CN106754292B - The single-stranded separators of DNA and separation method for pyrosequencing - Google Patents
The single-stranded separators of DNA and separation method for pyrosequencing Download PDFInfo
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Abstract
The invention discloses a kind of single-stranded separators of the DNA for pyrosequencing and separation method, the separator is the hollow filter post that filter membrane is provided with one, it is connected with that the DNA of affine connector is single-stranded to be separated after filtering through setting filter membrane in the separator, wherein the diameter of affine connector is more than the membrane filtration aperture.The separation method and separator that the present invention is provided enormously simplify device configuration, reduce equipment totle drilling cost, are effectively simplified operating procedure, shorten the operating time, reduce working strength, improve operating efficiency；The quality and efficiency separated is not only ensure that using the Filter column supporting with conventional centrifugal pipe, also causes separation process simply easily to realize that the filter membrane of Filter column is separated by the way that physics mode is single-stranded to DNA, difficulty and the condition requirement of separation is reduced.
Description
Technical field
The present invention relates to a kind of single-stranded separators of the DNA for pyrosequencing and separation method, belong to biotechnology neck
Domain.
Background technology
First, pyrosequencing techniques overview
Pyrosequencing techniques (Pyrosequencing) be grew up in 1987, based in DNA building-up processes
The sequencing technologies of pyrophosphoric acid (PPi) detection of release, pyrosequencing is reacted under a series of catalytic action of enzymes, its process
It is middle to produce the visible ray proportional to the aggregate number of deoxynucleotide triphosphates (dNTP), by can reach to visible detection
Determine the purpose of DNA sequence dna.However, pyrosequencing method needs to prepare the single-stranded masterplates of a certain amount of DNA, and masterplate DNA is single-stranded
Separating rate, purity, the complexity of separative efficiency and separation method affect invariably pyrosequencing it is ageing, can
The accuracy of operability and sequencing result, the cost of the single-stranded separation of masterplate DNA is also directly related to whole pyrosequencing
Cost, therefore, isolation technics single-stranded masterplate DNA have very important status during pyrosequencing, are related to Jiao
The popularization of phosphoric acid sequencing technologies and use, there is ineffable meaning for the development of pyrosequencing techniques.
2nd, the single-stranded isolation technics overviews of DNA
The single-stranded isolation technics of DNA is one of most common isolation technics in biomedical sector, it is adaptable to different nucleic acid samples
The various scale sequencings of DNA of product and probe device, are widely used in biology, medicine and pharmacology, preventive medicine, zoology and botany, agriculture
Animal husbandry, food and the field such as health, the energy and chemical industry, environmental monitoring and medical diagnosis and detection.In addition, suction single-stranded DNA
It is attached, extract with isolation technics water quality, water source, biomaterial, biological fluid (such as blood, serum, blood plasma, cerebrospinal fluid, urine,
Tear, sweat, digestive juice, seminal fluid, juice, tissue fluid, vomitus, excrement), tissue/cell and microbial lytic liquid, difference
Analysis detection, separation and the purifying of the biologies such as albumen, the nucleic acid in source, chemical molecular and medicine etc. and oligonucleotides, many
It is widely used in terms of the synthesis of peptide, lead compound and medicine, it is closely bound up with daily life, in biological medicine
Field has very important status.
The single-stranded separation methods of DNA and the deficiency of presence commonly used in biomedicine field are as follows：
1. thermal denaturation or alkali process.This kind of method is mainly heated double stranded PCR products or alkali process, due to DNA
Double-strand hydrogen bond under high temperature or a certain degree of alkaline environment can be broken so that DNA is changed into single-stranded.Although this kind of Method And Principle can
OK, it is simple to operate, but this kind of method is due to its separation rate and purity is relatively low and the gradually purifying not as single stranded DNA, is used for
DNA double-strand separation.
2.T7 reverse transcription methods.This kind of method is to add T7 promoters at an end of PCR primer 5 ', to purify PCR amplification productions
Thing is template, with the synthesizing single-stranded RNA of the external reverse transcription of t7 rna polymerase (Hughes, et.al., Nat.Biotechnol.,
2001,19：342-347).Although this kind of method principle is feasible, the single-stranded separation rates of DNA are higher, and obtain the single-stranded purity of DNA compared with
Height, but whole separation process need to be divided into two big steps completions, and operation is inconvenient, and the time is longer, and need to strictly control the dirt of RNase
Dye, therefore with certain limitation.
(Higuchi and Ochman, Nucleic.Acids Res., 1989,17 3. Exonucleolytic enzyme process：5865).By
It is phosphorylated in a PCR primer, PCR primer is when using exonuclease digestion, and the primer amplification chain being phosphorylated is not cut
Cut, enzyme is heated inactivation after digestion.This kind of method also needs purified pcr product, and separable programming is tediously long, and operation is also inconvenient, and
And the single-stranded pick-up rates of DNA depend on the activity of excision enzyme, uncontrollable factor is too strong, and the stability of experimental result is inadequate；Therefore, should
The implementation rate of the method for kind is not wide, and versatility is not high.
