Chip-based 5-hydroxymethylated cytosine detection method with high flux and high sensitivity

A technology for hydroxymethylated cytosine and sensitive detection is applied in the field of high-throughput and highly sensitive detection of 5-hydroxymethylated cytosine, and can solve the problems of false positive detection results, high detection cost, inaccurate detection results, and the like, To achieve the effect of high specificity, high sensitivity detection, high sensitivity and specificity

Inactive Publication Date: 2013-11-27
XUZHOU MEDICAL COLLEGE
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, for efficient detection of 5hmC status at specific sites in the genome, only limited 5hmC sites can be dete

Method used

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  • Chip-based 5-hydroxymethylated cytosine detection method with high flux and high sensitivity
  • Chip-based 5-hydroxymethylated cytosine detection method with high flux and high sensitivity

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Embodiment 1

[0034] To achieve 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 them to 20uM and fix them on a glass slide treated with acrylamide to form a microarray chip. Two open-loop detection probes are designed for each C site of the above 68 CCGG sequences, namely, open-loop detection probe I and open-loop detection probe II (the open-loop probe I is used to detect the non-hmC state, Open-loop probe II is used to detect hmC status. The main sequence of the open-loop detection probe is shown in Table 1.

[0036] (2) Treat 1ug of mouse spinal cord DNA with T4-BGT, that is, treat the genomic DNA of the mouse to be tested with β-glucosyltransferase, and sugar all 5-hydroxymethylated ...

Embodiment 2

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

[0042] (1) Design specific amplification primers with acrylamide modification at the 5'end for the first C sites of the 33 CGCG sequences, diluted to 20uM and fixed on the glass slide treated with acrylamide to form a microarray chip. Then, two open-loop detection probes are designed for each C site of the 33 CGCG sequences, namely, open-loop detection probe I and open-loop detection probe II (the open-loop probe I is used to detect non-hmC states). , Open-loop probe II is used to detect hmC status. The main sequence of the open-loop detection probe is shown in Table 1.

[0043] (2) Treat 1ug patient plasma DNA with T4-BGT, that is, treat the genomic DNA of the mouse to be tested with β-glucosyltransferase, and perform glycosylation of all 5-hydroxymethylated cytosines in the...

Embodiment 3

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

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

[0049] (2) Treat 1ug of mouse spinal cord DNA with T4-BGT, that is, treat the g...

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Abstract

The invention discloses a chip-based 5-hydroxymethylated cytosine detection method with high flux and high sensitivity. The method comprises steps of fixing rolling circle amplification primers to a chip to form a micro array; treating genome DNA to be detected by using beta-glucosyltransferase; subjecting all 5hmC in the genome DNA to galactosylated modification; treating the modified DNA by using sodium sulfite; placing open-loop probes used for detecting different C sites into the same reaction tube to carry out hybridization connection; subjecting products after connection and the chip to hybridization; subjecting specific primers of the fixed chip to rolling circle amplification by using the connected probe as the template; subjecting different fluorescence detection probes and the chip to hybridization; and determining whether hydroxymethylation happens to the different C sites of the genome DNA and the hydroxymethylation frequency according to fluorescence types and intensity of different matrix dots on the chip. The method has strong operationality, strong specificity, high sensitivity, accurate detection results, 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 (Kriaucionis and Heintz, 2009; Tahiliani et al., 2009), and it was also detected in other tissues of mammals With the existence of a large amount of 5hmC (Globisch 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 cont...

Claims

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Application Information

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