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Chemiluminescence reaction-based methylase detection probe, detection kit and detection method

A technology of methylase and chemiluminescence, which is applied in biochemical equipment and methods, measurement/inspection of microorganisms, DNA/RNA fragments, etc., can solve problems such as time-consuming, complicated pretreatment of gold nanoparticles, and unsafety

Inactive Publication Date: 2014-04-09
SHENZHEN INST OF ADVANCED TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

High-performance liquid chromatography requires a huge amount of material and manpower; radioactive labeling often requires the introduction of radioactive elements, which is unsafe; the pretreatment of gold nanoparticles is complex and time-consuming; fluorescence detection often has the disadvantage of low sensitivity

Method used

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  • Chemiluminescence reaction-based methylase detection probe, detection kit and detection method
  • Chemiluminescence reaction-based methylase detection probe, detection kit and detection method
  • Chemiluminescence reaction-based methylase detection probe, detection kit and detection method

Examples

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Embodiment 1

[0147] figure 1 Schematic diagram of the structure of the double-strand 1 formed by the 5' end side chain (stem-1) and the 3' end side chain (stem-2) of the hairpin probe provided in the embodiment of the present invention. The hairpin probe includes double-strand 1 and ring. Wherein, the double-stranded DNA1 has a methylase recognition site and a methylation-dependent endonuclease cleavage sequence, wherein the distance between the methylase recognition site and the terminal base of the loop is 2~8bp;

[0148] combine figure 1 Taking Dam methylase as an example, this embodiment provides a methylase detection probe based on chemiluminescence reaction, including the following steps:

[0149] (1) Synthesize the hairpin probe in vitro, the nucleotide sequence (5' end to 3') is shown in SEQ ID NO:12;

[0150] (2) Design primer-1 and primer-2 according to the sequence of the hairpin probe. As long as primer-1 includes the nicking enzyme cleavage site and the site complementary ...

Embodiment 2

[0161] Taking Dam methylase as an example, a flow chart of a DNA peroxidase-based methylase detection method is provided, as shown in figure 2 shown. Depend on figure 2 It can be seen that this detection method can be roughly divided into three steps. The first step is methylation and methylation-sensitive endonuclease digestion reaction: the sequence GATC on the stem structure of the hairpin probe can be specifically identified by the methyltransferase Dam To recognize and add a methyl group to the double-stranded A base; the methylated hairpin structure can be recognized and cut by endonuclease Dpn Ⅰ, resulting in a small hairpin structure and double-stranded DNA, due to The annealing temperature of the small hairpin structure and double-stranded DNA is too low, causing it to transform into single-stranded DNA at room temperature. The second step is the rolling circle amplification reaction and the nicking enzyme digestion reaction: the single-stranded DNA transformed in...

Embodiment 3

[0178] This embodiment provides a method for detecting methylase based on DNA peroxidase, which can directly obtain the concentration of methylase by preparing a standard curve, including the following steps:

[0179] Prepare the methylase into a solution of known concentration (the concentration of methylase is 0.025, 0.05, 0.1, 0.5, 1, 2, 2.5, 25, 50, 73, 120, 120, 200, 400 units per milliliter), Using the DNA peroxidase-based methylase detection kit provided in Example 2 and the detection method using the kit to detect the chemiluminescence signals of the methylase solutions at various concentrations, such as image 3 shown, and prepare a standard curve, such as Figure 4 shown. image 3 The relationship between different concentrations of methylase and the intensity of chemiluminescence is shown (the error bars represent the standard deviation of three experiments), and the concentration range is from 0.025 to 0.5 units per milliliter. Figure 4 The logarithmic value of ...

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Abstract

The invention provides a chemiluminescence reaction-based methylase detection probe, a detection kit and a detection method. The chemiluminescence reaction-based methylase detection probe provided by the invention is simple in design without being any labeled and modified. Meanwhile, according to the chemiluminescence reaction-based methylase detection probe, methylase and specific incision enzyme depending on methylation are used as a combination, a detection probe with a hairpin structure can be cut apart by depending an interaction of the methylase and the specific incision enzyme so as to generate a primer signal, and thus a chemiluminescence signal is generated. Moreover, any other action of the methylase to hairpin structures can not be recognized by incision enzyme outside the combination, and thus no primer signal can be generated, and therefore, no chemiluminescence signal is generated so that the specificity of the chemiluminescence reaction-based methylase detection probe is very high. In addition, according to the technical scheme adopted by the invention, two advantages of high ligase catalysis efficiency and remarkably enhanced chemiluminescence signal are serially connected, so that the detection sensitivity is greatly improved. The chemiluminescence reaction-based methylase detection probe is wide in application value on aspects of early diagnosis for diseases and screening of drugs.

Description

technical field [0001] The invention relates to the field of molecular detection, in particular to a chemiluminescent reaction-based methylase detection probe, detection kit and detection method. Background technique [0002] Methylases (such as Dam methylase, Dcm methylase, EcoK Ⅰ methylase, Sss Ⅰ methylase) have become important biomarkers for early diagnosis of many diseases including cancer, but Their low levels in cells make highly sensitive analysis of methylases challenging. [0003] Existing methylase detection techniques mainly include high performance liquid chromatography (HPLC), radioactive labeling, gold nanocolorimetric detection and fluorescence detection. High-performance liquid chromatography requires a huge amount of material and manpower; radiolabeling technology often requires the introduction of radioactive elements, which is unsafe; the pretreatment of gold nanoparticles is complex and time-consuming; fluorescence detection often has the disadvantage o...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/48C12N15/11
CPCC12Q1/6844C12Q2531/125C12Q2525/301C12Q2521/301
Inventor 曾亚平张春阳
Owner SHENZHEN INST OF ADVANCED TECH
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