4. denaturing high-performance liquid chromatography (denaturing high-performance liquid
Chromatography, DHPLC).Under conditions of partial denaturation, pass through heterozygosis and zygoid retention time in post
Difference, finds DNA mutation.Allogeneic dna sequence DNA double-strand is different from the melting properties of homologous dna double-strand, heterologous under the conditions of partial denaturation
Double-strand is because having the presence of mispairing district and more mutability, and retention time in the chromatography column is shorter than homoduplex, therefore is first eluted down
Come, bimodal or multimodal elution curve is shown as in chromatogram.Because a PCR primer is by biotin labeling, its PCR amplifications
Chain in DHPLC, can and another common chain separate (Dickman and Hornby, Anal.Biochem., 2000,284：
164-167).DNA needed for this method can directly be obtained in 15min from double stranded PCR products is single-stranded, but this kind of method
Implement to need supporting sufficiently expensive instrument, therefore be difficult to popularize all the time.
5. magnetic capture method.The surface of superparamagnetic nano particle is improved with nanometer technology and surface modification
Afterwards, it is prepared into superparamagnetism silica nanometer magnetic bead.The magnetic bead can specifically be recognized with nucleic acid molecules on micro interface and
Efficiently combine.Using the superparamagnetism of silica nanoparticle, in Chaotropic salt (guanidine hydrochloride, guanidinium isothiocyanate etc.) and outside
Plus in the presence of magnetic field, can be separated from the DNA and RNA in blood, animal tissue, food, pathogenic microorganism equal samples, so
Target to be obtained with NaOH processing single-stranded afterwards.This kind of method is simple to operate, the used time is short, and whole flow of extracting is divided into four steps, mostly may be used
To be completed in 36-40 minutes, and safety non-toxic, without using toxic reagents such as the benzene in conventional method, chloroforms, to experimental implementation
The injury of personnel is reduced, and meets modern environmental protection concept, and the magnetic bead specific binding single-stranded with DNA make it that the DNA extracted is single-stranded pure
Degree is high, concentration is big, but the coating magnetic bead used in this kind of method is costly, and needs, by magnetic frame separation, not only to separate
Cost is higher, also inconvenience, therefore limits the popularization of the technology to a certain extent.
6. asymmetric PCR.Above method is both needed to after PCR carry out extra processing, and asymmetric PCR can be expanded in PCR
While prepare DNA it is single-stranded.Conventional asymmetric PCR is normally expanded using the primer of two inequalities in the circulation of beginning
Increase.With the increase of circulation, measure few primer and gradually exhausted, and can to continue straight line amplification generation DNA single-stranded for the primer of excess
(Gyllensten and Erlich,Proc.Natl.Acad.Sci.U.S.A.,1988,85：7652-7656).This kind of method
With higher hybridization sensitivity and specificity, and ease-to-operate is stronger, but the ratio of its primer needs optimization, and non-spy
The chance increase of different amplification, in addition, the single-stranded separation processes of DNA need to rely on electrophoresis, separable programming is complicated, and electrophoresis is often visible
Smear, it is time-consuming and is not obvious.
Above-mentioned several separation methods are respectively provided with certain limitation, therefore, in order to meet to the operable of the single-stranded separation of DNA
Property and cost-effectiveness requirement, DNA of the prior art is single-stranded to use integrated extraction work station, by with streptavidin
Affine connector is combined with DNA double chain, and work station has suction filtration pin and supporting pump, and the affine connectors of DNA with reference to after pass through
Suction filtration absorption filter membrane bottom in suction filtration pin, work station is furnished with track and related system, and suction filtration pin is moved to Sheng by suction filtration after terminating
Have in NaOH disk, by alkali process solution double helix, it is single-stranded to obtain DNA, clean collect after suction filtration again.General 24 of suction filtration pin
(4*6) is one group, and the sample or reagent of sufficient amount are must assure that when using to ensure the normal operation of work station, therefore this
Collection mode single-stranded DNA is very dumb, can only add work station with fixed amount and be operated, and substantial amounts of loss is more
Produced in secondary suction filtration and transfer process, this is very unfavorable to micro-collection, and suction filtration pin group needs to be operated simultaneously, this
All there is certain volume requirement to each part of work station, whole work station space-consuming is very big.Huge system causes in DNA
In single-stranded separation operation process, micro- splitter needs liquid relief back and forth, and operation is very cumbersome, not only separation cycle length, efficiency
It is low, and integral device is expensive, causes the costly of the single-stranded separation of DNA, in addition it is also necessary to substantial amounts of reagent and other resources are expended,
It is extremely uneconomical.In addition, suction filtration pin is metal material in the work station, and it is expensive, re-used after often handling, therefore easily
Cause the cross pollution between residue, reliability is not high, the accuracy to separation and testing result can cause certain do
Disturb and influence.And it is adherent that portion of residual solution is had during solution extraction so that a certain amount of target DNA is single-stranded can not be micro-
Splitter is adsorbed, and is caused the DNA proportion of single chain reduction obtained, be have impact on separation rate, cause waste.Therefore, for pyrophosphoric acid
The single-stranded separation problems of DNA of the high quality and high efficiency of sequencing are urgently to be resolved hurrily.
The content of the invention
In view of the above-mentioned problems existing in the prior art, the present invention will be filtered for single-stranded pass through of the DNA of pyrosequencing
The mode of centrifugation is extracted, and the economization miniaturization for realizing a sample one collection is collected and efficiently low damaged there is provided a kind of
The single-stranded separation methods of DNA and device that are rapidly used for pyrophosphoric acid lost.
