CN102719426A - Method for extracting genomic DNA (deoxyribonucleic acid) in milk appropriate for large-scale genotype identification - Google Patents
Method for extracting genomic DNA (deoxyribonucleic acid) in milk appropriate for large-scale genotype identification Download PDFInfo
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Abstract
The invention discloses a method for extracting genomic DNA (deoxyribonucleic acid) in milk appropriate for large-scale genotype identification. Fresh milk is processed by improved technologies and methods such as a somatic cell enrichment technology, an SDS (Sodium Dodecyl Sulfonate) phenol splitting technology, a nucleic acid precipitation technology and the like, the acquisition quantity of the milk can be controlled between 10ml and 13ml, and the extracted genomic DNA has complete quality and better concentration and purity. At the same time, 500 bp even more than 1000 bp of long-segment gene sequences can be amplified in the extracted milk DNA, and large-scale genetic typing identification and subsequent molecular biology analysis can be carried out through the obtained sequence. The method has the characteristics of easiness in operation, low cost, high precision, convenient sampling, practicability and large scale, difficulties in blood and tissue sampling can be obviously avoided, stress response of cows can not be caused, and unnecessary loss brought to cow enterprises and farmers by other sampling methods is reduced. In addition, the method disclosed by the invention provides an advantageous theoretical foundation for controlling milk nutritional value, milk quality safety and milk production performance of the cows from a molecular level.
Description
Technical field
The invention belongs to biology field; The process for extracting that particularly relates to genomic dna in a kind of milk that is applicable to extensive genotype identification; This method can extract from 10mL-13mL fresh milk that quality is complete, concentration and purity genomic dna preferably; With this DNA is that template is carried out pcr amplification, can amplify the above long segment cow genome group dna sequence dna of 1000bp, and then carry out genotype identification and molecular biological analysis.
Background technology
In recent years, Protocols in Molecular Biology had become the favourable instrument in dairy products quality examination and the molecular nutrition research.Obtaining the high quality genomic dna is the basis of carrying out subsequent molecular such as food nutrition Function Identification, tracing back property of food, the variation of milk cow population genetic, molecular marker assisted selection breeding, paternity test, DNA Southern hybridization.At present, the material that separates milk cow DNA mainly comprises blood, muscle tissue, organs and tissues etc., and the blood sampling of milk bovine jugular vein is mainly adopted in the collection of blood, and the ear method of cutting is adopted in the collection of tissue.Find in the production practice, these modes all can to milk cow produce stress, influence the milk cow production level, certain technical difficulty is also arranged, need the professional to operate, and also be a no small loss for the diary farm, the resistance of sampling is very big.Therefore, it is imperative to seek more suitable material.
Somatocyte in the milk is made up of polymorph neutrophile leucocytes, scavenger cell, lymphocyte and a spot of mammary tissue epithelial cell etc. usually.Under the normal physiological situation, every ml milk has 20,000-200,000 individual cells approximately.Somatic number receives the influence of age and factors such as parity, stage lactation period, season, physical stress, individual character and milking operation.Milk cow has unique defense system, when the mammary system of milk cow receives the invasion and attack of different sorts bacterium and takes place to infect and during damage, bears through the immunologic mechanism of body and to get rid of the white corpuscle that infects with repairing the organization task of being damaged and will gather at this.Somatic number promptly rises to 300,000-1,000 ten thousand significantly in this mammary tissue excretory milk thereupon.Cell count in the blood also can be along with infectiosity is different and different, generally in 7,000,000-1,000 ten thousand scopes.Under equal volume, minimum somatocyte number is about 350 times of minimum somatocyte number in the milk in the blood, therefore from milk, extracts DNA difficulty relatively.Study demonstration through forefathers, the somatic number of average every ml milk reaches about 300,000, has promptly reached the cell concn that extracts DNA basically, and from milk, extracting DNA is feasible in theory.
In recent years; Some domestic and international few studies persons begin to probe into the method for from milk, extracting genomic dna; Tian Yu etc. combine separating milk somatocyte technology to extract DNA with high-temperature cracking method, Zhang Junwei etc. have carried out the optimization of this method, and Murphy etc. also use the phenol/chloroform method to extract DNA.But these method sampling amounts are bigger, all more than 50mL; And the DNA that extracts can only carry out the pcr amplification of 500bp with interior small segment gene order, is not suitable for extensive genotypic evaluation and molecular biological analysis.
