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
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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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