For achieving the above object, the technical solution adopted by the present invention is as follows：
The single-stranded separators of a kind of DNA for pyrosequencing, it is characterised in that the separator is that filter membrane is provided with one
Hollow filter post, be connected with that the DNA of affine connector is single-stranded to be separated after filtering through setting filter membrane in the separator, wherein parent
It is more than the membrane filtration aperture with the diameter of connector.In other words, the single stranded DNA after separation is in Filter column, affine
Connector may be selected it is any in the prior art can connect DNA microballoon, will not be described here.
Preferably, the filter membrane of hollow filter post is located at one end of Filter column.In other words, filter membrane constitutes the bottom of Filter column
End, the filtering surface of the single-stranded separators of DNA is located at splitter bottom, that is to say, that the single stranded DNA after separation is on filter membrane.
It is highly preferred that filter membrane diameter is more than the filtering surface diameter of Filter column, filter membrane completely encloses filtering surface, in Filter column
The interior filter cavity for forming a closing, is easy to stay in the single stranded DNA for being connected with affine connector to be collected above filter membrane.
As a preferred embodiment, filtering column bottom is provided inwardly with a circle boss along cylinder edge, filter membrane is fixed
Boss side is placed in, boss can play a part of fixed filter membrane position, and when filter membrane is built in Filter column, filter membrane is placed in boss
On, boss provides support and spacing for filter membrane, and when being wrapped in Filter column bottom outside filter membrane, boss plays position-limiting action.
Preferably, filter membrane is located at the non-end face of Filter column, and in other words, filter membrane is placed in any in addition to two ends in Filter column
Position.
It is highly preferred that in filter membrane in the middle part of Filter column, Filter column axle two ends centered on filter membrane are mirrored into being symmetrical arranged,
In other words, filter membrane is on the plane of symmetry of Filter column.
As a preferred embodiment, Filter column two ends are successively decreased setting towards filter membrane diameter, in other words, Filter column is whole
Section is hour-glass in shape on shape, and the diameter of Filter column is uniformly successively decreased by a diameter of of end face to the plane of symmetry.
In another preferred mode, filtering surface diameter and the Filter column caliber of Filter column, as long as ensureing filtering surface
Diameter and the diameter of Filter column port it is inconsistent, it is either large or small, when filtering surface diameter be less than Filter column port it is straight
Footpath, and be not uniformly successively decrease when, preferred filtration channel section is in dumbbell shaped or hourglass-shaped.
Another goal of the invention of the present invention is to provide a kind of using the single-stranded separation of the above-mentioned DNA for pyrosequencing
The single-stranded separators of DNA of device, collecting pipe is that one end open is closed in one end, and Filter column is in collecting pipe, and Filter column and receipts
The blind end bottom surface of collector is not fitted, and the cavity in collecting pipe in addition to Filter column is easy in separation process collect liquid.
Preferably, the end diameter of collecting pipe successively decreases, and end face is closed into dome shape；Centrifuge tube of the prior art can be with
Use, be not limited herein as the collecting pipe in the present invention.
Preferably, collecting pipe is provided with a lid, and the cap-shaped shape matches with collecting pipe opening shape and closes hollow filter pipe
End face, capping is easy to ensure that liquid does not leak outside in separation process, it is to avoid unnecessary loss extraneous splash droplet simultaneously
It cannot be introduced into that Filter column is single-stranded to DNA to be separated to be polluted Deng impurity；DNA after collection is single-stranded in collecting pipe, and covering tightly can
Directly to preserve, convenient use.
As a preferred embodiment, the external diameter of Filter column is equal to the internal diameter of collecting pipe, it can be made by frictional force
Filter post is maintained at a relatively steady state with collecting pipe, it is not necessary to which plus structural fixes for Filter column.
Under another embodiment, the external diameter of Filter column is less than the internal diameter of collecting pipe, it is necessary to regather the mouth of pipe inner edge of pipe
Provided with a boss or other similar structures, it is easy to provide support for Filter column, Filter column is fixed in collecting pipe.
The single-stranded separators of DNA in the present invention can coordinate collecting pipe to make the single-stranded separators of DNA to use, can also set on a large scale
It is placed in pyrosequencing instrument, is used as the single-stranded separation of DNA.
Single stranded DNA after separation is to be connected with the single-stranded of affinity body, it is necessary to which this chain can be washed to filter membrane on filter membrane
It is de-, another complementary strand without affine connector is contained in filtrate, it is necessary to can enter to the single stranded DNA in filtrate during backward sequencing
Row is collected.When needing to collect a chain of the band affine connector on filter membrane, collecting pipe can use the collecting pipe reclaimed,
Now collecting pipe only plays a part of Recycling of waste liquid, and reclaiming to use reduces cost, and environmental protection reduces the generation of white pollution.
Third object of the present invention is that providing one kind uses the single-stranded separators of above-mentioned DNA or the single-stranded separators of DNA
The single-stranded separation methods of the DNA for pyrosequencing, wherein in DNA double chain a chain is connected with affine connector, solves spiral shell
Supination is separated by filtration through the single-stranded separators of DNA or the single-stranded separators of DNA, and the DNA for then obtaining being connected with affine connector is mono-
Chain.
It is preferred that, when being filtered using the above-mentioned single-stranded separators of DNA, filtering, collecting pipe pipe are completed with unification centrifuge
Footpath matches the centrifuge.
