CN102533727B - Preparation method of developing Phaseolus vulgaris SSR primers by magnetic bead enrichment - Google Patents
Preparation method of developing Phaseolus vulgaris SSR primers by magnetic bead enrichment Download PDFInfo
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Abstract
The invention discloses a preparation method of developing Phaseolus vulgaris SSR primers by magnetic bead enrichment. The method comprises the following steps of: (1) digesting Phaseolus vulgaris genome DNA with Msel, and establishing a library by AFLP; (2) hybridizing the genome PCR library with a biotin-labeled repetitive sequence probe; (3) adding the hybridization mixed liquid into streptavidin-coated magnetic beads; (4) amplifying micro-satellite DNA fragments by PCR using eluent-purified micro-satellite DNA as DNA template; (5) linking the purified PCR amplification product on a T vector for cloning to obtain a DNA sequence with inserted fragments; and (6) repeating on the two sides of the core area to design primers, and screening primers to obtain effective microsatellite loci. The method is simple and feasible, increases efficiency of the primers, screens 20 pairs of effective primers from 48 pairs of primers, and provides guarantee for large-batch development of SSR primers.
Description
Technical field
The invention belongs to bioengineering field, relate to a kind of preparation method of enrichment with magnetic bead method exploitation Kidney bean SSR primer, this method is applicable to all Kidney bean kinds, strain or germ plasm resource material.
Background technology
Though the SSR mark is effective, the reliability height, its key point is the exploitation of SSR primer again.Only after the SSR primer of some amount is arranged, just may carry out the analysis of SSR mark to a certain species.Particularly for the living species that did not check order, for its DNA base sequence know little about it or the species known nothing can only utilize some special methods obtain this tumor-necrosis factor glycoproteins with and the flanking sequence on both sides design the SSR primer.The source of SSR primer is summarized and is got up to have following four kinds of approach: 1. closes document at present; 2. the primer of sibling species (for example belonging to together not of the same race); 3. database search method: in public databases such as GenBank, search the SSR sequence, according to flanking sequence design primer; 4. oneself screens SSR site both wings sequences Design primer from the research object genomic library.The 4th kind of method of species that checks order seldom at those great majority almost is unique channel, also is the most basic, effective means.The method that obtains the SSR site from genomic library has comprised classical way, concentration method and omission sieve storehouse method at present again.
Set up the genomic library of the species of studying, hybridize with it with the probe that contains SSR then, screening positive clone, order-checking is according to the dna sequence dna design primer of SSR both sides.This traditional method can be divided into two kinds again: the one, and a kind of is the big fragment gene group library of inserting of screening, and demonstrating with probe has the clone of strong hybridization signal to be further purified, and is divided into subclone, and another screening by hybridization contains the clone of tumor-necrosis factor glycoproteins then.Another kind is that genomic dna is become the small segment of 200-1500bp with digestion with restriction enzyme, also can use the ultrasonication genomic dna, and the fragment that obtains is littler relatively, and 200-600bp mends its flat with the big fragment of Klenow of dna polymerase i again.Make up and screen little insertion fragment gene group library.Two kinds of methods respectively have shortcoming, first kind to big subclone be difficult to the order-checking, second kind of yield that obtains the SSR positive colony is very low, has only 2%-3%.The efficient of this traditional classical method exploitation primer is very low, but simple to operate, easily grasps, and in existing acquired SSR mark, the classical way that utilizes more than 3/4ths is developed.
Concentration method has improved SSR clone's yield by setting up and the screening enriched microsatellite library.It is divided into again and utilizes the PCR primer contain SSR to carry out PCR enrichment (being the enrichment of primer method) and hybridize two kinds of methods of enrichment (being the hybrid method enrichment) with the probe that contains SSR.
The little satellite of primer method enrichment generally refers to use with the little satellite of purpose and repeats complementary oligonucleotide as primer, is reflected at before the library construction or makes up back enrichment microsatellite locus by PCR.Elaine etc. are material with the genomic dna of dog, adopt classical way to obtain the primary gene group library of a little insertion fragment earlier, Nucleotide (CA) n with 5 ' phosphorylation is that primer carries out a step PCR then, is transformed into bacterium, obtains one and contains (CA) n enriched library.The picking mono-clonal is cultivated the back at random changes film, hybridization, and screening again, the result who obtains is: 40%-50% is positive colony, has improved 50 times (Elaine et al., 1992) than before traditional method efficient.
Use more primer development strategy to be based on the enriching method of selective cross at present, representative is two kinds of hybridizing methods, is divided into nylon embrane method and enrichment with magnetic bead method according to employed medium difference.The nylon embrane method is come little satellite fragment in the enrichment genome by being adsorbed on many little satellite probe on the nylon membrane; The elution of bound fragment; Carry out pcr amplification; The connection carrier transformed into escherichia coli; The positive colony order-checking.Use the nylon embrane method, 6iovannim repeats probe with 11 little satellites and screen microsatellite locus from Saccharum spp, and bioaccumulation efficiency has improved 40%.The ultimate principle of enrichment with magnetic bead method is at first to hybridize with biotin labeling tumor-necrosis factor glycoproteins specific probe and with the genomic dna endonuclease bamhi, the characteristic that recycling vitamin H and streptavidin affinity are strong is carried out the enrichment that the tumor-necrosis factor glycoproteins target fragment is finished in magnetic force absorption with bag by the magnetic bead of streptavidin.Through the cloning and sequencing amount being declined to a great extent behind the enrichment with magnetic bead and the efficient of the fragment that obtains to have tumor-necrosis factor glycoproteins being increased substantially, therefore, become extremely effective means and being used widely rapidly of SSR primer development.Utilize the report of enrichment with magnetic bead method developing SSR primer to have much at present.
Lee Records-Original etc. cut the back with Pinus massoniana Lamb nuclear gene group DNA with Sau3A I enzyme and reclaim the 300-1000bp fragment, and jointing is hybridized behind the PCR with biotin labeled little satellite probe (AC) 15, (AG) 15; Use enrichment with magnetic bead to contain the dna fragmentation of microsatellite sequence.The sequence that obtains by behind the pcr amplification, is connected the pGEM-T carrier, be transformed into the competence intestinal bacteria, obtain the microsatellite sequence library.Directly increased in the library with the PCR method then, obtain 58 positive colonies, through sequencing analysis, obtain 33 of microsatellite sequences, successfully design Pinus massoniana Lamb micro-satellite primers 19 at last to (Lee Records-Original etc., 2007).
