CN101654671A - Elution separation method of nucleic acid hybridization molecules - Google Patents
Elution separation method of nucleic acid hybridization molecules Download PDFInfo
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Abstract
The invention discloses an elution separation method of nucleic acid hybridization molecules, relates to a separation method of nucleic acid sections, in particular to a separation method for mainly separating microsatellite DNA sections with different replication numbers and provides an elution separation method capable of improving the specificity of target nucleic acid sections, estimating melting points of hybrid molecules, separating the nucleic acid sections with different matching degrees and being compatible with the yield and the specificity of the nucleic acid hybridization molecules. The separation method comprises the following steps: hybridizing and fixing a nucleic acid capture probe containing a characteristic sequence and nucleic acid sections to be separated on a solid-phase substrate; bleaching by eluate to obtain a product, gradually improving the elution strength of the eluate, obtaining a product with higher and higher specificity; and analyzing the specificity ofproducts under different elution strengths to separately collect the products with different specificities and separate the nucleic acid sections of the same type.
Description
Technical field
The present invention relates to a kind of sorting method of nucleic acid fragment, particularly relate to a kind of being mainly used in and separate the different segmental sorting method of microsatellite DNA of multiplicity.
Background technology
In molecular biological research, often need catch the nucleic acid fragment that comprises its complementary sequence with nucleic acid probe that comprises particular sequence and the nucleic acid fragment hybridization for the treatment of sorting, and wash-out under certain conditions, to obtain qualified purpose fragment.For the nucleic acid fragment that is captured in by probe on the solid-phase matrix, under normal conditions, use the pre-wash-out of elutriant of sex change ability low (low temperature, high ionic strength) earlier, removing can not but be adsorbed on nucleic acid fragment on the solid-phase matrix by probe hybridization, uses the elutriant of sex change ability strong (high temperature, low ionic strength, alkalescence) will hybridize a wash-out recovery of nucleic acid fragment on probe again.
This method has been widely used at aspects such as mRNA purifying, subtractive hybridization, microsatellite DNA enrichments, especially for the segmental enrichment of microsatellite DNA, a lot of relevant reports is arranged both at home and abroad, mainly contains:
[1]HEIKE?HADRYS，JANNE?TIMM，BRUNO?STREIT?and?SANDRA?GIERE.A?panel?ofmicrosatellite?markers?to?study?sperm?precedence?patterns?in?the?emperor?dragonfly?Anax?imperator(Odonata：Anisoptera)[J].Molecular?Ecology?Notes，2007，7：296-298。
[2]JA?GALARZA，GF?TURNER，E?MACPHERSON，J?CARRERAS-CARBONELL?and?CRICO.Cross-amplification?of?10?new?isolated?polymorphic?microsatellite?loci?for?red?mullet(Mullus?barbatus)in?striped?red?mullet(Mullus?surmuletus)[J].Molecular?Ecology?Notes，2007，7：230-232。
[3]MASSIMILIANO?BABBUCCI，ANNA?MARIA?PAPPALARDO，VENERA?FERRITO，FEDERICA?BARBISAN，TOMASO?PATARNELLO?and?CONCETTA?TIGANO.Isolation?andcharacterization?of?eight?polymorphic?microsatellite?markers?in?Aphanius?fasciatus(Teleostei：Cyprinodontidae)[J].Molecular?Ecology?Notes，2007，7：293-295。
[4]R?UESUGI?and?MH?OSAKABE.Isolation?and?characterization?of?microsatellite?loci?in?thetwo-spotted?spider?mite，Tetranychus?urticae(Acari：Tetranychidae)[J].Molecular?Ecology?Notes，2007，7：209-292。
[5] Shen Fujun waits .Dynal enrichment with magnetic bead giant panda microsatellite marker. Acta Genetica Sinica, 2005,32 (5): 457-462.
[6] Xia Junhong, etc. the separation of the little satellite of yellowfin porgy genome. Chinese aquatic science, 2007,14 (2): 321-325.
[7] Li Qi, etc. little satellite clone's sharp separation of long oyster (Crassostrea gigas) and specificity analysis. Oceanologia et Limnologia Sinica, 2004,35 (4): 364-370.
[8] Zhou Jianlin, etc. the separation of bluish dogbean micro satellite DNA. Journal of Natural Science of Hunan Normal University, 2006,29 (1): 78-82.
[9] Liu Jun, etc. rare and endangered tree species hair toon microsatellite DNA separates and the SSR reaction system optimization. Chinese biological engineering magazine, 2006,26 (12): 50-55.