Preferably, when the single-stranded separators of DNA using filter membrane in Filter column bottom are separated, due to the DNA of use
The filter membrane of single-stranded separator only has a reaction chamber in Filter column bottom in the single-stranded separators of DNA, each step reaction exists
Carried out in separator, therefore the separation method used is specific as follows：
With reference to：DNA double chain fragment and appropriate affine connector are added in single-stranded separator, by the DNA pieces after amplification
Section is combined with affine connector；
Alkaline hydrolysis：Highly basic is added in DNA fragmentation after bonding, DNA double spiral is untied using highly basic, is obtained with affine connection
The DNA of body is single-stranded and its mixed liquor of complementary strand；
Double-strand is separated：Double-strand is separated, the DNA for being connected with affine connector is single-stranded and its mixed liquor of complementary strand is through filtering
Film is separated, after separation with affine connector DNA it is single-stranded stays on film, complementary strand by filter membrane, washed after filtering to
Less once；
Collect：Keep the filtering direction of the single-stranded separators of DNA constant, add in eluent to Filter column, suctioned out after piping and druming
DNA is single-stranded, is now located at because the DNA with affine connector is single-stranded on film, adds after eluent and stands, and gently piping and druming makes DNA
Single-stranded to suspend, sucking liquid is that to obtain DNA single-stranded.
As another preferred embodiment, when not separated using filter membrane in the Filter column of bottom, due to filter
Film in the both sides of film not there are two cavitys to be used as reaction chamber, the separation of use in the end face of Filter column, therefore Filter column
Method is specific as follows：
With reference to：DNA double chain fragment and appropriate affine connector are added in single-stranded separator, by the DNA pieces after amplification
Section is combined with affine connector；
Alkaline hydrolysis：Highly basic is added in DNA fragmentation after bonding, DNA double spiral is untied using highly basic, is obtained with affine connection
The DNA of body is single-stranded and its mixed liquor of complementary strand；
Double-strand is separated：Double-strand is separated, the DNA for being connected with affine connector is single-stranded and its mixed liquor of complementary strand is through filtering
Film is separated, after separation with affine connector DNA it is single-stranded stays on film, complementary strand by filter membrane, washed after filtering to
Less once；
Collect：By 180 ° of single-stranded separator rotations of DNA, add after eluent that to collect DNA single-stranded；With affine connector
Stayed in during the single-stranded separation of DNA on filter membrane, after Filter column two ends are exchanged, the single-stranded reversions of DNA are located under filter membrane, elution can directly by
DNA is single-stranded to be rinsed from filter membrane, is collected to collection device.It is only capable of drawing the mode collected in bottom different from filter membrane,
The elution process collection rate that filter membrane is exchanged behind direction is high, and membrane filter is put during two ends are symmetrical and easily facilitates exchange and separation receipts
Collect work, it may have the broader instrument scope of application.
This method focuses on to protect the situation for needing the DNA of the affine connector of collection belt single-stranded, and when needing collection, its is complementary
During chain, it is only necessary to be present in the sour neutralization of the single-stranded progress of DNA in filtrate after alkaline hydrolysis is filtered, pH value is neutralized to after collection environment with normal
Rule mode is collected, and will not be described here.
Different according to the structure of Filter column in the single-stranded collection methods of DNA of the present invention, operating method is different, and portion is not referred to
Divide and refer to prior art progress, be not limited herein.
There is following technique effect using the single-stranded separation methods of DNA and device that are used for pyrophosphoric acid：This method is simply easily grasped
Make, obtain target sample time short efficiency high, the collection single-stranded available for trace amount DNA and extraction, sample loss can almost be neglected
Slightly, using reagent is few, requirement to equipment is low, in separation process without suction pump, device configuration is enormously simplify, reduces and sets
Standby totle drilling cost, is effectively simplified operating procedure, shortens the operating time, reduce working strength, improve operating efficiency；Using
The Filter column supporting with conventional centrifugal pipe not only ensure that the quality and efficiency separated, also separation process simply easily be realized,
The filter membrane of Filter column is separated by the way that physics mode is single-stranded to DNA, reduces difficulty and the condition requirement of separation.Filter membrane is by equal
The material of matter with identical structure composition, can two-way exchange use, increase the designability of Filter column, can not only be made as
One end carries the hollow structure of filter membrane, enriches the structure and species of the single-stranded separators of DNA, is ensuring the premise of identical function
Under, add the application occasion of the DNA single-stranded separators of the present invention, it is to avoid product structure is single, and adaptability is significantly increased；
And structure symmetrical above and below can be used, it can up and down exchange and use cooperatively.The single-stranded separators of this DNA use cheap PC
Plastics, economic and practical, the design of infundibulate inner chamber is conducive to the aggregation and guiding of reaction solution so that reaction solution separation is more filled
Point more completely, the target DNA for obtaining higher proportion is single-stranded, it is ensured that higher separation rate, it is to avoid waste.In addition, this DNA
Single-stranded separator applies also for commercially available centrifuge tube, standardized designs so that the single-stranded separator versatilities of this DNA are extremely strong, fits
It is extremely wide with face, therefore its application prospect is very wide.
Brief description of the drawings
Fig. 1 is one to use preferred embodiment provided by the present invention for the single-stranded separators of DNA of pyrophosphoric acid.
Fig. 2 is another preferred embodiment of the single-stranded separators of DNA provided by the present invention for pyrophosphoric acid.
Fig. 3 is another preferred embodiment of the single-stranded separators of DNA provided by the present invention for pyrophosphoric acid.
Fig. 4 is another preferred embodiment of the single-stranded separators of DNA provided by the present invention for pyrophosphoric acid.
Fig. 5 is another preferred embodiment of the single-stranded separators of DNA provided by the present invention for pyrophosphoric acid.
