CN1401782A

CN1401782A - Process for extracting complete genome DNA of kelp

Info

Publication number: CN1401782A
Application number: CN 02111328
Authority: CN
Inventors: 周志刚; 胡远皆
Original assignee: Shanghai Ocean University
Current assignee: Shanghai Ocean University
Priority date: 2002-04-11
Filing date: 2002-04-11
Publication date: 2003-03-12
Anticipated expiration: 2022-04-11
Also published as: CN1195066C

Abstract

A process for extracting whole genom DNA of kelp includes washing raw material, treating with alginic acid lyase, removing part of polyose, settling separation to obtain unicells, digesting with CTAB, extracting in phenol and isopentanol, removing residual polyose, settling in isopropanol, critical drying, washing with cold alcohol, settling and vacuum drying. Its advnatages are simple process and low cost.

Description

Process for extracting complete genome DNA of kelp

The present invention relates to a kind of DNA extraction method, particularly relate to a kind of kelp spore body process for extracting complete genome DNA.

Not only have very high nuclease in the large-scale seaweed cell, and its cell walls contains a large amount of polysaccharide, and its form because of species different different.In the middle of red algae and brown alga, the polysaccharide of sulfur-bearing acidic group and carboxyl accounts for chief component.In the cell walls and the intercellular substance of ripe kelp spore body, also, cause the co-precipitation in the nucleic acid extraction process, thereby influenced the purity of nucleic acid because contain a large amount of polysaccharide.At present both at home and abroad in the middle of molecular level is carried out research to large-scale red, brown alga,, adopt the classical ultracentrifugal method of CsCl usually, but this method has relatively high expectations to experimental installation and operative technique, and spend higher in order to remove the interference of polysaccharide; Also have the researchist to adopt in addition such as purification process such as post filtrations, though preferably purifying genomic dna, in purge process, easily cause losing and damaging of genomic dna, and economical inadequately.If will carry out researchs such as population genetics, phyletic evolution, molecular mark, owing to need the laboratory sample of processing more, for above physics that adopts or chemical process, its loaded down with trivial details, expensive defective seems more and is unfavorable for the carrying out of experimental study.

The object of the present invention is to provide the method for a kind of economy, easy, easy row, in order to from various materials such as fresh, freezing kelp spore body or dry preserved specimen, to extract high-quality complete genome DNA.

For achieving the above object, the technical solution used in the present invention is:

1) cleans the kelp spore body with aseptic seawater, and in 12-14 ℃ of illumination box recovery 6-8 hour, then tissue is cut into the small pieces of 1-2mm.

2) in tissue, add enzymolysis solution (0.8M N.F,USP MANNITOL, 50mM trisodium citrate, 1% cellulase in 1: 8 ratio (g/ml), 0.5% macerozyme R-10, alginic acid lyase 0.2U/ml), be positioned on the shaking table (under 70 * g), 14 ℃ of dark conditions enzymolysis 6-8 hour.

3) gained dissociates and unicellularly filters through bolting silk (240 order), and with the centrifugal collection of 3000 * g.Extract damping fluid [3%CTAB (w/v), 1.4M NaCl, 20mM EDTA, 10mM Tris-Cl (pH7.8), 2% mercaptoethanol (w/v)], water-bath 45-60min in 1: 6 ratio (mg/ml) adding CTAB.

4) in above-mentioned system, add equal amounts of chloroform: amylalcohol, centrifugal (11000 * g 10min), gets supernatant, adds 2/3 volume cold isopropanol, places 20min for-20 ℃, centrifugal again (5100 * g, 10min).The gained precipitation precipitates with 70% cold washing with alcohol.

5) with resolution of precipitate in TE solution [10mM Tris-Cl (pH8.0), 1mM EDTA], 4 ℃ centrifugal, and (11000 * g 5min), gets supernatant.

6) add 1/3 volume 7.5M NH in the gained supernatant ₄The ethanol of Ac and 2.5 volumes 95% is placed 30min for-20 ℃, centrifugal (11000 * g, 15min), precipitation is cleaned with 70% cold ethanol, and vacuum-drying is standby in 4 ℃ of preservations.

