Colorimetric sensing method for detecting activity of DNA transmethylase based on DNA chain substituting circulation amplifying technology

A methyltransferase and cyclic amplification technology, applied in the field of colorimetric sensing, to achieve the effects of high sensitivity, simple and convenient visual detection, and low detection limit

Inactive Publication Date: 2015-06-10
NANCHANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no report on a colorimetric sensing method combining DNA strand disp...

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  • Colorimetric sensing method for detecting activity of DNA transmethylase based on DNA chain substituting circulation amplifying technology
  • Colorimetric sensing method for detecting activity of DNA transmethylase based on DNA chain substituting circulation amplifying technology
  • Colorimetric sensing method for detecting activity of DNA transmethylase based on DNA chain substituting circulation amplifying technology

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Embodiment 1

[0020] (1) Preparation of AuNPs: Add 49 mL of ultrapure water and 1 mL of 2% HAuCl in a round bottom flask 4 ·3H 2 O solution, after heating to make it boil, add 1 mL of trisodium citrate solution with a concentration of 5% by mass, continue to stir and keep boiling for 5 minutes, then let the solution naturally cool to room temperature under stirring, and then prepare a particle size of 13 nm and AuNPs at a concentration of 12 nM, at 4 o C is kept for later use;

[0021] (2) Preparation of AuNPs-labeled probe DNA: Add 5.5 nM activated probe DNA to 3 mL of the AuNPs solution prepared in step (1), shake on a shaker at room temperature for 12 hours, add phosphoric acid to adjust the phosphate radical in the solution The concentration of NaCl was 9 mM. After 30 minutes, the phosphate buffer solution was added, and the phosphate buffer solution was continued to be added six times in the next two days, so that the final concentration of NaCl was 0.3 mM, and aged overnight. Centr...

Embodiment 2

[0026] M.SssI activity detection

[0027] (1) Optimization of DNA I concentration, DNA I:DNA II, SAM concentration, and HpaII concentration

[0028] Figure 4 A is the effect of DNA I concentration on M.SssI activity detection. It can be seen from the figure that as the concentration of DNA I increases, the absorbance of the AuNPs solution decreases first and then reaches a stable trend whether with or without M.SssI. When the DNA I concentration was 40nM, the absorbance difference (ΔA) reached the maximum. Therefore, the optimal concentration of DNA I was chosen to be 40 nM. Figure 4 B is the effect of the ratio of DNA I to DNA II on the detection of M.SssI activity. In the present invention, the efficiency of strand substitution amplification depends to a certain extent on the ratio of DNA I to DNA II (DNA I:DNA II). The concentration of DNA I was fixed at 40 nM, and the DNA I:DNA II was gradually increased from 1:1 to 1:5. When the ratio of DNA I:DNA II was 1:3, the Δ...

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Abstract

The invention discloses a colorimetric sensing method for detecting activity of DNA transmethylase based on The DNA chain substituting circulation amplifying technology. The method is designed as that the neck to be recognized by CpG transmethylase M.SssI and HpaII restriction enzyme contains hairpin DNA I with the 5'-CCGG-3' sequence; the presence of M.SssI can be determined by determining whether the hairpin DNA I is methylated and whether the hairpin DNA can be cut by HpaII to cause gathering discoloration of AuNPs; the sensitive detection of M.SssI can be achieved by the colorimetric method.

Description

technical field [0001] The invention relates to a colorimetric sensing method for detecting DNA methyltransferase activity based on a DNA chain substitution cycle amplification technology, and belongs to the technical field of colorimetric sensing. Background technique [0002] DNA methylation plays a very important role in many normal cell growth processes including cell differentiation, transcriptional silencing, gene regulation, X chromosome inactivation and some other processes. Different methyltransferases can lead to different abnormal DNA methylation, and DNA methylation is closely related to many genetic diseases and cancers, making DNA methyltransferases potential targets and biomarkers for various cancer treatments. In malignant tumors, the abnormality of methyltransferase activity usually appears earlier than other phenomena, so it can be used for early diagnosis of cancer. Therefore, it is very necessary to develop sensitive detection methods for methyltransfera...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/48G01N21/78
CPCC12Q1/48C12Q1/68G01N21/78
Inventor 邱建丁钟兆花李志美梁汝萍
Owner NANCHANG UNIV
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