[10] Tong Guangxiang, etc. the structure and the analysis of taimen genome enriched microsatellite library. Chinese aquatic science, 2006,13 (2): 181-186.
The recovery method of the microsatellite DNA that above-mentioned research is adopted all is 3 traditional step methods: the pre-wash-out of capture probe hybridization, low strength sex change liquid and the wash-out of high strength sex change liquid reclaim.Each pre-wash-out step is different: pre-wash-out number of times from 4 times (document [4]) to 12 times (document [5]), to 68 ℃ (document [10]), (document [9] is to 3 * SSC (document [10]) from 0.06 * SSC for pre-elutriant ionic strength from 30 ℃ (document [7]) for pre-wash-out liquid temp.
The organic efficiency of above-mentioned research also has nothing in common with each other, but generally on the low side.Account for the statistics of the ratio of gross product according to specific purpose fragment, this ratio (document [1～10]) is respectively 18%, 9%, 11%, 47%, 9%, 60.4%, 20.5%, 19%, 34%, 36%, average less than 30%.And wherein the higher always pre-elutriant sex change ability of specificity ratio is strong: in document [6] and [10], the wash-out liquid temp is up to 58 ℃ with 68 ℃ in advance, and then the specificity ratio reaches 60.4% and 36%; In document [4] and [9], pre-elutriant ionic strength is low to moderate 0.1 * SSC and 0.06 * SSC, and then the specificity ratio reaches 47% and 34%.Nonspecific nucleic acid fragment is owing to can partly be remained with the probe imperfect hybridization.
Also there is the low problem of specificity in the specific purpose fragment that obtains in the above-mentioned research.Sequencing result shows, wherein the matching degree of Hen Dayibufen specific purpose fragment and probe is low, in document [4], the author uses the trapping nucleic acids probe of 30 Nucleotide of length, but the length overwhelming majority of the little satellite that obtains is less than 20 Nucleotide, the using value of this microsatellite locus is limited, mainly is because the allelotrope number is few, has limited its using value.This low specific nucleic acid fragment is owing to can be remained in a large number with the probe imperfect hybridization.
Above-mentioned research also in various degree existence repeats the problem of product.Generally speaking, in the middle of specific purpose fragment, always there be the repetition of 20% (document [4]) to the ratio of 30% (document [6]).Effectively solution is, the product break into portions that reclaims, collects specific purpose fragment respectively, just the ratio that repeats product can be dropped to 2% (document [5]).
The problem that needs to solve is:
The segmental ratio of the low and specific purpose of eluted product specificity is low, is the defective that traditional elution process exists.Because the sequence of the non-purpose nucleic acid fragment of part and purpose nucleic acid fragment is similar, thus can with the probe imperfect hybridization.These non-purpose nucleic acid fragments are the same with the purpose nucleic acid fragment, can be retained on the solid-phase matrix when pre-wash-out, and be eluted during wash-out in the second time, and the sorting of purpose nucleic acid fragment is had very big interference.Therefore, the sex change ability that improves pre-elutriant is very crucial to the specificity that improves the purpose nucleic acid fragment.But because the sequence similarity of many non-purpose nucleic acid fragments and purpose nucleic acid fragment, the fusing point of the hybrid molecule that the two and probe form is close, so in traditional elution process, the sex change ability of determining pre-elutriant is a selection of facing a difficult choice.The sex change ability is too low, can't effectively remove the non-purpose nucleic acid fragment of interferential, and the sex change ability is too high, and the purpose nucleic acid fragment is removed when pre-wash-out together.Therefore, the fusing point of the hybrid molecule of estimation different IPs acid fragment formation is extremely important to the sex change ability of determining pre-elutriant.
In addition, under a lot of situations because the purpose nucleic acid fragment itself is not single product but a big classification and probe mates fully or the nucleic acid fragment that not exclusively mates can be accepted.The nucleic acid fragment that matching degree is different need be reclaimed respectively, satisfies different using values, also helps to reduce simultaneously the ratio of repetition product.But these only rely on traditional elution process to finish.
Summary of the invention
The present invention aims to provide a kind of specificity of purpose nucleic acid fragment, the fusing point of estimation hybrid molecule, nucleic acid fragment that the sorting matching degree is different of improving, and takes into account the elution separation method of output and specific nucleic acid hybridization molecules.
