CN101403013A - Method for sifting macrobrachium nipponensis microsatellite mark with magnetic bead concentration method - Google Patents
Method for sifting macrobrachium nipponensis microsatellite mark with magnetic bead concentration method Download PDFInfo
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Abstract
The invention provides a method for screening microsatellite markers of Macrobrachium nipponensis by a magnetic beads enrichment method, including the following steps: the extraction of the genomic DNA of the Macrobrachium nipponensis; the hybridization of a probe and a target segment; the enrichment of magnetic beads; positive sequencing clone and the amplification of the genomic DNA according to primers which are related to the flanking sequences of the microsatellite. As the invention combines the magnetic beads enrichment and PRC amplification of bacterial liquid to carry out second-time screening, no isotope is needed. The method has the advantages of safety and high efficiency and low cost, short cycle, high reliability and the like. At the same time, in the enrichment process, the magnetic beads are repeatedly washed for a plurality of times, which can clean and remove microsatellite sequences which are not firmly adhered because of less repetition times. Therefore, the obtained microsatellite sequences are longer and are repeated for more times. The microsatellite sequences which are more frequently repeated are considered to have rich diversity at interspecies and intraspecies and can be widely applied to the diversity identification of germ plasm, the building of genetic maps, and QTL. Therefore, the method is one which screens the microsatellite markers of Macrobrachium nipponensis and can be easily popularized.
Description
Technical field
The invention belongs to the method for Macrobrachium nipponensis dna molecular genetic marker, relate in particular to a kind of method that adopts enrichment with magnetic bead method sharp separation, screening Macrobrachium nipponensis microsatellite marker.
Background technology
Macrobrachium nipponensis (Macrobrachium nipponensis) is commonly called as freshwater shrimp, belongs to Crustachia, Decapoda, and Palaemonidae, pond crayfish belongs to.It is strong to have adaptability, and it is wide to distribute, and feeding habits are assorted, and growth is fast, and characteristics such as culturing economic benefit height are important breed varieties of China's fresh water shrimps.Current research work majority to Macrobrachium nipponensis concentrates on growth characteristics (Liu Jun etc., 2003), karyotyping (Yang Wanxi and Zhou Hong, 2000) and the aspects such as (He Xugang and Gong Shiyuan, 2003) of growing seedlings.Because the genetic background data of Macrobrachium nipponensis quite has and is limited to the genetic information that obtains high information quantity so that understand the resource situation of Macrobrachium nipponensis more exactly, the research of carrying out dna molecular level more widely is very necessary.
Little satellite (Microsatellite) DNA claims simple repeated sequence (simple sequence repeat again, SSR), be a kind of be the sequence that reaches tens Nucleotide that repetition unit string joint group becomes by 1～6 Nucleotide, little satellite has very abundant polymorphism.Wherein repeat the most common (Powell etc., 1996) with dinucleotide.Because little satellite is a stochastic distribution in the eukaryotic gene group, as molecule marker very high polymorphism and codominance are arranged again, at species Diversity Detection (Nielsen and Heino, 2001), construction of genetic atlas (Nichols etc., 2001), sibship identifies that aspects such as (Zhang Yuguang etc., 2003) gains great popularity.Because the enrichment with magnetic bead method has the effect of efficiently concentrating microsatellite DNA, thereby is widely used in screening (Edwards etc., 1996 of microsatellite locus; Carleton etc. 2002).Appear in the newspapers in the domestic and international also end that is separated in of the little satellite of Macrobrachium nipponensis, this paper intends adopting the enrichment with magnetic bead method to isolate microsatellite molecular marker from the Macrobrachium nipponensis genome, for the germplasm of Macrobrachium nipponensis is protected, genetic improvement provides new technique means.
Macrobrachium nipponensis is as an important kind of China's freshwater aquiculture, and is less to its Genetic Diversity now.Rarely seen Jiang Su flies to wait (2006) utilization RAPD technology that the geographical population genetic of difference is made a variation and analyzes, but RAPD repeatability is relatively poor, and the quantity of information that is comprised is also less, can not well reflect the breeding true difference of sample sometimes; Sun Yuena etc. (2007) use the genetic construction that mtDNA has studied Macrobrachium nipponensis three colonies, and the mtDNA reaction is the matrilinear inheritance characteristic, polymorphism situation (Yang Guang etc., 2002) that can't authentic representative Macrobrachium nipponensis genomic level.Therefore, utilize repeatability such as little satellite is high and the codominance ratio is high molecule marker, can obtain more accurate and meticulousr conclusion for the research of Macrobrachium nipponensis population genetic polymorphism.
Separate microsatellite locus several different methods is arranged, as part library method (Partial genomic library), enriched library method (Enriched library), microsatellite sequence PCR partition method (PCR-based isolation of microsatellite arrays, PIMA) and AFLP microsatellite locus partition method (Fast isolation by AFLP of sequences containing repeats site, FIASCO) etc. (Xue Hui, 2005).Because separates the efficient of positive colony of method acquisition of microsatellite sequence with screening portion gene group library by classical structure very low, has only 1%～3% (Xue Hui, 2005), therefore for great majority research, seek the target that a kind of efficient simple separation method has just become research.
The enrichment with magnetic bead method is to develop the effective means of microsatellite molecular marker at present.Sun Xiaowen (2005), full winter jasmine etc. (2006) utilization enrichment with magnetic bead is respectively 86.3%, 97.19% in conjunction with the positive colony rate that twice screening of isotropic substance obtains.The positive colony rate that is higher than this experiment gained is 82.1%.But the above two all adopt isotropic substance to carry out programmed screening.Though abundance sensitivity is higher, higher because radioactivity is bigger to the laboratory requirement of shelter, and human body had harm, be difficult to popularize in an all-round way.
