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DNA methylation detection probe, and detection method and detection kit thereof

A technology for detecting probes and detection methods, which is applied in the field of molecular biology and can solve problems such as limited application and low sensitivity

Inactive Publication Date: 2014-02-19
SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

Based on surface-enhanced Raman spectroscopy and single-nucleotide amplification methods, limited applications due to low sensitivity

Method used

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  • DNA methylation detection probe, and detection method and detection kit thereof
  • DNA methylation detection probe, and detection method and detection kit thereof
  • DNA methylation detection probe, and detection method and detection kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Methylated DNA detection of H157 (human non-small cell lung cancer cell line) and H209 (human small cell lung cancer cell line) cell lines, such as Figure 4 shown.

[0054] 1. DNA extraction and digestion

[0055] 1.1 Cultivate two cell lines, H157 and H209, respectively. The p16 promoter region is hypermethylated in the normal H157 cell line, whereas these regions are not methylated in the normal H209 cell line.

[0056] Culture conditions: the two cell lines were respectively placed in DMEM medium containing 10% bovine serum albumin, and cultured in a humidified incubator containing 5% carbon dioxide at 37°C.

[0057] 1.2 DNA extraction

[0058] Genomic DNA of the above two cell lines was respectively extracted with a DNA extraction kit, and the absorbance of the DNA extraction solution at 260 nm was detected by a spectrophotometer, and the DNA concentration was converted.

[0059] 1.3 Treat the extracted DNA with two restriction enzymes, Pst I and BstE II, to di...

Embodiment 2

[0099] Specific detection of methylated genes

[0100] Previous studies have shown that methylation of cancer suppressor genes in p16 contributes to a variety of cancers. In order to prove the feasibility of this method, a piece of DNA was synthesized in this example, the sequence is the same as a piece of cancer gene in p16, the sequence is: GAG GGT GGG G m CG GAC m CG m C GTG m CGC T m CG G m CG GCTG (SEQ ID NO: 2), and the methylation of this DNA was detected. Such as Figure 15 As shown, the fluorescence intensity value obtained from 10nM methylated DNA is 698.8±25.3, which is 23 times higher than the fluorescence intensity value (30.2±5.5) obtained from 10nM unmethylated DNA, indicating that this method can be successfully used in formazan Detection of methylated DNA.

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Abstract

The invention relates to a DNA methylation detection probe, and a detection method and a detection kit thereof. The detection probe comprises a 3' end and a 5' end which are complementary to methylated DNA, and a specific sequence used for initiating an HRCA reaction in the middle. If DNA to be detected is the methylated DNA, the DNA to be detected can be completely complementarily paired with the detection probe, and the 3' end and the 5' end of detection probe are linked under the action of a subsequent DNA ligase to form annular DNA; and if the DNA to be detected is unmethylated DNA, the DNA to be detected cannot be completely complementarily paired with the detection probe, and no annular DNA is formed. The annular DNA can quantitatively analyze the methylation degree of the DNA to be detected through the subsequent HRCA reaction and signal detection, and the HRCA has a signal 10<9>-time amplification capability, so the detection ultrahigh-sensitivity requirement is guaranteed. By using the probe, there is no need to use an expensive fluorescently-labeled probe or carry out PCR amplification in the detection process, so the detection cost is substantially reduced. The detection method has no enzyme site requirements on the methylated DNA, so the method can be widely applied.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a DNA methylation detection probe, detection method and detection kit. Background technique [0002] With the completion of the Human Genome Project, one of the next major tasks will be to decipher the genetic system, or how a human cell uses genetic material to determine when and where a particular gene is expressed as it grows. DNA methylation has become an important part of the epigenetic system because of its ability to affect the genetic status of gene expression. DNA methylation in mammals mostly occurs on cytosine in CpG dinucleotides, that is, a methyl group is added at carbon 5 of the pyrimidine ring of cytosine. In normal cells, methylation mainly occurs in repetitive genomic regions, including genetic elements and satellite deoxynucleotides, whereas CpG islands associated with gene promoters and exons are usually unmethylated. However, mutational DNA methylation in t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/682C12Q2521/501C12Q2523/125
Inventor 张春阳曹岸萍
Owner SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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