Reference：
Collecting pipe 1；Collecting pipe boss 11；Filter column 2；Filter column boss 21；Connect band 3；Upper lid 4；Filter membrane 5；Separation is logical
Road 6.
Embodiment
The present invention is made with reference to embodiment and comparative example further in detail, intactly to illustrate.
Embodiment 1：
Unlike the separation method single-stranded existing DNA for pyrosequencing and device, DNA of the invention is mono-
Chain separation method uses the single-stranded separators of the DNA for pyrophosphoric acid as shown in Figure 1, including the single-stranded separators of DNA and collection
Pipe 1, the single-stranded separators of DNA are a Filter column 2, and separation process is as follows, and the single-stranded separators of DNA are remained out in separation process
The upward plumbness of mouth, wherein undisclosed partly can refer to prior art：
(1) combine：DNA double chain fragment and appropriate affine connector are added in single-stranded separator, after amplification
DNA fragmentation is combined with sepharose 4B, and the length of DNA fragmentation is 10~20kb, and DNA minimums applied sample amount should be not less than 500ng, agar
Sugared a diameter of 30 μm of pearl, surface is covered with biotin and Streptavidin, the DNA fragmentation after amplification and the coated fine jade of Streptavidin
Lipolysaccharide pearl spontaneously specifically binds；
(2) centrifuge：The DNA double chain after combining is drawn into Filter column 2, Filter column 2 is previously charged into collecting pipe 1, is put into
1min is centrifuged with 12000rmp after centrifuge, excess of solvent is removed；Wherein collecting pipe 1 is 1.5mL centrifuge tubes, uncovered；Filter column 2
For upper end open, the semi-enclosed cylinder in lower end, the upper end open outer rim of Filter column 2 is provided with boss 21, convex to be fixed on collecting pipe
On platform 11, the lower end of Filter column 2 is built-in with filter membrane 5, Filter column 2 a diameter of 4.5mm, a diameter of 4.7mm of filter membrane, by by filter membrane
It is allowed to be brought into close contact in press-in Filter column 2 by force and leaves no gaps；
(3) wash：Appropriate 70~80% ethanol is being added, the amplifing reagents such as the Taq enzyme of residual are washed away, gently blown and beaten mixed
Even rear 12000rmp centrifugations 1min；
(4) alkaline hydrolysis：Add 0.4M NaOH and 1M NaCl and untie DNA double chain spiral, obtain the DNA with affine connector mono-
The mixed liquor of chain and its complementary strand；
(5) double-strand is separated：Double-strand is separated, the DNA for being connected with affine connector is single-stranded and its mixed liquor of complementary strand is passed through
Filter membrane is separated, and the DNA with affine connector is single-stranded after separation is stayed on film, and complementary strand is washed by filter membrane after filtering
Wash at least one times, 12000rmp centrifugations 1min after mixing is gently blown and beaten during washing；
(6) pH value is adjusted：Appropriate elution buffer or ultra-pure water cleaning are added, the NaOH of residual is washed away, equilibrium ph is into
Property, 12000rmp centrifugations 1min after gently piping and druming is mixed；
(7) collect：Keep the filtering direction of the single-stranded separators of DNA constant, add eluent such as ultra-pure water etc. to Filter column 2
Interior, sucking-off DNA is single-stranded after piping and druming, is now located at because the DNA with affine connector is single-stranded on film, adds after eluent and stands,
Gently piping and druming makes the single-stranded suspensions of DNA, and sucking liquid is that to obtain DNA single-stranded,.DNA can detect that DNA is dense by nanodrop after collection
Degree, for pyrosequencing or 4 DEG C of sealing preserves.
Because the diameter of affine connector is more than the filter membrane diameter of the single-stranded separators of DNA, therefore filtered in single-stranded pass through of DNA
During film, the DNA of uncombined affine connector is single-stranded and other impurities can be by filter membrane, and combines the single-stranded quilt of affine connector
Stay on film and can not pass through, this filtering is physical.
Involved solution, parameter and other separation conditions in separation process in the present embodiment, in being the present embodiment
Preferred embodiment, it is any to be used equally for reference to experiment condition, parameter and the solution that prior art play respective action
The involved design parameter isolated and purified in process, the present embodiment and solution selection should not be used as to the present invention's in the present invention
Limitation.
As shown in Figure 1 to Figure 2, collecting pipe 1 is used to collect the waste liquid produced during centrifugation, and suggestion after each step is inclined in time
To prevent cross pollution, the upper pipes of collecting pipe 1 are cone as cylinder, lower end, and bottom is dome shape, can specifically be joined
See the 1.5mL centrifuge tubes used in DNA double chain purifying in the prior art, the NM concrete structure of Filter column 2 also can be found in existing
Have in technology DNA double chain purge process with the matching used screen pipe of collecting pipe, the external diameter of Filter column 2 is less than or equal to collecting pipe
1 internal diameter, the mouth of pipe inner edge of collecting pipe 1 is provided with a boss 11, for supporting Filter column.It is used as another preferred embodiment party
Formula, the upper lid 4 of collecting pipe configuration, upper lid 4 is connected on collecting pipe 1 by connect band 3, due to the connection of connect band 3, loads filtering
Upper lid also can be closely fastened on the boss 11 of collecting pipe 1 after post 2, and the effect of upper lid 4 is to prevent liquid from flying when liquid is centrifuged
Filter column 2 is spilt to cause damage and pollute.