The present invention includes following steps:
2) according to the characteristic sequence of the type definite kernel acid fragment of needed nucleic acid fragment, the nucleic acid capture probe;
2) trapping nucleic acids probe and the nucleic acid fragment annealing for the treatment of sorting are formed hybrid molecule, are fixed on then on the solid-phase matrix, perhaps earlier the trapping nucleic acids probe stationary on solid-phase matrix, and then with the nucleic acid fragment annealing formation hybrid molecule for the treatment of sorting;
3) earlier with the elutriant rinsing hybrid molecule of low eluting power, to obtain the nucleic acid fragment lower, improve eluting power afterwards gradually, to obtain the nucleic acid fragment higher with the probe matching degree with the probe matching degree;
4),, reclaim the elutriant in each step with the elutriant of a series of gradients of step 3) rinsing solid-phase matrix successively from minimum eluting power;
5) detect the nucleic acid fragment output of the recovery elutriant in each step, get rid of the recovery liquid of no product, in a series of recovery liquid of product were arranged, the highest eluting power was made as critical eluting power;
If the nucleic acid fragment specificity is required very high, then only collect the recovery product of going up under the critical eluting power, i.e. the specific nucleic acid fragment of mating most with probe is finished the elution separation of nucleic acid hybridization molecules;
If it is not very high that the nucleic acid fragment specificity is required, then continue following steps;
6) the recovery product with each gradient makes up the library respectively, the product of each gradient of methods analyst by order-checking of bacterium colony PCR, bacterium colony carrier or colony hybridization and the matching degree of probe, the fusing point of estimation hybrid molecule, the needed different eluting powers of nucleic acid fragment of the different matching degrees of understanding wash-out;
7) definite scope, and the scope of pairing eluting power because of the too low nucleic acid fragment that can't be suitable for of specificity, the upper bound of this eluting power is made as the lower critical eluting power;
8) reset the elutriant of a series of eluting power gradients to last critical eluting power from the lower critical eluting power, the gradient that wherein is lower than the lower critical eluting power can merge, the gradient that is higher than critical eluting power can merge, if need nucleic acid fragment be reclaimed respectively, then need to be provided with intensive gradient between lower critical eluting power and the last critical eluting power according to the height of nucleic acid fragment and nucleic acid probe matching degree; Otherwise, can merge the gradient between lower critical eluting power and the last critical eluting power;
9) capture probe that will comprise particular sequence and the nucleic acid fragment hybridization for the treatment of sorting are fixed on hybrid molecule on the solid-phase matrix then; Perhaps earlier capture probe is fixed on the solid-phase matrix, the nucleic acid fragment hybridization that to treat sorting then is on capture probe, then from the lower critical eluting power,, reclaim the elutriant in each step with the elutriant of a series of gradients of step 8) rinsing solid-phase matrix successively;
10) learn the specificity of the product of each gradient according to the result of step 6), collect the eluted product under the different gradients respectively, reach classification and sorting, be applied to different experiment demands, promptly finish the elution separation of nucleic acid hybridization molecules similar nucleic acid fragment.
In step 2) in, described solid-phase matrix can be selected from a kind of in nitrocellulose filter, cellulose acetate membrane, magnetic bead, silica gel, the silica bead etc.
In step 3), the elutriant of described low eluting power is preferably 6 * SSC under the room temperature, the described elutriant of high eluting power that improves eluting power gradually is preferably the ultrapure water under 98 ℃, the ionic strength of high eluting power is lower than the ionic strength of eluting power, perhaps the temperature of high eluting power is higher than the temperature of low eluting power, and perhaps the pH value of high eluting power is higher than the pH value of low eluting power.
In step 5), described nucleic acid fragment specificity requirement is very high to be meant that the matching degree of nucleic acid fragment and nucleic acid probe is 80%～100%.It is not that the very high matching degree of nucleic acid fragment and nucleic acid probe that is meant is less than 80% that described nucleic acid fragment specificity requires.
The mechanism that the present invention relates to is:
1) treat the nucleic acid fragment of sorting, comprise dna fragmentation and RNA fragment, and the derivative of DNA and RNA.These nucleic acid fragments can directly obtain from organism, also can make synthetic.
2) common characteristic of nucleic acid fragment on sequence for the treatment of sorting is characteristic sequence.Whether the existence of characteristic sequence is the foundation of identifying the nucleic acid fragment type, and the nucleic acid fragment with characteristic sequence is adopted, and does not have the nucleic acid fragment of characteristic sequence to adopt, and the long nucleic acid fragment of characteristic sequence preferentially adopts.For example: the polyA sequence is the characteristic sequence of mRNA, and the CA tumor-necrosis factor glycoproteins is the segmental characteristic sequence of CA/TG type microsatellite DNA, and it is the segmental characteristic sequence of GA/TC type microsatellite DNA that GA repeats.Characteristic sequence is the basis of nucleic acid capture probe.