Process for extracting complete genome DNA of kelp of the present invention is different from chemical processes such as mechanical-physical method such as traditional ultracentrifugation and purifying, and its advantage is that complete genome DNA is extracted material carries out a biological disposal upon earlier, to remove the pollution of polysaccharide.Employed material is not limited only to fresh kelp spore body, can also be the sample of ripe kelp spore body of refrigerated or drying preservation.And present method can also be applied to the extraction of large-scale brown, the full genome DAN of red algae such as other marine alga such as wakame, laver etc.The high quality genomic dna of gained can be widely used in every researchs such as marine alga population genetics, phyletic evolution, molecular marker breeding.

Describe the embodiment of the invention below in detail:

Embodiment one: get fresh ripe kelp spore body, clean the assorted algae of removal and other dirt settling with aseptic seawater, with scissors tissue is shredded 1-2mm, get 1 gram and put into the fine taper bottle of 8ml enzymolysis solution, add the alginic acid lyase again, make its final concentration reach 0.2U/ml, Erlenmeyer flask is placed on the shaking table that (70 * g) in the incubator of dark (14 ℃) about enzymolysis 6-8 hour.Take out then and remove the not fragment of enzymolysis with 240 purpose silk cover filterings, centrifugal (3000 * g) collections are unicellular, and clean 3-5 time with seawater, remove possible pollution.Get the unicellular of 30-100mg, add 0.6mlCTAB and extract damping fluid, 65 ℃ of temperature are bathed 45-60min, shake 3-5 time therebetween light and slowly, add the chloroform of equivalent again: primary isoamyl alcohol (v/v, 24: 1), shake gently, centrifugal under the room temperature (11000 * g, 10min), supernatant liquor is transferred in another new centrifuge tube, the Virahol that in centrifuge tube, adds 2/3 volume, place after 20 minutes for-20 ℃, centrifugal under the room temperature (5100 * g, 5min).Washing with alcohol precipitation with 70% is dissolved in 100-200 μ lTE damping fluid after the drying.4 ℃ centrifugal, and (11000 * g 5min), gets supernatant liquor and is dissolved in 1/3 volume 7.5M NH ₄The ethanol of Ac and 2.5 times of volumes 95% is placed more than the 30min for-20 ℃, centrifugal (11000 * g, 15min) after again with the cleaning of 70% ethanol, vacuum-drying, it is standby to be dissolved in an amount of sterilized water or the TE damping fluid 4 ℃ of preservations.

Embodiment two: get freezing kelp spore body or dry preserved specimen, put into aseptic seawater, recovered 24 hours in 12-14 ℃ of illumination box, the photoperiod is 12h/12h.Other step together

Embodiment one.

The kelp spore body that above embodiment explained is seen Fig. 1 and Fig. 2 through enzymolysis biological treatment and complete genome DNA.

Claims

1. process for extracting complete genome DNA of kelp is characterized in that following operation:

1) cleans the kelp spore body with aseptic seawater, add enzymolysis solution (N.F,USP MANNITOL 0.8M in 1: 8 ratio (g/ml), trisodium citrate 50mM, cellulase 1%, macerozyme R-100.5%, alginic acid lyase 0.2U/ml), be positioned on the shaking table (70 * g), under 14 ℃ of dark conditions enzymolysis 6-8 hour.

2) with 1) gained dissociates and unicellularly filters through bolting silk (240 order), and with the centrifugal collection of 3000 * g.Extract damping fluid [3%CTAB (w/v), 1.4M NaCl, 20mM EDTA, 10mM Tris-Cl (pH7.8), 2% mercaptoethanol (w/v)], water-bath 45-60min in 1: 6 ratio (mg/ml) adding CTAB.

3) to 2) add equal amounts of chloroform in the system: amylalcohol, centrifugal, get supernatant, add the cold isopropanol precipitation, centrifugal again.The gained precipitation is used washing with alcohol, is dissolved in TE solution [10mM Tris-Cl (pH8.0), 1mM EDTA], the centrifuging and taking supernatant.

4) 3) add 7.5M NH in the gained supernatant ₄Ac and 95% ethanol, low temperature is placed, and is centrifugal, with 70% cold ethanol cleaning, vacuum-drying, standby in 4 ℃ of preservations.

2. process for extracting complete genome DNA as claimed in claim 1 is characterized in that used DNA extraction material is fresh or refrigerated kelp spore body, also can be dry preserved specimen.

3. process for extracting complete genome DNA as claimed in claim 1 when it is characterized in that used DNA extraction material is freezing sporophyte or dry preserved specimen, should be used sea water immersion in advance.