CN102146375B - Method for massively extracting earthworm genome DNA - Google Patents

Abstract

The invention discloses a method for massively extracting earthworm genome DNA. The invention relates to extraction technology for earthworm genome DNA in soil pollution molecular diagnosis, and earthworm is a common model organism in molecular ecotoxicology research and soil pollution molecular diagnosis. The method is improved on the basis of a protease K method, the somite in 3 millimeters of the head of the earthworm is used as an initial sample for extracting, and the sample is directly ground and crushed in a centrifugal tube by adopting a glass pestle matched with the inner wall of the centrifugal tube; and a step of centrifugal operation is increased before phenol and chloroform extraction, so that the efficiency of earthworm tissue digestion is greatly improved, interference of sand in intestines and stomach of the earthworm on nucleic acid extraction is effectively avoided, and the whole extraction process is completed within 5 hours. By adopting the method, the success rate and the efficiency of earthworm genome extraction are improved, and the method has the advantages of saving time and saving labor; and the high-quality genome DNA obtained by the method is suitable for downstream experiments such as enzyme digestion, polymerase chain reaction (PCR) and the like.

Description

A kind of method of extraction earthworm genomic dna in enormous quantities

Technical field

The present invention relates to a kind of method of extracting the earthworm genomic dna, can be used for needs and obtain in enormous quantities, the research field of high quality earthworm genomic dna is like the molecular diagnosis field of soil pollution.

Background technology

Because sewage irrigation; The mankind's activities such as unreasonable landfill of chemical fertilizer and indiscriminate use of pesticide, industrial or agricultural solid waste; Soil pollution becomes more and more serious in China and world wide; Human diagnosis to soil pollution, detection and repair become and more and more pay attention to, and utilize biomarker just becoming a kind of very promising technique means as monitoring and diagnosis soil pollution.Earthworm is as a kind of important model animals in molecule ecotoxicology and the STUDY ON SPATIAL SOIL ECOLOGY; Be widely used in diagnosis, monitoring and the reparation of soil pollution, the needs of subject development and molecular diagnosis make the method for high efficiency extraction earthworm high quality genomic dna seem particularly important.At present, the method that relates to the genomic dna that extracts various biologies and various samples has a lot, like the SDS method, and CTAB method and Proteinase K method.Yet earthworm is not a model animals commonly used in the biological study, does not up to the present have a kind of suitable method to be used for extracting the earthworm genomic dna.Proteinase K is a kind of highly active protein enzyme of subtilisin class; Its principle is the protein that utilizes in protease K digesting tissue and the cell; Make the genomic dna that is aggregated on the karyomit(e) split away off and dissolve from histone; Through the imitative extracting of phenol, remove protein and other impurity then, obtain genomic dna through classical intermediate processing.Traditional Proteinase K method is used for the extracting genome DNA of animal tissues and cell (like mouse, people's cell and tissue).Yet earthworm but has its singularity, and its sediment charge is very high, and silt and impurity have suppressed the effect of Proteinase K, makes digestion not exclusively, and after the earthworm that contains silt usually was organized in and carries out protease K digesting, the Digestive system muddiness gave out a foul smell, dna degradation.In addition, the muscle protein content of earthworm tissue is very high, and this also makes protease K digesting become difficult, therefore before protease K digesting, tissue is carried out sufficient crushing grinding and seems particularly important.The crushing grinding of tissue has a variety of methods, like liquid nitrogen grinding, ultrasonication, and glass homogenizer homogenate etc., aforesaid method respectively has advantage, the field that respectively has it to be suitable for.Better to the effect of historrhexis like liquid nitrogen grinding, the low temperature of liquid nitrogen can suppress to organize the degraded of endogenous nucleicacidase to DNA simultaneously; The crushing effect of ultrasonication pair cell and tender tissue is better, and it is more convenient to cooperate instrument to use; The crushing effect of glass homogenizer homogenate method is also fine, but consuming time of a specified duration, is difficult to guarantee that DNA is not degraded.Liquid nitrogen grinding and glass homogenizer homogenate all be not suitable for the operation of sample in enormous quantities, and ultrasonication are not suitable for the fragmentation of tough and tensile tissue, and need being used of instrument owing to needs to shift sample.In sum, common Ginding process is defectiveness all, and wastes time and energy because need to shift sample, and the broken method of above-mentioned grinding has all satisfied not in the molecular diagnosis research of soil pollution the demand to high quality earthworm genomic dna in enormous quantities.