3) nucleic acid fragment for the treatment of sorting is a single chain molecule, is duplex molecule if treat the nucleic acid fragment of sorting, must make its sex change become single chain molecule so before hybridization with 70～99 ℃ high temperature.The trapping nucleic acids probe also is a single chain molecule.Treat the hybridization of can annealing of the nucleic acid molecule of sorting and trapping nucleic acids probe under certain condition, also sex change under certain condition separates, and this is the reversible process.And the trapping nucleic acids probe can be fixed on the solid-phase matrix, and this is irreversible process.For example: can combine with the solid-phase matrix of very high avidity with the band avidin with biotin labeled trapping nucleic acids probe.
4) hybridization forms double-stranded nucleic acid hybridization molecules to two nucleic acid molecule strands according to base complementrity paired principle, and the hydrogen bond between the base maintains the avidity between the two strands.Article two, the complementary degree of nucleic acid molecule strand is high more, and the paired zone is big more, and avidity is just high more, just needs high temperature that double-stranded sex change is separated, i.e. the fusing point height of nucleic acid hybridization molecules.If but the situation that the actual characteristic sequence for the treatment of the purpose nucleic acid molecule of sorting may exist curtailment or base to change, cause and trapping nucleic acids probe complementary pairing fully, so the avidity of purpose nucleic acid molecule and trapping nucleic acids probe descends, promptly the fusing point of nucleic acid hybridization molecules is low.
5) identical nucleic acid hybridization molecules, the fusing point in different media is different.In general, improve ionic strength, the fusing point of nucleic acid hybridization molecules is improved; Improve the pH value, the fusing point of nucleic acid hybridization molecules is reduced.Under the situation of homo(io)thermism, reduce ionic strength the fusing point of nucleic acid hybridization molecules is reduced, make to form double-stranded nucleic acid hybridization molecules sex change originally and separate, and make the isolating effect of nucleic acid hybridization molecules sex change similar by improving temperature.
6) solution of low temperature, high ionic strength can make low-melting nucleic acid hybridization molecules sex change separate, and dystectic nucleic acid hybridization molecules sex change is separated, and it is low to be called eluting power; The solution of high temperature, low ionic strength can make low melting point separate with dystectic nucleic acid hybridization molecules sex change, is called the eluting power height.
7) will treat that the purpose nucleic acid fragment of sorting and probe annealing form hybrid molecule.Wherein some nucleic acid fragment and probe matching degree are lower, and avidity is also low, and the hybrid molecule fusing point of formation is also low, only need the solution of low eluting power just can sex change to separate; And some nucleic acid fragment and probe matching degree are higher, and avidity is also high, and the hybrid molecule fusing point of formation is also high, need the solution of high eluting power just can sex change to separate.The scope of the matching degree of nucleic acid fragment can contain from the interval that is lower than 5% to 100%.
8) for the mixture of the different nucleic acid hybridization molecules of fusing point.With the solution rinsing of low eluting power, low-melting nucleic acid hybridization molecules sex change is separated earlier, the complementary degree of the nucleic acid molecule strand of acquisition is low, and specificity is low; Use the solution rinsing of high eluting power again, can make dystectic nucleic acid hybridization molecules sex change separate the complementary degree height of the nucleic acid molecule strand of acquisition, specificity height.Improve eluting power gradually, the specificity of the nucleic acid molecule strand of acquisition also improves thereupon gradually.
9) for the trapping nucleic acids probe of certain-length, the fusing point of the nucleic acid hybridization molecules of formation has a upper limit, is exactly the fusing point of the nucleic acid hybridization molecules of 100% complementary pairing.When eluting power reached this fusing point, all nucleic acid hybridization molecules can separate in sex change, continued to improve eluting power and can not obtain new nucleic acid strand.
It is segmental preferred that the elution separation method of nucleic acid hybridization molecules of the present invention is mainly used in specific nucleic acid, the fusing point of the nucleic acid fragment of different matching degrees and the estimation of eluting power, and the sorting of the nucleic acid fragment of different matching degrees.
