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Xanthine oxidase-deoxyribonucleic acid (DNA) complex for detecting target gene by uric acid instrument and preparation method and application of xanthine oxidase-deoxyribonucleic acid (DNA) complex in detection

A technology of xanthine oxidase and target detection, which is applied in the field of DNA detection, can solve the problems of expensive professional instruments and equipment, and achieve the effects of high selectivity detection, wide application prospects, and convenient operation

Inactive Publication Date: 2013-08-21
HUNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As widely used methods for detecting DNA, there are PCR (polymerase chain reaction) and DNA probe method. Although these two methods can achieve sensitive detection of target DNA, they both require expensive professional equipment and must be In a professional laboratory

Method used

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  • Xanthine oxidase-deoxyribonucleic acid (DNA) complex for detecting target gene by uric acid instrument and preparation method and application of xanthine oxidase-deoxyribonucleic acid (DNA) complex in detection
  • Xanthine oxidase-deoxyribonucleic acid (DNA) complex for detecting target gene by uric acid instrument and preparation method and application of xanthine oxidase-deoxyribonucleic acid (DNA) complex in detection
  • Xanthine oxidase-deoxyribonucleic acid (DNA) complex for detecting target gene by uric acid instrument and preparation method and application of xanthine oxidase-deoxyribonucleic acid (DNA) complex in detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] A xanthine oxidase-DNA complex (see figure 1 ), the xanthine oxidase-DNA complex is a long-chain molecular structure, one end of the molecule of the xanthine oxidase-DNA complex is xanthine oxidase, and the other end of the molecule of the xanthine oxidase-DNA complex is hybridizable to The auxiliary probe on the target gene to be detected, the xanthine oxidase and the auxiliary probe are separated by 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfosuccinimide The ester sodium salt is used as the connecting arm generated after the reaction of the raw material for connection, and the connecting arm includes a 4-(N-maleimidomethyl)cyclohexane group.

[0042] In this example, the specific connection method between the xanthine oxidase and the auxiliary probe through the connecting arm is as follows: the xanthine oxidase contains an amino group, the auxiliary probe is modified with a sulfhydryl group, and the 4-(N- The 1st position of the aliphatic ring of the male...

Embodiment 2

[0050] The following primer sequences will be used in this example (all DNA sequences were purchased from Shanghai Sangon Bioengineering Technology Co., Ltd.):

[0051] Target DNA 1: 5'-GGTTGTGAGGCGCTGCCCAAGCGA-3';

[0052] Capture probe 1: 5'-CGCCTCACAACCAAAAAAA-SH-3';

[0053] Auxiliary probe 1: 5'-HS-AAAAAAAAAAAATCGCTTGGGCAG-3';

[0054] Single base mismatch DNA1 (smDNA1): 5'-GGT TGT GAG GCG GTG CCC AAG CGA-3';

[0055] Double base mismatch DNA1 (dmDNA1): 5'-GGT AGT GAG GCG GTG CCC AAG CGA-3';

[0056] Three-base mismatch DNA1 (tmDNA1): 5'-GGTAGT GAG GCG GTG CCC TAG CGA-3';

[0057] Random sequence DNA1 (ranDNA1): 5'-TAC CAG TTGAGAACC CTGAAT CCG-3'.

[0058] like image 3 Shown, a kind of method utilizing the uric acid meter of the present invention to detect target DNA comprises the following steps:

[0059] (1) Wash the surface of the gold film with acetone, ethanol, and water respectively, then blow it dry with nitrogen, and spread the perforated silicone rubber sh...

Embodiment 3

[0070] The following primer sequences will be used in this example (all DNA sequences were purchased from Shanghai Sangon Bioengineering Technology Co., Ltd.):

[0071] Target DNA2: 5'-TGG GAG GAG TTG GGG GAG GAG ATT AGG TTAAAG GT-3';

[0072] Capture probe 2: 5'-TC CCC CAA CTC CTC CCA TTT TTT-SH-3';

[0073] Auxiliary probe 2: 5'-SH-TTT TTT TTT TTT ACC TTT AAC CTA ATC TCC-3';

[0074] Single base mismatch DNA2 (smDNA2): 5'-TGG GAG GAG TTG GGG GAG GAG ATT AGA TTAAAG GT-3';

[0075] Double base mismatch DNA2 (dmDNA2): 5'-TGG GAG GAG TTG GGG GAGAAGATTAGATTAAAG GT-3';

[0076] Three-base mismatch DNA2 (tmDNA2): 5'-TGG GAG GAA TTG GGG GAG AAG ATT AGA TTAAAG GT-3';

[0077] Random sequence DNA2 (ranDNA2): 5'-AAT TCA CAC CTG GGG GAG TAT TGC GGA GGAAGG TG-3'.

[0078] like image 3 As shown, a method for detecting target DNA using a uric acid meter of the present invention, except that target DNA1 in Example 2 is replaced by target DNA2, capture probe 1 is replaced by capture pr...

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Abstract

The invention discloses a xanthine oxidase-deoxyribonucleic acid (DNA) complex. According to the complex, one end of a molecule of the complex is xanthine oxidase, the other end of the molecule of the complex is an auxiliary probe, and the xanthine oxidase is connected with the auxiliary probe by a connecting arm which is generated after 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt (sulfo-SMCC) is used as a raw material to be reacted, wherein the connecting arm comprises 4-(N-maleimidomethyl) cyclohexane groups. The method for preparing thecomplex comprises the following steps of: mixing the auxiliary probe, a buffer solution and the like, incubating, oscillating and mixing the xanthine oxidase and the sulfo-SMCC for reacting, and mixing an auxiliary probe solution and a xanthine oxidase solution. According to the complex, target DNA can be detected by a uric acid instrument. A method for detecting the target DNA comprises the following steps of: pretreating a gold membrane, and adding a capture probe to construct a sensor; adding a target solution to be detected, hybridizing, washing, adding a complex solution, hybridizing andwashing; and adding xanthine, and detecting a reaction product by using the uric acid instrument. The uric acid instrument has the advantages of convenience for operation, high sensitivity and high selectivity.

Description

technical field [0001] The invention relates to the technical field of DNA detection, in particular to a compound that can be used for DNA detection, a preparation method thereof and a specific application in DNA detection. Background technique [0002] It has been a long-standing dream to develop a portable sensor for rapid, low-cost, and real-time quantitative detection of targets (such as ions, small molecules, DNA, proteins, viruses, etc.) at the residence or at the site of the incident. The development of such products is of great significance for scientific research, environmental monitoring, and disease diagnosis. In recent years, the research on portable testing instruments has made significant progress. Portable testing instruments such as uric acid meters and blood glucose meters have been commercialized, basically meeting the public's needs for certain diseases and personal disease diagnosis and prevention, and improving tens of thousands of diseases. counting th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11C12N15/10
Inventor 羊小海王青王柯敏刘芳朱红志翦立新
Owner HUNAN UNIV
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