BE889858A - PROCESS FOR THE SYNTHESIS OF POLYDESOXYNUCLEOTIDES - Google Patents

PROCESS FOR THE SYNTHESIS OF POLYDESOXYNUCLEOTIDES Download PDF

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Publication number
BE889858A
BE889858A BE1/10285A BE1010285A BE889858A BE 889858 A BE889858 A BE 889858A BE 1/10285 A BE1/10285 A BE 1/10285A BE 1010285 A BE1010285 A BE 1010285A BE 889858 A BE889858 A BE 889858A
Authority
BE
Belgium
Prior art keywords
emi
polydesoxynucleotides
synthesis
poly
mixture
Prior art date
Application number
BE1/10285A
Other languages
French (fr)
Original Assignee
Mta Kozponti Kemiai Ki
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Mta Kozponti Kemiai Ki filed Critical Mta Kozponti Kemiai Ki
Priority to BE1/10285A priority Critical patent/BE889858A/en
Publication of BE889858A publication Critical patent/BE889858A/en

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Enzymes And Modification Thereof (AREA)

Description

       

   <EMI ID=1.1> 

  
pH compris entre 6,5 et 9,5 et à une température comprise

  
 <EMI ID=2.1> 

  
 <EMI ID=3.1> 

  
 <EMI ID=4.1> 

  
 <EMI ID=5.1> 

  
 <EMI ID=6.1> 

  
 <EMI ID=7.1> 

  
Acide Research 4, 2767-2777 (1977). Dans ces procédés, la

  
 <EMI ID=8.1> 

  
 <EMI ID=9.1> 

  
au fonctionnement optimal de l'enzyme. Le mélange réactionnel est incubé et lorsque la réaction est achevée, l'enzyme est dénaturée (par exemple par chauffage de la solution) et la protéine d'enzyme précipitée est éliminée du mélange. Le polydésoxynucléotide produit présentant un poids moléculaire

  
 <EMI ID=10.1> 

  
Les polydésoxynucléotides obtenus de cette manière sont

  
 <EMI ID=11.1> 

  
physiques, chimiques, biochimiques et biologiques des acides nucléiques.

  
 <EMI ID=12.1> 

  
 <EMI ID=13.1>   <EMI ID=14.1> 

  
 <EMI ID=15.1> 

  
il faut donc une molécule motrice de poly d (A-T) . De cette

  
 <EMI ID=16.1> 

  
thétiser de novo du poly d (A-T), c'est-à-dire sans addition

  
 <EMI ID=17.1> 

  
 <EMI ID=18.1> 

  
 <EMI ID=19.1> 

  
 <EMI ID=20.1> 

  
réside en ce que l'enzysc ne peut être utilisée qu'une seule

  
 <EMI ID=21.1> 

  
 <EMI ID=22.1> 

  
La présente invention a pour but de pourvoir à une méthode qui supprime cet inconvénient.

  
La présente invention repose sur la constitution que ce

  
 <EMI ID=23.1> 

  
 <EMI ID=24.1> 

  
minée du mélange réactionnel par filtration et peut être

  
 <EMI ID=25.1> 

  
 <EMI ID=26.1> 

  
 <EMI ID=27.1>   <EMI ID=28.1>  

  
 <EMI ID=29.1>  

  
 <EMI ID=30.1> 

  
 <EMI ID=31.1>   <EMI ID=32.1> 

  
 <EMI ID=33.1>   <EMI ID=34.1> 

  
 <EMI ID=35.1> 

  
 <EMI ID=36.1> 

  
haut.

  
Le stockage et la réutilisation de l'enzyme immobilisée peuvent être réalisés de la façon suivante .

  
 <EMI ID=37.1> 

  
 <EMI ID=38.1> 

  
 <EMI ID=39.1> 

  
 <EMI ID=40.1> 

  
 <EMI ID=41.1> 

  
 <EMI ID=42.1> 

  
 <EMI ID=43.1> 

  
 <EMI ID=44.1> 

  
 <EMI ID=45.1> 

  
moin.

  
 <EMI ID=46.1>  <EMI ID=47.1>   <EMI ID=48.1>  gétt&#65533;e, <EMI ID=49.1>  tinue.

  
 <EMI ID=50.1> 

  
prend encore d'autres dispositions, qui ressortiront de la description qui va suivre.

  
L'invention sera mieux comprise à l'aide du complément

  
 <EMI ID=51.1> 

  
présente invention.

  
Il doit être bien entendu, toutefois, que ces exemples

  
 <EMI ID=52.1> 

  
 <EMI ID=53.1> 

  
aucune manière une limitation.

