CN109593755A - A method of for extracting genomic DNA in the animal sample saved - Google Patents
A method of for extracting genomic DNA in the animal sample saved Download PDFInfo
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- CN109593755A CN109593755A CN201811636752.1A CN201811636752A CN109593755A CN 109593755 A CN109593755 A CN 109593755A CN 201811636752 A CN201811636752 A CN 201811636752A CN 109593755 A CN109593755 A CN 109593755A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
- C12N15/102—Mutagenizing nucleic acids
- C12N15/1024—In vivo mutagenesis using high mutation rate "mutator" host strains by inserting genetic material, e.g. encoding an error prone polymerase, disrupting a gene for mismatch repair
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- 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
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
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- 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
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
Abstract
The present invention relates to technical field of molecular biology, and in particular to a method of for extracting genomic DNA in the animal sample saved.The present invention provides a kind of method for extracting genomic DNA in the animal sample saved, includes the steps that liquid is repaired in addition in the sample through cracking processing and carries out mutating alkali yl elimination and DNA damage reparation；The reparation liquid includes UNG enzyme.Genome DNA extracting method provided by the invention has the genomic DNA that high yield, high quality can be extracted from the limited animal sample of preservation, C > T and G > A artificial mutation are effectively eliminated, and the advantages such as the operating time is short, simple to operate, extraction cost is low, the per unit throughputs of extraction process are high, safety height of operation.
Description
Technical field
The present invention relates to technical field of molecular biology, and in particular to a kind of for extracting gene in the animal sample saved
The method of group DNA.
Background technique
In scientific research and clinical practice, the animal samples such as animal tissue of acquisition tend not to be examined immediately
Detection is tested, and is needed to carry out animal sample after processing appropriate in order to short-term or long-term preservation.Currently, the guarantor of animal tissue
Deposit mainly using dipped into formalin or by animal tissue be prepared as formalin fix, (the Formalin- of paraffin embedding
Fixed and Parrffin-Embedded, FFPE) sample.Formalin can occur with the amino of gal4 amino acid
Irreversible crosslinking is superior to other in terms of the integrality, antigen measurement property, tissue permeability that keep tissue cellularity
Fixer.Dipped into formalin fix or formalin-paraffin embedding physiology or pathology sample in it is wide comprising precious and source
General biomedical research material.Therefore, for a long time, dipped into formalin is fixed and formalin fixes-paraffin embedding skill
Art is used widely in clinical examination and medical scientific, and especially cytomorphology research and oncological pathology are ground
Study carefully.With the continuous development of this several years molecular diagnosis fields, which is also applied to the molecule of tumor disease more and more
Diagnosis.
Dipped into formalin is fixed and formalin fixes-paraffin-embedded tissue progress molecular biology research, is needed more
Carry out the nucleic acid especially extraction of DNA.But the crosslinked action of formalin can seriously affect the matter of the extraction of nucleic acid, nucleic acid
Amount and subsequent amplified reaction: the crosslinked action of formalin and paraffin can interfere the extraction of nucleic acid；Paraffin can hinder digestive juice
Infiltration to tissue influences tissue digestion and DNA is released so that protease inhibition K etc. digests the contact of ingredient with albumen in tissue
It puts；Formalin and paraffin can also inhibit the activity of archaeal dna polymerase, to influence PCR amplification.For these reasons, sample in addition
Product are rare and DNA is there may be condition in damaged, from dipped into formalin is fixed and formalin fix-paraffin-embedded tissue in mention
Take the yield of DNA usually extremely limited, quality is relatively low.Therefore, in order to eliminate formalin and paraffin to DNA extract and it is subsequent
The adverse effect of PCR amplification and DNA library building, it is necessary to guarantee do not increasing exogenous PCR inhibiting factor while subtracting as far as possible
Under the premise of the additional injuries of few DNA, thoroughly dewaxed to tissue, and remove the crosslinked action of formalin and paraffin, into
And it is purified into the genomic DNA of high quality, high yield.
