CN104278022B - A kind of preparation method and application of DNA capture probes - Google Patents
A kind of preparation method and application of DNA capture probes Download PDFInfo
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- CN104278022B CN104278022B CN201410464934.0A CN201410464934A CN104278022B CN 104278022 B CN104278022 B CN 104278022B CN 201410464934 A CN201410464934 A CN 201410464934A CN 104278022 B CN104278022 B CN 104278022B
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Abstract
The present invention provides a kind of preparation method and application of DNA capture probes, employ the probe preparation method that amplifying target genes interrupt biotin labeling at random, traditional tile type be instead of using the mentality of designing for covering at random to cover, compared with a large amount of DNA coverage goals regions are synthesized in conventional probe preparation method, the present invention only needs the amplimer for synthesizing target area, avoid the cost increase problem for being directly synthesized that probe is caused, thus without difference compared with the probe for preparing with conventional probe preparation method with specificity of the sensitivity for greatly reducing preparation cost and capturing.
Description
Technical field
The invention belongs to nucleic acid probe preparation field, and in particular to a kind of preparation method and application of DNA capture probes.
Background technology
Traditional mulberry lattice sequencing is extended using archaeal dna polymerase with reference to the primer on sequence template undetermined, until mixing one
Till planting chain termination nucleotide.Each reaction contains all four deoxynucleotide triphosphoric acid (dNTP), and is mixed into limitation not
With the dideoxyribonucleoside triphosphate (ddNTP) that color fluorescence is marked.As ddNTP lacks the 3-OH groups required for extending, make
The oligonucleotide of prolongation optionally terminates at G, A, T or C.Depending on terminating point is by corresponding double deoxidation in reacting.They
There is common starting point, but terminate on different nucleotides, capillary electrophoresis separation fragment of different sizes can be passed through,
The fluorescence labeling of observation different size piece degree is inferring sequence.
Second generation sequencing technologies mainly adopt the method being sequenced in synthesis, first with ultrasonic wave DNA samples to be measured
Originally small fragment, and different joints on the two ends of these small fragments are added is broken into, single-stranded DNA banks are constructed.These texts
Joint in storehouse is attached to solid phase surface, and carries out the amplification of bridge-type PCR on surface.Expand and cycles of denaturation through continuous, most
Each DNA fragmentation concentrates bunchy, very multiple copies of each beam containing single DNA template to enter on respective position eventually
The purpose of row this process is to realize amplifying the signal strength signal intensity of base, to reach the semaphore request needed for sequencing.Sequencing is anti-
During answering, add archaeal dna polymerase, adapter-primer and 4 kinds with base specific fluorescence mark in reaction system simultaneously
DNTP (such as Sanger PCR sequencing PCRs).The 3 '-OH of these dNTP are protected by chemical method, thus can only add one every time
dNTP.After dNTP is added on synthesis chain, all untapped free dNTP and archaeal dna polymerase can be washed away.Then,
Add and excite buffer solution needed for fluorescence, use LASER Excited Fluorescence signal, and the record of fluorescence signal is completed by optical device,
Optical signalling is converted into sequencing base using computer analysis finally.So after the completion of fluorescence signal record, chemistry is added
Reagent quenching fluorescence signal simultaneously removes 3 '-OH blocking groups of dNTP, to carry out the sequencing reaction of next round.
Since being born from 2005, second generation sequencing technologies illustrate wide application prospect.Can by scientist is sequenced
To see the important informations such as SNP, mutantional hotspot, genome structure variation, colony's polymorphism.In evolution field
Scientist explores the evolution modelling between species by colony's polymorphism analysis；Demonstrate,proved in microorganism field DNA sequencing parting
Lucid and lively prompt and accurate；And medical domain genome resurvey sequence have in terms of the relation of SNP and major disease is found important
Meaning.
