DD148955A1 - PROCESS FOR COUPLING NUCLEIC ACIDS TO TRAITER MATERIALS - Google Patents

Abstract

The invention relates to a chemical coupling method of nucleic acids to traeger materials, which is used for affinity chromatographic investigations. The goal is the stable coupling of nucleic acids to a carrier. It was surprisingly found that it is only in the acid-buffered pH range (5 to 6.5) that a coupling of the nucleic acids to cyanuric acid chloride-activated carrier materials that is stable under hybridization conditions is possible. The coupling method can also be used to transfer segregated nucleic acids to activated paper.

Description

Inventor: Dr. H.-D · Hunger

Method for coupling nucleic acids to support materials Field of application of the invention

The invention relates to a method for coupling nucleic acids to support materials, which in biochemistry for. affinity chromatographic investigations find application ·

Characteristics of known technical solutions The coupling of nucleic acids by known chemical methods (Ho Potuzak and PDG Dean (1978) Pebs-Lett. 88, 161-166; HH Weetall (Separation and Purification Methods, 2 (2) , 199-229 (1973 ))) leads to relatively unstable coupling products under hybridization conditions. They are therefore poorly suited for use in affinity chromatography of minor components of a nucleic acid mixture or for transfer experiments of nucleic acids from release gels by means of activated papers. Another known method with cyanuric chloride (S. Biagioni, R. Sisto, A. Perraro, P. Caiafa, C. Turano Analytical Biochemistry 8 pp., 616-619 (1978); NL Smith and H. M ₀ Lenhoff Analytical Biochemistry 61. »392 - 415 · (1974)) coupled only double-stranded DNA to crystalline cellulose. The coupling conditions do not allow stability of coupling under hybridization conditions.

Known coupling methods via diazobenzyloxymethyl groups (J.C. Alwine, D.-Kemp, B.A. Parker, J. Keizer, J.Renart, G.R. Stark, G.M., Methods in Enzymology, still in print) represent a relatively complicated chemical procedure.

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Object of the invention

The aim of the invention is to develop a chemically simple method for coupling nucleic acids to support materials,

Explanation of the invention of the invention

The object of the invention is to provide such a method that the binding of the nucleic acids to the carrier material is stable under hybridization and elution conditions. According to the invention, the carrier material used for the coupling of nucleic acids, preferably cellulose or paper activated with cyanuric chloride, is brought into contact with the nucleic acids in the weakly acidic buffered pH range, preferably 5-6 »5 (activation of the carrier material takes place in a mixture of dioxane / Syloi;) Surprisingly, it has been found that a coupling stable under hybridization conditions takes place only in the weakly acidic buffered pH range, preferably at pH = 6.1 and in the Y, -M Sorensen buffer.

A coupling to activated paper is used for the transfer of nucleic acids or their fragments from separating gels. The transfer of the nucleic acids from separating gels onto the paper takes place according to the technique described by Southern (6). The invention has the advantage that it is universally applicable. It can be DNA and RNA fragments of various sizes (single strand and double strand) are coupled. The coupling is stable under hybridization and elution conditions.

The invention will be explained in more detail by way of example.

Implementation example .

0.5 cm Whatman 540 paper shaken for 15 minutes in 3 M NaOH »The paper is placed in a 5 % cyanuric chloride solution in dioxane / xylene (1: 1) and shaken for 30 minutes at room temperature.

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The filter paper is washed for 10 minutes (2 × dioxane, 1 × dioxane glacial acetic acid / water (2/1/1), 1 × water)

 The coupling of the nucleic acids takes place "at room temperature in a 1/15 m Sörensen buffer pH 6.1, DNA concentration 100 / μg / ml ·

Covalently bond up to 20 / ug DITS / cm paper. Inactivation of existing residual groups takes place by means of 1 % ethanolamine in the coupling buffer

Claims (3)

-4- 2 18847 invention claim 1. A process for the coupling of nucleic acids to cyanuric chloride-activated carrier materials, characterized in that the carrier materials, preferably cellulose or paper, in contact with nucleic acids in weakly acidic, buffered pH range, preferably 5-6.5 , be brought. 2. The method according to item 1, characterized in that the coupling at pH 6.1 in τ, - performed Sörensen buffer , becomes,, · 3 · Method according to item 1, characterized in that the coupling is effected by means of Southern technology »