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Primers for detecting activity of telomerase

A technology of activity detection and telomerase, which is used in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc. The effect of increasing turnover

Inactive Publication Date: 2012-05-23
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

That is to say, this method requires the detected telomerase to have higher activity, which also affects the detection sensitivity to a certain extent.

Method used

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  • Primers for detecting activity of telomerase
  • Primers for detecting activity of telomerase
  • Primers for detecting activity of telomerase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1. Design of telomerase primers

[0047] The designed telomerase primer sequence is as follows: 5′-T GGG TA GGG C GGG TTA, the primer contains three groups of continuous G sequences (underlined parts), each group of continuous G sequences contains 3 G bases. In order to verify whether the designed primers can be used for the analysis of telomerase activity, GGG was introduced at the 3′-end of the primers to simulate the extension products of the primers under the action of telomerase, and its specific sequence is 5′-T GGG TA GGG C GGG TTA GGG. The feasibility of using the DNA sequence as a telomerase primer is tested by investigating the difference in peroxidase activity between the telomerase primer and the simulated extension product after being combined with hemin.

[0048] Specific steps are as follows:

[0049] Three tubes of reactions were prepared in parallel, and the total volume of the reaction solution was 100 μL. Its composition is as ...

Embodiment 2

[0051] Example 2. The designed primers are used for the detection of telomerase activity in leukemia HL-60 cells

[0052] The same telomerase primers as in Example 1 were used in this example. In this example, telomerase was extracted from HL-60 cells and the extracted telomerase was used as a primer for telomerase, and an inactivated extract was used as a negative control. For the sample to be tested with telomerase activity, the telomerase primer can be extended under the action of telomerase to generate the same sequence as the telomerase analog extension product in Example 1; and for the negative control, Telomerase primers cannot be extended. The detection of telomerase activity in the sample to be tested is carried out by comparing the difference in the peroxidase activity shown by the reaction system of the sample to be tested, the negative control and the hemin. The oxidation of 2,2'-amino-bis(3-ethyl-benzothiazoline-6-sulfonic acid) by hydrogen peroxide was monitore...

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Abstract

The invention provides novel primers for detecting the activity of telomerase. The sequences of the primers are as follows: 5'-loop sequence 1-Gj-loop sequence 2-Gn-loop sequence 3-Gm-loop sequence 4-3', wherein j, n and m are equal to 2-6, and the loop sequence 4 is as the same as the sequence of human telomerase, namely, the sequence is as follows: T, TT, TTA, TTAG or TTAGG. The primers comprise 3 groups of continues guanine (G) sequences. When the primers are taken as the primers of the telomerase, under the function of the telomerase, the 3'end of the primers can be extended to have one group of continuous G sequence, so that the design of the primers can meet the forming condition of G-tetrad structure, and the G-tetrad structure can be formed under a certain condition. The formed G-tetrad structure is combined with chlorhematin to show the activity of peroxidase, catalyze reaction that hydrogen peroxide oxidates 2, 2'-azyl-di (3-ethyl-benzothiazole-6-sulfonic acid), and detect the activity of the telomerase according to the change of a reaction system in the absorbance of lambada=422nm. The detection has simple operation, cheap reagent and high sensitivity.

Description

【Technical field】 [0001] The invention relates to a novel primer design scheme for detecting telomerase activity and belongs to the field of biotechnology. 【Background technique】 [0002] At present, cancer has become the number one killer of human health. Studies have found that cell immortalization is one of the important characteristics that distinguish tumor cells from normal cells, and is also the key to tumor growth and metastasis. Studies have found that the lifespan of cells is determined by the length of telomeres. Telomeres are special structures located at the ends of linear chromosomes in eukaryotic cells. In normal cells, as cell division proceeds, telomere DNA shortens continuously, and when it shortens to a critical value, cells stop differentiation and undergo programmed death. In most tumor cells, due to the existence of a special active enzyme, the length of the telomere can not be shortened with the increase of the number of cell divisions, and the telo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/25C12N15/11
Inventor 孔德明蔡莉莉沈含熙
Owner NANKAI UNIV