Due to often only to specific genome area (such as extron group) rather than full-length genome is interested, therefore can profit
Specific region is captured with gene trap probe, the DNA sequence dna for capturing out is sequenced.Prepared by probe is that capture is surveyed
Key link in sequence, the probe preparation method that commonly uses at present mainly take the side of " tile type covering design, artificial synthesized base "
Formula.Concrete grammar is：For the probe sequence that certain length is designed in region interested, each sequence is moved along gene location
Certain distance.Then synthesize above-mentioned sequence by the way of artificial synthesized in a large number.Typical representative has Chinese patent《A kind of line grain
The screening method of body ospc gene》(101270390 A of CN), for region interested in non-duplicate region design 60bp
Probe sequence, each sequence move design along gene location, move 3bp between probe, realize the tile type to target area
Cover；Then a large amount of synthesising probing needles of situ synthesis techniques are adopted.Other document [Calvo SE, Compton AG, Hershman
SG,Lim SC,Lieber DS,Tucker EJ,Laskowski A,Garone C,Liu S,Jaffe DB et al:
Molecular diagnosis of infantile mitochondrial disease with targeted next-
generation sequencing.Science translational medicine 2012,4(118):118ra110] in
Similar thinking is also used, simply probe length and distance is moved and is changed.
The maximum defect of " tile type covering design, artificial synthesized base " method is that cost is excessively high, and with target
The increase in region, probe synthesis cost steeply rise.
Content of the invention
It is an object of the invention to provide a kind of preparation method and application of DNA capture probes, can reduce probe preparation
Cost.
For reaching above-mentioned purpose, technical scheme below is present invention employs.
A kind of preparation method of DNA capture probes, comprises the following steps：
(1) special amplimer is designed for area-of-interest in genome, emerging using the amplimer amplification sense
Interesting region obtains amplified production；
(2) ultrasonically treated method is adopted to interrupt the amplified production for DNA fragmentation；
(3) biotin labeling is carried out to DNA fragmentation；
(4) after step (3), from the DNA fragmentation, the isolated DNA for being marked with biotin is single-stranded, obtains DNA and catches
Obtain probe.
The area-of-interest is human mitochondrial DNA.
Step (2) specifically include following steps：Amplified production (the human mitochondrial DNA amplified production of recovery) is diluted
To 10ng/L, then taking the amplified production after 100L dilutions and being put in Ultrasound Instrument carries out ultrasonically treated, the power output of Ultrasound Instrument
For 1000Hz, ultrasonically treated number of times is 90 times, and the single sonication time is 15 seconds, and ultrasonically treated twice is spaced 15 seconds.
Before carrying out biotin labeling, the DNA fragmentation that step (2) is obtained is purified according to clip size, is then carried out
Biotin labeling, the method for the purifying are comprised the following steps：To the 100L 10ng/L amplified productions processed through step (2)
Mixed after middle addition nucleic acid purification magnetic bead (Ampure bead), then stood 5min；It is placed in after standing on magnet stand, treats core
After acid purifying magnetic bead is adsorbed, inhales and abandon supernatant；Then nucleic acid purification magnetic bead is washed with 70% ethanol, dry nucleic acid purification after washing
Magnetic bead, is placed on magnet stand after the nucleic acid purification magnetic bead after being dried with water is resuspended, after nucleic acid purification magnetic bead is adsorbed, in transfer
Clearly, containing DNA fragmentation after purification in the supernatant.
The addition of the nucleic acid purification magnetic bead is that (addition of nucleic acid purification magnetic bead determines the DNA fragmentation for reclaiming to 90L
Size), the consumption of 70% ethanol uses 500～750L for washing every time.
Step (4) specifically include following steps：
A () adsorbs the successful DNA fragmentation of biotin labeling with the coated magnetic bead of Avidin (invitrogen)；
B () washs the coated magnetic bead of the Avidin after step (a) with sodium hydrate aqueous solution, by the DNA pieces
In section, the DNA of unmarked biotin is single-stranded removes from the coated magnetic bead of the Avidin；
C () is heated with the coated magnetic bead of the resuspended Avidin of deionized formamide and in 95 DEG C after step (b)
10min, makes the biotin separate with the Avidin, and the DNA for being then peeled off obtaining being marked with biotin is single-stranded.
The concentration of the sodium hydrate aqueous solution is 1.25mM (the alkaline too strong or too weak purpose for being unable to reach washing).
DNA capture probes prepared by the preparation method of above-mentioned DNA capture probes are being caught from people's genome sequencing library
Obtain the application in human mitochondrial DNA library.