Sun Xiaowen etc. cut grass carp (Ctenopharyngodon idella) genomic dna through the Sau3AI enzyme, reclaim purifying 400-900bp fragment, connect joint, make up in " genome PCR library ".Do probe and its hybridization with biotin labeled (CA) 15, hybridization complex is attached on the magnetic bead that is coated with Streptavidin, and through a series of washing process, enrichment contains the fragment of little satellite.Pcr amplification amplifies little satellite fragment, clone again and check order, conserved sequence design primer according to little satellite two ends, and carry out the secondary screening by hybridization by isotope-labeled probe (CA) 15, and obtaining 132 of positive colonies, resulting positive colony is through order-checking, 86.36% contains microsatellite sequence, obtain 130 microsatellite DNA sequences altogether, design SSR primer 83 is to (Sun Xiaowen etc., 2005).
As long as just can obtain being rich in the dna fragmentation of microsatellite locus by a few step simple operations, having lowered experimental cost from the document enrichment with magnetic bead method of report, shortened experimental period, and the success ratio height, is comparatively simple and effective SSR primer development method at present.
Kidney bean (Phaseolus vulgaris L.) is one of edible beans of cultivated area maximum in the world.Kidney bean originates in Central and South America, and the Kidney bean of Chinese cultivated is that the 15th century is directly introduced from America, and " Chinese " center " is considered to the secondary center of origin of Kidney bean, has abundant genetic diversity.Kidney bean is the important vegetable species of China, and cultivation history is long in China.Common kidney bean distributes the area extensively in China, and variety source is very abundant.Most of economize (city, the district) of China has Kidney bean to distribute, but mainly is distributed in provinces and regions such as Heilungkiang, the Inner Mongol, Shanxi, Shaanxi, Hubei, Sichuan, Guizhou, Yunnan.Chinese common kidney bean sown area is nearly 120.51 ten thousand hectares according to incompletely statistics.
Carried out at present economical character and identified and enroll the Chinese common kidney bean resource that eats beans variety source catalogue have 4480 parts, these resources have been gone into national germplasm storehouse and have been preserved.M.R.Escribano etc. have carried out the genetic diversity result of study to 18 economical characters that are planted in 56 Kidney bean kinds in the 4 kinds of varying environments in the Spain northwestward and have shown between kind all proterties all there were significant differences and most proterties has significant gene environment to make effect mutually.The morphological markers of 4 short living kinds of 13 semi-wild kinds of 43 the cultivar CIATs in 60 Kidney bean variety source Heilongjiang Province and Poland having been carried out cluster analysis result during Luan Fei etc., it is divided into the short non-hibernating eggs of two big classes is that overgrow kind and semi-wild kind of a class is combined into a class.
The Zhang Chihong of vegetable or flower institute of the Chinese Academy of Agricultural Sciences etc. analyzes 10 economical characters of 324 parts of common kidney bean germ plasm resources, the result shows, China's common kidney bean germ plasm resource has abundant form diversity, average diversity index is 1.632, be higher than materials overseas, and utilize 36 pairs of SSR primers that the genetic diversity of 332 parts of domestic common kidney beans, 16 parts of external common kidney beans and 29 parts of wild Kidney beans is analyzed.Xu Zhaosheng etc. have carried out the comprehensive characterization and evaluation in field to 38 parts of Kidney bean germplasms, to select the good excellent germplasm of comprehensive proterties, provide and produce and the scientific research utilization.Units such as the Chinese Academy of Agricultural Sciences Ins of Crop Varity Res, vegetable or flower institute, coastland, the Jiangsu Province institute of agricultural sciences, northeast agricultural university have carried out the collection of Kidney bean variety source, and resource carried out comprehensive evaluation, think that China's common kidney bean germ plasm resource has abundant form diversity, average diversity index is higher than materials overseas, the Germplasm Resources on Phaseolus Vulgaris hereditary basis is wideer, sibship is far away, heritable variation mainly is present in the population inside of overgrowing, Kidney bean becomes genetic diversity in population than horn of plenty, can be used as breeding material deposit germ plasm resource.And therefrom filter out the high elite germplasm material of strong, the tender pod protein content of a collection of disease resistance.
Molecular breeding comprises molecular marker assisted selection (MAS) and utilizes the genetic engineering means breeding.Molecular marker assisted selection is the key areas that molecular marking technique is used for crop improvement, is the product that traditional breeding technology and modern biotechnology combine.Have a lot of important characters (as output and the later stage leaf portion or fringe portion disease resistance etc.) only on ripe plant, just can show, therefore adopt traditional method in that after planting several months or several years all can not be selected it.And utilize molecule marker just can seedling (even to seed) to be detected, cultivate human and material resources and the financial resources that plant wastes thereby save greatly.
The SSR molecule marker has codominance, rich polymorphism in numerous molecule markers, numerous advantages such as wide distribute in the beans genome.At present just little satellite of known encoding sequence is screened and identify that little satellite of a large amount of non-coding regions is still waiting development and utilization.Therefore the present invention is that research object is opened the SSR primer of developing more Kidney bean kinds in the whole genome scope with Chinese Kidney bean.
In sum, Kidney bean is very big in China's demand, in vegetables, occupy an important position, the Germplasm Resources on Phaseolus Vulgaris of China is abundant simultaneously, genetic diversity is many, therefore is badly in need of the more SSR primer of exploitation, and molecular mark and conventional breeding are combined the process that could accelerate the Kidney bean breeding, cultivate new variety, realize society and economic benefit rapidly.
Summary of the invention
The objective of the invention is to be to provide a kind of preparation method of enrichment with magnetic bead method exploitation Kidney bean SSR primer, utilize paramagnetic particle method to carry out the SSR primer development, can screen the microsatellite sequence in the Kidney bean whole genome simply, rapidly, can develop a large amount of SSR primers at short notice, have only after the SSR primer of exploitation some amount, just may carry out the analysis of SSR mark to a certain species.Therefore, the present invention can provide guarantee for the realization of a large amount of SSR molecule marker of needs of work such as Kidney bean construction of genetic atlas and the assignment of genes gene mapping.