In sum, optimize setting up a kind of novel, the easy genome DNA extracting method that is suitable for extensive genotype identification, is one of problem of studying of applicant.
Summary of the invention
The objective of the invention is to; The process for extracting of genomic dna in a kind of milk that is applicable to extensive genotype identification is provided; This method can extract in 10mL-13mL fresh milk that quality is complete, concentration and purity genomic dna preferably; With this DNA is that template is carried out pcr amplification, can amplify the above long segment cow genome group dna sequence dna of 1000bp, and then carry out genotype identification and molecular biological analysis.
In order to realize above-mentioned task, the present invention is achieved through following technical scheme:
The process for extracting of genomic dna in a kind of milk that is applicable to extensive genotype identification is characterized in that this method is carried out as follows:
Step 1, centrifuge tube is prepared: chooses the 15mL centrifuge tube, in high-pressure sterilizing pot, sterilizes, subsequent use.
Step 2, milk collection: gather fresh milk 10mL-13mL to centrifuge tube, add 2 SRM 935as in each pipe, ice bag is taken back, and places-20 ℃ of preservations.
Step 3, milk somatic cell are separated and enrichment: the milk appearance of above-mentioned cryopreservation thawed in 4 ℃, and at 2500r/min, 4 ℃ of centrifugal 30min down.Scrape off the butterfat on centrifuge tube upper strata with little spoon, remove with the milk-protein of suction pipe with the middle layer, stay bottommost the beds of precipitation.Add 600 μ L phosphoric acid buffers (PBS) to centrifuge tube bottom, bottom settlings is beaten suspended and be transferred in the centrifuge tube of 1.5mL, the centrifugal 10min of 3500r/min normal temperature discards supernatant liquid, keeps bottom settlings.Add emulsifying agent 60 μ L, PBS 540 μ L to bottom settlings again, suspend fully with vibrator vibration to deposition, 40 ℃ of water bath with thermostatic control processing 10min slough the butterfat around the somatocyte; 3500r/min; The centrifugal 10min of normal temperature abandons supernatant, adds PBS 500 μ L suspension deposition; Make the enrichment of somatocyte deposition in 12000r/min low-temperature centrifugation 10min, abandon supernatant.
Step 4, milk somatic cell digestion: in the somatocyte deposition, add the DNA extraction damping fluid of 350 μ L, the SDS of 50 μ L, the Proteinase K of 10 μ L spends the night 56 ℃ of following water-bath digestion.
Step 5, the SDS phynol method extracts DNA: in order to extract DNA accurately and efficiently, this research adopts the SDS phynol method to extract DNA.At first, in digestion product, add the saturated phenol solution of isopyknic Tris, put upside down 10min back and forth, 12000r/min, low-temperature centrifugation 10min obtains supernatant; Supernatant is transferred in another 1.5mL centrifuge tube, added the mixture of isopyknic phenol, chloroform and primary isoamyl alcohol, phenol, chloroform and primary isoamyl alcohol are preparation in 25: 24: 1 by volume; Put upside down 10min back and forth, make resolution of precipitate, 12000r/min; Centrifugal 10min obtains supernatant; Supernatant is transferred in another 1.5mL centrifuge tube again, added the mixture of equal-volume chloroform and primary isoamyl alcohol, chloroform and primary isoamyl alcohol are preparation in 24: 1 by volume, put upside down 10min back and forth, make resolution of precipitate, 12000r/min, and centrifugal 10min obtains supernatant.
Step 6, nucleic acid deposition: supernatant is transferred to the centrifuge tube of another 1.5mL, add ice absolute ethyl alcohol (20 ℃) deposition of 2 times of volumes, jolting gently ,-20 ℃, leave standstill 30min, the centrifugal 10min of 12000r/min discards upper strata ethanol.It is 70% ice ethanol (20 ℃) washing that bottom settlings uses mass concentration, 12000r/min, and low-temperature centrifugation 10min carefully removes upper strata ethanol, and the ethanol that volatilizees fast adds the TE dissolving DNA of 25 μ L, and-4 ℃ of preservations are subsequent use.