In addition, since dipped into formalin is fixed and formalin fix-the limited material such as paraffin-embedded tissue DNA
Often there is the artificial mutation generated due to fixed and embedding conditions and long term storage in sequencing procedure.One among this
Specific question is that cytosine base deamination becomes Brdurd, so as to cause occurring C-T transformation in sequencing reaction.This is existing
The precise mechanism of elephant is still not clear, and possible explanation is that the deamination of cytimidine causes to produce uracil in the position, after
Person can match with adenine.In sequencing procedure, the result after this change can then be converted according to C > T is read.If in text
Chain information fails to retain in the building process of library, then two chains can be all sequenced, the at this moment artificial mutation be possibly shown as C > T or G >
A conversion.It therefore, is the accuracy for guaranteeing DNA sequencing, especially for (artificial mutation exists for the sequencing analysis of cancer sample
False positive may be detected as in such sample), when progress formalin is fixed or FFPE sample is sequenced, since starting is former
Expect that limited and false positive gene mutation relative frequency increases, avoids artificial mutation most important.
The process for dewaxing in mature kit is applied mainly to use the tastes such as dimethylbenzene pungent and endanger to human body at present
The evil biggish organic solvent of property dewaxes, and the process to dewax is cumbersome, and the reagent type used is more, and a sample time-consuming is extremely
It is 20 minutes few.Simultaneously as C-T conversion can be randomly generated in the fixed sample of formaldehyde, and there are cores during preservation for animal specimen
The loss of acid, and extracting method in the prior art, do not fix the generation of such artificial mutation, the damage of DNA and formaldehyde
Or extract the yield of nucleic acid in FFPE sample and effectively improved, therefore, high yield, high-quality can be obtained by needing to develop one kind
Measure and be capable of providing the extracting method of the genomic DNA of accurate DNA base information.
Summary of the invention
The technical issues of to solve in the prior art, the purpose of the present invention is to provide a kind of for extracting the animal saved
The method of genomic DNA in sample.
Animal sample will not usually test detection immediately after in vitro, and need that it is fixed, embed, fix
Cryo-conservation etc. is handled afterwards, in order to maintain the quality of animal sample during storage and transport.However, in fixation, embedding etc.
In treatment process, fixed and embedding conditions or long term storage often make the genomic DNA of animal tissue generate certain journey
The artificial base mutation such as the damage of degree and Brdurd, the yield for causing subsequent DNA to build library are lower and in sequencing reaction
In occur C > T or G > A conversion, influence sequencing accuracy.Inventor creatively has found, introduces in genome extraction process
The reparation step of UNG enzyme, can be effectively reduced the generation of DNA damage and artificial mutation.However it is prominent to eliminate base in addition UNG enzyme
While change, it is also possible to influence the yield and subsequent applications of genome, such as pcr amplification reaction or two generation sequencing library structures
It builds, therefore, the reagent dosage and reaction condition of step, and the condition of cracking processing is repaired by optimization, the present invention provides
Efficient animal sample Extraction Methods of Genome.
The present invention provides a kind of method for extracting genomic DNA in animal sample, including in the sample through cracking processing
It is added in product and repairs the step of liquid progress mutating alkali yl elimination is repaired with DNA damage；The reparation liquid includes UNG enzyme.
Further, inventors have found that UNG enzyme is added after sufficiently crack to sample, it can be improved the work of UNG enzyme
Use effect；And the suitable amounts of UNG enzyme and suitable treatment conditions can be while guaranteeing UNG enzymatic treatment efficiency, as far as possible
Reduce UNG enzyme for the extract yield of genomic DNA and the influence of quality and after continue the influence of library and amplification.
Specifically, the method for extracting genomic DNA in animal sample includes the following steps:
(1) sample dissociation: lysate is added in the sample and carries out cracking processing, isolated nucleic acid separating liquid；
(2) mutating alkali yl is eliminated and DNA damage reparation: the reparation liquid being added in the nucleic acid separating liquid and is repaired
It is multiple, the final concentration of 10~50U/ μ L of the UNG enzyme in the reaction system；
(3) separation and purified genomic dna.
Preferably, in above-mentioned steps (2), the mutating alkali yl is eliminated and DNA damage reparation is at 45~55 DEG C of incubations
Manage 1~2h.
To guarantee the abundant digestion of the ingredients such as protein in animal sample, cracking, in the present invention, the lysate includes to split
Solve buffer and Proteinase K；
Preferably, the final concentration of 15~50mg/mL of the Proteinase K in the reaction system；The lysis buffer packet
Include following component: lauryl sodium sulfate 1%~10%, 10~50mmol/L of guanidinium isothiocyanate 2~5mol/L, Tris-HCl,
EDTA0.01~0.2mol/L, Triton-X100 0.01%~0.1%；The pH of the lysis buffer is 7.5~8.0.