A kind of preparation method of enrichment with magnetic bead method exploitation Kidney bean SSR primer comprises the following steps:
1. the extraction of Kidney bean genomic dna:
Choose the Kidney bean kind with Kidney bean general feature.According to cultivation management and the rich water measure plantation of routine, treat that seedling grows 5-6 sheet leaf, get tender leaf 0.4 gram and utilize the CTAB method to extract genomic dna.
Described Kidney bean, claim again kidney bean (be commonly called as two season beans or string bean), pulse family section Phaseolus.Kidney bean suits in temperate zone and the plantation of tropical high altitude localities, and more cold-resistant happiness light belongs to cross-pollination Kidney bean, short day crop, the kidney bean well developed root system, and leaf green, alternate, heart-shaped, flower is the leaf flower of worm, raceme, the long 15-18 of bennet centimetre.Blooming, to bear pods few more.It is nutritious, and the protein content height namely is that vegetables are again grain, also can make cake and beans filling, is the important agricultural byproducts of foreign exchange earning.Kidney bean formal name used at school Kidney bean, the Papilionaceae Phaseolus.
2. extract the detection of genomic dna concentration:
The concentration of extracting genomic dna detects with spectrophotometer.The genomic dna concentration that can carry out the SSR primer development will reach 100ng-1ug/ul.
3. enzyme is cut genomic dna:
Carry out enzyme with the Kidney bean genomic dna of Mse I and cut, utilize AFLP amplification principle to add the single stranded oligonucleotide joint at the disconnected two ends of enzyme section, and use and carry out pcr amplification with the supporting joint primer of joint and amplify, create genome PCR library, purifying is carried out in the library.
4.Mse the I endonuclease bamhi adds joint:
Enzyme is cut DNA product Mse I joint (joint sequence: Oligo-MseI A5-TACTCAGGACTCAT-3 ', Oligo-Mse I B 5-GACGATGAGTCCTGAG-3 ') adding T4 DNAligase connects, behind the mixing, put 16 ℃ of thermostat water bath temperature and bathe and spend the night, be placed on then preserve in 4 ℃ of refrigerators standby.
5. the enzyme PCR that cuts connection increases in advance:
Cutting the product that connects with step 4 enzyme is template, is that primer carries out pcr amplification with MseI-N:5 '-GATGAGTCCTGAGTAAN-3 '.Reaction conditions: 1 circulation of the first step, 94 ℃ of 5min, second step 18 circulations, 94 ℃ of 1min, 53 ℃ of 1min, 72 ℃ of 1min, 72 ℃ of 10min of the 3rd step 1 circulation, 4 ℃ of preservations.Reaction product uses the 1%W/V agarose electrophoresis to detect, and the dispersive region just occurs and gets final product, as Fig. 1.
6.PCR pre-expansion volume increase thing and biotinylated probes hybridization:
Under suitable temperature (66-70 ℃), make PCR library and selected biotin labeled repetition (2 bases repeat 10-15 time, 3 or 4 bases repetition 5-8 time) sequence probe hybridize; Because the pan coating of magnetic bead has one deck Streptavidin, and this Streptavidin have very strong avidity with biology, therefore biotin labeled probe and the dna segment hybridization that contains the tumor-necrosis factor glycoproteins unit in conjunction with after, just can be adsorbed by magnetic bead at an easy rate, fix by non-strict wash-out and strict wash-out (seeing below) and with magnetic field, can remove other compositions of dna segment of not hybridized at an easy rate, thereby reach the purpose of highly enriched microsatellite sequence; Under the sex change condition, contain the microsatellite DNA of tumor-necrosis factor glycoproteins by the TE buffer solution elution; The elutriant purifying is later as dna profiling.
Hybridization system cumulative volume is 100 μ L, and parallel preparation 2 pipes (are respectively (AC) altogether
13(AG)
13) packing then, hybridize.Carry out at the PCR instrument, response procedures is 95 ℃ of sex change 5min; The suitableeest hybridization temperature (66-70 ℃) keeps 1-2h down.Add 300 μ L TEN100 solution after hybridization finishes in reaction system, it is standby that mixing is placed on 4 ℃ of preservations.The hybridization mixed solution that is added with 300 μ L TEN100 is joined through in the pretreated magnetic bead, (20-25 ℃ of room temperature, below identical) go up to place 30min, during stir lightly frequently or piping and druming solution, thereby avoid the more effectively DNA after the absorption hybridization of magnetic bead precipitation.(wash magnetic bead 3 times under room temperature with 400 μ L TEN1000, each 5min stirs or the piping and druming mixture frequently then the magnetic bead of hybridizing to be carried out non-strict wash-out.Be preheated to 55 ℃ TEN1000 solution washing 1 time after 3 rinsings again with 400 μ L) and strict washing (with 400 μ L, 0.2 * SSC (3M NaCl, 0.3M Na
3C
6H
5O
7.2H
2O; PH7.0, below identical)+0.1% (0.1g SDS, 100ml 2d H
2O, below identical) SDS solution washs magnetic bead 3 times under room temperature, each 5min stirs or the piping and druming mixture frequently.Be preheated to 0.2 * SSC+0.1%SDS solution washing 1 time of 55 ℃ after three rinsings again with 400 μ L.With the fixing magnetic bead of magnet stand, remove washings).Add 50 μ L TE8.0, inhale with pipettor and beat evenly, then in 95 ℃ of following water-bath 5min.Stir frequently or the piping and druming mixture.With the fixing magnetic bead of magnet stand, the sucking-off supernatant liquor is frozen in-20 ℃ rapidly, obtains target DNA fragment.Being substrate with the eluted product, is primer with MseI-N, is used for pcr amplification with 5 μ L target DNA wash-out fragments behind the purifying, amplification condition and the top PCR term harmonization that increases in advance, 30 circulations of increasing.
7. connection transformed clone:
Pcr amplification product behind the purifying is connected on the T carrier clones.Concrete operations connect test kit (available from Promega) working instructions according to the pMD 18-T carrier that TaKaRa company provides.The ligation system is 10 μ L, and its formation sees Table 1.Condition of contact is 16 ℃ of 1hr.
