NO310114B1 - Method of Preparation of DNA - Google Patents
Method of Preparation of DNA Download PDFInfo
- Publication number
- NO310114B1 NO310114B1 NO994144A NO994144A NO310114B1 NO 310114 B1 NO310114 B1 NO 310114B1 NO 994144 A NO994144 A NO 994144A NO 994144 A NO994144 A NO 994144A NO 310114 B1 NO310114 B1 NO 310114B1
- Authority
- NO
- Norway
- Prior art keywords
- dna
- lye
- salt
- lipid
- filtered
- Prior art date
Links
- 238000000034 method Methods 0.000 title claims description 16
- 238000002360 preparation method Methods 0.000 title description 5
- 102000053602 DNA Human genes 0.000 claims description 25
- 108020004414 DNA Proteins 0.000 claims description 25
- 150000002632 lipids Chemical class 0.000 claims description 19
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 12
- 150000003839 salts Chemical class 0.000 claims description 9
- 239000003960 organic solvent Substances 0.000 claims description 7
- 238000011282 treatment Methods 0.000 claims description 7
- 238000009835 boiling Methods 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- 239000002253 acid Substances 0.000 claims description 5
- 239000000706 filtrate Substances 0.000 claims description 5
- 239000012620 biological material Substances 0.000 claims description 3
- 239000000463 material Substances 0.000 claims description 3
- 239000003795 chemical substances by application Substances 0.000 claims 2
- 150000001298 alcohols Chemical class 0.000 claims 1
- 150000001335 aliphatic alkanes Chemical class 0.000 claims 1
- 150000001336 alkenes Chemical class 0.000 claims 1
- 239000000470 constituent Substances 0.000 claims 1
- 239000011368 organic material Substances 0.000 claims 1
- 238000001914 filtration Methods 0.000 description 11
- 239000002994 raw material Substances 0.000 description 8
- 235000013336 milk Nutrition 0.000 description 7
- 239000008267 milk Substances 0.000 description 7
- 210000004080 milk Anatomy 0.000 description 7
- 238000004519 manufacturing process Methods 0.000 description 5
- 150000003904 phospholipids Chemical class 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- 150000003626 triacylglycerols Chemical class 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 241000251468 Actinopterygii Species 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 230000002349 favourable effect Effects 0.000 description 2
- 238000002203 pretreatment Methods 0.000 description 2
- 238000007127 saponification reaction Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 241000276435 Gadus Species 0.000 description 1
- 238000003916 acid precipitation Methods 0.000 description 1
- 238000010306 acid treatment Methods 0.000 description 1
- 239000013068 control sample Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 235000020777 polyunsaturated fatty acids Nutrition 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 230000001932 seasonal effect Effects 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
Landscapes
- Saccharide Compounds (AREA)
Description
Den foreliggende oppfinnelse angår en metode for å fremstille DNA (deoxyribonukleinsyre) fra biologisk materiale. The present invention relates to a method for producing DNA (deoxyribonucleic acid) from biological material.
Metoden går i korthet ut på først å fjerne lipid f.eks. vha. løsningsmiddel før man videre i prosessen fremstiller DNA etter kjente tradisjonelle metoder. Da vil man bl.a. med kjente organiske løsningsmidler (f.esk. etanol) og etter koking av det avfettede råstoffet i lut oppnå en filtermasse med vesentlige bedre filtreringsegenskaper og et høyere utbytte av DNA enn uten den forutgående fettfjerningen. In short, the method involves first removing lipid, e.g. by solvent before DNA is produced further in the process according to known traditional methods. Then you will, among other things, with known organic solvents (e.g. ethanol) and after boiling the defatted raw material in lye, obtain a filter mass with significantly better filtration properties and a higher yield of DNA than without the previous fat removal.
Ved en tradisjonell metode for fremstilling av DNA, vil man koke det ubehandlede råstoffet i nasrvaer av lut og salt. Det vil da skje en forsåpning av det lipidet som er i råstoffet, og den resulterende masse er vanskelig å filtrere. Dette gir seg utslag i lang filtreringstid. Dette problemet løser man prosessteknisk ved bruk av kraftigere filfreringsbetingelser enn om massen var enkel å filtrere. Det er imidlertid prosessteknisk ønskelig å forbedre prosessen på In a traditional method for the production of DNA, the raw material will be boiled in a solution of lye and salt. A saponification of the lipid in the raw material will then occur, and the resulting mass is difficult to filter. This results in a long filtration time. This problem is solved technologically by using stronger filtration conditions than if the mass was easy to filter. However, it is technically desirable to improve the process
dette punktet. this point.
I tillegg er det ved tradisjonell metode et noe redusert utbytte av DNA i forhold til det maksimale. Ved å fjerne lipid før lutbehandlingen vil den reduserte forsåpningen muligens gjøre lutkokingen mer effektiv mht. å frigjøre DNA og derved resultere i et noe høyere utbytte. In addition, with the traditional method, there is a somewhat reduced yield of DNA compared to the maximum. By removing lipid before the lye treatment, the reduced saponification will possibly make the lye boiling more effective in terms of to release the DNA and thereby result in a somewhat higher yield.