Summary of the invention
The objective of the invention is to adopt isotropic substance to carry out the weak point of programmed screening in order to overcome above-mentioned existing enrichment with magnetic bead method, provide a kind of employing enrichment with magnetic bead to carry out programmed screening in conjunction with bacterium liquid pcr amplification, do not need to use isotropic substance, have safe and efficient, and the method for the sifting macrobrachium nipponensis microsatellite mark with magnetic bead concentration method that cost is low, the cycle is short, reliability is high.
The method of sifting macrobrachium nipponensis microsatellite mark with magnetic bead concentration method of the present invention comprises the genomic dna that extracts Macrobrachium nipponensis; Probe and the hybridization of purpose fragment; Enrichment with magnetic bead; Positive colony checks order and relates to the primer amplification genomic dna according to little satellite flanking sequence; The genomic dna of wherein said extraction Macrobrachium nipponensis may further comprise the steps:
1) gets the about 200mg of live body Macrobrachium nipponensis muscle tissue in the 1.5mLEppendorf pipe, shred, add 665 μ l extraction buffer (Tris.Cl:10mM, PH=8.0; EDTA:0.1mM PH=8.0), adds 35 μ lSDS (0.5%) and 10 μ l Proteinase Ks (20mg/mL) again, and 55 ℃ of water-bath digestion are spent the night;
2) add isopyknic saturated phenol (700 μ l) after DNA mixing instrument slowly mixes 30min, the centrifugal 10min extracting of refrigerated centrifuge 12000 commentaries on classics/min once;
3) get supernatant liquor and add isopyknic chloroform/primary isoamyl alcohol (24: 1) (700 μ l), after DNA mixing instrument slowly mixes 15min, once through the centrifugal 10min extracting of refrigerated centrifuge 12000 commentaries on classics/min;
4) get supernatant liquor and add isopyknic chloroform (700 μ l), after DNA mixing instrument slowly mixes 10min, once through the centrifugal 10min extracting of refrigerated centrifuge 12000 commentaries on classics/min;
5) get supernatant liquor, add the long-pending refrigerated dehydrated alcohol in advance of diploid, jiggle mixing after, the centrifugal 10min of 12000 commentariess on classics/min is deposited in DNA and manages at the end, abandons supernatant;
6) add 50 μ l ethanol (70%), washing precipitation twice is put into 37 ℃ of loft drier and is dried, and gets the Macrobrachium nipponensis genome DNA, adds 100 μ l sterilized water dissolving DNAs again, and 4 ℃ of refrigerators are preserved; Use DNA quantitative instrument quantitatively its concentration and purity, on sepharose, detect then.
The pulsating hybridization of described probe and purpose may further comprise the steps:
1) acquisition of Macrobrachium nipponensis genomic DNA fragment: get about 400ng Macrobrachium nipponensis genome DNA (7 μ l), at 50 μ l systems (NEB Buffer:5 μ l; BSA Buffer:0.5 μ l; DdH
2O:36.5 μ l) the restriction enzyme Mse I with 10 units in cut 3 hours in 37 ℃ of enzymes, then in 85 ℃ of water-bath 10min deactivation restriction endonucleases; Enzyme is cut back T4 ligase enzyme and is connected joint, and ligation is 15 μ l systems, and wherein enzyme is cut the about 11 μ l of product, comprising joint (50 μ m): 1.5 μ L; 10 * T4Ligase Buffe:1.5 μ L; The T4 ligase enzyme: 1 μ L is reflected at and spends the night 16 ℃ of following connections; Wherein, the DNA that is connected with joint obtains a plurality of copies through pcr amplification, and the reaction system of PCR is 25 μ L, wherein contains the connection product of ten times of dilutions: 5 μ l; 10 μ M primer: 1ul; The Mg2+:2 μ l of 25mmol/L; 10 * Taq buffer:2.5 μ l; 2.5mmol/L dNTP:1.2 μ l; Taqpolymerase:0.2 μ L; DdH
2O:13.1 μ l, the PCR reaction is 18 circulations, each circulation comprises: 94 ℃ of sex change 30s, 53 ℃ of annealing 1min, 72 ℃ are extended 1min, the preceding pre-sex change 5min of circulation first, 72 ℃ are extended 20min eventually after the last loop ends, and PCR pre-expansion product is standby at the rearmounted 4 ℃ of refrigerators of 1.2% agarose gel electrophoresis detection;
2) hybridization of genomic fragment and probe: above-mentioned pre-expansion product and the bioprobe Biotin----(AG) 13 that is connected with the DNA of joint hybridized 1.5hr in 68 ℃ in the molecular hybridization stove, hybridization is 100 μ l systems, comprising 6 * SSC+0.1%SDS:70 μ l; Biotin----(AG) 13 (40 μ M): 2 μ l; The pre-expansion product: 28 μ l, the hybridization solution of acquisition are probe and the pulsating hybridization solution of purpose, place 4 ℃ of refrigerators standby.