The elution separation method of nucleic acid hybridization molecules of the present invention is applied to the segmental enrichment of the little satellite of genomic dna, obtains a series of microsatellite DNA fragment.Different elution requirements can obtain the product of different matching degrees down, for repeating with the short-movie section is for little satellite hybrid molecule of probe sequence, higher matching degree means little satellite fragment that multiplicity is more, and the polymorphism of the microsatellite marker that little satellite fragment that multiplicity is many more can be developed is also high more.The microsatellite DNA fragment that obtains under the highest eluting power of gradient elution method has very high multiplicity, is applicable to the exploitation at highly polymorphic little satellite seat; Eluting power is low more, and the segmental multiplicity of the microsatellite DNA of acquisition is just few more, but output is high more; According to the analytical results of eluted product, learn the needed eluting power of microsatellite DNA fragment that the sorting multiplicity is different, design is more simplified and more accurate eluting power gradient; From the product of different gradients, collect the different microsatellite DNA fragment of multiplicity, can be applied to different purposes, reach the effect of classification and sorting.
Embodiment
Elect example as with the segmental branch of TG type microsatellite DNA, the implementation step of the elution separation method of nucleic acid hybridization molecules is:
1) hybridization.Sau3AI digests genomic dna with restriction enzyme, and connecting corresponding AFLP joint becomes the AFLP fragment.In the system of 50 μ L, with the AFLP fragment of 300ng and 5 ' the end band DNA probe labeled with biotin hybridization of 15pmol.This probe sequence is: 5 '-ATACACAC ACACACACAC ACACACACAC ACACACACACACACACACAC-3 ', and to catch the AFLP fragment that contains the TG tumor-necrosis factor glycoproteins.Hybridization is carried out in thermal cycler, and program is: 95 ℃ keep 10min down, and 0.3 ℃ of every min cooling is till 37 ℃.The hybridization product reclaims the test kit purifying with DNA, removes unnecessary probe.
2) catch.In the system of 100 μ L, the bag of hybridization product, 6 * SSC, 0.1%SDS, 0.01mg that contains step 1) is by the magnetic bead of streptavidin.43 ℃ of following hatching 30min, during vibration frequently prevent the magnetic bead cohesion of sinking.
3) magnetic bead rinsing., repeat 1 time at 60 ℃ of following rinsing magnetic bead 10min with the rinsing liquid 500 μ L of 6 * SSC, 0.1%SDS; , repeat 1 time at 60 ℃ of following rinsing magnetic bead 10min with the rinsing liquid 500 μ L of 1.5 * SSC, 0.1%SDS; With the rinsing liquid 500 μ L of 6 * SSC, no SDS rinsing magnetic bead 1min at room temperature, repeat 3 times.
4) the segmental gradient elution of purpose.At 64 ℃ of following rinsing magnetic bead 10min, collect elutriant with the elutriant 200 μ L of 1 * SSC; At 64 ℃ of following rinsing magnetic bead 10min, collect elutriant with the elutriant 200 μ L of 0.5 * SSC; At 64 ℃ of following rinsing magnetic bead 10min, collect elutriant with the elutriant 200 μ L of 0.2 * SSC; At 64 ℃ of following rinsing magnetic bead 10min, collect elutriant with the elutriant 200 μ L of 0.1 * SSC; At 64 ℃ of following rinsing magnetic bead 10min, collect elutriant with ultrapure water 200 μ L; At 69 ℃ of following rinsing magnetic bead 5min, collect elutriant with ultrapure water 200 μ L; At 74 ℃ of following rinsing magnetic bead 5min, collect elutriant with ultrapure water 200 μ L; At 79 ℃ of following rinsing magnetic bead 5min, collect elutriant with ultrapure water 200 μ L; At 84 ℃ of following rinsing magnetic bead 5min, collect elutriant with ultrapure water 200 μ L; At 89 ℃ of following rinsing magnetic bead 5min, collect elutriant with ultrapure water 200 μ L; At 94 ℃ of following rinsing magnetic bead 5min, collect elutriant with ultrapure water 200 μ L; At 98 ℃ of following rinsing magnetic bead 5min, collect elutriant with ultrapure water 200 μ L.
5) detection of recovery product.Each gradient is respectively got the recovery elutriant of 1 μ L and is done template, according to AFLP joint design primer, carries out pcr amplification, electrophoresis detection PCR product.1 * SSC, 0.5 * SSC, these 4 gradient of 0.2 * SSC, 0.1 * SSC have product, and electrophoresis detection shows that the PCR product is the DNA band of disperse, and the ultrapure water elutriant of 64 ℃ and above each temperature does not have product, and electrophoresis detection does not have band.