When handling paraffin-embedded sample, hydrodewaxing step and cleavage step are merged into a step and are incubated for processing step by the present invention
It carries out, i.e., when the sample is paraffin-embedded sample, the lysate further includes dewaxing liquid.
When carrying out the composition selection of dewaxing liquid, inventors have found that using the lesser hexadecane conduct of harm to the human body
The active constituent of dewaxing liquid can preferably dissolve the paraffin in paraffin-embedded sample, the sample handled through hexadecane, dewaxing
Thoroughly, the activation plays of UNG enzyme in subsequent reparation step can be advantageously promoted, and do not influence the subsequent application of genome,
It is therefore preferred that dewaxing liquid of the present invention includes hexadecane.
It is described when carrying out the extracting genome DNA of paraffin-embedded sample as a kind of preferred embodiment of the invention
Lysate is grouped as by following group: hexadecane, Proteinase K and lysis buffer.
Above-mentioned lysate can be divided into A liquid, B liquid, C liquid and each component is retained separately and is added:
A liquid: hexadecane；
B liquid: Proteinase K；
C liquid: lysis buffer.
Specifically, sample dissociation described in above-mentioned steps (1) includes the following steps:
(1) lysate, oscillation mixing, in 50~65 DEG C of incubation 1h~16h is added；
(2) 90~95 DEG C of incubation 40min~50min；
(3) aqueous phase solution containing nucleic acid is separated and collected.
The time of cracking processing in above-mentioned steps (1) can be according to sample size and different tissues within the above range
It is adjusted.
In above-mentioned steps (2), it can guarantee realizing preferable sample solution friendship in 90~95 DEG C of incubation 40min~50min
While connection, guarantee the integrality of genomic DNA；Incubation time is too short or lower temperature is incubated for, and will cause solution crosslinking and is not thorough,
The too long time then will affect the final integrality for extracting DNA.
In the present invention, separation and purified genomic dna can be used from the above-mentioned aqueous phase solution containing nucleic acid obtained
Genomic DNA isolation and purification method commonly used in the art carries out, such as column purification method, Beads enrichment method of purification, isopropanol precipitating method
Deng.
Wherein, arbitrary Genomic DNA Purification column in the prior art can be used in the column purification method；As of the invention
A kind of preferred embodiment, the column purification use the QIAamp MinElute columns of Qiagen.
Specifically, the separation and purified genomic dna include the following steps:
(1) RNA enzyme is added in the aqueous phase solution containing nucleic acid and removes RNA；
(2) genomic DNA is separated using nucleic acid extraction purification column, Beads enrichment or isopropanol precipitating；
(3) separate genomic DNA it is washed and dissolution to get.
As a kind of preferred embodiment of the invention, the genome DNA extracting method includes the following steps:
(1) lysate, oscillation mixing, in 55~60 DEG C of 1~4h of incubation are added in the sample；Isolated nucleic acid separation
Liquid；
When the sample is paraffin-embedded sample, the lysate includes hexadecane, Proteinase K and cracking buffering
Liquid；
When not containing paraffin in the sample, the lysate includes Proteinase K and lysis buffer；
Final concentration of 15~the 20mg/mL of the Proteinase K in the reaction system；
The lysis buffer include following component: lauryl sodium sulfate 1%~5%, guanidinium isothiocyanate 3~
5mol/L, Tris-HCl 10~15mmol/L, EDTA 0.05~0.1mol/L, Triton-X100 0.05%~0.1%；
The pH of the lysis buffer is 7.8~8.0；
(2) 90 DEG C of 40~45min of incubation, are centrifuged after being stored at room temperature, and draw the aqueous phase solution containing nucleic acid；
(3) be added repair liquid to the UNG enzyme final concentration of 10~15U/ μ L, mix, 50~55 DEG C be incubated for 1~
1.5h is centrifuged after being stored at room temperature；
(4) RNA enzyme is added in the solution that step (3) obtains to final concentration of 150~300mg/mL；It is stored at room temperature processing
1~10min；
(5) albumen is added and removes liquid, obtained suspension is transferred in nucleic acid purification post, it is purified to obtain genomic DNA；
The albumen removal liquid includes following component: 45~75mmol/L Tris-HCl, 100~120mmol/L NaCl,
30~60mmol/L disodium ethylene diamine tetraacetate, dehydrated alcohol 75%~95%.