Table 1DNA fragment and T carrier ligation system
In-70 ℃ of refrigerators, take out a pipe competent cell (M15,100 μ L), place ice bath to make it just melt (if the competent cell of made fresh can directly add and connect mixture) immediately.Add 5 μ L then and connect product, ice bath 30min.Ice bath finishes back 42 ℃ of heat shock 90sec, and ice bath 5min again adds the liquid LB substratum (not containing the ammonia benzyl) of 1mL precooling then, goes up recovery in 37 ℃ shaking tables (150rpm) and cultivates 45min.Recovery is cultivated and is finished the back in 4 ℃ of centrifugal 1min of 10000rpm, with sedimentation cell; Remove supernatant, residue 200-300 μ L substratum re-suspended cell.Add 4 μ L IPTG and 40 μ L X-gal on each LB flat board, coating is even, and per 100 μ L bacterium liquid are coated with a flat board, treats that liquid-absorbent finishes back (about 15min), is inverted in 37 ℃ spend the night (12-16hr) with flat board.
8. the picking of positive colony, cultivation, screening and order-checking:
Select positive colony and check order with the carrier universal primer, the dna sequence dna of the insertion segment that obtains is picked out suitable microsatellite DNA sequence.Cultured flat board (colony growth good and be evenly distributed) was placed some hours at 4 ℃, bacterium colony is fully developed the color.Be cloned in 500 μ L (the containing 50 μ LAmp) liquid nutrient medium with aseptic toothpick picking white.With 200rmp rotating speed overnight incubation, preserve bacterium liquid for 4 ℃ on 37 ℃ of shaking tables.
Get 1 μ L bacterium liquid and be used for pcr template.Amplification condition is: 95 ℃ of 5min; 94 ℃ of 40s; 54 ℃ of 30s; 72 ℃ of 1min; Circulate 30 times; 72 ℃ of 7min.Get 2 μ L products electrophoresis on the 1%W/V sepharose, with full automatic gel imaging analysis system log (SYSLOG) electrophoresis result; Electrophoresis on the sepharose, and with full automatic gel imaging analysis system log (SYSLOG) electrophoresis result.
To contain the positive colony that inserts fragment and size to fit (450-800bp) gets half bacterium liquid and send Beijing big-and-middle living development in science and technology company limited's order-checking of China (Fig. 2).Second half adds the 20%V/V sterile glycerol, behind the mixing in-70 ℃ of frozen backups.Check order with the M13+ universal primer, order-checking is carried out at ABI 3730XL DNA Sequencer, used reaction reagent is ABI PRISM BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit, and the useful length of every reaction can reach about 700-800bp.For the sequence that contains better repeated fragment, check order from other one section with the M13 primer, and forward and reverse sequencing result is spliced, to guarantee the accuracy of sequence.
9. the classification of sequential analysis and microsatellite locus and design:
With software SSRHunter (version 1.3.0; Li Qiang) from the sequence that obtains, finds out the microsatellite sequence that contains repeating unit.Instead wherein multiplicity more (2 bases repeat 10-15 time, 3 or 4 bases repetition 5-8 time), sequencing result is very good and the repeat region both wings have the sequence of enough conserved regions as microsatellite locus.Figure below shows dna sequence dna (Fig. 3) and the order-checking peak shape figure (Fig. 4) thereof of a desirable microsatellite locus
The standard that proposes according to Weber (1990) is divided into 3 classes with little satellite: perfect type (perfect), refer to not interrupt or near do not have the tumor-necrosis factor glycoproteins of other kind tumor-necrosis factor glycoproteins; Non-perfect type (imperfect), refer to 2 or above tumor-necrosis factor glycoproteins of the same race by 3 non repetitive sequences below the base the interval; Compound (compound), the tumor-necrosis factor glycoproteins that refers to a kind of tumor-necrosis factor glycoproteins and other kind by the non repetitive sequence below 3 the interval.
Earlier with the microsatellite sequence picked out with online tool VecScreen (
Http:// www.ncbi.nlm.nih.gov/VecScreen/) removal carrier sequence, mark the position of Mse I joint (A and B) then in sequence, use primer-design software Primer Premier 5.0 (Rozen and Skaletsky 1988) between two end connectors and core repeat region, to design forward and inverse PCR amplimer again, be used for the pcr amplification in corresponding site.
10. micro-satellite primers validity check:
The validity of each site primer is closed polymorphism screen, obtain effective microsatellite locus.Two quality of random choose dna profiling are preferably carried out pcr amplification to all synthetic primers, and the default temperature when annealing temperature is preferentially used the design primer is 50-62 ℃, if can not get amplified production or expanding effect is bad, then annealing temperature are adjusted.
Little satellite pcr amplification reaction system is 15 μ L, and it is constructed as follows table.Amplification condition is: 94 ℃ of 5min; 94 ℃ of 40s, Tm 30s, 72 ℃ of 40s, 35cycles; 72 ℃ of 7min.Pcr amplification product adds sample-loading buffer according to 5:1 with volume ratio, and 95 ℃ of sex change placed rapidly on ice after 5 minutes, and application of sample detects on 8% denaturing polyacrylamide gel then.In last sample, add 0.1gPBR322/Msp I Marker toward one of them well, as the relative molecular weight standard of amplified production.The method dyeing tape reading of dying with silver at last.The primer that can normally increase carries out result's tape reading of result's electrophoresis on polyacrylamide gel of pcr amplification to using the dna profiling of picking out under the suitableeest annealing temperature (50-62 ℃), the selection banding pattern is clear, has at least two or more allelic primer screenings to come out.Obtain 20 pairs of SSR primers that polymorphism is good altogether by this experiment.
Characteristics of the present invention: the enrichment with magnetic bead method be to utilize biotin labeling tumor-necrosis factor glycoproteins specific probe and hybridize with the genomic dna endonuclease bamhi, the characteristic that recycling vitamin H and streptavidin affinity are strong is carried out the enrichment that the tumor-necrosis factor glycoproteins target fragment is finished in magnetic force absorption with bag by the magnetic bead of streptavidin.Through the cloning and sequencing amount being declined to a great extent behind the enrichment with magnetic bead and the efficient of the fragment that obtains to have tumor-necrosis factor glycoproteins being increased substantially, therefore, become extremely effective means and being used widely rapidly of SSR primer development.Advantage of the present invention: just can obtain being rich in the dna fragmentation of microsatellite locus by a few step simple operations, lower experimental cost, shorten experimental period, and the success ratio height, be comparatively simple and effective SSR primer development method at present.The present invention altogether picking 105 positive colonies check order, wherein there are 48 sequencing sequences can design primer, 20 pairs of effective primers from 48 pairs of primers, have been obtained, effectively the primer yield be 19% (20/105) and Matthew W Blair etc. by genomic 120 the effective SSR primers that obtain to 3123 EST screening primers, effectively the primer yield is 3.8% (120/3123), this shows developing SSR primer efficient height of the present invention, and simple, can provide safeguard for large batch of developing SSR primer.