Summary of the invention

The objective of the invention is to solve traditional Proteinase K method extract the earthworm genome exist digestion not exclusively, problem such as Digestive system muddyly gives out a foul smell, the most of degraded of DNA and digestion time are long, a kind of novel method of extraction high quality earthworm genomic dna in enormous quantities is provided.

The present invention is on the basis of traditional Proteinase K method; High and the more tough and tensile characteristics to this biological tissue of earthworm muscle content; Through special sampling method, the direct grinding of glass grinding pestle and extra increase by go on foot centrifugally operated, have improved the efficient of historrhexis, protease K digesting and DNA purifying; Simplify the operation in downstream, improved the purity and the quality of genomic dna.

The method of extraction earthworm genomic dna in enormous quantities provided by the invention may further comprise the steps:

1st, special sampling point, the body segment of gripping earthworm head 3 mm (from pharyngeal about 10 body segments forward) as the original samples of extracting genome DNA, places 1.5 mL centrifuge tubes (eppendorf pipe);

2nd, the glass grinding pestle that is complementary of employing and centrifuge tube inwall directly grinds the sample of the 1st step gripping in centrifuge tube;

3rd, the sample that adopts improved Proteinase K method that the 2nd step was ground after the fragmentation carries out the extraction of genomic dna, obtain the earthworm genomic dna that will extract; Described improved Proteinase K method exactly before carrying out the imitative extracting of phenol, increases by a step centrifugally operated, makes residual solid impurity be deposited in the centrifuge tube bottom, thereby avoids the influence of solid impurity to subsequent operations, improves the quality and the purity of genomic dna.

Concrete leaching process:

One, the preparation of experiment material and reagent

The used glass grinding pestle of this novel method is available from market.The other materials that relates in the experiment is the common agents material in the molecular biology experiment.Preparation reference " molecular cloning laboratory manual " third edition of various solution:

SNET solution: 20 mmol/L Tris-Cl (pH8.0); 5 mmol/L EDTA (pH8.0); 400 mmol/L NaCl; 1% (m/V) SDS; Filter filtration sterilization with 0.43 μ m.

Lysate: per 500 uL lysates contain 480 uL SNET and 20 uL Proteinase Ks (20 mg/mL)

(phenol: chloroform: mixing solutions primary isoamyl alcohol):, and use 1M, the Tris damping fluid balance of pH 8.0 according to volume ratio 25:24:1 preparation.

(chloroform: mixing solutions primary isoamyl alcohol): prepare according to volume ratio 24:1.

75% (v/v) ethanolic soln: absolute ethyl alcohol and redistilled water are mixed according to volume ratio 3:1.

10 times of TE solution (pH8.0): 100 mmol/L Tris-Cl (pH 8.0), 10 mmol/L EDTA (pH 8.0) mix the back with above-mentioned solution and transfer pH to 8.0

1mol/L Tris-Cl (pH 8.0) damping fluid:, add concentrated hydrochloric acid 4.2 ml and transfer to pH8.0 with 80 mL dissolved in distilled water, 12.11 g Tris alkali

Proteinase K solution: it is 20 mg/ml that storage liquid contains Proteinase K concentration, and the working concentration of Proteinase K is 400 ug/ml.

RNase A solution: it is 20 mg/ μ L that storage liquid contains RNase A, and working concentration should >=20 ug/mg.

Two, operation steps:

1, disturb the genome leaching process for fear of silt, the about 30mg of tissue (about 3 mm of length) that when sampling, does not contain silt with tweezers gripping earthworm head is positioned in the 1.5 mL centrifuge tubes (eppendorf pipe), adds 50 uL lysates.

2, on ice, fully grind the earthworm tissue with the glass grinding pestle, make tissue crumble as far as possible.Add 450 uL lysates again, put upside down mixing.

3,55 ℃ of temperature are bathed 4 hours (being no more than 12 hours), thoroughly digest through Proteinase K, and the centrifuge tube planted agent is transparent weak yellow liquid.After finishing digestion, treat that temperature drops to room temperature, add the RNase A of 10 uL, 20 mg/ uL, 37 ℃ of temperature are bathed half a hour to remove RNA.

