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Probe used for identifying and detecting nucleic acid specificity

A specific recognition and probe technology, which is applied in the determination/inspection of microorganisms, DNA/RNA fragments, fluorescence/phosphorescence, etc., can solve the problems of high fluorescence background, limited single-base mutation recognition ability, complex design, etc., to achieve The effect of high hybridization efficiency, strong tolerance, and simple design

Active Publication Date: 2013-05-08
AMOYDX BIOTECHNOLOGY RES CENT CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the probes mentioned above generally have the disadvantages of complex design, high fluorescence background, and limited ability to recognize single-base mutations.

Method used

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  • Probe used for identifying and detecting nucleic acid specificity
  • Probe used for identifying and detecting nucleic acid specificity
  • Probe used for identifying and detecting nucleic acid specificity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1: Real-time PCR detects hepatitis B virus

[0042] In order to investigate the effectiveness of the protruding ring probe in real-time PCR detection, the PCR template is a series of diluted standard DNA samples. The total volume of the reaction system is 50 μl, including 5 μl 10×buffer (100 mM [NH 4 ] 2 SO 4 , 6700mM Tris-HCl pH 8.3), 1.5μl 25mM MgCl 2 , each dNTP 400 μM, upstream and downstream primers 0.4 μM, probe 0.1 μM, 2.0U Taq DNA polymerase, 5 μl template DNA. The real-time PCR reaction was carried out on the MX3000P real-time PCR instrument. The reaction conditions were: pre-denaturation at 94°C for 3 minutes, followed by 25s at 94°C, 20s at 58°C (detection of FAM and fluorescence signal), 15s at 72°C, a total of 35 cycles. Serial 10-fold serial dilution of standard DNA, H 2 O served as a negative control. The convex loop probe includes a nucleic acid sequence that is complementary to the amplified product. The upstream and downstream primer...

Embodiment 2

[0045] Example 2: Application of convex ring probes in the detection of SNPs

[0046] Select the HPA-4 gene of human platelet alloantigen (human platelet alloantigen, HPA), because there is a G>A mutation in the gene, resulting in the production of two platelet antigens, HPA-4a and HPA-4b, HPA-4a It is the wild type, and HPA-4b is the mutant type. Protrusion loop probes were designed for the gene sequences of the two antigens, and the difference between the two probes was only 1 base, and 3 typical samples with known genotypes were selected for testing. One is a pure and HPA-4a sample, one is a pure and HPA-4b sample, and one is a heterozygous sample containing both HPA-4a and HPA-4b. At the same time as the test, do a no (negative) sample, that is, H 2 O control. The first strand sequence of the wild-type (HPA-4a) probe is:

[0047] 5'-FAM-GGTGAGCTTT C GCATCTGGGT-3', the second strand sequence is 5'

[0048] -ACCCAGATGCAAAGCTCACC-3'-BHQ, mutant (HPA-4b) probe sequence i...

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Abstract

The invention discloses a probe used for identifying and detecting nucleic acid specificity. The probe consists of two oligonucleotides, wherein the 5' end of one oligonucleotide is marked with a fluorophor; and the 3' end of the other oligonucleotide is marked with a quencher; the first oligonucleotide is 1 to 3 non-complementary bases longer than the second oligonucleotide; the rest bases of the first oligonucleotide are completely complemented with the bases of the second oligonucleotide; the non-complementary bases of the first oligonucleotide is positioned in a middle region of a first chain; and under a certain condition, the two chains of the probes are combined into a dual-chain structure because of complementary bases so that the probe forms a secondary structure with a bulge loop. The probe of the invention has high specificity and can be used for the detection of target nucleic acid in real-time PCR.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a novel probe for nucleic acid recognition and detection. Background technique [0002] After the invention of nucleic acid polymerase chain reaction (PCR) technology, gene amplification and cloning become easier and easier. This technology can specifically amplify the target nucleic acid sequence to several million times within 1-3 hours. Now, due to the appearance of real-time PCR instrument, ordinary PCR no longer needs gel electrophoresis analysis, so there is no need for post-PCR operation. Once the PCR reaction program can judge the result immediately, it also minimizes the possibility of contamination of PCR products, so that Real-time PCR technology is more and more widely used clinically. [0003] In the early stage of real-time PCR nucleic acid detection technology, fluorescent dyes, such as SYBR GREEN and EVE GREEN, were used to indicate PCR reaction products. Although the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68G01N21/64C12N15/11
Inventor 阮力郑立谋陈琰
Owner AMOYDX BIOTECHNOLOGY RES CENT CO LTD
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