Preferred filter membrane material is that hole is preferably 10 μm between polyethylene microballoon, its microballoon in the present embodiment, less than parent
It with the diameter of connector, can will directly be stayed in by physical filtering with the single-stranded of affine connector on film, filter off not connected parent
With a chain of connector, its absorb-elute effect is good, and the DNA rate of recovery is high, low in raw material price environmental protection.
In order to which the filter membrane in the present embodiment is not moved in piping and druming or centrifugal process, in the present embodiment further preferably
Film pressing device is provided with above filter membrane, film pressing device includes pad and/or press mold frame.The liquid of purifying to be separated is first passed through after pad
Contacted with filter membrane, pad is preferably fibrous material, can tolerate soda acid and most of organic solvent, to most biomolecule not
Absorption can be produced.
Preferably in the top of pad, the side not contacted with filter membrane is additionally provided with press mold frame, press mold frame and filtering cylinder material
Material is identical, and filter membrane is compressed by mechanical pressure, filter membrane is not moved during the use such as piping and druming or centrifugation, causes to receive
Collection loss.
Embodiment 2：
As shown in figure 3, the present embodiment and embodiment 1 are differed only in, the Filter column 2 in embodiment 2 is symmetrical above and below
The hollow form cylinder of structure, filter membrane 5 is vertically positioned in the middle part of hollow cylinder, the Filter column 2 can two ends exchange and use, hollow cylinder
Internal diameter be 4.5mm, a diameter of 4.7mm of filter membrane, do not stay seam by the way that filter membrane 5 to be pressed into be allowed to be brought into close contact in Filter column 2 by force
The spacing squeeze-film mechanism (not shown) of filter membrane is added in gap, cylinder, displacement can not be carried out to fix filter membrane.Because the DNA is mono-
The chain separation device either still functionally two ends all same in structure, thus the single-stranded separators of the DNA need not distinguish into
Liquid end and outlet end, when in use two ends can arbitrarily select one and use.
Using the single-stranded separators of DNA as shown in Figure 3 specifically wrap for the single-stranded separation methods of the DNA of pyrosequencing
Include as follows, during separator single-stranded using DNA in the present embodiment, two ends can be carried out as needed exchange to use：
(1) combine：DNA double chain fragment and appropriate affine connector are added to the reaction of the opening upwards of single-stranded separator
In chamber, the DNA fragmentation after amplification is combined with sepharose 4B, and the length of DNA fragmentation is 15kb, and DNA minimum applied sample amounts should
Not less than 500ng, a diameter of 30 μm of sepharose 4B, surface is covered with biotin and Streptavidin, DNA fragmentation and chain after amplification
The mould coated sepharose 4B of Avidin is spontaneously specifically bound；
(2) centrifuge：Keep the single-stranded separator directions of DNA constant, draw the DNA double chain after combining into Filter column 2, filtering
Post 2 is previously charged into collecting pipe 1, is put into after centrifuge and is centrifuged 1min with 12000rmp, removes excess of solvent；
(3) wash：Keep the single-stranded separator directions of DNA constant, adding appropriate 70~80% ethanol, washing away residual
The amplifing reagents such as Taq enzyme, 12000rmp centrifugations 1min after gently piping and druming is mixed；
(4) alkaline hydrolysis：Keep the single-stranded separator directions of DNA constant, add 0.4M NaOH and 1M NaCl and untie DNA double chain spiral shell
Rotation, obtains that the DNA with affine connector is single-stranded and its mixed liquor of complementary strand；
(5) double-strand is separated：Keep DNA single-stranded separator directions constant, by the DNA for being connected with affine connector it is single-stranded and its
The mixed liquor of complementary strand is separated through filter membrane, and the DNA with affine connector is single-stranded after separation stays on film, and complementary strand leads to
Washed after filter membrane, filtering at least one times, 12000rmp centrifugations 1min after mixing is gently blown and beaten during washing；
(6) pH value is adjusted：Keep the single-stranded separator directions of DNA constant, add appropriate elution buffer or ultra-pure water cleaning, wash
Remove the NaOH of residual, equilibrium ph to neutrality, 12000rmp centrifugations 1min after gently piping and druming is mixed；
(7) collect：The single-stranded separator two ends of DNA are exchanged, eluent such as ultra-pure water etc. are added to the reaction chamber exchanged
Interior, is stayed on filter membrane, after the two ends of Filter column 2 are exchanged, DNA is single-stranded to be in during the single-stranded separation of DNA with affine connector
Under filter membrane, because filtering is only physical filtering, that is to say, that affine connector, which is stuck in outside aperture, to be passed through, and eluted after exchange
It can directly be rinsed DNA is single-stranded from filter membrane, collection liquid is added during collection, static more than 1mins can enter without piping and druming
Row elution step, centrifuge speed is no more than 10000rpm during elution, at least centrifuges 2min.DNA can pass through after collection
Nanodrop detects DNA concentration, carries out pyrosequencing or 4 DEG C of sealing preserves.It is only capable of drawing in bottom different from filter membrane and collects
Mode, the elution process collection rate that filter membrane is exchanged behind direction is high, and membrane filter is put during two ends are symmetrical and easily facilitates exchange
With separate and collect work, it may have the broader instrument scope of application.
The single-stranded separators of DNA in the present embodiment can also coordinate collecting pipe to be used as the single-stranded separators of DNA, separator
Provided with boss 21, it is easy to make the single-stranded separators of DNA be fixed in the collection mouth of pipe, for details, reference can be made to embodiment 1.
