CN110317805A - Nucleic acid fragment sorting and purifying reagent and method - Google Patents
Nucleic acid fragment sorting and purifying reagent and method Download PDFInfo
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Abstract
The invention discloses a nucleic acid fragment sorting and purifying reagent and a method, wherein the reagent comprises magnetic bead binding liquid, washing liquid and eluent, and can be used for sorting and purifying different sections of targeted nucleic acid fragments in a mixture sample, and the targeted nucleic acid fragment sorting step comprises the following steps: uniformly mixing a first nucleic acid mixture containing different sections with the magnetic bead binding solution, standing at room temperature, then placing on a magnetic frame, standing, removing supernate, washing and eluting; the volume mixing ratio of the first nucleic acid mixture to the magnetic bead binding solution is 1: 1.2-0.45. In addition, whether the second step of sorting is carried out or not can be selected according to the requirement, namely, the supernatant obtained after the first nucleic acid mixture is combined and separated with the magnetic beads is taken as a second nucleic acid mixture, and the second step of combining, separating, washing and eluting are carried out again; different sections of the target nucleic acid fragments can be sorted by adjusting the volume of the added magnetic bead binding solution. The reagent and the method provided by the invention can be used for downstream nucleic acid fragment analysis, second-generation sequencing platform library construction and the like.
Description
Technical field
The invention belongs to biological fields, and in particular to a kind of nucleic acid fragment sorting purified reagent and method.
Background technique
Britain biochemist Sanger has invented dioxygen and has terminated DNA sequencing method, and gene sequencing has greatly been pushed to develop
Industry prospect then gradually appears second generation sequencing technologies and third generation sequencing technologies, both technologies are commonly referred to collectively as next
For sequencing technologies (NGS), the application range of next generation's sequencing is wider, following several faces: 1, Denovo sequencing, resurvey sequence and other
DNA sequencing；2, RNA is sequenced；3, macro gene order-checking；4, epigenetics.No matter and next-generation sequencing needs is nucleic acid-templated
There is certain requirement from quality or quantity, and nucleic acid-templated clip size has important meaning to sequencing error is reduced
Justice.
The isolation and purification technology of nucleic acid is a basic technology of molecular biosciences industry.As Protocols in Molecular Biology is wide
General fields, the isolation and purification technologies of nucleic acid such as biology, medicine and its correlation of being applied to also is further developed.It is separating
Target nucleic acid is separated with other components in sample in purifying, such as protein pollutant or other nucleic acid, also commonly referred to as
Non-target nucleic acid.And for certain applications, the difference of clip size is to discriminate between the important evidence of target nucleic acid and non-target nucleic acid.
It is there are two ways to target nucleic acid of different size segment can be sorted at present, a kind of to use gel extraction, but in fact
Proved recipe method is cumbersome, and time-consuming, and there are certain risk for ultraviolet irradiation, and easily nucleic acid base are made to mutate.It is another
Kind method is described to using polyethylene glycol (PEG) and sodium chloride in patent US6534262 come precipitation and separation nucleic acid, is effectively sub-elected
Purpose nucleic acid segment, but need to handle the long period, it is complicated for operation, and when mixtures of nucleic acids Nucleic Acid is higher, just meeting
There is good recovery efficiency.Therefore quickly the purpose of the present invention is to propose to one, simply, the rate of recovery is high, moreover it is possible to realize high-throughput
A kind of nucleic acid fragment sorting purified reagent and method.
Summary of the invention
For the shortcoming for solving the prior art and method, the present invention proposes that a kind of nucleic acid fragment sorts purified reagent and side
Method, be it is a kind of i.e. quickly and the simple, rate of recovery and purity is high, and be able to achieve point of the high-throughput operation of automation liquor removing workstation
Select reagent and method.
The technical solution adopted by the invention is as follows:
A kind of nucleic acid fragment sorting purified reagent, includes following components: magnetic bead combination liquid, cleaning solution, eluent；
The magnetic bead combination liquid is the hydroxyethyl piperazineethanesulfonic acid of 4- containing 10-500mM, 0.5-2.5M guanidine hydrochloride, 1-50%
(W/V) and molecular weight ranges be 200-20000 polyethylene glycol, 1-50mM lithium chloride, 1-50mM magnesium chloride, 0.5-25% (V/
V dehydrated alcohol and 5-50mg/ml magnetic-particle)；
Eluent is no DNase and RNase enzyme sterile water or tris buffer or TE buffer.