The consumption of DNA capture probes is the 2～2.5% of the sequencing library quality.
Beneficial effects of the present invention are embodied in：
The probe preparation method of amplifying target genes-interrupt at random-biotin labeling is present invention employs, using covering at random
The mentality of designing of lid instead of traditional tile type and cover, with conventional probe preparation method in synthesize a large amount of DNA coverage goals areas
Domain is compared, and the present invention only needs the amplimer for synthesizing target area, it is to avoid be directly synthesized the cost increase problem that probe is caused,
Thus greatly reduce compared with preparation cost and the probe for preparing with conventional probe preparation method with specificity of sensitivity that captures
Without difference.
Description of the drawings
Fig. 1 is the capture effect figure of human mitochondrial DNA capture probe prepared by the present invention, wherein, A-D rows：Present invention system
The capture effect (four repetitions) of standby probe, E rows：The capture effect of the probe prepared using conventional method.
Specific embodiment
With reference to the accompanying drawings and examples the present invention is elaborated.
(1) preparation method of human mitochondrial DNA capture probe, comprises the following steps：
1. mitochondrial DNA full length sequence is expanded with three pairs of primers (1F and 1R, 2F and 2R, 3F and 3R)；
The condition of the amplification is：
(1) PCR reaction systems：
Archaeal dna polymerase used is that KOD FX, article No. KFX-101 spin in Japan.
Reaction system：
(2) PCR response procedures are：94 DEG C of 2 min of denaturation；94 DEG C of 30 s of denaturation, 60 DEG C of 30 s of annealing, 72 DEG C extend 8
min；40 circulations；72 DEG C of 15 min of extension.
The sequence of the primer is as shown in table 1：
Table 1
2. the amplified production equimolar of three pairs of primers is mixed, the fragment for being broken into about 200bp with Ultrasound Instrument；
Ultrasonically treated step and condition are：
The PCR primer (product mixtures of three pairs of primers) for reclaiming is diluted to 10ng/L；
Take 100L to add in EP pipes after dilution, ultrasonically treated, tune is then carried out using the new II cup type Ultrasound Instruments of sesame 98- in Ningbo
Whole power output be 1000Hz, 15 seconds on, 15 seconds off, 90 secondary responses；
The Ampure bead of 0.9 times of volume (90L) Beckman Kurt are added in ultrasonically treated backward EP pipes, fully mixed
After even, 5min is stood；
Then slightly centrifugation (use for laboratory palm centrifuge, without rotating speed and time requirement, as long as can will be bonded at tube wall and lid
Liquid on son is thrown to ttom of pipe, and this operational term is spin-down), EP pipes are placed in magnet stand after centrifugation, magnetic bead is treated
After (Ampure bead) is fully adsorbed, inhale and abandon supernatant；
70% ethanol of 700L washing magnetic bead (Ampure bead) (EP pipes are not removed from magnet stand) twice is subsequently adding, is washed
Magnetic bead is dried after washing；
Then 16L water is added in EP pipes, fully resuspended magnetic bead (Ampure bead)；
Then EP pipes are placed in magnet stand, after magnetic bead (Ampure bead) is fully adsorbed, shift the supernatant (DNA for containing
The size of fragment is about 200bp) preserve into new PCR pipe；
3. the fragment that air exercise is had no progeny carries out biotin labeling；
With reference to Roche High Prime products instructions.Article No. 1585649.
Size in PCR pipe is about the DNA fragmentation of 200bp after 95 DEG C of denaturation 5min, is rapidly inserted on ice；Cool down completely
Afterwards, 4L Biotin-high prime are added, is fully mixed, 37 DEG C of overnight incubations.
4. the successful DNA fragmentation of the coated Beads enrichment biotin labeling of 100L Avidins is used；(now biotin labeling
DNA fragmentation is combined on the coated magnetic bead of Avidin).
5. the coated magnetic bead of Avidin of DNA fragmentation is combined with 1.25mM sodium hydrate aqueous solutions 50L washings, is removed not
Marked is single-stranded；(now combine on the coated magnetic bead of Avidin be biotin labeling DNA single-stranded)
6., with the resuspended magnetic beads of deionized formamide 100L, 95 DEG C of heating 10min are made biotin be separated with Avidin and (are marked
Note has the single-stranded desorptions from the coated magnetic bead of Avidin of the DNA of biotin)；
DNA described in 7.Qiagen minElute kits 6 in system, products therefrom are biotin labeling
DNA probe, about 3g, i.e. capture probe.