Description of drawings
Fig. 1 is a kind of pre-expansion product synoptic diagram (the different cycle indexes of PCR).
" 1 " cuts the product that with joint be connected for different Kidney bean kinds through enzyme with " 2 " is template, is that primer carries out pcr amplification with MseI-N:5 '-GATGAGTCCTGAGTAAN-3 '; 14* wherein, 16*, 18*, 22* refer to PCR circulation 14 times, 16 times, 18 times, 22 times respectively.
Fig. 2 is a kind of bacterium liquid PCR product synoptic diagram.
Wells different among the figure are the result that primer carries out pcr amplification with M13 for being template with the different positive colonies of choosing.The band of choosing more than 500 checks order.
Fig. 3 is that a kind of AG repeats one of sequencing sequence.
This sequence is perfect sequence, and TC two bases repeat 13 times, and microsatellite sequence is between 474-499.
Fig. 4 is a kind of AG peak shape synoptic diagram that repeats to check order.
TC two bases repeat 13 times microsatellite sequence between 474-499, can design primer in its both sides, are exactly one of the SSR primer sequence that will develop of present method.
Embodiment
The present invention is described further below in conjunction with embodiment.
Embodiment 1:
A kind of preparation method of enrichment with magnetic bead method exploitation Kidney bean SSR primer the steps include:
A, extraction Kidney bean genomic dna carry out enzyme to genome and cut, and set up the AFLP-PCR library:
Kidney bean kind Qiao Yu (provide good Kidney bean kind by Wuhan City vegetables Science Institute, overgrow, the leaf look green, and pattern is pale yellow).This kind is the Kidney bean kind of high-quality, has the general feature of Kidney bean kind.According to cultivation management and the rich water measure plantation of routine, treat that seedling grows 5-6 sheet leaf, get tender leaf 0.4 gram and utilize the CTAB method to extract genomic dna.
(1) adds 4ml 3 * CTAB extracting solution in the Eppeddorf pipe of 10ml and (contain 0.2% beta-mercaptoethanol (W/V), preheating in 65 ℃ of water-baths.
(2) get 0.4 tender leaf that restrains Kidney bean and be put in the mortar, add suitable liquid nitrogen and repeatedly grind to form fine powder rapidly, change in the CTAB extracting solution of preheating, centrifuge tube is covered tightly to prevent the beta-mercaptoethanol volatilization.Put back to insulation (65 ℃) 40min in the water-bath, light rolling keeps shaking up several times in the insulating process.
(3) take out the Eppenddorf pipe, place rapid cool to room temperature on ice, add isopyknic chloroform/primary isoamyl alcohol (24: 1), slightly mix and fully, make it be mixed into milk sap up hill and dale, 4 ℃ of following centrifugal 12000rpm 10min draw supernatant liquor 1ml to be put in the 1.5ml centrifuge tube gently then, are put in-20 ℃ of refrigerators standby.
(4) add 1 times of 100% (V/V) ethanol that volume is ice-cold among the DNA that extracts above.
(5) 4 ℃, behind the centrifugal 10min of 10000rpm, carefully outwell supernatant liquor, the air-dry 5min of room temperature.
(6) add 1mlTE and 1 μ l RNase (10mg/ml), in 37 ℃, be incubated 40min.
(7) chloroform/the primary isoamyl alcohol extracting once to add equal-volume.The centrifugal 10min of 12000rpm draws supernatant liquor and changes in the another one Eppendorf pipe, adds sodium-acetate and 2 times of dehydrated alcohols that volume is ice-cold of 1/10 volume, places 1h for-20 ℃.
(8) the centrifugal 10min of 10000rpm carefully outwells supernatant liquor.
(9) the ethanol 1ml that adds 70% (V/V) washs the resuspended DNA of 150 μ l TE 3 times.
(10) be put in-20 ℃ of refrigerators and preserve.
The concentration of extracting genomic dna detects with spectrophotometer.Carry out enzyme with the Kidney bean genomic dna of MseI and cut, endonuclease reaction is handled 20min with deactivation MseI enzyme for 65 ℃ then at PCR instrument or 37 ℃ of reactions of water-bath 3h.The reaction system (NEB buffer 2:2.0 μ L, 100 * NEB BSA:0.2 μ L, MseI 10U/ul:0.5 μ L, DNA:10 μ L, the ddH that join 20ul
2O: mend to 20 μ L), enzyme is cut agarose and is detected.
Mse I joint sequence: Oligo-MseI A 5-TACTCAGGACTCAT-3 ', Oligo-MseI B5-GACGATGAGTCCTGAG-3 '.Preparation joint: Oligo-Mse I A and Oligo-Mse I B are made into 50 μ M stock solutions respectively, fully after the dissolving, take out each 50 μ L of equal-volume Oligo-MseI A and Oligo-MseI B, fully behind the mixing in 94 ℃ of sex change 5min, slowly cool to room temperature after the taking-up, be stored in-20 ℃ standby to concentration be 25uM.Linked system: total system 20ul (the 7ul enzyme is cut DNA product+1.5ul 10 * ligase buffer+4ul T4DNAl igase (3U/ul)+2.5ul MseI hybrid).Behind the mixing, put 16 ℃ of thermostat water bath temperature and bathe and spend the night, be placed on then preserve in 4 ℃ of refrigerators standby.
The PCR that B, enzyme are cut connection increases in advance:
(1) Mse I-N primer sequence 5 '-GAT GAG TCC TGA GTA AN-3 ' (N=A, T, G, C) totally four pairs form mix primer.
(2) reaction system that increases in advance
Pre-amplification reaction cumulative volume is 20 μ L, altogether parallel preparation 3 pipes.After the packing, carry out pcr amplification then.Amplified reaction constitutes as table 2:
The pre-amplification of table 2 reaction system
(3) the PCR condition that increases in advance: 94 ℃ of 5min; 94 ℃ of 1min, 53 ℃ of 1min, 72 ℃ of 1min; 72 ℃ of 7min.Design 3 groups of different cycle numbers (16/18/20cyles), every group differs 2 circulations, to determine the optimization amplification condition.