Further, the polyethylene glycol preferred molecular weight is 4000-10000, more preferably molecular weight 10000.
Further, the magnetic-particle is that hydrophilic macromolecule medium and active functional group hydroxyl are wrapped up in surface
The ferriferrous oxide particles of base, the particle have it is super pliable, and partial size be 1-3um.
Further, the pH of the magnetic bead combination liquid is 7-8.
Further, the pH of the eluent is 7-8.
A kind of nucleic acid fragment sorting purification process is realized based on above-mentioned nucleic acid fragment sorting purified reagent.Method is as follows:
By the first mixtures of nucleic acids containing different sections, liquid is mixed in conjunction with magnetic bead, after being placed at room temperature for, then is placed on magnetic frame and is stood, abandons
Supernatant washs magnetic-particle purification of nucleic acid with cleaning solution, then with the nucleic acid fragment on elution magnetic-particle；Described
The volumetric mixture ratio of first mixtures of nucleic acids liquid in conjunction with magnetic bead is 1:1.2-0.45.
In addition, liquid mixes in conjunction with magnetic bead in the first mixtures of nucleic acids, it is placed at room temperature for and is placed on magnetic frame after standing again, it can
To take its supernatant, as the second mixtures of nucleic acids, magnetic bead combination liquid is added again, mixes, after being placed at room temperature for, then is placed in magnetic force
It is stood on frame, abandons supernatant, wash magnetic-particle purification of nucleic acid with cleaning solution, then with the nucleic acid on elution magnetic-particle
Segment；The volume ratio of second mixtures of nucleic acids liquid in conjunction with the magnetic bead of addition is 1:0.2-0.5.
Further, first mixtures of nucleic acids can derive from: pcr amplification product, DNA are produced after mechanical shearing
Object, DNA are by product after fragmentation digestion or the nucleic acid product of NGS pre-treatment.
Reagent provided by the invention and method can be used for nucleic acid fragment analysis and the two generation microarray dataset library constructions in downstream
Deng.
Method of the invention has the advantage that
1) nucleic acid that this reagent and method are sorted can be directly used for building gene pool, the rate of recovery and purity is high；
2) this reagent and the nucleic acid speed of method sorting are fast, easy to operate, are suitably adapted for manual operations and automatic operation；
3) this reagent and method can cooperate semi-automatic and full-automatic liquor removing workstation, realize high-throughput sorting nucleic acid
Clip size.
Detailed description of the invention
Segment gradient sorts on Fig. 1 DNA.
Segment gradient sorts under Fig. 2 DNA.
The sorting of Fig. 3 DNA intermediate segment.
Specific embodiment
The present invention will be further described with reference to the accompanying drawing.
Segment gradient sorts on case study on implementation 1:DNA
Using contain 100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1kb,
The mixtures of nucleic acids of 1.2kb, 1.5kb, 2kb, 3kb carry out nucleic acid and sort template, sort the nucleic acid fragment of different sections.
Specific steps are as follows:
1, it combines: taking 7 1.5mlEP pipes, be separately added into 50ul mixtures of nucleic acids, it is then big according to target fragment need to be sorted
Small selection specific magnetic bead combination liquid product (being shown in Table 1), vortex mixing, is placed at room temperature for 5min, after EP pipe is placed in magnetic force
Frame stands 2min, abandons supernatant.
2, wash: taking 200ul cleaning solution whirlpool to mix magnetic bead, be placed at room temperature for 30s, after centrifuge tube is placed in magnetic frame
2min is stood, supernatant is abandoned, room temperature dries 2-5min.
3, it elutes: 20ul eluent being added into magnetic bead, suction, which is beaten, mixes magnetic bead, 56 DEG C of incubation 5min, then is placed in magnetic frame
On, it is kept completely separate to magnetic bead, supernatant is transferred to new EP and is managed.