En forutgående fjerning av lipid før lutbehandling vil muliggjøre anvendelse av dette lipidet. A prior removal of lipid before lye treatment will enable the use of this lipid.
I marint råstoff er lipidet særlig verdifullt pga. den gunstige sammensetningen av flerumettede fettsyrer. For fjerning av lipid fra råstoff til DNA-produksjon er alle kjente metoder aktuelle. In marine raw material, the lipid is particularly valuable because the favorable composition of polyunsaturated fatty acids. For the removal of lipid from raw material for DNA production, all known methods are applicable.
Mens triglyceridene kan være relativt enkle å fjerne fra biologisk materiale, er fosfolipidene langt vanskeligere å få trukket ut. Generelt vil man kunne bruke en kombinasjon av mekanisk/termisk metode for å fjerne lipid som har et høyt innhold av triglycerider, mens man med lipid rikt på fosfolipider vil bruke organiske løsningsmidler for å fjerne disse. Dette skyldes at fosfolipidene, som er hovedkomponenten i alle biologiske membraner, i liten grad fjernes sammen med triglyceridene ved f.eks. koking/pressing- While the triglycerides can be relatively easy to remove from biological material, the phospholipids are far more difficult to extract. In general, it will be possible to use a combination of mechanical/thermal methods to remove lipids that have a high content of triglycerides, while with lipids rich in phospholipids, organic solvents will be used to remove these. This is because the phospholipids, which are the main component of all biological membranes, are to a small extent removed together with the triglycerides by e.g. boiling/pressing
Råstoff som er særskilt aktuelt for produksjon av DNA er melke fra marine fiskearter. Denne melken inneholder 1,5 - 2% DNA, med naturlige årstidsvariasjoner. I tillegg inneholder melken 2-4% lipid, med variasjon i lipidinnholdet mellom ulike fiskearter. Dette lipidet er særskilt rikt på marine fosfolipider, med innhold opp til 75% fosfolipid av totallipid. Raw material that is particularly relevant for the production of DNA is milk from marine fish species. This milk contains 1.5 - 2% DNA, with natural seasonal variations. In addition, the milk contains 2-4% lipid, with variation in the lipid content between different fish species. This lipid is particularly rich in marine phospholipids, with a content of up to 75% phospholipid of total lipid.
I eksemplet nedenunder er det benyttet melke fra torsk { Gadus morhud) som råstoff og etanol som organisk løsningsmiddel. Resultatet i eksemplet viser en kraftig reduksjon i filtreringstid, 10 min for avfettet prøve mot 2 timer for ubehandlet, og det er oppnådd et høyere utbytte på 58% for avfettet prøve i forhold til ubehandlet. Vår metode er således nyskapende for fremstilling av DNA fra biologisk råstoff. In the example below, milk from cod {Gadus mother skin) is used as raw material and ethanol as organic solvent. The result in the example shows a strong reduction in filtration time, 10 min for degreased sample compared to 2 hours for untreated, and a higher yield of 58% has been achieved for degreased sample compared to untreated. Our method is thus innovative for the production of DNA from biological raw material.
EKSEMPEL EXAMPLE
Følgende forsøk demonstrerer effekten av fjerningen av lipid. The following experiment demonstrates the effect of the removal of lipid.
Eksempel 1 Example 1
DNA ble fremstilt fra 2 prøver å 200g grovoppdelt torskemelke. I prøve A ble lipid fjernet ved ekstraksjon med etanol, der den lipidholdige etanolrike vannfasen ble filtrert fra etter omrøring i ca. 10 min. Etter filtreringen ble DNA fremstilt fra prøve A og den ubehandlede prøve B på vanlig måte for fremstilling av DNA, ved tilsetting av vann til samme volum, tilsetting av salt og lut, koking av blandingen før filtrering og med etterfølgende syrefelling og lutbehandling. Det resulterende DNA ble tørket og analysert mht. kjemisk sammensetning i henhold til krav til kommersielle DNA-preparater. Resultatene er vist i tabell nedenfor. DNA was prepared from 2 samples of 200g coarsely divided cod milk. In sample A, lipid was removed by extraction with ethanol, where the lipid-containing ethanol-rich water phase was filtered from after stirring for approx. 10 minutes After the filtration, DNA was prepared from sample A and the untreated sample B in the usual way for the preparation of DNA, by adding water to the same volume, adding salt and lye, boiling the mixture before filtering and with subsequent acid precipitation and lye treatment. The resulting DNA was dried and analyzed for chemical composition according to requirements for commercial DNA preparations. The results are shown in the table below.