Step 7, the DNA quality examination: measure with ultraviolet spectrophotometer, the genomic dna concentration value that obtains is between the 12 μ g/ μ L-45 μ g/ μ L more than 80%, OD
260/280Value is between the 1.65-1.75, and agarose gel electrophoresis carries out band test strip neat and consistent, good in integrity, unobvious hangover and diffusing phenomenon.
Step 8, the selection of specific gene: the specific function gene of selecting ox at random is as research object, and in GenBank, searches this gene order, design specific primers.
Step 9, pcr amplification and order-checking: select suitable PCR system and reaction conditions, carry out pcr amplification, and with PCR product purification test kit reclaim, purifying, be sent to order-checking company and check order.
Step 10, gene type is identified: order-checking gained gene order can be carried out extensive gene type evaluation fully, carries out follow-up molecular biological analysis then.
The process for extracting of genomic dna in the milk that is applicable to extensive genotype identification of the present invention, sampling is convenient, and practical, simple to operate, scale is big, cost is low, and accuracy is high.Compared with prior art, the technique effect that is brought is:
1, can the consumption of fresh milk be controlled at 10mL-13mL, the 50mL that uses than people such as Tian Yu, Zhang Junwei significantly reduces, and the mass effect of extraction DNA obviously improves.In addition, to large-scale genetic analysis, need go out to collect milk appearance, the centrifuge tube of 50mL carries trouble than 15mL centrifuge tube far away; Under the condition of equal human and material resources, the milk appearance about every cow head collection 50mL can be gathered the scale of milk cow, be far smaller than every cow head and only gather the milk cow scale about 13mL.Therefore, fresh milk sampling of the present invention is convenient, quick, and the sampling scale increases.
2, adopt improving technology and method processing fresh milks such as somatocyte beneficiation technologies, SDS phenol cracking technique, nucleic acid precipitation technology, obtain the genomic dna concentration value and be between the 12 μ g/ μ L-45 μ g/ μ L OD
260/280Value is between the 1.65-1.75, and agarose gel electrophoresis carries out band test strip neat and consistent, good in integrity, unobvious hangover and diffusing phenomenon.Explain that the DNA that the present invention extracts can carry out the pcr amplification in later stage.
3, the DNA that extracts carries out pcr amplification as template, can amplify the above long segment cow genome group dna sequence dna of 1000bp.The general DNA major part of from milk, extracting can only be as the amplification template of ox chondriogen, thereby differentiates in the milk whether miserable false phenomenon is arranged, and can not carry out the gene sequencing that nuclear DNA can carry out.But gained genomic dna of the present invention both can be used as the amplification template of ox chondriogen, carried out the miserable false check and analysis of milk, and can carry out genotype identification and molecular biological analysis fully.
When using this method to extract milk DNA, avoided use blood and sample of tissue difficulty, also can not cause the milk cow stress response, sampling is convenient easy, has reduced additive method and has been sampled as the unnecessary loss that milk cow enterprise and raiser bring.Simultaneously, the DNA quality is complete, effect is better, and the 500bp that can increase, even bigger segmental gene order more than the 1000bp are for the molecular biological analysis of carrying out genetic component type and later stage has been established solid basis.The present invention is for providing favourable theoretical foundation from molecular level regulation and control nutritional value of milk, milk qualities safety and cow producing milk performance.
Description of drawings
Fig. 1 is the agarose gel electrophoresis figure that extracts DNA in the milk.From left to right 10 swimming lanes are respectively the DNA band that extracts in 100 ladder Marker and 9 milk.
Fig. 2 is to be template to extract the DNA that extracts in the milk, the gene order gel electrophoresis figure of amplification B2M1F and B2M1R primer.From left to right 10 swimming lanes are respectively 9 DNA sample amplification product bands and 100 ladder Marker.
Fig. 3 is to be template to extract the DNA that extracts in the milk, the gene order gel electrophoresis figure of amplification B2M2F and B2M2R primer.From left to right 10 swimming lanes are respectively 9 DNA sample amplification product bands and 100 ladder Marker.
Below in conjunction with the specific embodiment of specifically testing and the contriver provides the present invention is made more detailed description.
