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A chip-based method for high-throughput and high-sensitivity detection of 5-hydroxymethylated cytosine

A sensitive detection technology for hydroxymethylated cytosine, which is applied in the field of high-throughput and high-sensitivity detection of 5-hydroxymethylated cytosine, can solve the problems of high detection cost, false positive test results, inaccurate test results, etc. Achieve high sensitivity detection, high specificity, high sensitivity and specificity

Inactive Publication Date: 2016-01-06
XUZHOU MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for efficient detection of 5hmC status at specific sites in the genome, only limited 5hmC sites can be detected at present, or there are false positives in the test results, inaccurate test results and high cost of testing.

Method used

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  • A chip-based method for high-throughput and high-sensitivity detection of 5-hydroxymethylated cytosine
  • A chip-based method for high-throughput and high-sensitivity detection of 5-hydroxymethylated cytosine

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Experimental program
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Effect test

Embodiment 1

[0034]Realize the detection of the hydroxymethylated cytosine (hmC) status of the first C site (194-59366) of the 68 CCGG sequences of the BDNF gene (NT_039207.8) in the spinal cord of mice with chronic inflammatory pain.

[0035] (1) Design specific amplification primers with acrylamide modification at the 5' end for the first C sites of the above 68 CCGG sequences, dilute to 20uM and fix on acrylamide-treated glass slides to form a microarray chip. Two open-loop detection probes were designed for each C site of the above-mentioned 68 CCGG sequences, which are open-loop detection probe I and open-loop detection probe II (among them, open-loop detection probe I is used to detect non-hmC states, The open-loop probe II is used to detect the state of hmC. The main sequence of the open-loop detection probe is shown in List 1.

[0036] (2) Treat 1ug of mouse spinal cord DNA with T4-BGT, that is, treat the mouse genomic DNA to be detected with β-glucosyltransferase, and glycosylate ...

Embodiment 2

[0041] Realize the detection of the first C-site hydroxymethylated cytosine (hmC) status of the 33 CGCG sequences of the BDNF gene (AC_000143.1) in the plasma DNA of patients with chronic rheumatoid pain.

[0042] (1) Specific amplification primers with acrylamide modification at the 5' end were designed for the first C sites of the above 33 CGCG sequences, diluted to 20uM and fixed on acrylamide-treated glass slides to form a microarray chip. Then, two open-loop detection probes were designed for each C site of the above-mentioned 33 CGCG sequences, respectively, open-loop detection probe I and open-loop detection probe II (among them, open-loop detection probe I is used to detect non-hmC state , the open-loop probe II is used to detect hmC status. The main sequence of the open-loop detection probe is shown in List 1.

[0043] (2) Treat 1ug of the patient's plasma DNA with T4-BGT, that is, treat the genomic DNA of the mouse to be detected with β-glucosyltransferase, and glyco...

Embodiment 3

[0047] Combined detection of hmC status of the first C of 19 CCGG sequences (40945-59475) of the BDNF gene (NT_187012.1) and the first C of 10 CCGG sequences (771-15422) of the COMT gene (NT_187012.1) in spinal cord DNA of mice with chronic neuropathic pain .

[0048] (1) For the first C of the 19 CCGG sequences (40945-59475) of the above-mentioned BDNF gene and the first C of the 10 CCGG sequences (771-15422) of the COMT gene (NT_187012.1), respectively design the 5' end with acrylamide The modified specific amplification primers were diluted to 20uM and fixed on the acrylamide-treated glass slide to become a microarray chip. Two open-loop detection probes were designed for each C site, respectively, open-loop detection probe I and open-loop detection probe II (among them, open-loop detection probe I was used to detect non-hmC states, and open-loop detection probe II Used to detect the state of hmC. The main sequence of the open-loop detection probe is shown in List 1.

[0...

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Abstract

The invention discloses a chip-based method for detecting 5-hydroxymethylated cytosine with high throughput and high sensitivity. In the method, firstly, rolling circle amplification primers are fixed on the chip to form a microarray, and then β-glucosyl transfer Enzyme treatment of the genomic DNA to be detected, glycosylation modification of all 5hmC in the genomic DNA, and treatment of the modified DNA with sodium sulfite, and then placing the open-loop probes for detecting different C sites in the same reaction tube for hybridization ligation; The ligated product is hybridized with the chip, and the specific primers fixed on the chip are used for rolling circle amplification with the ligated probe as a template. Finally, different fluorescent detection probes are hybridized with the chip: according to the type and intensity of the fluorescence of different matrix points on the chip, the genome can be determined. Whether hydroxymethylation occurs at different C sites of DNA and its frequency. The method has strong operability, strong specificity, high sensitivity, accurate detection result, high detection efficiency, low detection cost and wide application range.

Description

technical field [0001] The invention belongs to gene chip technology, and especially designs a chip-based method for detecting 5-hydroxymethylated cytosine with high throughput and high sensitivity. Background technique [0002] As early as the 1970s, Penn et al. first discovered the presence of 5-hydroxymethylated cytosine (5hmC) in mammalian DNA (Pennetal., 1972). However, due to the backwardness of research methods, 5hmC has not received due attention. Until 2009, two scientists, Kriaucionis and Tahiliani, confirmed the presence of 5hmC in Purkinje cells, granule neurons and embryonic stem cells of mice (KriaucionisandHeintz, 2009; Tahilianie et al., 2009), and a large amount of 5hmC was also detected in other mammalian tissues (Globische et al., 2010), people realized the importance of 5hmC in regulating the physiology and biochemistry of the body. Therefore, 5hmC has become a hotspot in epigenetics research. However, how to regulate the content of 5hmC in the genome ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 曹君利潘志强李燕强郝凌云杨曦张松唐倩倩
Owner XUZHOU MEDICAL COLLEGE
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