Classifications

• • 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
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
  • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor

Definitions

• This invention is related to methods for the preparation of DNA (deoxyribonucleic acid) from biological materials.
• the method for the preparation of DNA from biological materials describes pre-treatment of the raw materials prior to conventional methods for DNA preparation.
• the pre- treatment is mixing the raw material with either acid, organic solvent or a mixture of acid and organic solvent.
• the pre-treatment may be followed by filtration to remove soluble materials, lipid extraction using conventional organic solvents (e.g. ethanol) to remove lipids and when using acid the raw material should be neutralised before heating and boiling the mixture in a alkaline solution.
• organic solvents e.g. ethanol
• the raw material is boiled in the presence of alkali and salt. This will result in saponification of the lipids present in the raw material, rendering the resulting mixture after boiling difficult to filtrate.
• the problem may to some extent be solved using high pressure filtering conditions, however, from a processing point of view it would advantageous if the alkaline raw material mixture after boiling was easy to filtrate.
• the yield of DNA is lower than available in the raw material.
• the pre-treatment the following boiling in alkali may be more efficient in solubilizing DNA, thereby resulting in a higher yield of DNA.
• lipids In marine raw materials the lipids present are valuable due co a favourable composition of polyunsaturated fatty acids (PUFA) .
• PUFA polyunsaturated fatty acids
• Removing lipids from marine raw materials, that are used for DNA preparation, can be achieved by a range of methods. Whereas triglycerides (TGA) may be removed efficiently by conventional mechanical methods, phospholipids (PL) are more difficult to remove.
• TGA triglycerides
• PL phospholipids
• organic solvents are used for removal of lipids rich in PL. This because the PL, which are located in biological membranes, are not removed by e.g. boiling/pressing.
• milt from cod Gadus morhua
• ethanol is used as the organic solvent .
• DNA was prepared from 2 samples of comminuted milt from cod, each 200g.
• sample A the milt was treated with acetic acid and ethanol and stirred for 10 min, after which the water phase rich in ethanol and containing PL was filtered off.
• sample B DNA was prepared from sample A and from sample B using standard procedure for DNA preparation. This includes addition of water to same volume (1000 ml) , adding soda and salt (pH 10-11) , boiling of mixture, filtration, followed by acid precipitation of DNA from the filtrate and alkali treatment of the DNA before final precipitation.
• the resulting DNA preparations were dried and analysed for chemical composition according to requirements for commercial preparations of DNA. The results are shown in table 1 below. Table 1.
• Table 1 Preparation of DNA from samples each 200 g of milt from cod (Ga us morhua) with and without pre-treatment with ethanol and removal of the ethanol rich water phase containing lipids .
• DNA was prepared according to the method described in example 2 using pre-treatment with acid and ethanol, with the additional step of neutralising the pre-treated mixture before heating. 1000 kg of comminuted milt from cod was pre-treated with 200 1, 60% acetic acid and 100 1 ethanol for 30 min, temperature 3-5 °C, followed by the addition of 67 kg NaOH to neutralise the mixture. This was heated to 100 °C during 1 hr using direct steam injection, additional 20 kg NaOH was added and the mixture was boiled for 1 hr before filtration and DNA precipitation as previously described.
• control 200 g of pre-treated comminuted milt was heated to 100 °C during 1 hr without neutralisation with NaOH.
• the pH was adjusted to pH 10,5 using NaOH, and the mixture was boiled for 1 hr. before filtration and subsequent DNA precipitation.

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• 2000
  • 2000-08-25 WO PCT/NO2000/000280 patent/WO2001014541A1/en not_active Application Discontinuation
  • 2000-08-25 EP EP00957150A patent/EP1206531A1/de not_active Withdrawn
  • 2000-08-25 AU AU68806/00A patent/AU6880600A/en not_active Abandoned
  • 2000-08-25 PE PE2000000880A patent/PE20010505A1/es not_active Application Discontinuation
• 2002
  • 2002-02-21 IS IS6277A patent/IS6277A/is unknown

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