Embodiment
Experiment material
1, sample source
112 healthy adult milk cows of Xi'an herding development company of modern agriculture comprehensive exploitation main office diary farm; It is about 13mL that every cow head is adopted the milk amount, wherein adds 2 SRM 935as as sanitas, in the centrifuge tube of the 15mL that packs into; Low temperature is taken back the laboratory ,-20 ℃ of preservations.
2, test reagent
(1) main agents
Tutofusin tris (Tris), saturated phenol of Tris and sodium laurylsulfonate (SDS) are Beijing ancient cooking vessel state biotech development center product; Proteinase K (Proteinase K), 10 * PCR buffer, dNTPs, 100bp marker and Taq archaeal dna polymerase are the precious biotech firm in Dalian product; SRM 935a, phenol, chloroform, primary isoamyl alcohol, absolute ethyl alcohol, agar gel, Proteinase K, NaCl, KCl, Na
2HPO
4, KH
2PO
4, Triton-X100, Tris-Cl, EDTA-Na, absolute ethyl alcohol, sodium lauryl sulphate (SDS), DNA marker, Witco 70, EB staining agent provide by the gloomy rich biological ltd in Xi'an.Agarose is available from Beijing Pu Bo Bioisystech Co., Ltd; PCR product purification test kit is available from sky, Beijing root biochemical technology ltd.
(2) solution preparation
Phosphoric acid buffer (PBS): take by weighing NaCl 8g, KCl 0.2g, Na respectively
2HPO
41.44g, KH
2PO
40.24g, various substance dissolves in 800mL zero(ppm) water, are transferred pH to 7.4, adding distil water is settled to 1L;
Emulsifying agent (OP): 90% Triton-X10020mL, 95% ethanol 125mL, 0.9g/L NaCl solution 855mL;
The DNA extraction damping fluid: NaCl 1M/L, Tris-Cl 0.5M/L, EDTA-Na 0.5M/L, pH transfer to 7.5-8.0;
The 20%SDS lysate: 20g SDS is dissolved in the 80mL distilled water in 68 ℃ of heating for dissolving, transfers to PH7.2 with dense HCl, be settled to 100mL, 4 ℃ of preservations are subsequent use;
TE solution: take by weighing pH respectively and be 8.0 1M/L Tris-Cl 2mL and 0.5M/L EDTA 0.4mL, its two kinds of solution are mixed, adding distil water is settled to 200mL, transfers pH to 8.0;
TAE electrophoretic buffer: take by weighing 24.2g Tris-Cl 10mL, 0.5M/L Na
2-EDTA, the glacial acetic acid of 5.71mL fully is settled to 1L after the dissolving, and pH transfers to 8-8.4.
3, key instrument
Ultra-high speed refrigerated centrifuge (U.S. Sigma company), low speed large capacity centrifuge (Anting Scientific Instrument Factory, Shanghai), supercentrifuge (Anting Scientific Instrument Factory, Shanghai), accurate micropipet (German Eppendorf), whirlpool mixed instrument (its woods Bel instrument Manufacturing Co., Ltd of Haimen City), full automatic gel imaging analysis instrument (Shanghai Peiqing Science Co., Ltd), grads PCR appearance (BLO-RAD), and instrument such as water-bath, microwave oven, electrophoresis apparatus, palm type whizzer.
Embodiment 1: the extraction of DNA and quality examination in the milk
1, milk somatic cell separates and enrichment
Above-mentioned cryopreservation milk appearance is thawed for 4 ℃, at 2500r/min, 4 ℃ of following centrifugal 30min.With little spoonful of butterfat that scrapes off the centrifuge tube upper strata, remove with the milk-protein of suction pipe the middle layer, stay the deposition of one deck of bottommost.Add 600 μ L PBS to centrifuge tube bottom, bottom settlings is beaten suspended and be transferred in the centrifuge tube of 1.5mL, the centrifugal 10min of 3500r/min normal temperature discards supernatant liquid, keeps bottom settlings.Add emulsifying agent 60 μ L, PBS 540 μ L to bottom settlings again, suspend fully with vibrator vibration to deposition, 40 ℃ of water bath with thermostatic control processing 10min slough the butterfat around the somatocyte; 3500r/min; The centrifugal 10min of normal temperature abandons supernatant, adds PBS 500 μ L suspension deposition; Make the enrichment of somatocyte deposition in 12000r/min low-temperature centrifugation 10min, abandon supernatant.