As the preferred embodiment of the present invention, the nucleic acid purification post in above-mentioned steps (5) is QIAamp MinElute
Columns, the purifying is according to the progress of the method for the specification of QIAamp MinElute columns.
In the present invention, the sample is the animal sample saved.The animal sample includes animal tissue, animal body fluid
Deng.
Preferably, the sample is to fix through formalin or the animal sample through paraffin embedding.
The beneficial effects of the present invention are: the present invention repairs step by introducing UNG enzyme, repairs animal sample at preservation
The base mutation and DNA damage occurred during reason is repaired the reagent dosage and reaction condition of step by optimization, improved
While the treatment effeciency of UNG enzyme, as far as possible reduction repair process for the extract yield of genomic DNA and the influence of quality and
Influence for follow-up library building and PCR amplification.In addition, sample fixed for formaldehyde, by optimizing cleavage step；For stone
Wax embedded samples, by screening most suitable dewaxing liquid ingredient and optimizing the dewaxing and cleavage step of sample, developing can be from formaldehyde
High yield, high quality, low damage, the genome that C > T and G > A artificial mutation will not be introduced are extracted in fixed or paraffin-embedded sample
The method of DNA.
The extraction of genome DAN, the extraction total amount and purity of obtained genomic DNA are carried out using the method provided by the present invention
It is significantly better than traditional extracting method；It can be preferably due to reducing the DNA damage for saving sample and mutation, genomic DNA
It is connect with linker DNA and carries out subsequent amplified reaction, therefore, produced using the library for extracting obtained genomic DNA building
Amount is also significantly greater than traditional extracting method.Genome DNA extracting method provided by the invention has can be from the limited of preservation
Animal sample in extract high yield, high quality genomic DNA, effectively eliminate C > T and G > A artificial mutation, and operating time
The advantages such as short, simple to operate, extraction cost is low, the per unit throughputs of extraction process are high, the safety height of operation.
Specific embodiment
The preferred embodiment of the present invention is described in detail below in conjunction with embodiment.It will be appreciated that following real
Providing merely to play the purpose of explanation for example is applied, is not used to limit the scope of the present invention.The skill of this field
Art personnel without departing from the spirit and purpose of the present invention, can carry out various modifications and replace to the present invention.
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Embodiment 1
The present embodiment provides a kind of extracting methods of the genomic DNA of Animal Histological Section sample, extract sample point
It Wei not the surgical tissue paraffin patch from the breast tissue of breast cancer patients and the left lung tissue from lung cancer patient
Puncturing tissue paraffin patch, the specific method is as follows:
(1) area > 10mm × 10mm, about 5-10 μm of thickness of tissue paraffin section de sample are scraped with clean knife blade
This, is transferred in ready 1.5mL centrifuge tube, and 12000rpm is centrifuged 3min, and the tissue of scraping is centrifuged to tube bottom；
(2) 160 μ L dewaxing liquids (dewaxing liquid is hexadecane) are added in the above-mentioned centrifuge tube equipped with paraffin-embedded tissue,
If sample size is big or paraffin component is more, the dewaxing liquid doubled can be used and handled；
(3) Proteinase K is added to final concentration of 20mg/mL, the 200 μ l lysis buffer (formulas of lysis buffer are added
It is as follows: lauryl sodium sulfate 5% (mass percentage), guanidinium isothiocyanate 5mol/L, Tris-HCl 15mmol/L,
EDTA0.05mol/L, Triton-X1000.05% (volumn concentration)；To improve quality, volumn concentration and mole dense