4, agarose gel electrophoresis analysis is carried out, electrophoretogram is shown in Fig. 1.
The magnetic bead combination liquid is 425mM4- hydroxyethyl piperazineethanesulfonic acid, and 2M guanidine hydrochloride, 30% molecular weight is 10000 poly-
Ethylene glycol, 50mM lithium chloride, 25mM magnesium chloride, 25% dehydrated alcohol, 50mg/ml magnetic particle.
The cleaning solution is 70% dehydrated alcohol.
The eluent is pH 8.0TE.
The corresponding sorting nucleic acid fragment of the magnetic bead combination liquid of 1 different volumes of table
| Magnetic bead combination liquid Product ratio | 1.2× | 0.8× | 0.7× | 0.6× | 0.55× | 0.5× | 0.45× |
| DNA fragmentation size | 100bp- 3.0kb | 200bp- 3.0kb | 300bp- 3.0kb | 500bp- 3.0kb | 800bp- 3.0kb | 2.0kb- 3.0kb | 0bp |
From electrophoretogram as can be seen that in a certain range, as the volume of magnetic bead combination liquid is reduced, the segment of sorting is more next
It is bigger, meet the requirement of sorting.
Segment gradient sorts under case study on implementation 2:DNA
Using contain 100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1kb,
The mixtures of nucleic acids of 1.2kb, 1.5kb, 2kb, 3kb carry out nucleic acid and sort template, sort the nucleic acid fragment of different sections.
Specific steps are as follows:
1, it combines (segment in removal) for the first time: taking 6 1.5mlEP pipes, be separately added into 50ul mixtures of nucleic acids, then root
Specific magnetic bead combination liquid product (being shown in Table 2) is selected according to target fragment size need to be sorted, vortex mixes, and is placed at room temperature for 5min, ties
EP pipe is placed in magnetic frame after beam and stands 2min, it is spare that supernatant is transferred to new EP pipe.
2, second combine (in conjunction with lower segment): according to target fragment size select specific magnetic bead combination liquid accumulate (see
Table 2), be added in the supernatant of transfer, vortex mix, be placed at room temperature for 5min, after by EP pipe be placed in magnetic frame stand
2min abandons supernatant.
3, wash: taking 200ul cleaning solution whirlpool to mix magnetic bead, be placed at room temperature for 30s, after that EP pipe is placed in magnetic frame is quiet
1min is set, supernatant is abandoned, room temperature dries 2-5min.
4, it elutes: 20ul eluent being added into magnetic bead, suction, which is beaten, mixes magnetic bead, 56 DEG C of incubation 5min, then is placed in magnetic frame
On, it is kept completely separate to magnetic bead, supernatant is transferred to new EP and is managed.
5, agarose gel electrophoresis analysis is carried out, electrophoretogram is shown in Fig. 2.
The magnetic bead combination liquid is 425mM4- hydroxyethyl piperazineethanesulfonic acid, and 2M guanidine hydrochloride, 30% molecular weight is 10000 poly-
Ethylene glycol, 50mM lithium chloride, 25mM magnesium chloride, 25% dehydrated alcohol, 50mg/ml magnetic particle.
The cleaning solution is 70% dehydrated alcohol.
The eluent is pH 8.0TE.
The corresponding sorting nucleic acid fragment of the magnetic bead combination liquid (two steps) of 2 different volumes of table
| First volume ratio | 1.2× | 0.9× | 0.8× | 0.75× | 0.7× | 0.65× |
| Second volume ratio | 0.2× | 0.5× | 0.6× | 0.65× | 0.7× | 0.75× |
| DNA fragmentation size | 100bp | 100-200bp | 100-300bp | 100-400bp | 100-500bp | 100-700bp |
In table: the first volume ratio refers to the volume of the magnetic bead combination liquid and mixtures of nucleic acids that are added in combination for the first time
Than the second volume ratio is the volume ratio of magnetic bead combination liquid and the supernatant of transfer in second of combination.
From electrophoretogram as can be seen that the nucleic acid fragment of the available different sections of various combination.