2, milk somatic cell digestion
The DNA extraction damping fluid that in the somatocyte deposition, adds 350 μ L, the SDS of 50 μ L, the Proteinase K of 10 μ L spends the night 56 ℃ of following water-bath digestion.
3, the SDS phynol method extracts DNA
In order to extract DNA accurately and efficiently, this research adopts the SDS phynol method to extract DNA.At first, in digestion product, add the saturated phenol solution of isopyknic Tris, put upside down 10min back and forth, 12000r/min, low-temperature centrifugation 10min obtains supernatant; Supernatant is transferred in another 1.5mL centrifuge tube, added the mixture of isopyknic phenol, chloroform and primary isoamyl alcohol, phenol, chloroform and primary isoamyl alcohol are the 25:24:1 preparation by volume; Put upside down 10min back and forth, make resolution of precipitate, 12000r/min; Centrifugal 10min obtains supernatant; Supernatant is transferred in another 1.5mL centrifuge tube again, added equal-volume chloroform and iso pentane alcohol mixture, chloroform and primary isoamyl alcohol for the 24:1 preparation, are put upside down 10min by volume back and forth, make resolution of precipitate, 12000r/min, and centrifugal 10min obtains supernatant.
4, nucleic acid deposition
Supernatant is transferred to the centrifuge tube of another 1.5mL, add ice absolute ethyl alcohol (20 ℃) deposition of 2 times of volumes, jolting gently ,-20 ℃, leave standstill 30min, the centrifugal 10min of 12000r/min discards upper strata ethanol.Bottom settlings is with 70% ice ethanol (20 ℃) washing, 12000r/min, and low-temperature centrifugation 10min carefully removes upper strata ethanol, and the ethanol that volatilizees fast adds the TE dissolving DNA of 25 μ L, and-4 ℃ of preservations are subsequent use.
5, DNA quality examination
Measure DNA concentration and purity OD respectively with ultraviolet spectrophotometer
260/280Value, the milk sample DNA concentration value that calculates more than 80% according to formula is between the 12 μ g/ μ L-45 μ g/ μ L OD
260/280Value is between the 1.65-1.75.Carry out band with 1% agarose gel electrophoresis and detect, partial results is seen Fig. 1, DNA band neat and consistent, and good in integrity is not obviously trailed and diffusing phenomenon.
Embodiment 2: ox B2M gene B2M1F and amplification of B2M1R primer PCR and genotype identification
1, the selection of specific gene
Select ox B2 microglobulin gene B2M (beta-2-microglobulin) at random, its gene order in GenBank is numbered NC_007308.
2, primer design and synthetic
According to ox B2M gene order design primer, and give birth to worker bio-engineering corporation by Shanghai and synthesize:
Upstream primer (B2M1F): 5 ' CAT CTG TCT TTC CCT GCC GC 3 '
Downstream primer (B2M1R): 5 ' CTA CAG CCT TCC TCA TCT CCC CT 3 '.
3, PCR reaction
(1) be that template is carried out pcr amplification with ox DNA, comprise following solution or reagent in the 10 μ L reaction systems:
(2) above-mentioned solution is mixed, through groping PCR renaturation temperature repeatedly, finally confirming 65 ℃ is annealing temperature, and the PCR reaction conditions is following:
95 ℃, preparatory sex change 5min; 94 ℃, sex change 30sec, 65 ℃, annealing 35sec, 72 ℃ are extended 30sec, 32 circulations; 72 ℃ are extended 10min.
(3) select 9 DNA samples to carry out pcr amplification altogether as amplification template.Reaction is got PCR reaction solution (5 μ L) and is carried out agarose gel electrophoresis after finishing, and detects the PCR product.Obtain being included by first of primer B2M1F and B2M1R amplification that subarea, the second exon district, second include the subarea, the second exon district and the 3rd includes the amplified fragments that the subarea is formed, the length of this amplified fragments is the 1019bp sequence, sees Fig. 2.
4, PCR product order-checking
With the amplified production of this primer with PCR product purification test kit reclaim, purifying, and extension increasing sequence carried out two-way order-checking.The sequencing result of 9 DNA sample amplification products is consistent with the sequence height of ox B2M gene NC_007308 announcement among the NCBI.
