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Single-stranded nucleic acid for detecting nucleic acid or protein in real time and detection method using same

A real-time detection, single-nucleic acid technology, applied in biochemical equipment and methods, measurement devices, and microbial determination/inspection, etc., can solve the problems of difficult to analyze SNP, difficult to apply endpoint genotyping, reduced accuracy, etc., and achieve savings. Analyze the effects of time and expense

Inactive Publication Date: 2019-03-01
NURIBIO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] Third, it is difficult to analyze SNPs using CataCleave probes
That is, it is difficult to apply the CataCleave probe to the conventional endpoint genotyping (Endpoint Genotyping), and the accuracy of the method of directly hybridizing with the point mutation site to confirm it decreases.

Method used

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  • Single-stranded nucleic acid for detecting nucleic acid or protein in real time and detection method using same
  • Single-stranded nucleic acid for detecting nucleic acid or protein in real time and detection method using same
  • Single-stranded nucleic acid for detecting nucleic acid or protein in real time and detection method using same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0169] Embodiment 1: Utilize the real-time nucleic acid analysis of single nucleic acid of the present invention

[0170]In order to measure cytokeratin-18 (CK-18, cytokeratin-18), cytokeratin-19 (CK-19, cytokeratin-19) and 3-phosphate glyceraldehyde dehydrogenase (GAPDH, Glyceraldehyde 3-phosphatedehydrogenase) Whether the gene is expressed, as described in Table 1 below, the single nucleic acid (promer) and primer (primer) of the present invention were entrusted to Integrated DNA Technologies (IDT, Integrated DNA Technologies, USA) for manufacture (refer to Table 1). Among them, for a single nucleic acid (promer), fluorescein succinimidyl ester (FAM, fluoresceinsuccinimidyl ester) is attached to the 5' end, and 3IABkFG is attached to the 3' end. In order to distinguish ribonucleotide (RNA) from deoxyribose nucleus Nucleotide (DNA), preceded by the sequence, is indicated by the subscript "r". As a comparison group, the primer to be used in the SYBR method was entrusted to ID...

Embodiment 2

[0177] Example 2: Mutation assay using single nucleic acid of the present invention

[0178] Using the single nucleic acid (promer) of the present invention, it is determined whether G12D and G12V of the KRAS gene (gene) are mutated. Specifically, entrusted to IDT, the single nucleic acid (promer) and reverse primer (reverse primer) of the present invention were manufactured according to the following Table 3. For the single nucleic acid (promer), fluorescein succinyl was attached to the 5' end. Fluorescein succinimidyl ester (FAM, fluorescein succinimidyl ester), with 3IABkFG attached at the 3' end, in order to distinguish ribonucleotide (RNA) and deoxyribonucleotide (DNA), in front of the sequence, it is indicated by subscript "r".

[0179] table 3

[0180]

[0181]

[0182] In addition, total DNA was obtained from SW620 (BioSample: SAMN05292430) and LS174T cell line (BioSample: SAMN03701686) for the KRAS gene to be measured. (Please inform NCBI of the source.) The w...

Embodiment 3

[0188] Example 3: Optimal Size Determination of Single Nucleic Acids of the Invention

[0189] The OPN1MW gene (Themedium-wave-sensitive opsin-1 gene, Themedium-wave-sensitive opsin-1gene) located on the long arm of the X chromosome (Xq28) is a gene related to color weakness and color blindness, and mutations occur at 4 nucleotide positions , thus being found to be red-green weak. The mutation positions are C282A, T529C, T607C, and G989A.

[0190] Accordingly, in order to confirm whether the mutation based on the length of the single nucleic acid (promer) of the present invention was detected, the present inventors produced a DNA sequence inserted into the 241st to 1030th nucleotide (see the DNA sequence of SEQ ID NO: 13) of the OPN1MW T529C mutant gene. After plasma, after dilution at concentrations of 10fM, 1fM, 100aM, and 10aM, each 1 μl was removed, and as shown in Table 4 below, five kinds of single nucleic acids and reverse primers manufactured by IDT were used for dete...

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Abstract

The present invention relates to a single-stranded nucleic acid (promer) that has the structure of X-Y-Z with a detectable marker attached to both ends or inside thereof and which is used as a primerand a probe during real-time detection of a nucleic acid or a protein, and a method for detecting a nucleic acid and a protein in real time, comprising the steps of amplifying a target nucleic acid tobe detected with the promer and then quantitatively measuring cleaved fragments. The method for detecting a nucleic acid or a protein in accordance with the present invention employs a small amount of oligos compared to conventional detection methods, and does not need a probe at an additional position for real-time identification, thereby detecting a nucleic acid or protein to be detected in real time at lower cost and with a simpler process. In addition, the method allows for the identification of a mutation at a Y locus through the step of amplification after the digestion of the Y locus of the promer and is capable of multi-detecting nucleic acids or proteins in a number exceeding the number of fluorescent materials combined with the promer, thereby being usefully available for prognostic diagnosis and the diagnosis of various diseases.

Description

technical field [0001] The present invention relates to a single nucleic acid (promer) that can be used as both a primer and a probe for real-time detection of nucleic acid or protein, and a method for real-time detection of nucleic acid or protein using the single nucleic acid (promer). Background technique [0002] The method for real-time detection of nucleic acid or protein, as described in the art, widely used microarray (microaray), reverse transcription polymerase chain reaction (Real-time polymerase chain reaction, RT-PCR; Quantitative real-time Polylmerase chain reaction, qRT-PCR) detection method, NASBA, RCA, TDMA and other isothermal amplification methods. [0003] This nucleic acid or protein detection method, for real-time detection, requires forward / reverse primers (primer) and intercalator type (Intercalator type) dsDNA-binding agents (dsDNA-binding agents) or probe type ( probe type) Taqman Probe (Taqman Probe), Molecular Beacon (Molecular Beacon), MGB probe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N33/533
CPCC12Q1/34G01N33/582C12Q1/6804C12Q1/6853C12Q2521/327C12Q2525/121C12Q2525/186C12Q2561/113C12Q2563/107C12Q2565/1025C12Q2521/301C12Q2525/131C12Q2531/113C12Q1/6818C12Q1/6876G01N33/533C12Q2525/161C12Q2525/204C12Q2600/156
Inventor 南泳铉
Owner NURIBIO
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