4, for avoiding the sightless solid impurity of naked eyes to disturb DNA extraction, the centrifugal 1min of 12000 rpm makes indigested solid impurity be deposited in 1.5 mL centrifuge tubes (eppendorf pipe) bottom.

5, the imitative extracting of phenol, in 1.5 mL centrifuge tubes (eppendorf pipe) adding 500 uL (phenol: chloroform: mixing solutions primary isoamyl alcohol), centrifuge tube is placed horizontally on the shaking table, normal temperature, 200 commentaries on classics/min shake 15 min-30 min.

6,1.5 mL centrifuge tubes (eppendorf pipe) are taken out the centrifugal 5min-10min of 12000 rpm from shaking table.

7, carefully draw supernatant in another 1.5 mL centrifuge tube (eppendorf pipe), add 500 uL (chloroform: mixing solutions primary isoamyl alcohol), centrifuge tube is placed horizontally on the shaking table, normal temperature, 200 commentaries on classics/min shake 15 min-30 min.

8,1.5 mL centrifuge tubes (eppendorf pipe) are taken out the centrifugal 5min-10min of 12000 rpm from shaking table.

9, carefully draw supernatant in another 1.5 mL centrifuge tube (eppendorf pipe), add the Virahol of the precooling on ice of 1 times of volume then, leave standstill 15 min-1h after putting upside down mixing.

10,4 ℃, the centrifugal 15min collecting precipitation of 12000 rpm.

11, abandon supernatant, blot all liquid as far as possible with micropipet.Add 1 mL75% (v/v) ethanolic soln washing DNA deposition, turn upside down.Centrifugal 1 min of 12000 rpm.

12, repeating step 11: with the washing precipitation once more of 1 mL75% (v/v) ethanolic soln.

13, abandon supernatant, blot all liquid as far as possible with micropipet.With the uncovered placement 10 min-15 min of centrifuge tube.

14, the redistilled water dissolving DNA deposition that adds TE solution (pH8.0) or the pH 8.0 of 100 uL.Promptly obtain earthworm genomic dna solution, this DNA is fit to carry out enzyme and cuts and subsequent operationss such as PCR.

The present invention has following beneficial effect:

1) in historrhexis's step, replaced glass homogenizer homogenate or liquid nitrogen grinding with the glass grinding pestle, reduced the consuming time and cost of experiment, improved workmanship when reducing workload.Use the supporting 1.5 mL centrifuge tubes of glass grinding pestle (eppendorf pipe) to grind, taken into account grinding and ice bath simultaneously and operated, and saved the operation of transferred material and cleaning glass homogenizer (mortar) in original homogenate or the liquid nitrogen grinding process;

2) sampling method of uniqueness has been avoided the pollution of silt impurity, and experience shows that the body segment of the about 0.3mm of earthworm head (from pharyngeal about 10 body segments forward) does not contain silt.Therefore get the long body segment of earthworm head 0.3mm as parent material, can avoid the silt impurity pollution problem in the genome leaching process.Again because the sequence of genomic dna any organ with organize in all be the same, so special sampling point does not influence the quality of extraction;

3) after the protease K digesting; Centrifugal process before the imitative extracting of phenol has increased the separation efficiency between solid impurity and the DNA; Behind protease K digesting, still have some solid impurities residual inevitably, in subsequent operations, bring interference for fear of these solid impurities; Before the imitative extracting of phenol, carry out the centrifugal solid impurity that can make of a step and be fixed on the centrifuge tube bottom, made things convenient for subsequent operations.

Description of drawings

Fig. 1 is the glass grinding pestle;

Fig. 2 grinds in 1.5 mL centrifuge tubes with the glass grinding pestle;

Fig. 3 is that 50 mg samples are with the appearance of Digestive system digestion after 4 hours;

Fig. 4 utilizes the present invention's agarose gel electrophoresis of 24 earthworm tissue samples of batch extracting genomic dna simultaneously; Success rate of extracting reaches 100%, all can extract the genomic dna of the about 100 ng/ μ L of concentration, and electrophoresis result shows that dna fragmentation more than 20 Kb, does not have hangover, and disperse or RNA pollute.

