CN104278022A - Preparation method and application of DNA capturing probe - Google Patents
Preparation method and application of DNA capturing probe Download PDFInfo
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- CN104278022A CN104278022A CN201410464934.0A CN201410464934A CN104278022A CN 104278022 A CN104278022 A CN 104278022A CN 201410464934 A CN201410464934 A CN 201410464934A CN 104278022 A CN104278022 A CN 104278022A
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Abstract
The invention provides a preparation method and application of a DNA capturing probe. The preparation method of the probe comprises the steps of amplifying a target gene; randomly breaking; and labeling with biotin. The traditional tile coverage is replaced with the design thought of random coverage; compared with the traditional probe preparation method in which a great number of DNA coverage target regions are needed to be synthesized, the preparation method provided by the invention has the advantages that only amplification primers of the target regions are needed to be synthesized, and the problem of cost increase caused by direct synthesis of the probe is avoided, so that the preparation cost is greatly reduced and the capturing sensitivity and specificity are same as those of a probe prepared by using the traditional probe preparation method.
Description
Technical field
The invention belongs to nucleic acid probe preparation field, be specifically related to a kind of preparation method and application of DNA capture probe.
Background technology
The order-checking of tradition mulberry lattice utilizes archaeal dna polymerase to extend the primer be combined on sequence template undetermined, until mix a kind of chain termination nucleotide.Each reaction contains all four kinds of deoxynucleotide triphosphoric acids (dNTP), and the fluorescently-labeled dideoxyribonucleoside triphosphate of the different colours being mixed into limitation (ddNTP).Because ddNTP lacks the 3-OH group required for extending, the oligonucleotide of prolongation is optionally stopped at G, A, T or C place.Terminating point is determined by two deoxidation corresponding in reacting.They have common starting point, but terminate on different Nucleotide, and the fragment varied in size by capillary electrophoresis separation, observes the fluorescent mark of different big or small slice degree to infer sequence.
The method that s-generation sequencing technologies mainly adopts synthesis limit, limit to check order, first utilizes ultrasonic wave that DNA sample to be measured is broken into small segment, and joints different on the two ends of these small segments are added, construct single-stranded DNA banks.Joint in these libraries is attached to solid phase surface, and carries out bridge-type pcr amplification on surface.Through constantly increasing and cycles of denaturation, final each DNA fragmentation concentrates bunchy on respective position, the a lot of part copies of each bundle containing single DNA template, the object of carrying out this process is to realize the strength of signal of base being amplified, to reach the semaphore request needed for order-checking.In sequencing reaction process, in reaction system, add archaeal dna polymerase, adapter-primer and the 4 kinds of dNTP (as Sanger sequencing) with base specific fluorescence mark simultaneously.The 3 '-OH of these dNTP protect by chemical process, thus can only add a dNTP at every turn.Be added to after on synthesis chain at dNTP, all untapped free dNTP and archaeal dna polymerase can be washed away.Then, then add the damping fluid needed for fluorescence excitation, use LASER Excited Fluorescence signal, and complete the record of fluorescent signal by optical device, finally utilize Computer Analysis that optical signalling is converted into order-checking base.After such fluorescent signal record completes, then add chemical reagent quenching fluorescence signal and remove dNTP 3 '-OH blocking group, the sequencing reaction of next round can be carried out.
Since being born from 2005, s-generation sequencing technologies illustrates wide application prospect.The important informations such as the variation of single nucleotide polymorphism, mutantional hotspot, genome structure, colony's polymorphism can be seen by order-checking scientist.The evolution modelling between species is explored by colony's polymorphism analysis evolution field scientist; Be proved to be quick and accurate at microorganism field DNA sequencing somatotype; And have great significance in the relation finding SNP and major disease in medical field genome sequence of resurveying.
Due to often only to specific genome area (such as exon group) instead of full-length genome interested, gene trap probe therefore can be utilized to catch specific region, the DNA sequence dna of catching out is checked order.Probe preparation catches key link in order-checking, and probe preparation method conventional at present mainly takes the mode of " tile type covering design, synthetic base ".Concrete grammar is: for the probe sequence of interested zone design certain length, each sequence moves a certain distance along gene location.Then the mode of synthetic is adopted to synthesize above-mentioned sequence in a large number.Typical representative has Chinese patent " a kind of screening method of mitochondrial disease gene " (CN 101270390 A), for the probe sequence of the non-duplicate zone design 60bp in interested region, each sequence moves design along gene location, move 3bp between probe, realize covering the tile type of target area; Then a large amount of synthesising probing needle of situ synthesis techniques is adopted.Document [Calvo SE in addition, 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 also use similar thinking, just probe length and move distance and change to some extent.
The maximum defect of " tile type covering design, synthetic base " method is that cost is too high, and along with the increase of target area, probe synthesis cost sharply rises.