8. human mitochondrial DNA library is captured from people's genome sequencing library with the DNA probe of biotin labeling, specifically
As follows：
The captured object of this probe is to have built the high-throughput sequencing library for completing；
The probe for preparing (the 2～2.5% of sequencing library quality) is mixed with 100～500ng sequencing libraries, in PCR
Hybridize 24h under the conditions of reaction buffer；
The coated magnetic bead of Avidin is added, Avidin is fully combined with biotin；
The library that non-specific binding is washed off with wash buffer；
The 1.25mM NaOH aqueous solution is added, is separated captured library double-strand, and is reclaimed the single-stranded library under washing；
Enter performing PCR amplification to single-stranded library.Capture effect is as shown in Figure 1.Referring to probe prepared by Fig. 1, the inventive method
The probe capture effect that capture effect is prepared with conventional method is compared, and has all reached covering and covering to purpose region 100%
Degree is more uniform, and the 1% of cost deficiency conventional method.
Advantage of the invention maximum be can ensure capture rate and specific under the premise of, substantially reduce probe and be prepared into
This.As a example by being published in a scientific paper on Science translational medicine by 2012, synthesize in text
The target synthesis region about 4.1Mbp of probe.Described in text, the cost of rough calculation only synthesising probing needle will be more than 12,000,000
Yuan.And the method for the present invention is used, prepare needed for same probe less than 100,000 yuans, and capture effect phase therewith
When.
Claims (7)
1. a kind of preparation method of DNA capture probes, it is characterised in that：Comprise the following steps：
(1) special amplimer is designed for area-of-interest in genome, expand the region of interest using amplimer
Domain obtains amplified production；
(2) ultrasonically treated method is adopted to interrupt the amplified production for DNA fragmentation
Amplified production is diluted to 10ng/ μ L, the amplified production after 100 μ L dilutions is then taken and is put in Ultrasound Instrument and carries out ultrasonic place
Reason, the power output of Ultrasound Instrument is 1000Hz, and ultrasonically treated number of times is 90 times, and the single sonication time is 15 seconds, surpasses twice
It is spaced 15 seconds between sonication；
(3) biotin labeling is carried out to DNA fragmentation
DNA fragmentation is rapidly inserted on ice after 95 DEG C of denaturation 5min；After cooling down completely, 4 μ LBiotin-high prime are added,
Fully mix, 37 DEG C of overnight incubations；
(4) after step (3), from the DNA fragmentation, the isolated DNA for being marked with biotin is single-stranded, obtains DNA captures and visits
Pin；
The area-of-interest is human mitochondrial DNA.
2. a kind of preparation method of DNA capture probes according to claim 1, it is characterised in that：Before carrying out biotin labeling,
The DNA fragmentation that step (2) is obtained is purified according to clip size, biotin labeling, the method for the purifying is then carried out
Comprise the following steps：Carry out after nucleic acid purification magnetic bead is added in the 100 μ L 10ng/ μ L amplified productions processed through step (2)
Mix, then stand 5min；It is placed in after standing on magnet stand, after nucleic acid purification magnetic bead is adsorbed, inhales and abandon supernatant；Then with 70%
Ethanol washs nucleic acid purification magnetic bead, dries nucleic acid purification magnetic bead after washing, and the nucleic acid purification magnetic bead after being dried with water is resuspended is rearmounted
On magnet stand, after nucleic acid purification magnetic bead is adsorbed, supernatant is shifted, containing DNA fragmentation after purification in the supernatant.
3. a kind of preparation method of DNA capture probes according to claim 2, it is characterised in that：The nucleic acid purification magnetic bead
Addition be 90 μ L, the consumption of 70% ethanol uses 500～750 μ L for washing every time.