Fig. 5 is the present invention and the genomic agarose gel electrophoresis of traditional method for extracting earthworm.Swimming lane 1: the earthworm genomic dna that this ditty is extracted, band is clear, about 20 Kb of size, no disperse or hangover; Swimming lane 2: traditional Proteinase K method is extracted the earthworm genomic dna, and degraded is serious; The maximum band of swimming lane 3:DNA maker is 15000 bp.

Fig. 6 is that earthworm genomic dna that the present invention extracts is the agarose gel electrophoresis figure that template is carried out the specific amplification products behind the PCR.Swimming lane 1,2: fragment length is about the specific amplification fragment of 3Kb; Swimming lane 3:DNA Maker, maximum band are 15000 bp.

Fig. 7 is that the earthworm genomic dna that the present invention extracts is the reacted agarose gel electrophoresis figure of substrate RAPD (randomly amplified polymorphic DNA mark).Swimming lane 1,2: enzyme is cut after product; Swimming lane 3:DNA Maker, maximum band are 15000 bp.

Embodiment

Embodiment 1

One, the preparation of experiment material and reagent

Triumphant curtain bio tech ltd is buied glass grinding pestle (Fig. 1) from Beijing, and the other materials that relates in the experiment is the common agents material in the molecular biology experiment.Preparation reference " molecular cloning laboratory manual " third edition of various solution:

SNET solution: 20 mmol/L Tris-Cl (pH8.0); 5 mmol/L EDTA (pH8.0); 400 mmol/L NaCl; 1% (m/V) SDS; Filter filtration sterilization with 0.43 μ m.

Lysate: per 500 uL lysates contain 480 uL SNET and 20 uL Proteinase Ks (20 mg/mL)

Phenol: chloroform: primary isoamyl alcohol mixing solutions:, and use 1M, the Tris damping fluid balance of pH 8.0 according to volume ratio 25:24:1 preparation.

Chloroform: primary isoamyl alcohol mixing solutions: prepare according to volume ratio 24:1.

75% (v/v) ethanolic soln: absolute ethyl alcohol and redistilled water are mixed according to volume ratio 3:1.

10 times of TE solution (pH8.0): 100 mmol/L Tris-Cl (pH 8.0), 10 mmol/L EDTA (pH 8.0) mix the back with above-mentioned solution and transfer pH to 8.0

1mol/L Tris-Cl (pH 8.0) damping fluid:, add concentrated hydrochloric acid 4.2 ml and transfer to pH8.0 with 80 mL dissolved in distilled water 12.11g Tris alkali

Proteinase K solution: it is 20 mg/ml that storage liquid contains Proteinase K concentration, and the working concentration of Proteinase K is 400 ug/ml.

RNase A solution: it is 20 mg/ μ L that storage liquid contains RNase A, and working concentration should >=20 ug/mg.

Two, operation steps:

1, disturb the genome leaching process for fear of silt, the about 30mg of tissue (about 3 mm of length) that when sampling, does not contain silt with tweezers gripping earthworm head is positioned in the 1.5 mL centrifuge tubes (eppendorf pipe), adds 50 uL lysates.

2, as shown in Figure 2, fully grind the earthworm tissue on ice with the glass grinding pestle, make tissue crumble as far as possible.Add 450 uL lysates again, put upside down mixing.

3,55 ℃ of temperature are bathed 4 hours (being no more than 12 hours); Thoroughly digest through Proteinase K; The centrifuge tube planted agent is transparent weak yellow liquid (as shown in Figure 3, the sample standard deviations in two 1.5 mL centrifuge tubes have obtained complete digestion, transparent and do not have a macroscopic solid impurity).After finishing digestion, treat that temperature drops to room temperature, add the RNase A of 10 uL, 20 mg/ uL, 37 ℃ of temperature are bathed half a hour to remove RNA.

4, for avoiding the sightless solid impurity of naked eyes to disturb DNA extraction, the centrifugal 1min of 12000 rpm makes indigested solid impurity be deposited in 1.5 mL centrifuge tubes (eppendorf pipe) bottom.

5, the imitative extracting of phenol, in 1.5 mL centrifuge tubes (eppendorf pipe) adding 500 uL (phenol: chloroform: mixing solutions primary isoamyl alcohol), centrifuge tube is placed horizontally on the shaking table, normal temperature, 200 commentaries on classics/min shake 15 min-30 min.