Exemple 1

  
 <EMI ID=54.1> 

  
industrielle, lyophilisé, pendant 15 minutes à la température ambiante dans 60 mM de tampon phosphate de potassium (pH 7,4). Le gel gonflé est versé dans une colonne de verre (1,5 X 18 cm) équipée d'une enveloppe thermostable et d'un filtre de

  
 <EMI ID=55.1> 

  
 <EMI ID=56.1> 

  
 <EMI ID=57.1> 

  
 <EMI ID=58.1> 

  
 <EMI ID=59.1> 

  
 <EMI ID=60.1>   <EMI ID=61.1> 

  
 <EMI ID=62.1> 

  
 <EMI ID=63.1> 

  
gel en la levant avec une quantité supplémentaire de 10 ml de

  
 <EMI ID=64.1> 

  
l'ensemble du tampon à dix reprises au moyen d'uno pompe p6ri-

  
 <EMI ID=65.1> 

  
 <EMI ID=66.1> 

  
10 ml). On démarre la circulation du système, on rejette les 2-3 premiers millilitres du mélange (vérifiés par spectrophotométrie), puis on fait circuler le mélange au moyen d'une

  
 <EMI ID=67.1> 

  
est suivie par mesure de la radioactivité de la substance insoluble dans les acides.

  
Après 24 heures d'incubation, la quantité de polymère

  
 <EMI ID=68.1> 

  
de [<3>H] dAMP dans le produit insoluble dans les acides. 45 %

  
 <EMI ID=69.1> 

  
culaire est éliminée par dialyse. La quantité de poly d (AT) pur de haut poids moléculaire est de 2,2 mg.

  
zxomlo 2

  
 <EMI ID=70.1>   <EMI ID=71.1> 

  
 <EMI ID=72.1> 

  
 <EMI ID=73.1> 

  
 <EMI ID=74.1> 

  
 <EMI ID=75.1> 

  
Ex<BBpla 3 '

  
 <EMI ID=76.1> 

  
trats sont incorporée dans la matière polymère. Le poly

  
 <EMI ID=77.1> 

  
(supérieur à 200 000 Daltons) ; la quantité obtenue est de 2,12 mg.

  
Ainsi que cola ressort de ce qui précède, l'invention

  
 <EMI ID=78.1> 

  
 <EMI ID=79.1> 

  
de façon plus explicite ; elle en embrasse au contraire

  
 <EMI ID=80.1> 

  
 <EMI ID=81.1> 

  
portée, de la présente invention.



   <EMI ID = 1.1>

  
pH between 6.5 and 9.5 and at a temperature between

  
 <EMI ID = 2.1>

  
 <EMI ID = 3.1>

  
 <EMI ID = 4.1>

  
 <EMI ID = 5.1>

  
 <EMI ID = 6.1>

  
 <EMI ID = 7.1>

  
Acide Research 4, 2767-2777 (1977). In these processes, the

  
 <EMI ID = 8.1>

  
 <EMI ID = 9.1>

  
optimal functioning of the enzyme. The reaction mixture is incubated and when the reaction is complete, the enzyme is denatured (e.g. by heating the solution) and the precipitated enzyme protein is removed from the mixture. The product polydeoxynucleotide having a molecular weight

  
 <EMI ID = 10.1>

  
The polydeoxynucleotides obtained in this way are

  
 <EMI ID = 11.1>

  
physical, chemical, biochemical and biological nucleic acids.

  
 <EMI ID = 12.1>

  
 <EMI ID = 13.1> <EMI ID = 14.1>

  
 <EMI ID = 15.1>

  
a motor molecule of poly d (A-T) is therefore required. Of this

  
 <EMI ID = 16.1>

  
de novo thetiser of poly d (A-T), i.e. without addition

  
 <EMI ID = 17.1>

  
 <EMI ID = 18.1>

  
 <EMI ID = 19.1>

  
 <EMI ID = 20.1>

  
is that the enzysc can only be used for one

  
 <EMI ID = 21.1>

  
 <EMI ID = 22.1>

  
The object of the present invention is to provide a method which eliminates this drawback.

  
The present invention is based on the constitution that this

  
 <EMI ID = 23.1>

  
 <EMI ID = 24.1>

  
mined from the reaction mixture by filtration and can be

  
 <EMI ID = 25.1>

  
 <EMI ID = 26.1>

  
 <EMI ID = 27.1> <EMI ID = 28.1>

  
 <EMI ID = 29.1>

  
 <EMI ID = 30.1>

  
 <EMI ID = 31.1> <EMI ID = 32.1>

  
 <EMI ID = 33.1> <EMI ID = 34.1>

  
 <EMI ID = 35.1>

  
 <EMI ID = 36.1>

  
high.