(4) the electrophoresis check of pre-expansion volume increase thing
Get 3 μ LPCR reaction product, add 1 μ L sample-loading buffer, behind the mixing in the 1%W/V agarose gel electrophoresis.Successful sign is to occur a smear zone (200-800bp) in the electrophoresis product.The cycle number (18) that makes the smear zone just occur is best amplification cycles number.
C, PCR pre-expansion volume increase thing and biotinylated probes hybridization:
(1) hybridization system and condition
Hybridization system cumulative volume is 100 μ L, and parallel preparation 2 pipes (are respectively (AC) altogether
13(AG)
13) packing then, hybridize.Hybridization diagram of system 3:
Table 3 hybridization constitution system
Carry out at the PCR instrument, response procedures is 95 ℃ of sex change 5min; The suitableeest hybridization temperature (68 ℃) keeps 1h down.Add 300 μ L TEN100 solution after hybridization finishes in reaction system, it is standby that mixing is placed on 4 ℃ of preservations.
(2) magnetic bead hybridization and washing
1. the pre-treatment of magnetic bead
With the 2mg magnetic bead with the careful washing of 300 μ L TEN100 solution 3 times, each 5min, when each washing finishes with the fixing magnetic bead of magnet stand, sucking-off supernatant liquor.After washing finishes, with 40 μ L TEN100 solution suspension magnetic beads, place 4 ℃ of preservations standby.
2. magnetic bead hybridization
The hybridization mixed solution that is added with 300 μ L TEN100 is joined through in the pretreated magnetic bead, places 30min on the room temperature, during stir lightly frequently or piping and druming solution, thereby avoid the more effectively DNA after the absorption hybridization of magnetic bead precipitation.
3. magnetic bead washing
A. after magnetic bead absorption finishes, with the fixing magnetic bead of magnet stand, remove the hybridization mixed solution.
B. non-strict wash-out (nonstringency wash): wash magnetic bead 3 times under room temperature with 400 μ L TEN1000, each 5min stirs or the piping and druming mixture frequently.Be preheated to 55 ℃ TEN1000 solution washing 1 time after 3 rinsings again with 400 μ L.
C. strict wash-out (stringency wash): wash magnetic bead 3 times under room temperature with 400 μ L, 0.2 * SSC+0.1%SDS solution, each 5min stirs or the piping and druming mixture frequently.Be preheated to 0.2 * SSC+0.1%SDS solution washing 1 time of 55 ℃ after 3 rinsings again with 400 μ L.With the fixing magnetic bead of magnet stand, remove washings.
D. the wash-out of target DNA fragment: add 50 μ L TE8.0, with pipettor inhale beat even, then in 95 ℃ of following water-bath 5min.Stir frequently or the piping and druming mixture.With the fixing magnetic bead of magnet stand, the sucking-off supernatant liquor is frozen in-20 ℃ rapidly.
D. the purifying of target DNA fragment and amplification:
Being substrate with twice eluted product, is primer with MseI-N, is used for pcr amplification with 5 μ L target DNA wash-out fragments behind the purifying, amplification condition and the top PCR term harmonization that increases in advance, 30 circulations of increasing.
(1) carrier connects
Concrete operations connect the test kit working instructions according to the pMD 18-T carrier that TaKaRa company provides.The ligation system is 10 μ L, and it constitutes as table 4.Condition of contact is 16 ℃ of 1hr.
Table 4DNA fragment and T carrier ligation system
(2) conversion of connection product
In-70 ℃ of refrigerators, take out a pipe competent cell (M15,100 μ L), place ice bath to make it just melt (if the competent cell of made fresh can directly add and connect mixture) immediately.Add 5 μ L then and connect product, ice bath 30min.Ice bath finishes back 42 ℃ of heat shock 90sec, and ice bath 5min again adds the liquid LB substratum (not containing the ammonia benzyl) of 1mL precooling then, goes up recovery in 37 ℃ shaking tables (150rpm) and cultivates 45min.Recovery is cultivated and is finished the back in 4 ℃ of centrifugal 1min of 10000rpm, with sedimentation cell; Remove supernatant, residue 200-300 μ L substratum re-suspended cell.Add 4 μ L IPTG and 40 μ L X-gal on each LB flat board, coating is even, and per 100 μ L bacterium liquid are coated with a flat board, treats that liquid-absorbent finishes back (about 15min), is inverted in 37 ℃ spend the night (12-16hr) with flat board.
The picking of E, positive colony, cultivation and screening:
(1) picking of positive colony, cultivation
A. generally clone's bacterium colony of each flat board is controlled about 200, conveniently to select positive colony.Cultured flat board (colony growth good and be evenly distributed) was placed some hours at 4 ℃, made bacterium colony fully develop the color (positive colony is white, and empty plasmid be blueness or light blue).
B. be cloned in 500 μ L (the containing 50 μ LAmp) liquid nutrient medium with aseptic toothpick picking white.
C.37 on ℃ shaking table with 200rmp rotating speed overnight incubation, preserve the bacterium liquid for 4 ℃
(2) PCR check positive colony
Get 1 μ L bacterium liquid and be used for pcr template.Amplification reaction system is 15 μ L, does negative control simultaneously, and the reaction system formation sees Table 5.Amplification condition is: 95 ℃ of 5min; 94 ℃ of 40s; 54 ℃ of 30s; 72 ℃ of 1min; Circulate 30 times; 72 ℃ of 7min.
Table 5 is for detection of the PCR reaction system of positive colony
Get 2 μ L products electrophoresis on 1% sepharose, and with full automatic gel imaging analysis system log (SYSLOG) electrophoresis result.To contain the positive colony that inserts fragment and size to fit (450-800bp) gets half bacterium liquid and send the order-checking of Beijing China big-and-middle living development in science and technology company limited.Second half adds 20% sterile glycerol, behind the mixing in-70 ℃ of frozen backups.Check order with the M13+ universal primer, order-checking is carried out at ABI 3730XL DNA Sequencer, used reaction reagent is ABI PRISM BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit, and the useful length of every reaction can reach about 700-800bp.For the sequence that contains better repeated fragment, check order from other one section with the M13 primer, and forward and reverse sequencing result is spliced, to guarantee the accuracy of sequence.