6) optimum segmental screening.Get rid of the gradient of no product, 0.1 * SSC is the highest eluting power.Reclaim the PCR product under 0.1 * SSC eluting power, connect the pMD19-T carrier, transform, be prepared into the enriched library of base series TG tumor-necrosis factor glycoproteins.Be PCR and detect the library with arrange in pairs or groups the respectively primer (5 '-TGTGTGTGTGTGTGTGTGTG-3 ') of characteristic sequence of the little satellite of CA/TG type of the universal primer M13/-47 of pMD19 carrier sequence and M13/-48, find that 100% bacterium colony contains the TG tumor-necrosis factor glycoproteins, shows as the very strong positive.Simultaneously, the result of carrier order-checking confirms that all bacterium colonies all contain the TG tumor-necrosis factor glycoproteins, and the TG multiplicity is much in 23 times.In the product that non-gradient elution method obtains, really contain the TG multiple less than 50%, wherein major part is again because multiplicity possesses the value of exploitation microsatellite marker very little and not, with respect to similar non-gradient elution method, the gradient elution method that the present invention uses shows high specificity.
7) the fusing point analysis of hybrid molecule.The detected result of step 5) shows: base series A C repeats 23 times probe and the 100% coupling hybrid molecule that base series TG repeats 23 times and above dna fragmentation forms, about 64 ℃ of the fusing point under 0.1 * SSC eluting power.This eluting power is last critical eluting power, can obtain the most perfect product.Under 64 ℃ ultrapure water and the above higher eluting power, probe is sex change fully, can't obtain product.Under 64 ℃ 0.2 * SSC and the lower eluting power, the product of acquisition is that TG repeats to be lower than 23 times and the dna fragmentation of probe imperfect hybridization, confirms this point by library scanning.Wherein under the eluting power of 0.2 * SSC of 64 ℃, the TG of the product of acquisition repeats more than 10 times.And under the eluting power of 0.5 * SSC of 64 ℃ and 1 * SSC, the TG of the product of acquisition repeats a lot of less thaies 10 times.So, 0.5 * SSC of 64 ℃ are made as the lower critical eluting power.Therefore, 64 ℃ ultrapure water and above higher eluting power gradient can be cancelled or merge; The following eluting power gradient of 0.5 * SSC of 64 ℃ can merge; Can be divided into more intensive gradient below the eluting power of 0.1 * SSC of 64 ℃, scan the needed different eluting powers of product of learning different matching degrees, satisfy meticulousr analysis and sorting requirement by the library.
8) classification and sorting of similar dna fragmentation.Interpretation of result according to the scanning of step 7) library, under the eluting power of 0.2 * SSC of 64 ℃, though containing the TG multiplicity, product is lower than 23, but can satisfy the requirement that microsatellite locus is developed, therefore, product under the eluting power of 0.1 * SSC of the eluting power of 0.2 * SSC of 64 ℃ and 64 ℃ is respectively applied for and makes up the library, develops different microsatellite locus.Though this step make can not 100% and the probe coupling have still but very that the nucleic acid fragment of high value is adopted, with respect to step 6), this step is farthest improving output basic the assurance under the specific prerequisite of product.With respect to similar non-gradient elution method, the gradient elution sorting method that the present invention uses makes the similarity degree of different nucleic acid fragments according to its characteristic sequence and probe sequence, reclaimed by different gradient elutions, realization has been alleviated the puzzlement that repeated cloning causes in the library to a certain extent to the sorting of similar nucleic acid fragment.If sorting is had higher requirement, can be between 0.1 * SSC and 0.2 * SSC, and insert more eluting power gradient between 0.2 * SSC and the 0.5 * SSC, realize meticulousr sorting.
Below provide the application example of the elution separation method of nucleic acid hybridization molecules.
(1) the segmental sorting of leopard line gill sour jujube perch genome TG type microsatellite DNA.Adopt 1 * SSC, 0.5 * SSC, 0.1 * SSC, 4 gradient of ultrapure water in the time of 64 ℃, per 5 ℃ are provided with a ultrapure water gradient more than 64 ℃, up to 98 ℃.The PCR detected result shows that 1 * SSC, 0.5 * SSC, these 3 gradient of 0.1 * SSC have product, does not have product in the ultrapure water elutriant of 64 ℃ and above each temperature.Be prepared into the enriched library of TG tumor-necrosis factor glycoproteins with 0.1 * SSC gradient product, 95% bacterium colony contains the TG tumor-necrosis factor glycoproteins in the library, and the result of carrier order-checking confirms that the TG multiplicity is much in 16 times.Be prepared into the enriched library of TG tumor-necrosis factor glycoproteins with the mix products of the gradient of 0.5 * SSC and 1 * SSC, the bacterium colony less than 30% in the library contains the TG tumor-necrosis factor glycoproteins, and the result of carrier order-checking confirms that the TG multiplicity is many below 12 times.The microsatellite sequence of the enriched library of 0.1 * SSC is developed as molecule marker, is applied to the research of leopard line gill sour jujube perch population genetics.
