EP1206531A1

EP1206531A1 - A method for preparation of dna from biological material

Info

Publication number: EP1206531A1
Application number: EP00957150A
Authority: EP
Inventors: Terje Strom; Jens-Petter Jostensen; Per-Ivar Larsen
Original assignee: JOSTENSEN JENS PETTER; LARSEN PER IVAR
Current assignee: JOSTENSEN JENS PETTER; LARSEN PER IVAR
Priority date: 1999-08-26
Filing date: 2000-08-25
Publication date: 2002-05-22
Also published as: IS6277A; PE20010505A1; WO2001014541A1; AU6880600A

Abstract

The present invention is related to a method for the preparation of DNA (deoxyribonucleic acid) from biological materials. The method for 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 an alkaline solution. By this pre-treatment other substances may be extracted from the raw material prior to DNA preparation, the filtering properties of the raw material mixture after boiling in alkaline are improved and the resulting yield of DNA is higher than without the pre-treatment of the raw material.

Description

A METHOD FOR PREPARATION OF DNA FROM BIOLOGICAL MATERIAL

Field of the invention

This invention is related to methods for the preparation of DNA (deoxyribonucleic acid) from biological materials.

Background

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. By this pre-treatment other substances may be extracted from the raw material prior to DNA preparation, the filtering properties of the raw material mixture after boiling in alkaline are improved and the resulting yield of DNA is higher than without the pre-treatment of the raw material.

By traditional methods for preparation of DNA 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.

By traditional methods the yield of DNA is lower than available in the raw material. By the pre-treatment the following boiling in alkali may be more efficient in solubilizing DNA, thereby resulting in a higher yield of DNA.

The pre-treatment and subsequent filtration and/or lipid extraction using organic solvent prior to boiling in alkali may open up for preparation of other substances, hereby lipids. In marine raw materials the lipids present are valuable due co a favourable composition of polyunsaturated fatty acids (PUFA) . 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. Generally a combination of mechanical methods in combination with heat treatment are used for removing lipids with a high content of TGA, while 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.

Fish milts from marine species are particularly used for preparation of DNA. The DNA contents are 1,5 - 2%, with natural seasonal variations. In addition, the milts contain 2-4% of lipids, which are rich in PL up to 75% of total lipids.

In the examples below milt from cod ( Gadus morhua) is used as raw material and ethanol is used as the organic solvent .

Examples

The following examples demonstrates the pre-treatment and removal of lipids prior to DNA preparation.

Example 1

DNA was prepared from 2 samples of comminuted milt from cod, each 200g. In 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. After filtration, 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. 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 .

There were no significant differences in the chemical composition of the two DNA preparations, and all analytical values were within the requirements for commercial preparations of DNA.

It was initially thought that the removal of lipids, by extraction with e.g. ethanol prior to boiling in alkali, was necessary for the improved results. However, subsequent experiments have shown that the pre-treatment with acids, organic solvents or a mixture of acids and organic solvent followed by addition of alkali and salt and boiling of the alkaline mixture also give good results. This is demonstrated in example 2.

Example 2

The same method for DNA preparation as used in example 1 was followed, i.e. with pre-treatment of the milt, however, without removal of the ethanol rich water phase before boiling in alkaline and subsequent precipitation, washing and drying of the resulting DNA. The results are shown in table 2. Table 2. Preparation of DNA from samples, each 200 g of milt from cod(Gadus morhua) with and without pre-treatment with ethanol and a mixture of acetic acid and ethanol.

As seen, the best result was obtained with a. mixture of acid and ethanol, however, improved result was also obtained by pre-treatment with ethanol alone. The effect of acid in the pre-treatment mixture is clearly demonstrated by improved filtration time, increased volume of filtrate from filtration after boiling in alkali and a 3-fold increase in yield compared to control without pre-treatment There were no significant differences in the chemical composition of the three DNA preparations, and all analytical values were within the requirements for commercial preparations of DNA.

Example 3

Large scale experiments (i.e. 1 ton of raw material) have shown that when using pre-treatment with acid it is necessary to neutralise the resulting mixture before heating and boiling in alkali (pH 10-11) . Without this neutralisation DNA will partly be destroyed due to the effect of acid hydrolysis of DNA during the long heating time (approximately 1 hr) . This is demonstrated in the experiments below.

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.

In the 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.

Table 3. Preparation of DNA from milt from cod (Gadus orhua) with and without neutralisation after pre-treatment with a mixture of acetic acid and ethanol before heating to 100 °C

Claims

P A T E N T C L A I M S

1. A method for preparation of DNA (deoxyribonucleic acid) from biological material by boiling the raw material in the presence of alkali and salt evenly distributed with the raw material, whereby the resulting mixture is filtered and DNA is recaptured by precipitation after acidifying the filtrate, c h a r a c t e r iz e d b y pre-treatment of the evenly distributed material before addition of alkali and salt using acids, using organic solvents or using a mixture of acids and organic solvents.

2. The method according to claim 1, in which the pre- treated raw is filtered, and lipids and other valuable substances are collected from the filtrate.

3. The method according to claim 1, in which alkali and salt are added to the pre-treated raw material before boiling, filtration and precipitation of DNA from the filtrate .

4. The method according to claims 1 and 2, in which as organic solvents are used alkanes, alkenes or alcohols, particularly ethanol.

5. The method according to claims 1,2 and 4, in which acid is used in pre-treatment of raw material the resulting mixture is neutralised prior to preparation of DNA.