Degree is calculated on the basis of the total volume of lysate buffer), vortex vibrates 10s, brief centrifugation；(after Proteinase K is added,
Solution layering, upper layer is dewaxing liquid, and lower layer is Proteinase K)；It is incubated for 1h in 56 DEG C of constant temperature blending instruments, until tissue cracking completely；
(4) above-mentioned cracking is organized in 90 DEG C of incubation 40min (placing into sample when metal bath is warming up to 90 DEG C)；It will
Sample takes out, and is stored at room temperature 5min；12000rpm is centrifuged 1min, draws lower layer's solution (about 95 μ L) to new 1.5mL centrifuge tube
In (slow imbibition not be drawn onto upper layer dewaxing liquid)；
(5) 115 μ L RNase-Free Water, vortex are added in aqueous phase solution obtained above and vibrate 10s, wink
When be centrifuged；The reparation liquid (reparation liquid is UNG enzyme) of 35 μ L is added, so that the final concentration of 15U/ μ L of UNG enzyme in the reaction system,
Vortex vibrates 10s, and brief centrifugation, 50 DEG C are incubated for 1 hour；
After (6) 50 DEG C are incubated for, it is stored at room temperature 2min, 12000rpm room temperature is centrifuged 1min；
(7) when sample is cooled to room temperature, 2 μ L RNase A (100mg/ml) are added, RNase A is in the reaction system
Final concentration of 150mg/mL, vortex vibrate 10s, and brief centrifugation is stored at room temperature 2min；
(8) 200 μ L buffer 1 (bufferAL that buffer1 is QIAmp DNAFFPE Tissue Kit) are added,
Vortex vibrates 10s, brief centrifugation；250 μ L dehydrated alcohols are added, vortex vibrates 10s, and 12000rpm is centrifuged 1min；
(9) suspension in centrifuge tube is transferred to QIAamp MinElute columns, about 740 μ L；12000rpm centrifugation
Filtered fluid suction in the collecting pipe of lower section is transferred to QIAamp MinElute columns, 12000rpm centrifugation by 1min
1min；
(10) the QIAamp MinElute columns that centrifugation finishes is placed in new collecting pipe, is separately added into 500 μ L
Buffer2 (the Buffer AW1 that buffer2 is QIAmp DNAFFPE Tissue Kit), 12000rpm is centrifuged 1min, discards
Filtered solution in collecting pipe；
(11) 250 μ L dehydrated alcohols are added, vortex vibrates 10s, and 12000rpm is centrifuged 1min, discards the filter in collecting pipe
Cross liquid；12000rpm is centrifuged 2min, discards the filtered solution in collecting pipe；
(12) the QIAamp MinElute columns that centrifugation finishes is placed in new 1.5mL centrifuge tube, is stored at room temperature
5min；
(13) 55 μ L eluents are added in QIAamp MinElute columns, and (eluent is QIAmp DNAFFPE
The Elution Buffer of Tissue Kit), it is stored at room temperature 2min, 12000rpm is centrifuged 2min, obtains genomic DNA.
Embodiment 2
The present embodiment provides the extracting methods that a kind of formaldehyde impregnates the genomic DNA of animal tissue, provide with embodiment 1
The difference of method is only that step (1) and step (2), and in the present embodiment, (1) and step (2) are merged into the step of embodiment (1)
Step (1): impregnating operation tissue block with clean knife blade scraping formaldehyde, be transferred in ready 1.5ml centrifuge tube,
12000rpm is centrifuged 3min, and the tissue of scraping is centrifuged to tube bottom.Remaining step is same as Example 1.The present embodiment mentions
Sampling is originally for from the formaldehyde of the breast tissue of mammary cancer surgery patient immersion surgical tissue.
Embodiment 3
The present embodiment provides a kind of extracting methods of the genomic DNA of animal Pleural effusions, with the method for the offer of embodiment 1
Difference is only that step (1) and step (2), and in the present embodiment, (1) and step (2) merge into step the step of embodiment (1)
(1): take 200 μ l Pleural effusions into ready 1.5ml centrifuge tube, 12000rpm be centrifuged 3min, by the tissue of scraping be centrifuged to
Tube bottom.Remaining step is same as Example 1.The sample that extracts of the present embodiment is the Pleural effusions from lung cancer patient.
