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Whole genome hydroxymethylation conversion reagent and application thereof

A hydroxymethylation, whole-genome technology, applied in the field of whole-genome hydroxymethylation conversion reagents, can solve the problems of unbalanced bases, large DNA damage, inconvenient operation, etc., and achieves simple components, low cost, and genome ratio. high effect

Pending Publication Date: 2022-07-12
GENETALKS BIO TECH CHANGSHA CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, the DNA damage after bisulfite treatment is large, the proportion of AT in the built library is >80%, and the bases are seriously unbalanced. On-board detection needs to be mixed with other genome-wide libraries for sequencing, which is inconvenient to operate

Method used

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  • Whole genome hydroxymethylation conversion reagent and application thereof
  • Whole genome hydroxymethylation conversion reagent and application thereof
  • Whole genome hydroxymethylation conversion reagent and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043]Example 1 Whole genome hydroxymethylation conversion reagent

[0044] In this example, a genome-wide hydroxymethylation conversion reagent is prepared, including an oxidizing agent and a reducing agent, and the oxidizing agent is K 2 RuO 4 , by a concentration of 15mM K 2 RuO 4 and 50mM NaOH, and the reducing agent is a 2-picoline borane solution with a concentration of 5mol / L.

[0045] (1) The formulation of the oxidizing agent is shown in Table 1.

[0046] Table 1

[0047]

[0048] (2) The formula of reducing agent is shown in Table 2.

[0049] Table 2

[0050]

Embodiment 2

[0051] Example 2 Whole genome hydroxymethylated non-bisulfite sequencing library

[0052] This embodiment provides a genome-wide hydroxymethylation non-bisulfite sequencing library. The schematic diagram of the whole-genome hydroxymethylation conversion reagent and the flow chart of the library construction operation are as follows: Figure 1-2 As shown, the specific construction method is:

[0053] 1. Preparation of methylation and hydroxymethylation positive standards

[0054] The positive standard used in the present invention is an artificially synthesized sequence containing methylation and hydroxymethylation sites. The specific sequence is as follows, / i5medC / represents methylated cytosine, / 55hydmeC / represents hydroxymethylation The modified cytosine, whose sequence is shown in Table 3, was synthesized by Shanghai Shenggong.

[0055] table 3

[0056]

[0057] The above-synthesized sequence was quantitatively diluted to 100 μM with 10 mM Tris-HCl (pH 8.0) accordi...

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Abstract

The invention discloses a whole genome hydroxymethylation conversion reagent and application thereof, the reagent comprises an oxidizing agent and a reducing agent, the oxidizing agent comprises a metal oxide, and the reducing agent comprises 2-methylpyridine borane. The whole-genome hydroxymethylation conversion reagent can specifically recognize and convert 5hmC, only hydroxymethylation-modified cytosine is converted into T, methylation-modified cytosine is not converted, and the problems that in the prior art, a methylation sequencing library constructed based on bisulfite conversion is unbalanced in basic group, high in specificity and high in specificity are solved. And the kit has the advantages of simple components, low cost and high efficiency.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a whole genome hydroxymethylation conversion reagent and application thereof. Background technique [0002] DNA methylation is a major form of epigenetic modification of genomic DNA. Studies have shown that DNA methylation is closely related to cancer and can be used as an important indicator for early diagnosis and prognosis evaluation of tumors. In the past, mammalian DNA methylation was thought to be very stable, but an increasing number of studies have shown that this process is dynamically reversible. Therefore, in the past ten years, people have been trying to find the reverse reaction of DNA methylation, that is, direct DNA demethylation, but have not been successful. The discovery of the sixth base, 5-hydroxy-methylcytosine (5hmC), provides a new idea for the study of DNA demethylation. At present, the rapid development of related analysis, detection and sequenc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6869C40B50/00
CPCC12Q1/6869C40B50/00
Inventor 朱碧银王琳燕陈香王煜
Owner GENETALKS BIO TECH CHANGSHA CO LTD
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