Embodiment 3：
As shown in figure 4, the single-stranded separators of another DNA for pyrosequencing that embodiment 3 provides for the present invention
Preferred embodiment, as different from Example 2, in the embodiment the single-stranded separators of DNA Filter column 2 use space round platform
Type structure, filter membrane 5 is arranged at the public top surface of two circular platform type Filter columns 2, top surface diameter and the filter membrane 5 of circular platform type Filter column 2
Diameter it is consistent, filter membrane both sides are provided with squeeze-film mechanism (not shown), and displacement can not be carried out to fix filter membrane.Truncated cone-shaped is filtered
The side wall of the open lower end of post 2 is provided with boss 21, it is ensured that timing can be fixed on the boss 11 of collecting pipe 1 at two ends.
The step of using the separation method of the separator described in the present embodiment with described in embodiment 2, is identical, only in knot
Improved on structure, this improvement is not unique.
The single-stranded separators of DNA in the present embodiment can also coordinate collecting pipe to be used as the single-stranded separators of DNA, separator
Provided with boss 21, it is easy to make the single-stranded separators of DNA be fixed in the collection mouth of pipe, for details, reference can be made to embodiment 1.
Embodiment 4：
As shown in figure 5, the single-stranded separators of another DNA for pyrosequencing that embodiment 4 provides for the present invention
Preferred embodiment, as illustrated, as different from Example 2, in order to preferably assemble with guiding reaction solution, make reaction solution point
From more fully more completely, filtering dead angle is evaded, the embodiment is additionally arranged a split tunnel 6, described point on the basis of embodiment 2
It is arranged between two Filter columns 2 and is connected with the two from passage 6, two Filter columns 2 and split tunnel 6 concentrically axis, filter membrane 5
On the plane of symmetry for being arranged on split tunnel 6, filter membrane both sides are provided with squeeze-film mechanism (not shown), can not be carried out with fixing filter membrane
Displacement, the middle part of two Filter columns 2 is provided with boss 21, it is ensured that two ends can be fixed on the boss 11 of collecting pipe 1 to timing.
One end 201 that Filter column 2 is connected with split tunnel 6 is rounding mesa-shaped, and the two collectively forms a funnel-form space
Structure, the basal diameter of rounding you 201 is consistent with the internal diameter of Filter column 2, and top surface diameter is consistent with the diameter of split tunnel 6, should
Plant the aggregation and guiding that are provided with beneficial to reaction solution of structure so that reaction solution separation is more fully more complete.Therefore, the embodiment
In the single-stranded separators of DNA on the basis of embodiment 2 cause DNA it is single-stranded be separated by filtration more thoroughly, separative efficiency is significantly carried
It is high.
The step of using the separation method of separator affiliated in the present embodiment with described in embodiment 2, is identical, only exists
Improved in structure, this improvement is not unique.
The single-stranded separators of DNA in the present embodiment can also coordinate collecting pipe to be used as the single-stranded separators of DNA, separator
Provided with boss 21, it is easy to make the single-stranded separators of DNA be fixed in the collection mouth of pipe, for details, reference can be made to embodiment 1.
Embodiment 5：
The present embodiment is uniquely differed only in embodiment 4, and filter membrane 5 can be placed in one any perpendicular to split tunnel 6
Position, two Filter columns 2 are hollow dissymmetrical structure, and the diameter of filter membrane is slightly larger than split tunnel, and filter membrane both sides are provided with press mold
Mechanism (not shown), displacement can not be carried out to fix filter membrane.
The step of using the separation method of separator affiliated in the present embodiment with described in embodiment 2, is identical, only exists
Improved in structure, this improvement is not unique.
The single-stranded separators of DNA in the present embodiment can also coordinate collecting pipe to be used as the single-stranded separators of DNA, separator
Provided with boss 21, it is easy to make the single-stranded separators of DNA be fixed in the collection mouth of pipe, for details, reference can be made to embodiment 1.
Finally be necessary described herein be：Above example is served only for making further detailed to technical scheme
Ground explanation, it is impossible to be interpreted as limiting the scope of the invention, those skilled in the art is according to the above of the invention
Some the nonessential modifications and adaptations made belong to protection scope of the present invention.
Claims (13)
1. the single-stranded separators of a kind of DNA for pyrosequencing, it is characterised in that the separator is provided with filter membrane in one
Hollow filter post, is connected with that the DNA of affine connector is single-stranded to be separated after filtering through setting filter membrane in the separator, wherein affine
The diameter of connector is more than the membrane filtration aperture.
2. the single-stranded separators of the DNA according to claim 1 for pyrosequencing, it is characterised in that hollow filter post
Filter membrane be located at Filter column one end.
3. the single-stranded separators of the DNA according to claim 2 for pyrosequencing, it is characterised in that filter membrane diameter is big
In the filtering surface diameter of Filter column.
4. the single-stranded separators of the DNA according to claim 1 for pyrosequencing, it is characterised in that filter membrane was located at
Filter the non-end face of post.
5. the single-stranded separators of the DNA according to claim 4 for pyrosequencing, it is characterised in that be located in filter membrane
In the middle part of Filter column, Filter column axle two ends centered on filter membrane are mirrored into being symmetrical arranged.
6. the single-stranded separators of the DNA according to claim 5 for pyrosequencing, it is characterised in that Filter column two ends
Successively decrease setting towards filter membrane diameter.
