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Modified fluorescent probe and application thereof

A technology of probes and oligonucleotide probes, applied in the field of modified fluorescent probes, which can solve the problems of low detection specificity and accuracy, difficult PCR, and many DNA damages and breaks

Active Publication Date: 2020-11-20
SINGLERA HEALTH TECH SHANGHAI LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The DNA sequence treated with bisulfite has poor base balance and more DNA damage and fragmentation, so PCR detection is more difficult than conventional detection, and detection specificity and accuracy are low

Method used

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  • Modified fluorescent probe and application thereof
  • Modified fluorescent probe and application thereof
  • Modified fluorescent probe and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0097] Example 1: The present invention designs probes for methylation-specific detection

[0098] Use bisulfite-treated reference DNA to verify primer specificity. First, in this example, the target DNA in the ASCL4 gene region was selected as the detection site, and the internal reference gene ACTB was designed, and primers and probes were designed respectively. Then, the gradient was configured with 100%, 50%, 25%, and 10% ratios of methylated DNA (Qiagen 59655) to unmethylated DNA (Qiagen 59665), and the total amount of DNA was 4 ng. Finally, the designed primers were used to amplify the DNA in each gradient DNA mixture in two replicates. Refer to Table 1 for the sequences of the primers and the modified probes of the present invention used to detect ASCL4, as well as the unmethylated specific primers and probes used to detect the internal reference gene ACTB. Among them, ASCL4 uses the invention to design methylation-specific probes, and the internal reference gene uses...

Embodiment 2

[0107] Embodiment 2: The design probe of the present invention compares with conventional Taqman probe

[0108] Compared with the detection method in Example 1, the detection performances of the designed probes of the present invention and conventional probes were compared. Wherein the routine probe sequence of ASCL4 is: TCGGCGTTTTCGTTTAGTAGC.

[0109] The gradient configuration was 100%, 50%, 25%, 10% ratio of methylated DNA (Qiagen 59655) to unmethylated DNA (Qiagen 59665), and the total amount of DNA was 4 ng. The designed primers were used to amplify the DNA of each gradient DNA mixture, which was repeated twice.

[0110] result

[0111] The results show that under the same template gradient, the probe of the present invention produces a better amplification curve than the conventional probe, showing a lower fluorescence background ( Figure 3A-3D ), higher fluorescence increments and smaller Ct values ​​( Figures 4A-4D ), and can significantly improve the amplificati...

Embodiment 3

[0114] Embodiment 3: The present invention design probe is used for the detection of colorectal tissue sample

[0115] Compared with the test method in Example 1, using the ASCL4 probe sequence designed by the present invention, and the internal reference detection primers and probe sequences in Example 1, 10 white blood cell DNA, paracancerous tissue DNA, and intestinal adenoma tissue were detected. DNA and colorectal cancer tissue DNA.

[0116] Described detection comprises the steps:

[0117] Ten DNA samples each were obtained from blood cells, paracancerous tissue, high-grade adenoma tissue, and colorectal cancer tissue (ie, a total of 40 samples). The commercial kit QIAamp DNA Mini Kit was used to extract blood cell DNA according to the instructions; for paracancerous tissue, high-grade adenoma tissue and colorectal cancer tissue DNA, the commercial kit QIAamp DNA FFPETissue Kit was used to extract according to the instructions;

[0118] Use the commercial bisulfite conve...

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Abstract

The invention provides an oligonucleotide probe. The oligonucleotide comprises an oligonucleotide molecule and a marker binding to the oligonucleotide molecule, wherein the oligonucleotide molecule comprises a self-complementary region which can form a hairpin structure, a target nucleic acid recognition region and a target nucleic acid recognition analogue region; and the marker provides a detectable signal when the probe is in a non-hybridized form, but weakens or substantially does not provide a detectable signal when the probe hybridizes with a complementary nucleic acid, or the marker provides a detectable signal when the probe hybridizes with a complementary nucleic acid, but weakens or substantially does not provides a detectable signal when the probe is in a non-hybridized form.

Description

technical field [0001] The invention relates to the field of detection of nucleic acid molecules, in particular to the field of fluorescent probes, more specifically including modified fluorescent probes, their preparation methods and applications. Background technique [0002] Fluorescent probe technology is based on fluorescence resonance energy transfer and is used for qualitative and quantitative detection of nucleic acids. The nucleotide sequence containing a specific sequence contains a pair of fluorescent substances, one of which is an energy donor and the other is an energy acceptor. When the donor and the acceptor are close enough in space, the fluorescence energy generated by exciting the donor is absorbed by the acceptor, and the detector only obtains a background fluorescence signal. In the process of fluorescent PCR detection, the qualitative and quantitative detection of the target nucleic acid is carried out by positively correlating the content of the target...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/6816C12Q1/6858
CPCC12Q1/6816C12Q1/6858C12Q2563/107C12Q2525/301C12Q2531/113C12Q2521/101
Inventor 王辉刘蕊何东华
Owner SINGLERA HEALTH TECH SHANGHAI LTD
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