Used magnetic bead, magnet stand and the matched reagent of described enrichment with magnetic bead test is PROMEGA company and produces, and its experiment may further comprise the steps:
1) magnetic bead pre-treatment: with 1～2mg magnetic bead with the careful washing of the TNE100 of 300 μ L 3 times, when washing at every turn with the fixing magnetic bead of magnetic force frame, after washing finishes with the resuspended magnetic bead of 300 μ L TNE100, to magnetic bead smooth till, standby;
2) enrichment with magnetic bead: standby hybridization solution all is transferred in the pretreated magnetic bead pipe, during constantly blow and beat solution with pipettor, avoid magnetic bead precipitation, make hybridization solution and magnetic bead fully contact the efficient of raising enrichment as far as possible;
3) magnetic bead wash-out:
A: after enrichment absorption finishes, use fixedly magnetic bead of magnetic force frame, shift mixed solution and place the new centrifuge tube of sterilization, be labeled as I;
B: non-specific wash-out: in the magnetic bead pipe, add TEN1000 (400 μ l) and under room temperature, wash magnetic bead, wash altogether 3 times, each 5 minutes, stir and blow and beat gently washings frequently with pipettor, fixing magnetic bead on the magnetic force frame shifts mixed solution and places the new centrifuge tube of sterilization at last, is labeled as II respectively, III, IV;
C: specificity wash-out: in the magnetic bead pipe, add (0.2 * SSC+0.1 SDS) (400 μ l) and under room temperature, wash magnetic bead, wash altogether 3 times, each 5 minutes, constantly blow and beat washings gently with pipettor, fixing magnetic bead on the magnetic force frame shifts mixed solution and places the new centrifuge tube of sterilization at last, and mark is counted V respectively, VI, VII; Add 0.5 SSC (400 μ l) again and wash magnetic bead 1 time in the magnetic bead pipe under room temperature, method is the same, shifts mixed solution at last and places the new centrifuge tube of sterilization, is labeled as VIII;
D: the segmental wash-out of purpose: in the magnetic bead pipe, add TE (50 μ L) evenly with pipettor piping and druming, constantly piping and druming is even during placing 95 ℃ of incubators then, use fixedly magnetic bead of magnetic force frame behind the 5min, the sucking-off supernatant liquor is transferred in the new centrifuge tube of sterilization rapidly again, repeat wash-out three times, be labeled as IX respectively, X, XI;
E: contain the amplification of the dna segment of microsatellite sequence: respectively the enrichment eluted product that is numbered I～XI that obtains is carried out augmentation detection, reaction system is 25 μ L, and wherein eluted product respectively: 5 μ L; The primer of 10 μ M: 1ul; The Mg2+:2 μ l of 25mmol/L; 10 * Taq buffer:2.5 μ l; 2.5mmol/L dNTP:1.2 μ l; Taq polymerase:0.2 μ l; DdH
2O:13.1 μ l.The PCR reaction is 25 circulations, and each circulation comprises: 94 ℃ of sex change 30s, and 53 ℃ of annealing 1min, 72 ℃ are extended 1min, the preceding pre-sex change 5min of circulation first, 72 ℃ are extended 20min eventually after the last loop ends.Product detects at 1.2% agarose gel electrophoresis, and that eluted product that defines product increases again, guarantees that the product amount reaches more than the 100 μ L, and 4 ℃ of refrigerators of product are standby;
F: contain the purifying of the dna fragmentation amplified production of microsatellite sequence, test kit is that the production of worker bio-engineering corporation is given birth in Shanghai, and step is as follows:
(a) after PCR finishes, from the PCR reaction tubes, reaction solution is moved in the clean 1.5mLEppendorf centrifuge tube, adds the Solution BS mixing of 4 times of volumes, no matter sample what, minimumly need to add 400uL Solution BS;
(b) the 3S post is put into collection tube, mixed solution is transferred in the post, do not cover the centrifuge tube lid, room temperature was placed 2 minutes; Cover centrifugal lid, use desk centrifuge high speed centrifugation (10000rpm) 1 minute;
(c) outwell waste liquid in the collection tube, the 3S post is put into same collection tube, add 600 μ l Wash Solution, high speed centrifugation (10000rpm) 1 minute;
(d) repeating step (3) once;
(e) outwell waste liquid in the collection tube, the 3S post is put into same collection tube, high speed centrifugation (10000rpm) 2 minutes;
(f) the 3S post is put into new 1.5mL centrifuge tube, central authorities add 30 μ l ddH at 3S post film
2O does not cover the centrifuge tube lid, and room temperature was placed 2 minutes;
(g) cover the centrifuge tube lid, 10000rpm high speed centrifugation 1 minute, the liquid in the centrifuge tube is the dna fragmentation of recovery, can use or be stored in-20 ℃ immediately.
Described positive colony checks order and carries out amplifying genom DNA according to little satellite distinguished sequence design primer and may further comprise the steps:
1) the pulsating external connection of target DNA: the pMD18-T support agent box of test usefulness is that Takara company produces, and the ligation system is 8 μ l, wherein Buffer:4 μ l; PMD18-T:0.4 μ l; PCR purified product: 3.6 μ l; 16 ℃ of connections are spent the night, and it is standby to connect 4 ℃ of refrigerators of product;
2) conversion of connection product: the competent cell test kit of test usefulness is that Takara company produces, and will connect product and be transformed among the efficient competent cell DH5 α, and concrete steps are as follows:
(A) 10 μ l DNA are connected liquid and join (50 μ l competent cells need 25ng DNA) in the aseptic centrifuge tube of the 1.5mL that competent cell is housed, rotate with the mixing content ice bath 30min gently;
(B) pipe is put into the circulator bath 90s that heats in advance to 42 ℃, do not shaken test tube, put into ice bath then rapidly, make cell cooling 2-3min;
(C) every pipe adds 400 μ l LB nutrient solutions, 37 ℃ of shaking table incubation 45min.Make the antibiotics resistance marker gene of bacteria resuscitation and expression plasmid coding;
(D) competent cell that will transform is transferred on AMP+ (penbritin) flat board of using 4 μ l isopropylthio-(IPTG) solution (200mg/mL) and the coating of 40 μ l X-gal solution (20mg/mL) in advance;
(E) flat board is placed room temperature 1hr, be absorbed until liquid;
(F) be inverted flat board,, bacterium colony can occur behind the 12-16h in 37 ℃ of cultivations;
3) select positive colony and order-checking: the single white colony of picking is inoculated in the 800 μ l LB substratum (having contained 100 μ g/mLAmp) from the AMP+ flat board, cultivates 5 hours on 37 ℃ of shaking tables, makes bacterium liquid PCR then.Reaction is 25 μ l system, wherein bacterium liquid: 5 μ l; The primer of 10 μ M: 1ul; The Mg2+:2 μ l of 25mmol/L; 10 * Taq buffer:2.5 μ l; 2.5mmol/L dNTP:1.2 μ l; Taq polymerase:0.2 μ l; DdH
2O:13.1 μ l.The PCR reaction is 25 circulations, and each circulation comprises: 94 ℃ of sex change 30s, and 53 ℃ of annealing 1min, 72 ℃ are extended 1min, the preceding pre-sex change 5min of circulation first, 72 ℃ are extended 20min eventually after the last loop ends.Product detects at 1.2% agarose gel electrophoresis, and the positive clone of band limpid in sight is arranged, the order-checking of positive colony bacterium liquid sample presentation.