Det var ingen signifikant forskjell i den kjemiske sammensetningen av DNA prøvene, som begge hadde analyseverdier innenfor grensene for kommersiell DNA preparater. There was no significant difference in the chemical composition of the DNA samples, both of which had analysis values within the limits of commercial DNA preparations.
Hvis det ikke er ønskelig å gjenvinne lipidene fra den lipidinnholdende fase så har det overraskende vist seg at det er mulig å utelate filtreringen etter ekstraksjon med eksempelvis etanol og umiddelbart tilsette salt og lut og koke blandingen før filtrering. If it is not desirable to recover the lipids from the lipid-containing phase, it has surprisingly been shown that it is possible to omit the filtration after extraction with e.g. ethanol and immediately add salt and lye and boil the mixture before filtration.
Det har vist seg at behandlingene av det finfordelte materiale, før koking med lut og salt, også kan foretas med en syrebehandling eller med en behandling med det organiske oppløsningsmiddel i nærvær av en syre. Fordelen med dette er naturligvis å unngå et prosesstrinn og likevel oppnå gode filtreringstider, spesielt med et surgjort organisk oppløsningsmiddel, hvilket fremgår av det etterfølgende eksempel 2. It has been shown that the treatments of the finely divided material, before boiling with lye and salt, can also be carried out with an acid treatment or with a treatment with the organic solvent in the presence of an acid. The advantage of this is naturally to avoid a process step and still achieve good filtration times, especially with an acidified organic solvent, which is evident from the following example 2.
Eksempel 2 Example 2
Den samme standardiserte metode for fremstilling av DNA ble fulgt, hvor 200 g melke ble forbehandlet før tilsetning av lut (pH 10 - 11), salt og vann (sluttvolum = 1000 ml) og deretter kokt, avkjølt og filtrert. DNA ble felt ved syre-tilsetning, vasket, tørket og veiet for bestemmelse av utbytte. Resultatene fremgår av den etterfølgende tabell 2. The same standardized method for the preparation of DNA was followed, where 200 g of milk was pre-treated before the addition of lye (pH 10 - 11), salt and water (final volume = 1000 ml) and then boiled, cooled and filtered. DNA was precipitated by acid addition, washed, dried and weighed for yield determination. The results appear in the following table 2.
Som det fremgår vil forbehandlingen føre til kortere filtreringstid, større filtratvolum og øket utbytte av DNA. As can be seen, the pre-treatment will lead to a shorter filtration time, a larger filtrate volume and an increased yield of DNA.
Spesielt synes kombinasjonen av organisk oppløsningsmiddel og syre å gi et gunstig resultat med hensyn til filtreringstid, filtratvolum og utbytte som er ca. 3 ganger så høyt som for kontrollprøven, dvs. hvor den finfordelte melke ble behandlet direkte med lut og salt uten forbehand-ling . In particular, the combination of organic solvent and acid seems to give a favorable result with regard to filtration time, filtrate volume and yield, which is approx. 3 times as high as for the control sample, i.e. where the finely divided milk was treated directly with lye and salt without pre-treatment.
Claims (4)
Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
NO994144A NO310114B1 (en) | 1999-07-19 | 1999-08-26 | Method of Preparation of DNA |
PCT/NO2000/000280 WO2001014541A1 (en) | 1999-08-26 | 2000-08-25 | A method for preparation of dna from biological material |
PE2000000880A PE20010505A1 (en) | 1999-08-26 | 2000-08-25 | A METHOD FOR THE PREPARATION OF DNA FROM BIOLOGICAL MATERIAL |
EP00957150A EP1206531A1 (en) | 1999-08-26 | 2000-08-25 | A method for preparation of dna from biological material |
AU68806/00A AU6880600A (en) | 1999-08-26 | 2000-08-25 | A method for preparation of dna from biological material |
IS6277A IS6277A (en) | 1999-08-26 | 2002-02-21 | Method of preparing DNA from organic matter |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
NO993532A NO993532D0 (en) | 1999-07-19 | 1999-07-19 | Method of Preparation of DNA |
NO994144A NO310114B1 (en) | 1999-07-19 | 1999-08-26 | Method of Preparation of DNA |
Publications (3)
Publication Number | Publication Date |
---|---|
NO994144D0 NO994144D0 (en) | 1999-08-26 |
NO994144L NO994144L (en) | 2001-01-22 |
NO310114B1 true NO310114B1 (en) | 2001-05-21 |
Family
ID=26648988
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
NO994144A NO310114B1 (en) | 1999-07-19 | 1999-08-26 | Method of Preparation of DNA |
Country Status (1)
Country | Link |
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NO (1) | NO310114B1 (en) |
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1999
- 1999-08-26 NO NO994144A patent/NO310114B1/en not_active IP Right Cessation
Also Published As
Publication number | Publication date |
---|---|
NO994144L (en) | 2001-01-22 |
NO994144D0 (en) | 1999-08-26 |
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