(2) adjustment of the fusing point analysis of hybrid molecule and elutriant gradient.Learn according to the segmental separation results of leopard line gill sour jujube perch genome TG type microsatellite DNA: in the time of 64 ℃, the 100% coupling hybrid molecule that probe that the AC repetition is 23 times and TG repeat 23 times and above dna fragmentation forms can not unwind in 0.5 * SSC fully, can unwind fully in 0.1 * SSC.This is convenient to adjust the setting of elutriant gradient: add the elutriant gradient of a 0.2 * SSC between 0.5 * SSC and 0.1 * SSC, merge each ultrapure water gradient more than 64 ℃.
(3) the segmental sorting of striped mottled bamboo shark genome TG type microsatellite DNA.Adjust the setting of elutriant gradient according to the segmental separation results of leopard line gill sour jujube perch genome TG type microsatellite DNA: adopt 1 * SSC, 0.5 * SSC, 4 gradient of 0.2 * SSC, 0.1 * SSC in the time of 64 ℃, one 98 ℃ ultrapure water gradient is set more than 64 ℃.The PCR detected result shows that 1 * SSC, 0.5 * SSC, these 4 gradient of 0.2 * SSC, 0.1 * SSC have product, and do not have product in the ultrapure water elutriant.Be prepared into the enriched library of TG tumor-necrosis factor glycoproteins with 0.1 * SSC gradient product, 100% bacterium colony contains the TG tumor-necrosis factor glycoproteins in the library, and the result of carrier order-checking confirms that the TG multiplicity is much in 22 times.Be prepared into the enriched library of TG tumor-necrosis factor glycoproteins with 0.2 * SSC gradient product, 85% bacterium colony contains the TG tumor-necrosis factor glycoproteins in the library, and the result of carrier order-checking confirms that the TG multiplicity is many between 10 to 22 times.Be prepared into the enriched library of TG tumor-necrosis factor glycoproteins with the mix products of the gradient of 0.5 * SSC and 1 * SSC, the bacterium colony less than 30% in the library contains the TG tumor-necrosis factor glycoproteins, and the result of carrier order-checking confirms that the TG multiplicity is many below 12 times.Microsatellite sequence with the enriched library of 0.1 * SSC and 0.2 * SSC is developed as molecule marker respectively, is applied to the research of striped mottled bamboo shark population genetics.
Claims (6)
1. the elution separation method of nucleic acid hybridization molecules is characterized in that may further comprise the steps:
1) according to the characteristic sequence of the type definite kernel acid fragment of needed nucleic acid fragment, the nucleic acid capture probe;
2) trapping nucleic acids probe and the nucleic acid fragment annealing for the treatment of sorting are formed hybrid molecule, are fixed on then on the solid-phase matrix, perhaps earlier the trapping nucleic acids probe stationary on solid-phase matrix, and then with the nucleic acid fragment annealing formation hybrid molecule for the treatment of sorting;
3) earlier with the elutriant rinsing hybrid molecule of low eluting power, to obtain the nucleic acid fragment lower, improve eluting power afterwards gradually, to obtain the nucleic acid fragment higher with the probe matching degree with the probe matching degree;
4),, reclaim the elutriant in each step with the elutriant of a series of gradients of step 3) rinsing solid-phase matrix successively from minimum eluting power;
5) detect the nucleic acid fragment output of the recovery elutriant in each step, get rid of the recovery liquid of no product, in a series of recovery liquid of product were arranged, the highest eluting power was made as critical eluting power;
If the nucleic acid fragment specificity is required very high, then only collect the recovery product of going up under the critical eluting power, i.e. the specific nucleic acid fragment of mating most with probe is finished the elution separation of nucleic acid hybridization molecules;
If it is not very high that the nucleic acid fragment specificity is required, then continue following steps;
6) the recovery product with each gradient makes up the library respectively, the product of each gradient of methods analyst by order-checking of bacterium colony PCR, bacterium colony carrier or colony hybridization and the matching degree of probe, the fusing point of estimation hybrid molecule, the needed different eluting powers of nucleic acid fragment of the different matching degrees of understanding wash-out;
7) definite scope, and the scope of pairing eluting power because of the too low nucleic acid fragment that can't be suitable for of specificity, the upper bound of this eluting power is made as the lower critical eluting power;
8) reset the elutriant of a series of eluting power gradients to last critical eluting power from the lower critical eluting power, the gradient that wherein is lower than the lower critical eluting power merges, the gradient that is higher than critical eluting power merges, if need nucleic acid fragment be reclaimed respectively, then need to be provided with intensive gradient between lower critical eluting power and the last critical eluting power according to the height of nucleic acid fragment and nucleic acid probe matching degree; Otherwise, merge the gradient between lower critical eluting power and the last critical eluting power;
9) capture probe that will comprise particular sequence and the nucleic acid fragment hybridization for the treatment of sorting are fixed on hybrid molecule on the solid-phase matrix then; Perhaps earlier capture probe is fixed on the solid-phase matrix, the nucleic acid fragment hybridization that to treat sorting then is on capture probe, then from the lower critical eluting power,, reclaim the elutriant in each step with the elutriant of a series of gradients of step 8) rinsing solid-phase matrix successively;
10) learn the specificity of the product of each gradient according to the result of step 6), collect the eluted product under the different gradients respectively, reach classification and sorting, be applied to different experiment demands, promptly finish the elution separation of nucleic acid hybridization molecules similar nucleic acid fragment.
