Detection probe for methylation locus, preparation method of detection probe, detection kit and detection method

A technology for detecting probes and methylation, which is applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc. Avoid the impact of sequencing depth, avoid quality degradation, and achieve high detection efficiency

Inactive Publication Date: 2019-11-15
SHENZHEN YHLO BIOTECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these two methods still have some limitations
[0004] Due to the specificity of restriction endonuclease recognition of the base sequence, the restriction enzyme digestion method can only analyze the recognition sequence of the restriction endonuclease, and the detection range is limited by the recognition of the restriction endonuclease
In addition, the interpretation of the results of this method is based on the difference in the electrophoretic bands of the methylated and non-methylated fragments after enzyme digestion. Methylation, but cannot accurately determine the exact site of methylation
[0005] The bisulfite conversion method relies on base conversion and cloning and sequencing, so the test results are affected by whether the conversion is complete and the depth of sequencing, and the process of building a library during cloning and sequencing is complicated, which can easily lead to degradation of the target sequence
Therefore, it is time consuming and less accurate

Method used

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  • Detection probe for methylation locus, preparation method of detection probe, detection kit and detection method
  • Detection probe for methylation locus, preparation method of detection probe, detection kit and detection method
  • Detection probe for methylation locus, preparation method of detection probe, detection kit and detection method

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preparation example Construction

[0052] Gold nanoparticles were synthesized by reducing tetrachloroauric acid (III) trihydrate with trisodium citrate dihydrate. Specifically, 4.5 mL of 1%-2% (w / v) citrate and 45.5 mL of 0.01%-0.03% (w / v) HAuCl 4 Respectively preheat to the reaction temperature of 100°C to 110°C. After reaching a stable temperature for 30 minutes, add 1% to 2% citrate to 0.01% to 0.03% HAuCl 4 middle. After reacting for 20 minutes to 30 minutes, cool to room temperature and filter to obtain gold nanoparticles.

[0053] Further, 4.5 mL of 1% (w / v) citrate and 45.5 mL of 0.01% (w / v) HAuCl 4 Each was preheated to a reaction temperature of 107°C. After reaching a stable temperature for 30 min, add 1% citrate to 0.01% HAuCl 4 middle. After reacting for 30 minutes, cool to room temperature and filter to obtain gold nanoparticles.

[0054] When incubating nano gold and nucleotide fragments, the molar ratio of the first fragment to nano gold is 1:1-1.5:1; the molar ratio of the second fragment ...

Embodiment 1

[0081] DNA extraction

[0082] A commercial DNA extraction kit (Sangon Bioengineering (Shanghai) Co., Ltd.) was used to extract DNA from 24 samples according to the instructions of the kit. Among them, 24 samples were obtained from 4 cases of stage I lung cancer. Whole blood samples from patients, whole blood samples from 4 patients with stage II lung cancer, whole blood samples from 4 patients with stage III lung cancer, whole blood samples from 4 patients with stage IV (all with bronchoscopy and pathological test results), whole blood samples from 4 healthy people Samples and 4 cases of deionized water samples, whole blood samples were provided by Shenzhen People's Hospital. The 24 samples were numbered 1-24 respectively. The specific processing operation of each sample is roughly the same, and the processing of the deionized water sample is only different from the processing of the whole blood sample. Among them, the specific operation of each whole blood sample is as fol...

Embodiment 2

[0087] Preparation of detection probes

[0088] (1) Clean the glassware and magnetic stirring bar with aqua regia solution, and rinse thoroughly with Milli-Q ultrapure water before use.

[0089] (2) Mix 4.5 mL of 1% (w / v) sodium citrate and 45.5 mL of 0.01% (w / v) HAuCl 4 Each was preheated to a reaction temperature of 107°C. After reaching a stable temperature for 30 min, 1% sodium citrate was added to 0.01% HAuCl 4 middle. After reacting for 30 minutes, the solution that completed the reaction was cooled to room temperature in the air, and filtered to obtain gold nanoparticles with a particle size of 25 nm. Store at 4°C for later use.

[0090] (3) Entrust General Biosystems (Anhui) Co., Ltd. to synthesize the first fragment and the second fragment. Wherein, the sequence of the first fragment is shown in SEQ ID No.1, the sequence of the second fragment is shown in SEQ ID No.2, the 3' end of the first fragment has a thiol group, and the 5' end of the second fragment has a ...

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Abstract

The invention relates to a detection probe for a methylation locus, a preparation method of the detection probe, a detection kit and a detection method. The detection probe for the methylation locus comprises a nucleotide segment used for being bound with an area around a methylation high mutation area, and nanogold or nanoparticles connected with the nucleotide segment. When the detection probe for the methylation locus is applied to detection for the methylation locus, the detection efficiency and accuracy can be improved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a detection probe of a methylation site, a preparation method thereof, a detection kit and a detection method. Background technique [0002] Due to the regulatory effect of DNA methylation on gene expression and silencing of cell differentiation, embryonic development, genetic diseases and tumorigenesis, DNA methylation has become the most valuable first sequence after restriction fragment polymorphism and DNA point mutation. Three generations of genetic markers. [0003] The current detection methods of DNA methylation mainly include restriction enzyme digestion method and bisulfite conversion method. However, these two methods still have certain limitations. [0004] Due to the specificity of restriction endonucleases for base sequence recognition, the restriction enzyme digestion method can only analyze the sequence recognized by restriction endonucleases, and the detection rang...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/6886C12N15/11
CPCC12Q1/6883C12Q1/6886C12Q2600/156
Inventor 阙艳鹏钱纯亘胡鹍辉
Owner SHENZHEN YHLO BIOTECH
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