Comparative example 1
This comparative example provides a kind of extracting method of the genomic DNA of Animal Histological Section sample, for according to QIAmp
The method of DNAFFPE Tissue Kit kit specification carries out, and samples sources used are identical with embodiment 1, specific side
Method is as follows:
(1) it takes paraffin surgical tissue to be sliced 6, about 5-10 μm of area > 10mm × 10mm, thickness, is scraped with knife blade
Tissue samples are loaded in 1.5ml clean centrifuge tube；
(2) use dimethylbenzene lost-wax process, every pipe be added the west 1ml how mountain, be acutely vortexed 10sec；- 12,000rpm centrifugation
1min abandons supernatant, adds 1ml dimethylbenzene, be acutely vortexed 10sec；12,000rpm (~13,400 × g) are centrifuged 2min, in abandoning
Clearly；
(3) 1ml dehydrated alcohol is added in above-mentioned pipe, is vortexed and mixes 10sec；12,000rpm (~13,400 × g) centrifugation
1min abandons supernatant；
(4) 12,000rpm (~13,400 × g) are centrifuged 2min, remove remaining dehydrated alcohol with liquid-transfering gun, are placed at room temperature for
5-10min, sufficiently volatilization ethyl alcohol；
(5) 200 μ l buffer ATL are added and 20 μ l Proteinase Ks, vortex oscillator sufficiently vibrates 1min；56 DEG C
In water-bath.It is incubated overnight, until tissue cracking completely finishes.
(6) sample is placed in 90 DEG C of water-baths, is incubated for 1h；
(7) when sample is cooled to room temperature, 100mg RNase A is added, to remove RNA remaining in solution；
(8) 200 μ l buffer AL and 250 μ l absolute alcohols are added in each sample, and vortex vibrates immediately
It mixes well；
(9) suspensions whole in centrifuge tube are moved into QIAamp MinElute column, 12000rpm is centrifuged 1min, will under
Filtered solution and collecting pipe in square collecting pipe abandon together；
(10) adsorption column that centrifugation finishes is placed in new collecting pipe and is separately added into 200 μ l AW1, after covering lid
12000rpm is centrifuged 1min, discards the filtered solution in collecting pipe；
(11) adsorption column that centrifugation finishes is placed in new collecting pipe, 500 μ l AW2 is separately added into, after covering lid
12000rpm is centrifuged 1min, discards the filtered solution in collecting pipe；12000rpm is centrifuged 2min, discards the filtered solution in collecting pipe；
(12) adsorption column that centrifugation finishes is placed in clean 1.5ml centrifuge tube, is stored at room temperature 5min；
(13) 55 μ l NFW are added in QIAamp MinElute columns；Be stored at room temperature 2min, 12,000rpm from
Heart 2min.
Comparative example 2
This comparative example provides a kind of extracting method of the genomic DNA of formaldehyde immersion animal tissue, provides with comparative example 1
The difference of method is only that step (1)~step (4), and in this comparative example, (1)~step (4) is merged into the step of comparative example (1)
Step (1): operation tissue block is impregnated with clean knife blade scraping formaldehyde, is transferred in ready 1.5mL centrifuge tube.Its
Remaining step is identical as comparative example 1.Samples sources used in this comparative example are identical with embodiment 2.
Comparative example 3
This comparative example provides a kind of extracting method of the genomic DNA of animal Pleural effusions, the method provided with comparative example 1
Difference is only that step (1)~step (4), and in this comparative example, (1)~step (4) merges into step the step of comparative example (1)
(1): take 200 μ l Pleural effusions into ready 1.5mL centrifuge tube, 12000rpm be centrifuged 3min, by the tissue of scraping be centrifuged to
Tube bottom.Remaining step is identical as comparative example 1.Samples sources used in this comparative example are identical with embodiment 3.
The extraction total amount and quality analysis of 1 genomic DNA of experimental example
Be respectively adopted nonodrop and3.0Fluorometer detection utilizes Examples 1 to 3 and comparative example 1~3
The quality and extraction total amount for the genomic DNA that extracting method is extracted, the results are shown in Table 1, the results showed that, utilize embodiment
The quality (A260/280 and A260/230) for the genomic DNA that 1~3 extracting method is extracted is significantly better than comparative example 1
~3 genomic DNAs extracted using conventional reagents box extracting method, extract to obtain using the extracting method of Examples 1 to 3
Genomic DNA concentration and extract total amount and extracted using conventional reagents box extracting method from being significantly higher than comparative example 1~3
The genomic DNA arrived.It can be seen that genome DNA extracting method provided by the invention is applicable not only to paraffin embedding preservation
The extraction of the genomic DNA of animal tissue is mentioned in the genome for the animal sample for impregnating the preservations such as tissue for Pleural effusions, formaldehyde
When taking, dewaxing liquid only need to be saved, can similarly obtain the genomic DNA of high yield and high quality.