The method of sifting macrobrachium nipponensis microsatellite mark with magnetic bead concentration method of the present invention owing to adopt enrichment with magnetic bead to carry out programmed screening in conjunction with bacterium liquid pcr amplification, does not need to use isotropic substance, has safe and efficiently, and cost is low, the cycle short, the reliability advantages of higher.In the process of enrichment, magnetic bead is through repeated washing repeatedly, some can be adhered to not firm microsatellite sequence because of repetition number is less and clean and remove, and causes the microsatellite sequence that is obtained longer, and multiplicity is more.And the more microsatellite sequence of multiplicity is considered to all have abundant polymorphism between planting and in planting, and can be widely used among the evaluation of germplasm diversity, genetic map construction, the QTL.Therefore present method is a kind of method of the screening Macrobrachium nipponensis microsatellite marker that is easy to promote.
Embodiment:
1, extract the genomic dna of Macrobrachium nipponensis, method is as follows:
Get the about 200mg of live body Macrobrachium nipponensis muscle tissue in the 1.5mLEppendorf pipe, shred, add 665 μ l extraction buffer (Tris.Cl:10mM, PH=8.0; EDTA:0.1mM PH=8.0), adds 35 μ lSDS (0.5%) and 10 μ l Proteinase Ks (20mg/mL) again,, 55 ℃ of water-bath digestion are spent the night.Add isopyknic saturated phenol (700 μ l) after DNA mixing instrument slowly mixes 30min, the centrifugal 10min extracting of refrigerated centrifuge 12000 commentaries on classics/min once.Get supernatant liquor and add isopyknic chloroform/primary isoamyl alcohol (24: 1) (700 μ l), after DNA mixing instrument slowly mixes 15min, once through the centrifugal 15min extracting of refrigerated centrifuge 12000 commentaries on classics/min.Get supernatant liquor and add isopyknic chloroform (700 μ l), after DNA mixing instrument slowly mixes 10min, once through the centrifugal 10min extracting of refrigerated centrifuge 12000 commentaries on classics/min.Get supernatant liquor, add the long-pending refrigerated dehydrated alcohol in advance of diploid, jiggle mixing after, the centrifugal 10min of 12000 commentariess on classics/min is deposited in DNA and manages at the end, abandons supernatant.Add 50 μ l ethanol (70%), washing precipitation twice is put into 37 ℃ of loft drier and is dried, and adds 100 μ l sterilized water dissolving DNAs again, and 4 ℃ of refrigerators are preserved.
2, the pulsating hybridization of probe and purpose, method is as follows:
(1) acquisition of Macrobrachium nipponensis genomic DNA fragment: get about 400ng Macrobrachium nipponensis genome DNA (7 μ l), at 50 μ l systems (NEB Buffer:5 μ l; BSA Buffer:0.5 μ l; DdH
2O:36.5 μ l) the restriction enzyme Mse I with 10 units in cut 3 hours in 37 ℃ of enzymes, then in 85 ℃ of water-bath 10min deactivation restriction endonucleases.Enzyme is cut back T4 ligase enzyme and is connected joint, and ligation is 15 μ l systems, and wherein enzyme is cut product: 11 μ l; Joint (50 μ m): 1.5 μ L; 10 * T4 LigaseBuffe:1.5 μ L; T4 ligase enzyme: 1 μ L; Connect under being reflected at 16 ℃ and spend the night.The DNA that is connected with joint obtains a plurality of copies through pcr amplification, and the reaction system of PCR is 25 μ L, wherein contains the connection product of ten times of dilutions: 5 μ l; 10 μ M primer: 1ul; The Mg2+:2 μ l of 25mmol/L; 10 * Taq buffer:2.5 μ l; 2.5mmol/L dNTP:1.2 μ l; Taq polymerase:0.2 μ L; DdH
2O:13.1 μ l.The PCR reaction is 18 circulations, and each circulation comprises: 94 ℃ of sex change 30s, and 53 ℃ of annealing 1min, 72 ℃ are extended 1min, the preceding pre-sex change 5min of circulation first, 72 ℃ are extended 20min eventually after the last loop ends.It is standby that PCR pre-expansion product detects rearmounted 4 ℃ of refrigerators at 1.2% agarose gel electrophoresis;
(2) hybridization of genomic fragment and probe: with pre-expansion product and bioprobe Biotin----(AG) 13 in the molecular hybridization stove in 68 ℃ of hybridization 1.5hr, hybridization is 100 μ l systems, comprises 6 * SSC+0.1%SDS:70 μ l; Biotin----(AG) 13 (40 μ M): 2 μ l; Pre-expansion product: 28 μ l.The 4 ℃ of refrigerators of hybridization solution that obtain are standby;
3, enrichment with magnetic bead, method is as follows:
(1) magnetic bead pre-treatment: with 1～2mg magnetic bead with the careful washing of the TNE100 of 300 μ L 3 times, when washing at every turn with the fixing magnetic bead of magnetic force frame, after washing finishes with the resuspended magnetic bead of 300 μ L TNE100, to magnetic bead smooth till, standby;
(2) enrichment with magnetic bead: standby hybridization solution all is transferred in the pretreated magnetic bead pipe, during constantly blow and beat solution with pipettor, avoid magnetic bead precipitation, make hybridization solution and magnetic bead fully contact the efficient of raising enrichment as far as possible;
(3) magnetic bead wash-out:
A: after enrichment absorption finishes, use fixedly magnetic bead of magnetic force frame, shift mixed solution and place the new centrifuge tube of sterilization, be labeled as I;
B: non-specific wash-out: in the magnetic bead pipe, add TEN1000 (400 μ l) and under room temperature, wash magnetic bead, wash altogether 3 times, each 5 minutes, stir and blow and beat gently washings frequently with pipettor, fixing magnetic bead on the magnetic force frame shifts mixed solution and places the new centrifuge tube of sterilization at last, is labeled as II respectively, III, IV;
C: specificity wash-out: in the magnetic bead pipe, add (0.2 * SSC+0.1 SDS) (400 μ l) and under room temperature, wash magnetic bead, wash altogether 3 times, each 5 minutes, constantly blow and beat washings gently with pipettor, fixing magnetic bead on the magnetic force frame shifts mixed solution and places the new centrifuge tube of sterilization at last, and mark is counted V respectively, VI, VII; Add 0.5 SSC (400 μ l) again and wash magnetic bead 1 time in the magnetic bead pipe under room temperature, method is the same, shifts mixed solution at last and places the new centrifuge tube of sterilization, is labeled as VIII;
D: the segmental wash-out of purpose: in the magnetic bead pipe, add TE (50 μ L) evenly with pipettor piping and druming, constantly piping and druming is even during placing 95 ℃ of incubators then, use fixedly magnetic bead of magnetic force frame behind the 5min, the sucking-off supernatant liquor is transferred in the new centrifuge tube of sterilization rapidly again, repeat wash-out three times, be labeled as IX respectively, X, XI;
E: contain the amplification of the dna segment of microsatellite sequence: respectively the enrichment eluted product that is numbered I～XI that obtains is carried out augmentation detection, reaction system is 25 μ L, and wherein eluted product respectively: 5 μ L; The primer of 10 μ M: 1ul; The Mg2+:2 μ l of 25mmol/L; 10 * Taq buffer:2.5 μ l; 2.5mmol/L dNTP:1.2 μ l; Taq polymerase:0.2 μ l; DdH
2O:13.1 μ l.The PCR reaction is 25 circulations, and each circulation comprises: 94 ℃ of sex change 30s, and 53 ℃ of annealing 1min, 72 ℃ are extended 1min, the preceding pre-sex change 5min of circulation first, 72 ℃ are extended 20min eventually after the last loop ends.Product detects at 1.2% agarose gel electrophoresis, and that eluted product that defines product increases again, guarantees that the product amount reaches more than the 100 μ L, and 4 ℃ of refrigerators of product are standby;
F: contain the purifying of the dna fragmentation amplified production of microsatellite sequence, test kit is that the production of worker bio-engineering corporation is given birth in Shanghai, and step is as follows:
(a) after PCR finishes, from the PCR reaction tubes, reaction solution is moved in the clean 1.5mLEppendorf centrifuge tube, adds the Solution BS mixing of 4 times of volumes, no matter sample what, minimumly need to add 400uL Solution BS;
(b) the 3S post is put into collection tube, mixed solution is transferred in the post, do not cover the centrifuge tube lid, room temperature was placed 2 minutes; Cover centrifugal lid, use desk centrifuge high speed centrifugation (10000rpm) 1 minute;
(c) outwell waste liquid in the collection tube, the 3S post is put into same collection tube, add 600 μ l Wash Solution, high speed centrifugation (10000rpm) 1 minute;
(d) repeating step (3) once;
(e) outwell waste liquid in the collection tube, the 3S post is put into same collection tube, high speed centrifugation (10000rpm) 2 minutes;
(f) the 3S post is put into new 1.5mL centrifuge tube, central authorities add 30 μ l ddH2O at 3S post film, do not cover the centrifuge tube lid, and room temperature was placed 2 minutes;
(g) cover the centrifuge tube lid, 10000rpm high speed centrifugation 1 minute, the liquid in the centrifuge tube is the dna fragmentation of recovery, can use or be stored in-20 ℃ immediately.