The classification of F, sequential analysis and microsatellite locus and design:
With software SSRHunter (version 1.3.0; Li Qiang) from the sequence that obtains, finds out the microsatellite sequence that contains repeating unit.Earlier the microsatellite sequence of picking out is removed the carrier sequence with online tool VecScreen, mark the position of Mse I joint (A and B) then in sequence, use primer-design software Primer Premier 5.0 (Rozen and Skaletsky again, 1988) design forward and inverse PCR amplimer between two end connectors and core repeat region are for the pcr amplification in corresponding site.When the design primer, (primer length is between 18-27bp, and optimal length is 20bp for relevant parameter inaction default value; The amplification annealing temperature is between 50-63 ℃, and optimum temperuture is 60 ℃; GC content (GC%) is between 20-80%, and optimum content is 50%; Amplified production length is between 100-500bp, and optimal length is 200bp).
G, micro-satellite primers validity check:
(1) little satellite pcr amplification
Two quality of random choose dna profiling are preferably carried out pcr amplification to all synthetic primers, and the default temperature when annealing temperature is preferentially used the design primer is 50-62 ℃, if can not get amplified production or expanding effect is bad, then annealing temperature are adjusted.
Little satellite pcr amplification reaction system is 15 μ L, and it constitutes as table 6.Amplification condition is: 94 ℃ of 5min; 94 ℃ of 40s, Tm 30s, 72 ℃ of 40s, 35cycles; 72 ℃ of 7min.
The little satellite amplification reaction system of table 6
(2) pcr amplification product detects
Pcr amplification product added sample-loading buffer according to 5: 1 with volume ratio, and 95 ℃ of sex change placed rapidly on ice after 5 minutes, and application of sample detects on 8% denaturing polyacrylamide gel then.In last sample, add 0.1g PBR322/Msp I Marker toward one of them well, as the relative molecular weight standard of amplified production.The method dyeing tape reading of dying with silver at last.The concrete operations step is as follows:
1. wash sheet glass.With brush sheet glass is cleaned oven dry, clean 3 times with alcohol again before the encapsulating.
2. silication and anti-silication sheet glass.The silication agent (200 μ L peel off silane+2mL 95% ethanol) for preparing is poured on the short slab, even with the rapid wiping of filter paper; The anti-silication agent (the affine silane of 9 μ L+18 μ L glacial acetic acid+2mL, 95% ethanol) that configures is poured on the long slab, even with the rapid wiping of filter paper.In ventilating kitchen, dried then 15 minutes.
3. encapsulating.Sheet glass is installed, tilt to be put on the flat desktop slightly.Add the urea gel monomer that 60mL configures in a small beaker, 400 μ L 10%AP shake up slowly encapsulating of back behind the 40 μ L TEMED, avoid occurring bubble.
4. gel.The offset plate horizontal positioned, the anti-comb that inserts was placed 3 hours.
5. prerunning.Comb is extracted, offset plate and comb are cleaned, install offset plate at electrophoresis chamber, last groove and following groove are respectively irritated 1 * TBE 400mL.The permanent power electrophoresis of 45w 1 hour to 45 ℃.
6. go up the sample electrophoresis.Stop prerunning before the last sample earlier, will go up sample top blast wash clean with rifle, insert good comb then.Every hole point sample 3 μ L, 45w electrophoresis 1.5 hours.
7. silver dyes.The first step is pulled down the acetum of putting into 1.5L10% with long slab and is fixed 30 minutes; Second step, remove stationary liquid, use rinsed with deionized water 3 times, each two minutes; In the 3rd step, dyeing is 30 minutes in containing 1 ‰ Silver Nitrate dye liquors of 1.5 ‰ formaldehyde solutions; The 4th step, rinsed with deionized water 10 seconds; In the 5th step, developing solution (3% sodium carbonate solution) colour developing is clear to being with; The 6th goes on foot, and stationary liquid is poured in the colour developing liquid stopped showing.
8. take a picture and preserve.With digital camera the offset plate that developed the color is taken a picture.
(3) screening in polymorphic micro-satellite site
The primer that can normally increase carries out result's tape reading of result's electrophoresis on polyacrylamide gel of pcr amplification to using the dna profiling of picking out under the suitableeest annealing temperature, the selection banding pattern is clear, has at least two or more allelic primer screenings to come out.