7. the single-stranded separators of the DNA according to claim 6 for pyrosequencing, it is characterised in that the mistake of Filter column
Filtering surface diameter and Filter column caliber.
8. a kind of single-stranded separators of DNA, it is characterised in that including any described single-stranded separators of DNA of claim 1~7
With a collecting pipe, collecting pipe is that one end open is closed in one end, and Filter column is in collecting pipe, and the closing of Filter column and collecting pipe
Do not fit end bottom surface.
9. a kind of single stranded DNA separation method, it is characterised in that：A chain in DNA double chain is connected with affine connector, uncoiling
By the single-stranded separator filterings point of DNA described in any described single-stranded separators of DNA of claim 1~7 or claim 8
From the DNA for then obtaining being connected with affine connector is single-stranded.
10. the single-stranded separation methods of DNA according to claim 9, it is characterised in that：It is mono- using the DNA described in claim 8
When chain separation device is filtered, filtering, centrifuge described in collecting pipe match of pipe diameter are completed with unification centrifuge.
11. the single-stranded separation methods of DNA according to claim 9, it is characterised in that using the DNA described in claim 2
During single-stranded separator, separation method is specific as follows：
A is combined：DNA double chain fragment and appropriate affine connector are added in single-stranded separator, by the DNA fragmentation after amplification with
Affine connector is combined；
B alkaline hydrolysis：Highly basic is added in DNA fragmentation after bonding, DNA double spiral is untied using highly basic, is obtained with affine connector
DNA is single-stranded and its mixed liquor of complementary strand；
C double-strands are separated：Double-strand is separated, the DNA for being connected with affine connector is single-stranded and its mixed liquor of complementary strand is through filter membrane
Separated, the DNA with affine connector is single-stranded after separation is stayed on film, and complementary strand is washed at least by filter membrane after filtering
Once；
D is collected：Keep the filtering direction of the single-stranded separators of DNA constant, add in eluent to Filter column, DNA is suctioned out after piping and druming
Single-stranded, the DNA now with affine connector is single-stranded on film, adds after eluent and stands, and gently piping and druming makes DNA single-stranded outstanding
Floating, sucking liquid is that to obtain DNA single-stranded.
12. the single-stranded separation methods of DNA according to claim 9, it is characterised in that using the DNA described in claim 5
During single-stranded separator, separation method is specific as follows：
A is combined：DNA double chain fragment and appropriate affine connector are added in single-stranded separator, by the DNA fragmentation after amplification with
Affine connector is combined；
B alkaline hydrolysis：Highly basic is added in DNA fragmentation after bonding, DNA double spiral is untied using highly basic, is obtained with affine connector
DNA is single-stranded and its mixed liquor of complementary strand；
C double-strands are separated：Double-strand is separated, the DNA for being connected with affine connector is single-stranded and its mixed liquor of complementary strand is through filter membrane
Separated, the DNA with affine connector is single-stranded after separation is stayed on film, and complementary strand is washed at least by filter membrane after filtering
Once；
D is collected：The single-stranded separator two of DNA is exchanged, collection DNA after eluent is added single-stranded；DNA with affine connector
Stayed in during single-stranded separation on filter membrane, after Filter column two ends are exchanged, DNA is single-stranded to be under filter membrane, and elution can be directly single-stranded by DNA
Rinse, collected to collection device from filter membrane.
13. a kind of single-stranded separation methods of DNA for the DNA single-stranded complementary chains for collecting affine connector, it is characterised in that using as weighed
Profit requires the single-stranded separators of DNA or the single-stranded separators of DNA as claimed in claim 8 described in 1, and filter is collected after alkaline hydrolysis filtering
Liquid, it is single-stranded that acid collects DNA after neutralizing.
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| CN201710061927.XA Active CN106754292B (en) | 2017-01-12 | 2017-01-27 | The single-stranded separators of DNA and separation method for pyrosequencing |
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| CN201710076466.3A Active CN107299054B (en) | 2017-01-12 | 2017-02-13 | Control system and control method of DNA sequencing device |
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| CN109880742A (en) * | 2019-03-15 | 2019-06-14 | 廖祥菊 | A kind of biological inoculum packing save set |
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| CN112033743B (en) * | 2020-07-16 | 2024-04-30 | 上海市东方医院（同济大学附属东方医院） | Parkinsonism fecal fluid extraction device |
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| CN114453146B (en) * | 2022-04-12 | 2022-06-14 | 深圳人体密码基因科技有限公司 | Centrifugal device for gene detection of self-separation solution |
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| CN106754343A (en) | 2017-05-31 |
| CN107299054B (en) | 2020-09-01 |
| CN107299054A (en) | 2017-10-27 |
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| CN107312710B (en) | 2021-06-25 |
| CN106754313A (en) | 2017-05-31 |
| CN106754343B (en) | 2017-10-31 |
| CN106754292A (en) | 2017-05-31 |
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| CN106591107B (en) | 2019-04-12 |
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Effective date of registration: 20210408 Address after: 201600 room 710, Building 29, No.1, Lane 618, Dingyuan Road, Songjiang District, Shanghai Patentee after: Fist (Shanghai) Biotechnology Co.,Ltd. Address before: 3 / F, Lingyun Photoelectric Industrial Park, No.3, liufangyuanheng Road, Donghu New Technology Development Zone, Wuhan City, Hubei Province, 430074 Patentee before: Wuhan feisite Medical Equipment Co.,Ltd. |