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Fluorescence polarization screening method

A technology of fluorescence polarization and fluorescent substances, which is applied in the fields of fluorescence/phosphorescence, biochemical equipment and methods, and material analysis through optical means, and can solve problems such as limited and lack of applications

Inactive Publication Date: 2001-05-30
NOVO NORDISK AS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Until the late 1970s, this characteristic of fluorescent molecules has not attracted enough attention, which may be due to the lack of technically fine enough devices that can measure the emitted polarized light and the polarization change in it, so its application is limited.

Method used

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Examples

Experimental program
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Embodiment 1

[0141] The invention is illustrated by the following non-limiting examples: Example 1: Labeled oligoxyloglucan

[0142] The oligoxyloglucan was end-labeled with sulforhodamine as described by Fry et al. (1997). Example 2: Labeling of xyloglucan by partial chemical oxidation

Embodiment 2

[0143] After stirring for 7 hours, xyloglucan (0.50 g) was dissolved in water (60 ml) at 60°C. The solution was cooled to room temperature, and an aqueous solution (10 ml) containing sodium metaperiodate (15 mg) (Merck, Germany) was added. The mixture was left overnight in the dark, and then dialyzed against demineralized water (molecular weight cut-off of 12,000-14,000) and the receiving solution was changed frequently. 25 mg of fluorescein-5-thiosemicarbazide (Molecular Probes, OR, USA) was dissolved in 1 ml of DMF (Sigma, MO, USA), and this orange solution was added to the dialyzed polymer (approximately 60 ml of solution). The solution was stirred in the dark for 18 hours. The polymer was precipitated by adding 96% ethanol (20 ml), filtered on Miracloth (Calbiochem, Germany) to collect the wet polymer, and washed with a mixture of water and ethanol (1:1) until A colorless filtrate was obtained. Finally, the labeled yellow xyloglucan was washed with absolute ethanol and air-dri...

Embodiment 3

[0144] Arabinan (0.50 g) was dissolved in water (10 ml), and an aqueous solution (5 ml) containing sodium metaperiodate (15 mg) was added. The mixture was left in the dark overnight, then diluted with water (15 ml), the solution was dialyzed against water (molecular weight cut-off 12,000-14,000) and the receiving solution was frequently replaced. 25 mg of fluorescein-5-thiosemicarbazide (Molecular Probes, OR, USA) was dissolved in 1 ml of DMF, and this orange solution was added to the dialyzed polymer. The solution was stirred in the dark for 36 hours, and then the solution was divided into two parts. One part was concentrated to a smaller volume. The polymer was precipitated by adding 90% ethanol. It is estimated that the marking degree is 0.7% (from 492nm). Calculate the absorption value). Example 4: Labeling of polygalacturonic acid

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Abstract

A method for screening for a nucleotide sequence encoding for a biological compound comprising measuring the fluorescence polarization of a fluorescent substance reacting with the biological compound expressed by an expression system comprising the nucleotide sequence.

Description

Invention field [0001] The present invention relates to a method for screening nucleotide sequences encoding biological compounds in gene expression systems, such as gene libraries or in vitro expression systems. This method uses fluorescence polarization (FP) characteristics to detect biological compounds produced in the expression system by reacting with fluorescent substances. Fluorescence detection is very sensitive and therefore suitable for detecting very small amounts of biological compounds. However, the use of fluorescence polarization assays is particularly useful because the technique enables sensitive detection and can strongly prevent signal scattering or quenching. The use of fluorescence polarization can also perform uniform screening tests, that is, it is not necessary to separate the reaction product of the biological compound and the fluorescent substance from the unreacted fluorescent substance. Background of the invention [0002] The phenomenon of fluorescenc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N15/10C12P19/04C12Q1/34C12Q1/40C12Q1/44C12Q1/48C12Q1/68G01N21/64
CPCC12Q1/40G01N2333/924C12N15/1086C12Q1/34C12Q1/48C12Q1/44G01N2333/9108G01N21/6445G01N2333/918
Inventor L·康斯巴克K·S·乔根森J·瓦尔伯乔恩C·T·乔根森T·L·胡萨姆S·厄恩斯特S·莫勒
Owner NOVO NORDISK AS
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