Claims (1)
1. the preparation method of an enrichment with magnetic bead method exploitation Kidney bean SSR primer comprises the following steps:
The extraction of A, Kidney bean genomic dna:
Choose the Kidney bean kind, according to cultivation management and the rich water measure plantation of routine, seedling grows 5-6 sheet leaf, gets tender leaf 0.4 gram and utilizes the CTAB method to extract genomic dna;
B, the detection of extracting genomic dna concentration:
The concentration of extracting genomic dna detects with spectrophotometer, and the genomic dna concentration of carrying out the SSR primer development will reach 100ng-1ug/ul;
C, enzyme are cut genomic dna:
Carry out enzyme with the Kidney bean genomic dna of Mse I and cut, utilize AFLP amplification principle to add the single stranded oligonucleotide joint at the disconnected two ends of enzyme section, and use and carry out pcr amplification with the supporting joint primer of joint and amplify, create genome PCR library, purifying is carried out in the library;
D, Mse I endonuclease bamhi add joint:
Enzyme is cut DNA product Mse I joint: joint sequence Oligo-MseI A5-TACTCAGGACTCAT-3 ', Oligo-MseI B5-GACGATGAGTCCTGAG-3 ' adds T4DNAligase and connects, behind the mixing, put 16 ℃ of thermostat water bath temperature and bathe and spend the night, be placed on then preserve in 4 ℃ of refrigerators standby;
The PCR that E, enzyme are cut connection increases in advance:
Cutting the product that connects with step D enzyme is template, be that primer carries out pcr amplification with MseI-N:5 '-GATGAGTCCTGAGTAAN-3 ', reaction conditions: 1 circulation of the first step, 94 ℃ of 5min, second step 18 circulations, 94 ℃ of 1min, 53 ℃ of 1min, 72 ℃ of 1min, 72 ℃ of 10min of the 3rd step 1 circulation, 4 ℃ of preservations, reaction product use the 1%W/V agarose electrophoresis to detect;
F, PCR pre-expansion volume increase thing and biotinylated probes hybridization:
Under 66-70 ℃, make PCR library and selected biotin labeled repetitive probe hybridize; The pan coating of magnetic bead has one deck Streptavidin, and biotin labeled probe is hybridized after the combination with the dna fragmentation that contains the tumor-necrosis factor glycoproteins unit, fixes by washing process and with magnetic field, contains the microsatellite DNA of tumor-necrosis factor glycoproteins by the TE buffer solution elution under the sex change condition; The elutriant purifying is later as dna profiling;
Hybridization system cumulative volume is 100 μ L, and parallel preparation 2 pipe packing are hybridized altogether, carry out at the PCR instrument, and response procedures is 95 ℃ of sex change 5min; Hybridize 66-70 ℃ and keep 1-2h down, after finishing, hybridization in reaction system, adds 300 μ L TEN100 solution, it is standby that mixing is placed on 4 ℃ of preservations, the hybridization mixed solution that is added with 300 μ L TEN100 is joined through in the pretreated magnetic bead, place 30min on the room temperature, magnetic bead to hybridization carries out wash-out then, SDS solution washs magnetic bead 3 times under room temperature, each 5min is preheated to 0.2 * SSC+0.1%SDS solution washing 1 time of 55 ℃ again with 400 μ L after three rinsings, add 50 μ L TE8.0, beat evenly with the pipettor suction, in 95 ℃ of following water-bath 5min, frozen in-20 ℃ then, obtain target DNA fragment, being substrate with the eluted product, is primer with MseI-N, is used for pcr amplification with 5 μ L target DNA wash-out fragments behind the purifying, the PCR of amplification condition and the step e term harmonization that increases in advance, 30 circulations of increasing;
G, connection transformed clone:
Pcr amplification product behind the purifying is connected on the T carrier clones, operation connects the test kit working instructions according to the pMD18-T carrier, the ligation system is 10 μ L, condition of contact is 16 ℃ of 1hr, in-70 ℃ of refrigerators, take out a pipe competent cell, place ice bath to melt, add 5 μ L and connect product, ice bath 30min, ice bath finish back 42 ℃ of heat shock 90sec, ice bath 5min again, the liquid LB substratum that adds the 1mL precooling then, 45min is cultivated in recovery on 37 ℃ shaking table, and recovery is cultivated and finished the back in 4 ℃ of centrifugal 1min of 10000rpm, with sedimentation cell; Remove supernatant, residue 200-300 μ L substratum re-suspended cell adds 4 μ L IPTG and 40 μ L X-gal on each LB flat board, and coating is even, and per 100 μ L bacterium liquid are coated with a flat board, treat that liquid-absorbent finishes after, flat board is inverted in 37 ℃ spends the night;
The picking of H, positive colony, cultivation, screening and order-checking:
Selecting positive colony checks order with the carrier universal primer, the dna sequence dna of the insertion segment that obtains, pick out the microsatellite DNA sequence, with cultured flat board 4 ℃ of placements, make the bacterium colony colour developing, be cloned in the 500 μ L liquid nutrient mediums with aseptic toothpick picking white, with 200rmp rotating speed overnight incubation, preserve bacterium liquid for 4 ℃ on 37 ℃ of shaking tables;
Get 1 μ L bacterium liquid and be used for pcr template, amplification condition is: 95 ℃ of 5min; 94 ℃ of 40s; 54 ℃ of 30s; 72 ℃ of 1min; Circulate 30 times; 72 ℃ of 7min get 2 μ L products electrophoresis on the 1%W/V sepharose, with full automatic gel imaging analysis system log (SYSLOG) electrophoresis result;
To contain the positive colony that inserts fragment and size and get half bacterium liquid order-checking, second half adds 20%V/V bacterium glycerine, behind the mixing in-70 ℃ of frozen backups, check order with the M13+ universal primer, order-checking is carried out at ABI3730XL DNA Sequencer, used reaction reagent is ABI PRISM BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit, the useful length of every reaction reaches 700-800bp, check order from other one section with the M13 primer, and forward and reverse sequencing result is spliced;
The classification of J, sequential analysis and microsatellite locus and design:
From the sequence that obtains, find out the microsatellite sequence that contains repeating unit with software SSRHunter, order-checking, the standard that proposes according to Weber is divided into 3 classes with little satellite: perfect type, refer to not interrupt or near do not have the tumor-necrosis factor glycoproteins of other kind tumor-necrosis factor glycoproteins; Non-perfect type, refer to 2 or above tumor-necrosis factor glycoproteins of the same race by 3 non repetitive sequences below the base the interval; Compound, the tumor-necrosis factor glycoproteins that refers to a kind of tumor-necrosis factor glycoproteins and other kind by the non repetitive sequence below 3 the interval, the microsatellite sequence of picking out is removed the carrier sequence, mark the position of Mse I joint in sequence, between two end connectors and core repeat region, design forward and inverse PCR amplimer with primer-design software Primer Premier5.0 again, be used for the pcr amplification in corresponding site;
K, micro-satellite primers check:
Validity and polymorphism to each site primer are screened, obtain effective microsatellite locus, select two dna profilings all synthetic primers are carried out pcr amplification, default temperature when annealing temperature is used the design primer is 50-62 ℃, little satellite pcr amplification reaction system is 15 μ L, and amplification condition is: 94 ℃ of 5min; 94 ℃ of 40s, Tm30s, 72 ℃ of 40s, 35cycles; 72 ℃ of 7min, pcr amplification product adds sample-loading buffer according to 5:1 with volume ratio, 95 ℃ of sex change were placed on ice in 5 minutes, application of sample detects on 8% denaturing polyacrylamide gel, in last sample, add 0.1g PBR322/Msp I Marker toward one of them well, the primer of normal amplification is to carrying out result's tape reading of result's electrophoresis on polyacrylamide gel of pcr amplification under 50-62 ℃ of annealing with the dna profiling of picking out, the selection banding pattern is clear, has at least two or more allelic primer screenings to come out.
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| CN106119404A (en) * | 2016-08-30 | 2016-11-16 | 中国科学院南海海洋研究所 | A kind of Hong Kong Concha Ostreae three base repeats primer and the screening technique of microsatellite marker |
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