5, genotype identification
Seqman instrument with DNAStar software is analyzed the sequencing result of 9 samples, finds that whole extension increasing sequence does not have base to undergo mutation, and genotype is same genotype, explains that this fragment gene sequence is very conservative.
Embodiment 3: ox B2M gene B2M2F and amplification of B2M2R primer PCR and genotype identification
1, primer design and synthetic
According to ox B2M gene order design primer, and give birth to worker bio-engineering corporation by Shanghai and synthesize, wherein:
Upstream primer (B2M2F): 5 ' GGC TTT CCC AGC ATC ACT AAC 3 '
Downstream primer (B2M2R): 5 ' TCA CAG CAC CAC CAA ACT TAT CT 3 '.
2, PCR reaction
(1) be that template is carried out pcr amplification with ox DNA, 10 μ L reaction systems are with embodiment two.
(2) solution with the PCR reaction system mixes, and through groping PCR renaturation temperature repeatedly, finally confirming 60 ℃ is annealing temperature, and the PCR reaction conditions is following:
95 ℃, preparatory sex change 5min; 94 ℃, sex change 30sec, 60 ℃, annealing 30sec, 72 ℃ are extended 30sec, 30 circulations; 72 ℃ are extended 10min.
(3) select 9 DNA samples to carry out pcr amplification altogether as amplification template.Reaction is got PCR reaction solution (5 μ L) and is carried out agarose gel electrophoresis after finishing, and detects the PCR product.Obtain being included by the 3rd of primer B2M2F and B2M2R amplification the amplified fragments in subarea, the length of this amplified fragments is the 729bp sequence, sees Fig. 3.
3, PCR product order-checking
With the amplified production of this primer with PCR product purification test kit reclaim, purifying, and extension increasing sequence carried out backward sequencing.The sequencing result of 9 DNA sample amplification products is consistent with the sequence height of ox B2M gene NC_007308 announcement among the NCBI.
4, genotype identification
Seqman instrument with DNAStar software is analyzed the sequencing result of 9 samples; Find that whole extension increasing sequence has 6 mutational sites; Lay respectively at 36bp, 168bp, 186bp, 362bp, 592bp and 597bp site in this section sequence, called after: A36G, G168T, G186A, A362G, G592T and G597C.6 sites can form 36=729 genotype in theory, explain that the conservative type of this section sequence is not strong.
Claims (2)
1. the process for extracting of genomic dna in the milk that is applicable to extensive genotype identification is characterized in that, comprises the following steps:
Step 1, centrifuge tube is prepared: adopts the 15mL centrifuge tube, in high-pressure sterilizing pot, sterilizes, subsequent use;
Step 2, the milk collection: gather fresh milk 10mL-13mL to centrifuge tube, add 2 SRM 935as in each pipe, ice bag is taken back, and places-20 ℃ of preservations;
Step 3, milk somatic cell are separated and enrichment: the milk appearance of above-mentioned cryopreservation thawed in 4 ℃, and at 2500r/min, 4 ℃ of centrifugal 30min down; With little spoonful of butterfat that scrapes off the centrifuge tube upper strata, remove with the milk-protein of suction pipe the middle layer, keep one deck deposition of bottommost; Add 600 μ L phosphoric acid buffers (PBS) to centrifuge tube bottom, bottom settlings is beaten suspended and be transferred in the centrifuge tube of 1.5mL, the centrifugal 10min of 3500r/min normal temperature discards supernatant liquid, keeps bottom settlings; Add the PBS that 60 μ L fly emulsifying agent, 540 μ L to bottom settlings again, suspend fully with vibrator vibration to deposition, 40 ℃ of water bath with thermostatic control processing 10min slough the butterfat around the somatocyte; 3500r/min; The centrifugal 10min of normal temperature abandons supernatant, adds PBS 500 μ L suspension deposition; Make the enrichment of somatocyte deposition in 12000r/min low-temperature centrifugation 10min, abandon supernatant;
Step 4, milk somatic cell digestion: in the somatocyte deposition, add the DNA extraction damping fluid of 350 μ L, the SDS of 50 μ L, the Proteinase K of 10 μ L spends the night 56 ℃ of following water-bath digestion;