6,1.5 mL centrifuge tubes (eppendorf pipe) are taken out the centrifugal 5min-10min of 12000 rpm from shaking table.

7, carefully draw supernatant in another 1.5 mL centrifuge tube (eppendorf pipe), add 500 uL (chloroform: mixing solutions primary isoamyl alcohol), centrifuge tube is placed horizontally on the shaking table, normal temperature, 200 commentaries on classics/min shake 15 min-30 min.

8,1.5 mL centrifuge tubes (eppendorf pipe) are taken out the centrifugal 5min-10min of 12000 rpm from shaking table.

9, carefully draw supernatant in another 1.5 mL centrifuge tube (eppendorf pipe), add the Virahol of the precooling on ice of 1 times of volume then, leave standstill 15 min-1h after putting upside down mixing.

10,4 ℃, the centrifugal 15min collecting precipitation of 12000 rpm.

11, abandon supernatant, blot all liquid as far as possible with micropipet.Add 1 mL75% (v/v) ethanolic soln washing DNA deposition, turn upside down.Centrifugal 1 min of 12000 rpm.

12, repeating step 11: with the washing precipitation once more of 1 mL75% (v/v) ethanolic soln.

13, abandon supernatant, blot all liquid as far as possible with micropipet.With the uncovered placement 10 min-15 min of centrifuge tube.

14, the redistilled water dissolving DNA deposition that adds TE solution (pH8.0) or the pH 8.0 of 100 uL.Promptly obtain earthworm genomic dna solution, this DNA is fit to carry out enzyme and cuts and subsequent operationss such as PCR.

After the batch extracting, electrophoresis result such as Fig. 4, the genomic dna quality of all 24 earthworm tissue samples that obtain through present method is better, protein or RNA do not occur and pollutes.For relatively the present invention and traditional albumen K method are extracted the effect of earthworm genomic dna; The genomic dna that simultaneously two kinds of methods is obtained carries out agarose gel electrophoresis: show like Fig. 5; The genomic dna quality that present method is extracted is better, and clip size is about 20 Kb, does not have protein; RNA or other small molecules pollute, and the earthworm genomic dna that traditional Proteinase K method is extracted degraded serious (Fig. 5).Through UV spectrophotometer measuring, the OD260/OD280 of the genomic dna that present method obtains between 1.8 to 1.9, OD260/OD230 >=2.0, showing does not have protein, the pollution of RNA and other small-molecule substance.And the earthworm genomic dna that traditional Proteinase K method is extracted is second-rate, and OD260/OD280 has only 1.5, and OD260/OD230＜2.0 show to exist protein and small molecular weight impurity to pollute.Fig. 6; Fig. 7 shows that extracting the genomic dna that obtains with present method is the regular-PCR that carries out of reactant and the result of RAPD (randomly amplified polymorphic DNA mark); All obtain gratifying result, this has also proved the genomic dna reliable in quality that present method is extracted from another point of view.

The result of embodiment 1 shows that the earthworm genomic dna quality that traditional Proteinase K method is extracted is not good, and signs of degradation is arranged, and exists protein and other small molecules to pollute simultaneously; And present method is extracted the earthworm genomic dna obtain, and though from the success rate of extracting of batch sample with extract the quality aspect, its result is gratifying.

Claims (1)

1. method of extracting the earthworm genomic dna in enormous quantities is characterized in that comprising 3 concrete steps of this method:

1st, the body segment at gripping earthworm head 3 mm places as the original samples of extracting genome DNA, places 1.5 mL centrifuge tubes;

2nd, the glass grinding pestle that is complementary of employing and centrifuge tube inwall directly grinds fragmentation to the sample of the 1st step gripping in centrifuge tube;

3rd, the sample that adopts improved Proteinase K method that the 2nd step was ground after the fragmentation carries out the extraction of genomic dna, obtain the earthworm genomic dna that will extract; Described improved Proteinase K method exactly before carrying out the imitative extracting of phenol, increases by a step centrifugally operated, makes residual solid impurity be deposited in the centrifuge tube bottom, thereby avoids the influence of solid impurity to subsequent operations, improves the quality and the purity of genomic dna.

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