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Terminal-phosphate-labeled nucleotides and methods of use

A phosphate radical and labeling technology, which is applied in the direction of chemical instruments and methods, biochemical equipment and methods, microbial determination/inspection, etc., can solve problems such as dynamic range limitation

Active Publication Date: 2007-05-30
GLOBAL LIFE SCI SOLUTIONS USA LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But because the quenching is not absolute, the dynamic range of this assay is limited

Method used

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  • Terminal-phosphate-labeled nucleotides and methods of use
  • Terminal-phosphate-labeled nucleotides and methods of use
  • Terminal-phosphate-labeled nucleotides and methods of use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0124] Preparation of γ-(4-trifluoromethylcoumarinyl)ddGTP (γCF3coumarin-ddGTP)

[0125] ddGTP (200 μl, 46.4mM solution, purity>96%) was co-evaporated together with anhydrous dimethylformamide (DMF, 2×0.5ml), and dicyclohexylcarbodiimide (DCC, 9.6mg, 5 eq), the mixture was co-evaporated with anhydrous DMF (0.5ml), then the residue was treated in anhydrous DMF (0.5ml), and the mixture was stirred overnight. About 20% of uncyclized triphosphate (probably from hydrolysis of the cyclic trimethylphosphate on the column) remained. Another 2 equivalents of DCC were added to the mixture, and after stirring for 2 hours, 7-hydroxy-4-trifluoromethylcoumarin (4-trifluoromethylumbelliferone, 42.7 mg, 20 equivalents) and triethylamine ( 26 μl, 20 equivalents), the mixture was stirred at room temperature, and after 2 days, HPLC (0-30% acetonitrile / 15 minutes in 0.1M triethylammonium acetate (TEAA), 30-50% acetonitrile / 5 min and 50-100% acetonitrile / 10 min, C18 3.9 x 150 mm column, flow 1 m...

Embodiment 2

[0130] Preparation of γ-(3-cyanocoumarinyl)ddATP (γ-CNcoumarin-ddATP)

[0131]ddATP (100 μl, 89 mM solution, >96%) was co-evaporated with anhydrous DMF (2×1 ml), to which was added DCC (9.2 mg, 5 equiv), the mixture was stirred, and then co-evaporated with anhydrous DMF (1 ml), The residue was treated with anhydrous DMF (0.5ml) and 7-hydroxy-3-cyanocoumarin (33.3mg, 20eq) and TEA (25p.1, 20eq) were added after the reaction was stirred overnight at room temperature. After stirring the mixture at room temperature for 1 day a major product (55% at 254 nm) was observed at 8.1 min and another minor product was observed at 10 min (approximately 10%), no major changes were observed after one day.

[0132] The reaction mixture was concentrated on a rotary evaporator, the residue was extracted with 3 x 2 ml water and filtered, the aqueous solution was concentrated and purified on C-18 using 0-30% acetonitrile (pH 6.7) in 0.1 M TEAB / 30 min, 30 -50% acetonitrile / 10min, flow rate 15ml / mi...

Embodiment 3

[0136] Preparation of 8-9H(1,3-dichloro-9,9-dimethylacridin-2-on-7-yl)-dideoxythymidine-5′-tetraphosphate (ddT4P-DDAO)

[0137] Co-evaporate ddTTP (100gel, 80mM solution) and anhydrous dimethylformamide (DMF, 2×1ml), add dicyclohexylcarbodiimide (8.3mg. DMF (1 ml) was co-evaporated, the residue was treated with anhydrous DMF (1 ml), the reaction was stirred at room temperature overnight, HPLC showed mostly cyclized triphosphate (-82%). Concentrate the reaction mixture, wash the residue three times with anhydrous diethyl ether, redissolve in anhydrous DMF, and concentrate to dryness with a rotary evaporator. The residue was leached and stirred at 40°C for one week, HPLC showed the formation of a new product with a desirable UV profile at 11.96 minutes. (HPLC method: 0.30% acetonitrile (pH 7) in 0.1 M triethylammonium acetate / 15 min, 30-50% acetonitrile / 5 min, Novapak C-18 3.9 x 150 mm column, 1 ml / min). LCMS (ES-) also showed the main peak 834 of the M-1 peak. The reaction m...

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Abstract

The present invention relates to improved methods of detecting a target using a labeled substrate or substrate analog. The methods comprise reacting the substrate or substrate analog in an enzyme-catalyzed reaction which produces a labeled moiety with independently detectable signal only when such substrate or substrate analog reacts. The present invention, in particular, describes methods of detecting a nucleic acid in a sample, based on the use of terminal-phosphate-labeled nucleotides as substrate for nucleic acid polymerases. The methods provided by this invention utilize a nucleoside polyphosphate, dideoxynucleoside polyphosphate, or deoxynucleoside polyphosphate analogue which has a colorimetric dye, chemiluminescent, or fluorescent moiety, a mass tag or an electrochemical tag attached to the terminal-phosphate. When a nucleic acid polymerase uses this analogue as a substrate, an enzyme-activatable label would be present on the inorganic polyphosphate by-product of phosphoryl transfer. Cleavage of the polyphosphate product of phosphoryl transfer via phosphatase leads to a detectable change in the label attached thereon. When the polymerase assay is performed in the presence of a phosphatase, there is provided a convenient method for real-time monitoring of DNA or RNA synthesis and detection of a target nucleic acid.

Description

[0001] related application [0002] This application claims priority to US Patent Application Serial No. 10 / 358,818 (filed February 5, 2003), which is a continuation-in-part of US Patent Application Serial No. 10 / 113,030 (filed April 1, 2002). field of invention [0003] The present invention relates to improved methods for assaying targets using labeled substrates or substrate analogs. The improvement comprises reacting a substrate or substrate analog in an enzyme-catalyzed reaction which produces a labeled moiety with an independently detectable signal only when said substrate or substrate analog reacts. In particular, the invention relates to a method for the determination of polynucleotides in a sample based on the use of terminal-phosphate-labeled nucleotides comprising three or more phosphates as substrates for nucleic acid polymerases. The labels used are dyes that undergo a chemical change and become fluorescent or color-producing reagents only by the action of a poly...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C07H19/10C07H19/20C07H21/00C12QC12Q1/00C12Q1/42G01N33/52G01N33/58
CPCC07H19/10C12Q1/6816C12Q1/6851C07H21/00C07H19/20C12Q1/6823C09B57/02C09B15/00C09B11/24C09B69/109C12Q2521/525
Inventor C·福勒S·库马A·索德J·纳尔逊
Owner GLOBAL LIFE SCI SOLUTIONS USA LLC
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