Step 5, the SDS phynol method extracts DNA: at first, in digestion product, adds the saturated phenol solution of isopyknic Tris, puts upside down 10min back and forth, 12000r/min, low-temperature centrifugation 10min obtains supernatant; Supernatant is transferred in another 1.5mL centrifuge tube, added isopyknic phenol, chloroform and iso pentane alcohol mixture, wherein, phenol, chloroform and primary isoamyl alcohol be 25 ︰, 24 ︰, 1 preparation by volume; Put upside down 10min back and forth, make resolution of precipitate, 12000r/min, centrifugal 10min obtains supernatant; Supernatant is transferred in another 1.5mL centrifuge tube again, added isopyknic chloroform and iso pentane alcohol mixture, wherein, chloroform and primary isoamyl alcohol volume ratio are 24 ︰ 1, put upside down 10min back and forth, make resolution of precipitate, 12000r/min, and centrifugal 10min obtains supernatant;
Step 6, nucleic acid deposition: supernatant is transferred to the centrifuge tube of another 1.5mL, add-20 ℃ of ice absolute ethyl alcohols depositions of 2 times of volumes, jolting is gently left standstill 30min under-20 ℃ of conditions, and the centrifugal 10min of 12000r/min discards upper strata ethanol; It is-20 ℃ of ice washing with alcohol of 70% that bottom settlings uses mass concentration, 12000r/min, and low-temperature centrifugation 10min carefully removes upper strata ethanol, and volatilization ethanol adds the TE dissolving DNA of 25 μ L, and-4 ℃ of preservations are subsequent use;
Step 7, the DNA quality examination: measure with ultraviolet spectrophotometer, the genomic dna concentration value that obtains is between the 12 μ g/ μ L-45 μ g/ μ L more than 80%, purity OD
260/280Value is between the 1.65-1.75, and agarose gel electrophoresis carries out band test strip neat and consistent, good in integrity, unobvious hangover and diffusing phenomenon;
Step 8, the selection of specific gene: the specific function gene of selecting ox at random is as research object, and in GenBank, searches this gene order, design specific primers;
Step 9, pcr amplification and order-checking: select suitable PCR system and reaction conditions, carry out pcr amplification, and with PCR product purification test kit reclaim, purifying, be sent to order-checking company and check order;
Step 10, gene type is identified: order-checking gained gene order, can carry out extensive gene type and identify, carry out follow-up molecular biological analysis then.
2. the method for claim 1 is characterized in that, the DNA that is extracted is used for 500bp with interior short segments sequence amplification, perhaps is used for the above fragment sequence amplification of 500bp, perhaps is used for the above long segment sequence amplification of 1000bp.
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| CN104164416A (en) * | 2013-05-16 | 2014-11-26 | 长江大学 | Method for high efficiently extracting nucleic acid from fresh milk |
| WO2015016763A1 (en) * | 2013-07-29 | 2015-02-05 | Delaval Holding Ab | A method of preparing a milk sample, and a device configured to be used when preparing a milk sample |
| CN105018607A (en) * | 2015-07-07 | 2015-11-04 | 陕西师范大学 | DNA quality-based method for identification of fat and protein content in milk |
| CN105018606A (en) * | 2015-07-07 | 2015-11-04 | 陕西师范大学 | DNA quality based method for evaluation of milk freshness |
| CN105018607B (en) * | 2015-07-07 | 2018-10-26 | 陕西师范大学 | A method of based on Oil content and Protein content in DNA Quality Identification milk |
| CN105018606B (en) * | 2015-07-07 | 2018-10-30 | 陕西师范大学 | A method of based on DNA Quality Identification milk freshness |
| CN107365857A (en) * | 2017-08-17 | 2017-11-21 | 陕西师范大学 | Based on DNA gel electrophoretogram and mtDNA:The method that nDNA values differentiate fresh milk and reconstituted milk |
| CN107365857B (en) * | 2017-08-17 | 2020-07-28 | 陕西师范大学 | Method for identifying fresh milk and recovered milk based on DNA gel electrophoresis chart and mtDNA (deoxyribonucleic acid). nDNA value |
| CN108396025A (en) * | 2018-01-23 | 2018-08-14 | 安徽微分基因科技有限公司 | A method of extracting microorganism MetaDNA from lacto's sample |
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