The sorting of case study on implementation 3:DNA intermediate segment
Using contain 100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1kb,
The mixtures of nucleic acids of 1.2kb, 1.5kb, 2kb, 3kb carry out nucleic acid and sort template, sort the nucleic acid fragment of different sections.
Specific steps are as follows:
1, it combines (segment in removal) for the first time: taking 7 1.5mlEP pipes, be separately added into 50ul mixtures of nucleic acids, then root
Specific magnetic bead combination liquid product (being shown in Table 3) is selected according to target fragment size need to be sorted, vortex mixes, and is placed at room temperature for 5min, ties
EP pipe is placed in magnetic frame after beam and stands 2min, it is spare that supernatant is transferred to new EP pipe.
2, it combines (in conjunction with intermediate segment) second: specific magnetic bead combination liquid product is selected according to target fragment size
(being shown in Table 3), is added in the supernatant of transfer, vortex mix, be placed at room temperature for 5min, after by EP pipe be placed in magnetic frame stand
2min abandons supernatant.
3, wash: taking 200ul cleaning solution whirlpool to mix magnetic bead, be placed at room temperature for 30s, after that EP pipe is placed in magnetic frame is quiet
1min is set, supernatant is abandoned, room temperature dries 2-5min.
4, it elutes: 20ul eluent being added into magnetic bead, suction, which is beaten, mixes magnetic bead, 56 DEG C of incubation 5min, then is placed in magnetic frame
On, it is kept completely separate to magnetic bead, supernatant is transferred to new EP and is managed.
5, agarose gel electrophoresis analysis is carried out, electrophoretogram is shown in Fig. 3.
The magnetic bead combination liquid is 425mM4- hydroxyethyl piperazineethanesulfonic acid, and 2M guanidine hydrochloride, 30% molecular weight is 10000 poly-
Ethylene glycol, 50mM lithium chloride, 25mM magnesium chloride, 25% dehydrated alcohol, 50mg/ml magnetic particle.
The cleaning solution is 70% dehydrated alcohol.
The eluent is pH 8.0TE.
The corresponding sorting nucleic acid fragment of the magnetic bead combination liquid (two steps) of 3 different volumes of table
| First volume ratio | 1.2× | 0.8× | 0.7× | 0.6× | 0.55× | 0.5× | 0.45× |
| Second volume ratio | 0.2× | 0.4× | 0.2× | 0.2× | 0.2× | 0.2× | 0.2× |
| DNA fragmentation size | 100bp | 100-200bp | 200-400bp | 300-800bp | 300bp-1.0kb | 400bp-3.0kb | 500bp-3.0kb |
From electrophoretogram as can be seen that the nucleic acid fragment of the centre portion in the available mixing nucleic acid of various combination.
Claims (9)
1. a kind of nucleic acid fragment sorts purified reagent, which is characterized in that include following components: magnetic bead combination liquid, cleaning solution, elution
Liquid；The magnetic bead combination liquid is the hydroxyethyl piperazineethanesulfonic acid of 4- containing 10-500mM, 0.5-2.5M guanidine hydrochloride, 1-50% (W/V)
And molecular weight ranges are the nothing of the polyethylene glycol of 200-20000,1-50mM lithium chloride, 1-50mM magnesium chloride, 0.5-25% (V/V)
Water-ethanol and 5-50mg/ml magnetic-particle；Eluent buffers for no DNase and RNase enzyme sterile water or tris buffer or TE
Liquid.
2. nucleic acid fragment as described in claim 1 sorts purified reagent, which is characterized in that the preferred molecule of the polyethylene glycol
Amount is 4000-10000, more preferably molecular weight 10000.
3. nucleic acid fragment as described in claim 1 sorts purified reagent, it is characterised in that: the magnetic-particle is surface packet
Wrap up in the ferriferrous oxide particles of hydrophilic macromolecule medium and active functional group hydroxyl, the particle have it is super pliable,
And partial size is 1-3um.
4. nucleic acid fragment as described in claim 1 sorts purified reagent, which is characterized in that the pH of magnetic bead combination liquid is 7-8.
5. nucleic acid fragment as described in claim 1 sorts purified reagent, which is characterized in that the pH of eluent is 7-8.