4, positive colony checks order and relates to the primer amplification genomic dna according to little satellite flanking sequence, and method is as follows:
1) the pulsating external connection of target DNA: the pMD18-T support agent box of test usefulness is that Takara company produces, and the ligation system is 8 μ l, wherein Buffer:4 μ l; PMD18-T:0.4 μ l; PCR purified product: 3.6 μ l; 16 ℃ of connections are spent the night, and it is standby to connect 4 ℃ of refrigerators of product;
2) conversion of connection product: the competent cell test kit of test usefulness is that Takara company produces, and will connect product and be transformed among the efficient competent cell DH5 α, and concrete steps are as follows:
(A) 10 μ l DNA are connected liquid and join (50 μ l competent cells need 25ng DNA) in the aseptic centrifuge tube of the 1.5mL that competent cell is housed, rotate with the mixing content ice bath 30min gently;
(B) pipe is put into the circulator bath 90s that heats in advance to 42 ℃, do not shaken test tube, put into ice bath then rapidly, make cell cooling 2-3min;
(C) every pipe adds 400 μ l LB nutrient solutions, 37 ℃ of shaking table incubation 45min.Make the antibiotics resistance marker gene of bacteria resuscitation and expression plasmid coding;
(D) competent cell that will transform is transferred on AMP+ (penbritin) flat board of using 4 μ l isopropylthio-(IPTG) solution (200mg/mL) and the coating of 40 μ l X-gal solution (20mg/mL) in advance;
(E) flat board is placed room temperature 1hr, be absorbed until liquid;
(F) be inverted flat board,, bacterium colony can occur behind the 12-16h in 37 ℃ of cultivations;
3) select positive colony and order-checking: the single white colony of picking is inoculated in the 800 μ l LB substratum (having contained 100 μ g/mLAmp) from the AMP+ flat board, cultivates 5 hours on 37 ℃ of shaking tables, makes bacterium liquid PCR then.Reaction is 25 μ l system, wherein bacterium liquid: 5 μ l; The primer of 10 μ M: 1ul; The Mg2+:2 μ l of 25mmol/L; 10 * Taq buffer:2.5 μ l; 2.5mmol/L dNTP:1.2 μ l; Taq polymerase:0.2 μ l; DdH
2O:13.1 μ l.The PCR reaction is 25 circulations, and each circulation comprises: 94 ℃ of sex change 30s, and 53 ℃ of annealing 1min, 72 ℃ are extended 1min, the preceding pre-sex change 5min of circulation first, 72 ℃ are extended 20min eventually after the last loop ends.Product detects at 1.2% agarose gel electrophoresis, and the positive clone of band limpid in sight is arranged, the order-checking of positive colony bacterium liquid sample presentation.
5, design primer amplification genomic dna: mainly be on the basis of order-checking, utilize the conservative property of the sequence of microsatellite DNA both sides, utilization primer-design software primer 5.0 design Auele Specific Primers, be used to increase little satellite fragment in this site, design of primers adopts following rigorous degree: (a) primer length is 19-25mer; (b) GC content 40%-60%; (c) annealing temperature 45-65 degree; (d) expection PCR product length is 100-250bp.
By aforesaid method, can within a week, obtain the little satellite sign of a large amount of Macrobrachium nipponensis.It is a kind of method of the screening Macrobrachium nipponensis microsatellite marker that is easy to promote.
Claims (5)
1, a kind of method of sifting macrobrachium nipponensis microsatellite mark with magnetic bead concentration method comprises the genomic dna that extracts Macrobrachium nipponensis; Probe and the hybridization of purpose fragment; Enrichment with magnetic bead; Positive colony checks order and relates to the primer amplification genomic dna according to little satellite flanking sequence; The genomic dna that it is characterized in that described extraction Macrobrachium nipponensis may further comprise the steps:
1) gets the about 200mg of live body Macrobrachium nipponensis muscle tissue in the 1.5mLEppendorf pipe, shred, add 665 μ l extraction buffer (Tris.Cl:10mM, PH=8.0; EDTA:0.1mM PH=8.0), adds 35 μ lSDS (0.5%) and 10 μ l Proteinase Ks (20mg/mL) again, and 55 ℃ of water-bath digestion are spent the night;
2) add isopyknic saturated phenol (700 μ l) after DNA mixing instrument slowly mixes 30min, the centrifugal 10min extracting of refrigerated centrifuge 12000 commentaries on classics/min once;
3) get supernatant liquor and add isopyknic chloroform/primary isoamyl alcohol (24: 1) (700 μ l), after DNA mixing instrument slowly mixes 15min, once through the centrifugal 15min extracting of refrigerated centrifuge 12000 commentaries on classics/min;
4) get supernatant liquor and add isopyknic chloroform (700 μ l), after DNA mixing instrument slowly mixes 10min, once through the centrifugal 10min extracting of refrigerated centrifuge 12000 commentaries on classics/min;
5) get supernatant liquor, add the long-pending refrigerated dehydrated alcohol in advance of diploid, jiggle mixing after, the centrifugal 10min of 12000 commentariess on classics/min is deposited in DNA and manages at the end, abandons supernatant;
6) add 50 μ l ethanol (70%), washing precipitation twice, put into 37 ℃ of loft drier dry the Macrobrachium nipponensis genome DNA, add 100 μ l sterilized water dissolving DNAs again, 4 ℃ of refrigerators are preserved; Use DNA quantitative instrument quantitatively its concentration and purity, on sepharose, detect then.
2,, it is characterized in that the pulsating hybridization of probe and purpose may further comprise the steps according to the method for the described sifting macrobrachium nipponensis microsatellite mark with magnetic bead concentration method of claim 1:
1) acquisition of Macrobrachium nipponensis genomic DNA fragment: get about 400ng Macrobrachium nipponensis genome DNA (7 μ l), at 50 μ l systems (NEB Buffer:5 μ l; BSA Buffer:0.5 μ l; DdH2O:36.5 μ l) the restriction enzyme Mse I with 10 units in cut 3 hours in 37 ℃ of enzymes, then in 85 ℃ of water-bath 10min deactivation restriction endonucleases; Enzyme is cut back T4 ligase enzyme and is connected joint, and ligation is 15 μ l systems, and wherein enzyme is cut the about 11 μ l of product, comprising joint (50 μ m): 1.5 μ L; 10 * T4Ligase Buffe:1.5 μ L; The T4 ligase enzyme: 1 μ L is reflected at and spends the night 16 ℃ of following connections; Wherein, the DNA that is connected with joint obtains a plurality of copies through pcr amplification, and the reaction system of PCR is 25 μ L, wherein contains the connection product of ten times of dilutions: 5 μ l; 10 μ M primer: 1ul; The Mg2+:2 μ l of 25mmol/L; 10 * Taq buffer:2.5 μ l; 2.5mmol/L dNTP:1.2 μ l; Taq polymerase:0.2 μ L; DdH2O:13.1 μ l, the PCR reaction is 18 circulations, each circulation comprises: 94 ℃ of sex change 30s, 53 ℃ of annealing 1min, 72 ℃ are extended 1min, the preceding pre-sex change 5min of circulation first, 72 ℃ are extended 20min eventually after the last loop ends, and PCR pre-expansion product is standby at the rearmounted 4 ℃ of refrigerators of 1.2% agarose gel electrophoresis detection;
2) hybridization of genomic fragment and probe: above-mentioned pre-expansion product and the bioprobe Biotin----(AG) 13 that is connected with the DNA of joint hybridized 1.5hr in 68 ℃ in the molecular hybridization stove, hybridization is 100 μ l systems, comprising 6 * SSC+0.1%SDS:70 μ l; Biotin----(AG) 13 (40 μ M): 2 μ l; The pre-expansion product: 28 μ l, the hybridization solution of acquisition are probe and the pulsating hybridization solution of purpose, place 4 ℃ of refrigerators standby.