2. the elution separation method of nucleic acid hybridization molecules as claimed in claim 1 is characterized in that in step 2) in, described solid-phase matrix is selected from a kind of in nitrocellulose filter, cellulose acetate membrane, magnetic bead, silica gel, the silica bead.
3. the elution separation method of nucleic acid hybridization molecules as claimed in claim 1, it is characterized in that in step 3), the elutriant of described low eluting power is 6 * SSC under the room temperature, and the described elutriant of high eluting power that improves eluting power gradually is the ultrapure water under 98 ℃.
4. the elution separation method of nucleic acid hybridization molecules as claimed in claim 1, it is characterized in that in step 3), the ionic strength of high eluting power is lower than the ionic strength of eluting power, perhaps the temperature of high eluting power is higher than the temperature of low eluting power, and perhaps the pH value of high eluting power is higher than the pH value of low eluting power.
5. the elution separation method of nucleic acid hybridization molecules as claimed in claim 1 is characterized in that in step 5), and described nucleic acid fragment specificity requirement is very high to be meant that the matching degree of nucleic acid fragment and nucleic acid probe is 80%～100%.
6. the elution separation method of nucleic acid hybridization molecules as claimed in claim 1 is characterized in that in step 5), and it is not that the very high matching degree of nucleic acid fragment and nucleic acid probe that is meant is less than 80% that described nucleic acid fragment specificity requires.
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| CN103757107A (en) * | 2014-01-08 | 2014-04-30 | 江南大学 | Detection probe of bacillus strains |
| CN107893067A (en) * | 2016-09-29 | 2018-04-10 | 北京茗天基因科技有限公司 | The method based on solid phase carrier film enrichment target nucleic acid for high-flux sequence |
| CN109913539A (en) * | 2017-12-13 | 2019-06-21 | 浙江大学 | A kind of targeted capture HLA gene order and the method being sequenced |
| CN114717326A (en) * | 2022-03-10 | 2022-07-08 | 中国科学院南海海洋研究所 | SSR (simple sequence repeat) marker of plectropomus leopardus as well as amplification primer and application of SSR marker |
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- 2009-08-07 CN CN2009101123525A patent/CN101654671B/en not_active Expired - Fee Related
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103757107A (en) * | 2014-01-08 | 2014-04-30 | 江南大学 | Detection probe of bacillus strains |
| CN103757107B (en) * | 2014-01-08 | 2017-01-11 | 江南大学 | Detection probe of bacillus strains |
| CN107893067A (en) * | 2016-09-29 | 2018-04-10 | 北京茗天基因科技有限公司 | The method based on solid phase carrier film enrichment target nucleic acid for high-flux sequence |
| CN109913539A (en) * | 2017-12-13 | 2019-06-21 | 浙江大学 | A kind of targeted capture HLA gene order and the method being sequenced |
| CN114717326A (en) * | 2022-03-10 | 2022-07-08 | 中国科学院南海海洋研究所 | SSR (simple sequence repeat) marker of plectropomus leopardus as well as amplification primer and application of SSR marker |
| CN114717326B (en) * | 2022-03-10 | 2024-03-19 | 中国科学院南海海洋研究所 | SSR (simple sequence repeat) marker of Perch gill and amplification primer and application thereof |
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