6. a kind of nucleic acid fragment sorts purification process, it is characterised in that: be based on nucleic acid piece as described in any one in claim 1-5
Section sorting purified reagent is realized.
7. nucleic acid fragment as claimed in claim 6 sorts purification process, which is characterized in that method is as follows: will contain different sections
The first mixtures of nucleic acids in conjunction with magnetic bead liquid mix, after being placed at room temperature for, then be placed on magnetic frame and stand, supernatant is abandoned, with washing
Liquid washing magnetic-particle purification of nucleic acid is washed, then with the nucleic acid fragment on elution magnetic-particle；First nucleic acid is mixed
The volumetric mixture ratio for closing object liquid in conjunction with magnetic bead is 1:1.2-0.45.
8. nucleic acid fragment as claimed in claim 7 sorts purification process, which is characterized in that in the first mixtures of nucleic acids and magnetic bead
It is mixed in conjunction with liquid, is placed at room temperature for and is placed on magnetic frame again after standing, its supernatant can be taken, as the second mixtures of nucleic acids, then
Secondary addition magnetic bead combination liquid mixes, after being placed at room temperature for, then is placed on magnetic frame and stands, and abandons supernatant, washs magnetism with cleaning solution
Particle purification of nucleic acid, then with the nucleic acid fragment on elution magnetic-particle；Second mixtures of nucleic acids and addition
The volume ratio of magnetic bead combination liquid is 1:0.2-0.5.
9. nucleic acid fragment as claimed in claim 7 or 8 sorts purification process, which is characterized in that the first nucleic acid mixing
Object derives from: pcr amplification product, DNA are by product after mechanical shearing, DNA by product after fragmentation digestion or NGS pre-treatment
Nucleic acid product.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110904095A (en) * | 2019-12-12 | 2020-03-24 | 杭州倍强医药科技有限公司 | Method for improving small fragment nucleic acid purification yield |
| CN111154750A (en) * | 2020-01-13 | 2020-05-15 | 苏州行知康众生物科技有限公司 | Method for targeted enrichment of plasma target free DNA |
| CN115612685A (en) * | 2022-11-03 | 2023-01-17 | 杭州联川基因诊断技术有限公司 | Magnetic bead reagent for nucleic acid purification or fragment screening and application thereof |
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| CN104419638A (en) * | 2013-09-06 | 2015-03-18 | 精工爱普生株式会社 | Cartridge for nucleic acid amplification reaction |
| CN106893721A (en) * | 2017-02-16 | 2017-06-27 | 广东氪生物技术有限公司 | A kind of kit screened for nucleic acid purification or fragment and its application method |
| CN107312773A (en) * | 2017-06-13 | 2017-11-03 | 天根生化科技（北京）有限公司 | The combination liquid of purification of nucleic acid and its application |
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2019
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104419638A (en) * | 2013-09-06 | 2015-03-18 | 精工爱普生株式会社 | Cartridge for nucleic acid amplification reaction |
| CN106893721A (en) * | 2017-02-16 | 2017-06-27 | 广东氪生物技术有限公司 | A kind of kit screened for nucleic acid purification or fragment and its application method |
| CN107312773A (en) * | 2017-06-13 | 2017-11-03 | 天根生化科技（北京）有限公司 | The combination liquid of purification of nucleic acid and its application |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110904095A (en) * | 2019-12-12 | 2020-03-24 | 杭州倍强医药科技有限公司 | Method for improving small fragment nucleic acid purification yield |
| CN110904095B (en) * | 2019-12-12 | 2021-08-06 | 杭州倍强医药科技有限公司 | Method for improving small fragment nucleic acid purification yield |
| CN111154750A (en) * | 2020-01-13 | 2020-05-15 | 苏州行知康众生物科技有限公司 | Method for targeted enrichment of plasma target free DNA |
| CN115612685A (en) * | 2022-11-03 | 2023-01-17 | 杭州联川基因诊断技术有限公司 | Magnetic bead reagent for nucleic acid purification or fragment screening and application thereof |
| CN115612685B (en) * | 2022-11-03 | 2023-08-29 | 杭州联川基因诊断技术有限公司 | Magnetic bead reagent for nucleic acid purification or fragment screening and application thereof |
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