The total amount and quality versus's table for the genomic DNA that 1 different type sample of table is extracted using distinct methods
| Serial number | Extracting method | Sample type | Extract total amount | A260/280 | A260/230 |
| Sample 1 | Comparative example 2 | Formaldehyde impregnates surgical tissue | 145 | 0.48 | -0.4 |
| Sample 1 | Embodiment 2 | 1145 | 1.96 | 2.12 | |
| Sample 2 | Comparative example 3 | Pleural effusions | 4260 | 0.67 | -0.92 |
| Sample 2 | Embodiment 3 | 6880 | 1.65 | 2.02 | |
| Sample 3 | Comparative example 1 | Surgical tissue paraffin patch | 2250 | 1.12 | -0.43 |
| Sample 3 | Embodiment 1 | 6800 | 1.78 | 2.24 | |
| Sample 4 | Comparative example 1 | Puncturing tissue paraffin patch | 89.6 | 1.02 | -0.02 |
| Sample 4 | Embodiment 1 | 260 | 1.79 | 2.02 |
The data of each sample are the average value of independent repeated trials three times in table 1.
Experimental example 2 is analyzed using the yield and the sequencing frequency of mutation in the genomic DNA building DNA sequencing library extracted
By the DNA of the genomic DNA extracted using Examples 1 to 3 and 1~3 extracting method of comparative example, use is identical
Library construction Kit and identical DNA library construction method (banking process of this field routine) carry out library construction, it is right
The yield in library is detected, and the results are shown in Table 2, the results showed that, it is extracted using the extracting method of Examples 1 to 3
The library yield of the building of genomic DNA is all remarkably higher than what comparative example 1~3 was extracted using conventional reagents box extracting method
Genomic DNA, the initial amount that the genomic DNA obtained due to each sample extraction is used to build library is identical, builds all during library
Experimental condition is identical, the volume variance of final library be mainly the genomic DNA extracted due to distinct methods damage or
There are the degree of mutation it is different caused by, therefore, the above result shows that, the genome provided using the embodiment of the present invention 1~3
The method that the degree of injury for the genomic DNA that DNA extraction method obtains is significantly lower than comparative example 1~3, it was demonstrated that by introducing UNG
The reparation step of enzyme can obviously repair the damage or mutation of genomic DNA in sample.
The genomic DNA that table 2 is extracted using distinct methods is used to construct the library yield in library
Choose the hand of the breast tissue of the above-mentioned breast cancer patients extracted using the extracting method of embodiment 1 and comparative example 1
The library of the genomic DNA building of art tissue paraffin patch sample carries out machine sequencing, and sequencing data analyzes result such as 3 institute of table
Show, the results show that the cosmic variation that the genomic DNA that 1 extracting method of embodiment is extracted detects is substantially less than and compares
The genomic DNA that 1 extracting method of example is extracted, it was demonstrated that the reparation step by being introduced into UNG enzyme can be eliminated obviously in sample
The base mutation frequency of genomic DNA, DNA plerosis damage.
The base mutation for the genomic DNA building sequencing library that table 3 is extracted using distinct methods is analyzed
Although above having used general explanation, specific embodiment and test, the present invention is made to retouch in detail
It states, but on the basis of the present invention, it can be made some modifications or improvements, this is apparent to those skilled in the art
's.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to claimed
Range.
Claims (10)
1. a kind of method for extracting genomic DNA in the animal sample saved, which is characterized in that be included in and handled through cracking
Sample in be added repair liquid carry out mutating alkali yl eliminate and DNA damage repair the step of；The reparation liquid includes UNG enzyme.
2. the method according to claim 1, wherein including the following steps:
(1) sample dissociation: lysate is added in the sample and carries out cracking processing, isolated nucleic acid separating liquid；
(2) mutating alkali yl is eliminated and DNA damage reparation: the reparation liquid being added in the nucleic acid separating liquid and is repaired；Institute
State the final concentration of 10~50U/ μ L of UNG enzyme in the reaction system；
(3) separation and purified genomic dna.
3. according to the method described in claim 2, it is characterized in that, the mutating alkali yl is eliminated and DNA damage reparation is 45
~55 DEG C of incubations handle 1~2h.