3. the method for sifting macrobrachium nipponensis microsatellite mark with magnetic bead concentration method according to claim 2 is characterized in that used magnetic bead, magnet stand and the matched reagent of enrichment with magnetic bead test is PROMEGA company and produces, and its experiment may further comprise the steps:
1) magnetic bead pre-treatment: with 1～2mg magnetic bead with the careful washing of the TNE100 of 300 μ L 3 times, when washing at every turn with the fixing magnetic bead of magnetic force frame, after washing finishes with the resuspended magnetic bead of 300 μ L TNE100, to magnetic bead smooth till, standby;
2) enrichment with magnetic bead: standby hybridization solution all is transferred in the pretreated magnetic bead pipe, during constantly blow and beat solution with pipettor, avoid magnetic bead precipitation, make hybridization solution and magnetic bead fully contact the efficient of raising enrichment as far as possible;
3) magnetic bead wash-out:
A: after enrichment absorption finishes, use fixedly magnetic bead of magnetic force frame, shift mixed solution and place the new centrifuge tube of sterilization, be labeled as I;
B: non-specific wash-out: in the magnetic bead pipe, add TEN1000 (400 μ l) and under room temperature, wash magnetic bead, wash altogether 3 times, each 5 minutes, stir and blow and beat gently washings frequently with pipettor, fixing magnetic bead on the magnetic force frame shifts mixed solution and places the new centrifuge tube of sterilization at last, is labeled as II respectively, III, IV;
C: specificity wash-out: in the magnetic bead pipe, add (0.2 * SSC+0.1 SDS) (400 μ l) and under room temperature, wash magnetic bead, wash altogether 3 times, each 5 minutes, constantly blow and beat washings gently with pipettor, fixing magnetic bead on the magnetic force frame shifts mixed solution and places the new centrifuge tube of sterilization at last, and mark is counted V respectively, VI, VII; Add 0.5 SSC (400 μ l) again and wash magnetic bead 1 time in the magnetic bead pipe under room temperature, method is the same, shifts mixed solution at last and places the new centrifuge tube of sterilization, is labeled as VIII;
D: the segmental wash-out of purpose: in the magnetic bead pipe, add TE (50 μ L) evenly with pipettor piping and druming, constantly piping and druming is even during placing 95 ℃ of incubators then, use fixedly magnetic bead of magnetic force frame behind the 5min, the sucking-off supernatant liquor is transferred in the new centrifuge tube of sterilization rapidly again, repeat wash-out three times, be labeled as IX respectively, X, XI;
E: contain the amplification of the dna segment of microsatellite sequence: respectively the enrichment eluted product that is numbered I～XI that obtains is carried out augmentation detection, reaction system is 25 μ L, and wherein eluted product respectively: 5 μ L; The primer of 10 μ M: 1ul; The Mg2+:2 μ l of 25mmol/L; 10 * Taq buffer:2.5 μ l; 2.5mmol/L dNTP:1.2 μ l; Taq polymerase:0.2 μ l; DdH
2O:13.1 μ l.The PCR reaction is 25 circulations, and each circulation comprises: 94 ℃ of sex change 30s, and 53 ℃ of annealing 1min, 72 ℃ are extended 1min, the preceding pre-sex change 5min of circulation first, 72 ℃ are extended 20min eventually after the last loop ends.Product detects at 1.2% agarose gel electrophoresis, and that eluted product that defines product increases again, guarantees that the product amount reaches more than the 100 μ L, and 4 ℃ of refrigerators of product are standby;
F: contain the purifying of the dna fragmentation amplified production of microsatellite sequence, test kit is that the production of worker bio-engineering corporation is given birth in Shanghai, and step is as follows:
(a) after PCR finishes, from the PCR reaction tubes, reaction solution is moved in the clean 1.5mLEppendorf centrifuge tube, adds the Solution BS mixing of 4 times of volumes, no matter sample what, minimumly need to add 400uL Solution BS;
(b) the 3S post is put into collection tube, mixed solution is transferred in the post, do not cover the centrifuge tube lid, room temperature was placed 2 minutes; Cover centrifugal lid, use desk centrifuge high speed centrifugation (10000rpm) 1 minute;
(c) outwell waste liquid in the collection tube, the 3S post is put into same collection tube, add 600 μ l Wash Solution, high speed centrifugation (10000rpm) 1 minute;
(d) repeating step (3) once;
(e) outwell waste liquid in the collection tube, the 3S post is put into same collection tube, high speed centrifugation (10000rpm) 2 minutes;
(f) the 3S post is put into new 1.5mL centrifuge tube, central authorities add 30 μ l ddH at 3S post film
2O does not cover the centrifuge tube lid, and room temperature was placed 2 minutes;
(g) cover the centrifuge tube lid, 10000rpm high speed centrifugation 1 minute, the liquid in the centrifuge tube is the dna fragmentation of recovery, can use or be stored in-20 ℃ immediately.
