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Method and kit for detecting 5-hydroxymethyl cytosine in DNA through boric acid mediated polymerase chain reactions

A technology of hydroxymethylcytosine and a kit, applied in the field of biological detection, can solve problems such as inability to distinguish gene sequences

Active Publication Date: 2015-09-23
食药环检验研究院(山东)集团有限公司 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The detection kit is limited by the enzyme cutting site, that is, the 5ghmC site must be located on the enzyme cutting site to be detected, and it cannot distinguish multi-site gene sequences

Method used

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  • Method and kit for detecting 5-hydroxymethyl cytosine in DNA through boric acid mediated polymerase chain reactions
  • Method and kit for detecting 5-hydroxymethyl cytosine in DNA through boric acid mediated polymerase chain reactions
  • Method and kit for detecting 5-hydroxymethyl cytosine in DNA through boric acid mediated polymerase chain reactions

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1. Using boric acid-mediated polymerase chain reaction kit to detect 5hmC of specific gene sequence of genomic DNA

[0041] The present invention uses a boric acid-mediated polymerase chain reaction kit to analyze the genomic DNA of mouse embryonic stem cells rich in 5hmC (5hmC content is 800 pieces / 10 6 C) The specific gene sequence 5hmC was identified and analyzed.

[0042] A highly enriched gene region of 5hmC was detected using this kit. In the results of mouse embryonic stem cell genomic DNA high-throughput sequencing (Illumina ChIP-Seq, GSE43262), three PCR amplification regions (INTRON_PAX5_1, INTRON_PAX5_2, INTRON_PAX5_3) of the B cell transcription factor related gene PAX5 were selected, and UTR-5_SRR was selected as the Quantitative PCR internal reference genes, design specific primers for different gene sequences, and then sequentially perform glucosylation and 2-CB-PBA treatment on the genomic DNA, and use the designed specific primers for PCR amplificati...

Embodiment 2

[0044] Example 2.5hmC quantitative analysis

[0045] Taking the sample treated with 2-CB-PBA as an example, the negative control probe (5hmC-ds100mer) and the positive control probe (5ghmC-ds100mer) are mixed in different proportions, and the molar concentration ratios of 5ghmC: 5hmC are selected as: 0:1, 1:16, 1:8, 1:4, 1:2, 1:1, 2:1, 4:1, and 1:0, and the mixed template concentration is maintained at 5.0nM. Then use this mixture as a PCR amplification template to investigate the 5ghmC-ds100mer concentration and ΔC t The relationship between the values ​​so that the curve C can be amplified by PCR t The value change is used to quantitatively analyze 5ghmC. Attached Figure 5 a and Figure 5 As shown in b, as the concentration of 5ghmC increases, ΔC t The value increases from 0 to 6.0, and the linear relationship between the two is satisfied: ΔC t =6.14+4.87log[5ghmC / ds100mer](R 2 =0.97). Although ΔC t Value increases, but the amount of PCR amplification product is gradually dec...

Embodiment 3

[0046] Example 3. Comparison of boric acid-mediated polymerase chain reaction kit and restriction endonuclease-binding PCR detection kit (ie MspI enzymatic hydrolysis)

[0047] The restriction endonuclease MspI recognizes the CCGG sequence, and its digestion specificity is changed by glucosylation: MspI recognizes and cleaves 5mC and 5hmC, but cannot cleave 5ghmC; when the 5ghmC site is on the CCGG sequence, it can The cleaved 5hmC site was converted into a non-cleavable 5ghmC site, so that the sequence was preserved by PCR amplification. Therefore, the restriction endonuclease method is restricted by various conditions such as restriction sites.

[0048] First, use the positive control probe (5ghmC-ds100mer) as the PCR amplification template to investigate the PCR amplification conditions after 2-CB-PBA treatment and MspI digestion. After the probe is digested by MspI, C t The value does not change significantly, that is, the enzymatic hydrolysis treatment does not affect the PCR...

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Abstract

The invention discloses a method and kit for detecting 5-hydroxymethyl cytosine in DNA through boric acid mediated polymerase chain reactions. On the basis of glucosylation reactions, boric acid and derivative reagents can carry out covalent effect with vicinal diol in glycosylated 5-hydroxymethyl cytosine; thus the volume of the duplicated base unit is prominently increased, the polymerase amplification reactions are effectively inhibited; boric acid mediated polymerase chain amplification reactions are provided, and the 5-hydromethyl cytosine in specific gene / sections can be specifically recognized. The invention further provides a kit on the basis of the method, and the kit can rapidly and sensitively detect the 5-hydroxymethyl cytosine and can be applied to various cell analysis. Compared with the conventional detection kits, the provided kit is not limited by the cleavage sites, and can be used to analyze and identify 5-hydroxymethyl in various sequences. The kit has a wide application prospect, and has an important meaning for the further research on the biological functions of 5-hydroxymethyl cytosine.

Description

Technical field [0001] The invention relates to the technical field of biological detection, in particular to a method and a kit for quickly, accurately and efficiently detecting and locating 5-hydroxymethylcytosine in a specific gene / fragment. Background technique [0002] 5-hydroxymethylcytosine (5hmC) is the sixth base found after 5-methylcytosine (5mC) and has been detected in many mammalian tissues and cells . Compared with 5mC, the content of 5hmC is lower (In 2009, the Heintz research group used thin-layer chromatography and mass spectrometry and other analytical methods to detect 5hmC in human and mouse brain and embryonic stem cells, and the cortex and brain stem regions are rich in 5hmC. The content of 5hmC in Purkinje cells accounts for about 0.6% of the total nucleotides, and about 0.2% in granular cells; S. Kriaucionis, N. Heintz, Science, 2009, 324: 929-930), but the same is true for 5hmC Has a very important biological function. [0003] Existing studies have show...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 汪海林赵超赵柏林李翠平
Owner 食药环检验研究院(山东)集团有限公司
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