4. a kind of preparation method of DNA capture probes according to claim 1, it is characterised in that：Step (4) specifically wrap
Include following steps：
A () adsorbs the successful DNA fragmentation of biotin labeling with the coated magnetic bead of Avidin；
B () washs the coated magnetic bead of the Avidin after step (a) with sodium hydrate aqueous solution, by the DNA fragmentation
The DNA of unmarked biotin is single-stranded to be removed from the coated magnetic bead of the Avidin；
C () heats 10min with the coated magnetic bead of the resuspended Avidin of deionized formamide and in 95 DEG C after step (b),
The biotin is made to separate with the Avidin, the DNA for being then peeled off obtaining being marked with biotin is single-stranded.
5. a kind of preparation method of DNA capture probes according to claim 4, it is characterised in that：The NaOH is water-soluble
The concentration of liquid is 1.25mM.
6. the DNA capture probes that prepared by a kind of preparation method of DNA capture probes as claimed in claim 1 are from people's full-length genome
The application in capture human mitochondrial DNA library in sequencing library.
7. the DNA capture probes that according to claim 6 prepared by a kind of preparation method of DNA capture probes are from people's full genome
The application in human mitochondrial DNA library is captured in group sequencing library, it is characterised in that：The consumption of DNA capture probes is the sequencing
The 2～2.5% of Library Quality.
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| CN105779590A (en) * | 2016-03-16 | 2016-07-20 | 北京迈基诺基因科技有限责任公司 | Capture probe set and kit for detecting human mitochondrial genes |
| CN105671176A (en) * | 2016-03-16 | 2016-06-15 | 北京迈基诺基因科技有限责任公司 | Probe set and kit for detecting ALK fusion breaking point |
| CN106591288A (en) * | 2016-12-13 | 2017-04-26 | 上海美迪维康生物科技有限公司 | Second-generation sequencing library building technology based on probe capturing |
| CN107236727B (en) * | 2017-05-31 | 2021-09-14 | 江苏为真生物医药技术股份有限公司 | Preparation method of single-stranded probe for polygene capture sequencing |
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| CN109609610A (en) * | 2018-11-27 | 2019-04-12 | 江苏苏博生物医学科技南京有限公司 | A method of self-control probe in detecting BRCA gene |
| CN109810973A (en) * | 2019-03-19 | 2019-05-28 | 深圳因合生物科技有限公司 | A method of gDNA simulation cfDNA is interrupted using ultrasonic wave |
| CN110305945A (en) * | 2019-07-09 | 2019-10-08 | 中国人民解放军第四军医大学 | A kind of free Mitochondrial DNA Mutation detection technique based on two generation sequencing technologies |
| US20210032702A1 (en) * | 2019-07-31 | 2021-02-04 | The General Hospital Corporation | Lineage inference from single-cell transcriptomes |
| CN113122612B (en) * | 2019-12-31 | 2023-06-27 | 深圳华大智造科技股份有限公司 | Preparation method and application of DNA capture probe for capturing and sequencing target area |
| CN113046346A (en) * | 2021-03-18 | 2021-06-29 | 深圳人体密码基因科技有限公司 | Preparation method of genome capture probe |
| CN114480739A (en) * | 2022-02-18 | 2022-05-13 | 武汉凯德维斯医学检验实验室有限公司 | Method for preparing human papilloma virus capture probe set and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1712544A (en) * | 2004-06-14 | 2005-12-28 | 蔡剑平 | Magnetic capture of SARS coronary virus and PCR detection therewith |
| CN101864489A (en) * | 2010-06-09 | 2010-10-20 | 北京大学 | Method for gathering foreign DNA in transgenosis product |
| CN102839168A (en) * | 2012-07-31 | 2012-12-26 | 深圳华大基因研究院 | Nucleic acid probe, and preparation method and application thereof |
-
2014
- 2014-09-12 CN CN201410464934.0A patent/CN104278022B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1712544A (en) * | 2004-06-14 | 2005-12-28 | 蔡剑平 | Magnetic capture of SARS coronary virus and PCR detection therewith |
| CN101864489A (en) * | 2010-06-09 | 2010-10-20 | 北京大学 | Method for gathering foreign DNA in transgenosis product |
| CN102839168A (en) * | 2012-07-31 | 2012-12-26 | 深圳华大基因研究院 | Nucleic acid probe, and preparation method and application thereof |
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