4, the method for sifting macrobrachium nipponensis microsatellite mark with magnetic bead concentration method according to claim 3 is characterized in that described positive colony order-checking and carries out amplifying genom DNA according to little satellite distinguished sequence design primer may further comprise the steps:
1) the pulsating external connection of target DNA: the pMD18-T support agent box of test usefulness is that Takara company produces, and the ligation system is 8 μ l, wherein Buffer:4 μ l; PMD18-T:0.4 μ l; PCR purified product: 3.6 μ l; 16 ℃ of connections are spent the night, and it is standby to connect 4 ℃ of refrigerators of product;
2) conversion of connection product: the competent cell test kit of test usefulness is that Takara company produces, and will connect product and be transformed among the efficient competent cell DH5 α, and concrete steps are as follows:
(A) 10 μ l DNA are connected liquid and join (50 μ l competent cells need 25ng DNA) in the aseptic centrifuge tube of the 1.5mL that competent cell is housed, rotate with the mixing content ice bath 30min gently;
(B) pipe is put into the circulator bath 90s that heats in advance to 42 ℃, do not shaken test tube, put into ice bath then rapidly, make cell cooling 2-3min;
(C) every pipe adds 400 μ l LB nutrient solutions, 37 ℃ of shaking table incubation 45min.Make the antibiotics resistance marker gene of bacteria resuscitation and expression plasmid coding;
(D) competent cell that will transform is transferred on AMP+ (penbritin) flat board of using 4 μ l isopropylthio-(IPTG) solution (200mg/mL) and the coating of 40 μ l X-gal solution (20mg/mL) in advance;
(E) flat board is placed room temperature 1hr, be absorbed until liquid;
(F) be inverted flat board,, bacterium colony can occur behind the 12-16h in 37 ℃ of cultivations;
3) select positive colony and order-checking: the single white colony of picking is inoculated in the 800 μ l LB substratum (having contained 100 μ g/mL Amp) from the AMP+ flat board, cultivates 5 hours on 37 ℃ of shaking tables, makes bacterium liquid PCR then.Reaction is 25 μ l system, wherein bacterium liquid: 5 μ l; The primer of 10 μ M: 1ul; The Mg2+:2 μ l of 25mmol/L; 10 * Taq buffer:2.5 μ l; 2.5mmol/L dNTP:1.2 μ l; Taq polymerase:0.2 μ l; DdH2O:13.1 μ l.The PCR reaction is 25 circulations, and each circulation comprises: 94 ℃ of sex change 30s, and 53 ℃ of annealing 1min, 72 ℃ are extended 1min, the preceding pre-sex change 5min of circulation first, 72 ℃ are extended 20min eventually after the last loop ends.Product detects at 1.2% agarose gel electrophoresis, and the positive clone of band limpid in sight is arranged, the order-checking of positive colony bacterium liquid sample presentation.
5, the method for sifting macrobrachium nipponensis microsatellite mark with magnetic bead concentration method according to claim 4, it is characterized in that designing the primer amplification genomic dna mainly is on the basis of order-checking, utilize the conservative property of the sequence of microsatellite DNA both sides, utilization primer-design software primer 5.0 design Auele Specific Primers, be used to increase little satellite fragment in this site, design of primers adopts following rigorous degree: (a) primer length is 19-25mer; (b) GC content 40%-60%; (c) annealing temperature 45-65 degree; (d) expection PCR product length is 100-250bp.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101864489A (en) * | 2010-06-09 | 2010-10-20 | 北京大学 | Method for gathering foreign DNA in transgenosis product |
| CN106350606A (en) * | 2016-11-25 | 2017-01-25 | 河北大学 | Microsatellite marker applied to growth trait analysis of Macrobrachium nipponense and application thereof |
| CN106434974A (en) * | 2016-11-22 | 2017-02-22 | 河北大学 | Microsatellite primer for macrobrachium nipponensis diversity analysis and application thereof |
| CN107686862A (en) * | 2017-10-29 | 2018-02-13 | 信阳学院 | A kind of method of freshwater fish screening microsatellite sequence |
| CN108588065A (en) * | 2018-05-07 | 2018-09-28 | 江苏中洋集团股份有限公司 | A kind of Penaeus Vannmei viral DNA extracting method |
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2008
- 2008-11-18 CN CNA2008102348112A patent/CN101403013A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101864489A (en) * | 2010-06-09 | 2010-10-20 | 北京大学 | Method for gathering foreign DNA in transgenosis product |
| CN106434974A (en) * | 2016-11-22 | 2017-02-22 | 河北大学 | Microsatellite primer for macrobrachium nipponensis diversity analysis and application thereof |
| CN106434974B (en) * | 2016-11-22 | 2019-07-19 | 河北大学 | A kind of micro-satellite primers and application thereof for Macrobrachium nipponensis diversity analysis |
| CN106350606A (en) * | 2016-11-25 | 2017-01-25 | 河北大学 | Microsatellite marker applied to growth trait analysis of Macrobrachium nipponense and application thereof |
| CN107686862A (en) * | 2017-10-29 | 2018-02-13 | 信阳学院 | A kind of method of freshwater fish screening microsatellite sequence |
| CN108588065A (en) * | 2018-05-07 | 2018-09-28 | 江苏中洋集团股份有限公司 | A kind of Penaeus Vannmei viral DNA extracting method |
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