4. according to the method in claim 2 or 3, which is characterized in that the lysate includes lysis buffer and protease
K；
Preferably, the final concentration of 15~50mg/mL of the Proteinase K in the reaction system；The lysis buffer includes such as
Lower component: lauryl sodium sulfate 1%~10%, 10~50mmol/L of guanidinium isothiocyanate 2~5mol/L, Tris-HCl, EDTA
0.01~0.2mol/L, Triton-X100 0.01%~0.1%；
The pH of the lysis buffer is 7.5~8.0.
5. according to the described in any item methods of claim 2~4, which is characterized in that when the sample is paraffin-embedded sample,
The lysate further includes dewaxing liquid.
6. according to the method described in claim 5, it is characterized in that, the dewaxing liquid includes hexadecane.
7. according to the described in any item methods of claim 2~6, which is characterized in that the sample dissociation includes the following steps:
(1) lysate, oscillation mixing, in 50~65 DEG C of incubation 1h~16h is added；
(2) 90~95 DEG C of incubation 40min~50min；
(3) aqueous phase solution containing nucleic acid is separated and collected.
8. according to the described in any item methods of claim 2~7, which is characterized in that the separation and purified genomic dna include
Following steps:
(1) RNA enzyme is added in the aqueous phase solution containing nucleic acid and removes RNA；
(2) genomic DNA is separated using nucleic acid extraction purification column, Beads enrichment or isopropanol precipitating；
(3) separate genomic DNA it is washed and dissolution to get.
9. described in any item methods according to claim 1~8, which comprises the steps of:
(1) lysate, oscillation mixing, in 55~60 DEG C of 1~4h of incubation are added in the sample；Isolated nucleic acid separating liquid；
When the sample is paraffin-embedded sample, the lysate includes hexadecane, Proteinase K and lysis buffer；
When not containing paraffin in the sample, the lysate includes Proteinase K and lysis buffer；
Final concentration of 15~the 20mg/mL of the Proteinase K in the reaction system；
The lysis buffer include following component: lauryl sodium sulfate 1%~5%, 3~5mol/L of guanidinium isothiocyanate,
Tris-HCl 10~15mmol/L, EDTA 0.05~0.1mol/L, Triton-X100 0.05%~0.1%；
The pH of the lysis buffer is 7.8~8.0；
(2) 90 DEG C of 40~45min of incubation, are centrifuged after being stored at room temperature, and draw the aqueous phase solution containing nucleic acid；
(3) be added it is described repair liquid to the UNG enzyme final concentration of 10~15U/ μ L, mix, 50~55 DEG C be incubated for 1~
1.5h is centrifuged after being stored at room temperature；
(4) RNA enzyme is added in the solution that step (3) obtains to final concentration of 150~300mg/mL；Be stored at room temperature processing 1~
10min；
(5) albumen is added and removes liquid, obtained suspension is transferred in nucleic acid purification post, it is purified to obtain genomic DNA；
Albumen removal liquid includes following component: 45~75mmol/L Tris-HCl, 100~120mmol/L NaCl, 30~
60mmol/L disodium ethylene diamine tetraacetate, dehydrated alcohol 75%~95%.
10. described in any item methods according to claim 1~9, which is characterized in that the sample is the animal sample saved；
It is preferably fixed through formalin or the animal sample through paraffin embedding.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1155153B1 (en) * | 1999-02-22 | 2006-01-04 | Quantovir AB | Method and kit for early cancer prediction |
| CN102230016A (en) * | 2011-06-22 | 2011-11-02 | 山东省眼科研究所 | Loop-mediated isothermal amplification primers and kit for detecting acanthamoeba |
-
2018
- 2018-12-29 CN CN201811636752.1A patent/CN109593755A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1155153B1 (en) * | 1999-02-22 | 2006-01-04 | Quantovir AB | Method and kit for early cancer prediction |
| CN102230016A (en) * | 2011-06-22 | 2011-11-02 | 山东省眼科研究所 | Loop-mediated isothermal amplification primers and kit for detecting acanthamoeba |
Non-Patent Citations (1)
| Title |
|---|
| 鲍洪波等: "尿嘧啶N糖基化酶(UNG)的研究进展", 《中国生物工程杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113106085B (en) * | 2020-01-09 | 2023-10-13 | 创想生物科技有限公司 | Nucleic acid separation method and nucleic acid separation system |
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