect the genotype of the single nucleotide polymo...

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Abstract

The invention provides a structure of a double-stranded oligonucleotide nucleic acid probe, a method of use and application thereof in nucleic acid fluorescence qualitative and quantitative analysis,medical diagnosis and life science researches. The double-stranded oligonucleotide nucleic acid probe is composed of two complete or partially base-complementary oligonucleotide strands, the end of each oligonucleotide strand can be connected to a fluorescent group or a corresponding fluorescent quenching group, and the two oligonucleotide probe strands can hybridize with a target nucleic acid sequence to be tested. The double-stranded oligonucleotide nucleic acid probe has complete fluorescence quenching, high detection sensitivity, no specific extension to form a primer dimer and high specificity, and can be widely used for fields of nucleic acid detection such as pathogen detection, drug resistance analysis, life science research and the like.

Description

technical field [0001] The invention relates to a biological detection technology tool, specifically a double-stranded oligonucleotide nucleic acid probe and its use method and its application in life science research such as gene fluorescence qualitative, quantitative analysis, medical diagnosis, etc., belonging to gene detection technology field. Background technique [0002] Real-time fluorescent PCR technology was launched by Applied Biosystems in 1996. It refers to the method of adding fluorescent groups to the PCR reaction system, using the accumulation of fluorescent signals to monitor the entire PCR process in real time, and finally performing qualitative and quantitative analysis of unknown templates through standard curves. Because of its accuracy, speed, and sensitivity, it is internationally recognized as one of the most effective means for the laboratory confirmation of pathogenic microorganisms related to infectious diseases. played an irreplaceable role. [...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/686C12N15/11
CPCC12Q1/686C12Q2561/113C12Q2563/107C12Q2521/107C12Q1/6876C12Q2565/1015C12Q1/6806C12Q2533/107
Inventor 王升启刘琪琦张影刘丽艳赵怡
Owner ACADEMY OF MILITARY MEDICAL SCI
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