  
Storage and reuse of the immobilized enzyme can be accomplished as follows.

  
 <EMI ID = 37.1>

  
 <EMI ID = 38.1>

  
 <EMI ID = 39.1>

  
 <EMI ID = 40.1>

  
 <EMI ID = 41.1>

  
 <EMI ID = 42.1>

  
 <EMI ID = 43.1>

  
 <EMI ID = 44.1>

  
 <EMI ID = 45.1>

  
less.

  
 <EMI ID = 46.1> <EMI ID = 47.1> <EMI ID = 48.1> gétt &#65533; e, <EMI ID = 49.1> continuous.

  
 <EMI ID = 50.1>

  
takes other steps, which will become apparent from the description which follows.

  
The invention will be better understood using the complement

  
 <EMI ID = 51.1>

  
present invention.

  
It should be understood, however, that these examples

  
 <EMI ID = 52.1>

  
 <EMI ID = 53.1>

  
no way a limitation.

Example 1

  
 <EMI ID = 54.1>

  
industrial, lyophilized, for 15 minutes at room temperature in 60 mM potassium phosphate buffer (pH 7.4). The swollen gel is poured into a glass column (1.5 X 18 cm) equipped with a thermostable envelope and a filter.

  
 <EMI ID = 55.1>

  
 <EMI ID = 56.1>

  
 <EMI ID = 57.1>

  
 <EMI ID = 58.1>

  
 <EMI ID = 59.1>

  
 <EMI ID = 60.1> <EMI ID = 61.1>

  
 <EMI ID = 62.1>

  
 <EMI ID = 63.1>

  
lifting the gel with an additional 10 ml of

  
 <EMI ID = 64.1>

  
the entire buffer ten times using a p6ri pump

  
 <EMI ID = 65.1>

  
 <EMI ID = 66.1>

  
10 ml). We start the circulation of the system, we reject the first 2-3 milliliters of the mixture (verified by spectrophotometry), then we circulate the mixture by means of a

  
 <EMI ID = 67.1>

  
is followed by measuring the radioactivity of the substance insoluble in acids.

  
After 24 hours of incubation, the amount of polymer

  
 <EMI ID = 68.1>

  
of [<3> H] dAMP in the acid insoluble product. 45%

  
 <EMI ID = 69.1>

  
is removed by dialysis. The amount of pure poly d (AT) of high molecular weight is 2.2 mg.

  
zxomlo 2

  
 <EMI ID = 70.1> <EMI ID = 71.1>

  
 <EMI ID = 72.1>

  
 <EMI ID = 73.1>

  
 <EMI ID = 74.1>

  
 <EMI ID = 75.1>

  
Ex <BBpla 3 '

  
 <EMI ID = 76.1>

  
trats are incorporated into the polymeric material. The poly

  
 <EMI ID = 77.1>

  
(greater than 200,000 Daltons); the amount obtained is 2.12 mg.

  
As cola emerges from the above, the invention

  
 <EMI ID = 78.1>

  
 <EMI ID = 79.1>

  
more explicitly; on the contrary, she embraces

  
 <EMI ID = 80.1>

  
 <EMI ID = 81.1>

  
scope of the present invention.

Claims (1)

<EMI ID=82.1>  <EMI ID = 82.1> <EMI ID=83.1>  <EMI ID = 83.1>
BE1/10285A 1981-08-05 1981-08-05 PROCESS FOR THE SYNTHESIS OF POLYDESOXYNUCLEOTIDES BE889858A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
BE1/10285A BE889858A (en) 1981-08-05 1981-08-05 PROCESS FOR THE SYNTHESIS OF POLYDESOXYNUCLEOTIDES

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
BE1/10285A BE889858A (en) 1981-08-05 1981-08-05 PROCESS FOR THE SYNTHESIS OF POLYDESOXYNUCLEOTIDES
BE889858 1981-08-05

Publications (1)

Publication Number Publication Date
BE889858A true BE889858A (en) 1982-02-05

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
BE1/10285A BE889858A (en) 1981-08-05 1981-08-05 PROCESS FOR THE SYNTHESIS OF POLYDESOXYNUCLEOTIDES

Country Status (1)

Country Link
BE (1) BE889858A (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992015674A3 (en) * 1991-03-02 1992-12-10 Tepnel Medical Ltd Dna -or rna- modifying enzymes immobilized to supports

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992015674A3 (en) * 1991-03-02 1992-12-10 Tepnel Medical Ltd Dna -or rna- modifying enzymes immobilized to supports

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Legal Events

Date Code Title Description
RE Patent lapsed

Owner name: MTA KOZPONTI KEMIAI KUTATO INTEZETE

Effective date: 19880831