Summary of the invention
The object of the present invention is to provide a kind of preparation method and application of DNA capture probe, probe preparation cost can be reduced.
For achieving the above object, present invention employs following technical scheme.
A preparation method for DNA capture probe, comprises the following steps:
(1) design special amplimer for area-of-interest in genome, utilize the amplimer described area-of-interest that increases to obtain amplified production;
(2) method of supersound process is adopted to interrupt as DNA fragmentation by described amplified production;
(3) biotin labeling is carried out to DNA fragmentation;
(4) after step (3), be separated from described DNA fragmentation and obtain the DNA single chain being marked with vitamin H, obtain DNA capture probe.
Described area-of-interest is human mitochondrial DNA.
Described step (2) specifically comprises the following steps: amplified production (the human mitochondrial DNA amplified production of recovery) is diluted to 10ng/L, then the amplified production got after 100L dilution is put into ultrasonic apparatus and is carried out supersound process, the output rating of ultrasonic apparatus is 1000Hz, supersound process number of times is 90 times, the single sonication time is 15 seconds, 15 seconds, interval between twice supersound process.
Before carrying out biotin labeling, according to clip size, purifying is carried out to the DNA fragmentation that step (2) obtains, then biotin labeling is carried out, the method of described purifying comprises the following steps: mix add nucleic acid purification magnetic bead (Ampure bead) in the 100L 10ng/L amplified production processed through step (2) after, then leaves standstill 5min; Leaving standstill is placed on magnet stand, after nucleic acid purification magnetic bead is adsorbed, inhales and abandons supernatant; Then use 70% washing with alcohol nucleic acid purification magnetic bead, dry nucleic acid purification magnetic bead after washing, the nucleic acid purification magnetic bead after drying with water is resuspended is placed on magnet stand, and after nucleic acid purification magnetic bead is adsorbed, transfer supernatant, containing the DNA fragmentation after purifying in this supernatant.
The add-on of described nucleic acid purification magnetic bead is 90L (add-on of nucleic acid purification magnetic bead determines the size of the DNA fragmentation reclaimed), and the consumption of 70% ethanol is each washing use 500 ~ 750L.
Described step (4) specifically comprises the following steps:
A () adsorbs the successful DNA fragmentation of biotin labeling with the magnetic bead (invitrogen) of avidin bag quilt;
B (), after step (a), washs the magnetic bead of described avidin bag quilt with aqueous sodium hydroxide solution, removed by the magnetic bead of the DNA single chain of vitamin H unmarked in described DNA fragmentation from described avidin bag quilt;
C (), after step (b), in 95 DEG C of heating 10min, makes described vitamin H be separated with described avidin with the magnetic bead of the resuspended described avidin bag quilt of deionized formamide, is then separated and obtains the DNA single chain being marked with vitamin H.
The concentration of described aqueous sodium hydroxide solution is 1.25mM (the too strong or too weak object that all cannot reach washing of alkalescence).
DNA capture probe prepared by the preparation method of above-mentioned DNA capture probe is in the application of catching human mitochondrial DNA library from people's genome sequencing library.
The consumption of DNA capture probe is 2 ~ 2.5% of described sequencing library quality.
Beneficial effect of the present invention is embodied in:
Present invention employs amplifying target genes-interrupt-biotin labeled probe preparation method at random, utilize the random mentality of designing covered to instead of traditional tile type to cover, with synthesize compared with a large amount of DNA coverage goal region in conventional probe preparation method, the present invention only needs the amplimer synthesizing target area, avoid the cost increase problem that direct synthesising probing needle causes, thus greatly reduce preparation cost and there is no difference compared with the probe prepared of the susceptibility of catching and specificity and conventional probe preparation method.
Accompanying drawing explanation
Fig. 1 is the capture effect figure of human mitochondrial DNA capture probe prepared by the present invention, and wherein, A-D is capable: the present invention prepares the capture effect (repeating for four times) of probe, and E is capable: the capture effect of the probe adopting traditional method to prepare.
Embodiment
Below in conjunction with drawings and Examples, the present invention is elaborated.
(1) preparation method of human mitochondrial DNA capture probe, comprises the following steps:
1. to increase Mitochondrial DNA full length sequence with three pairs of primers (1F and 1R, 2F and 2R, 3F and 3R);
The condition of described amplification is:
(1) PCR reaction system:
Archaeal dna polymerase used is that KOD FX spins in Japan, article No. KFX-101.
Reaction system:
(2) PCR response procedures is: 94 DEG C of denaturation 2 min; 94 DEG C of sex change 30 s, 60 DEG C of annealing 30 s, 72 DEG C extend 8 min; 40 circulations; 72 DEG C extend 15 min.
The sequence of described primer is as shown in table 1:
Table 1
2., by mole mixings such as the amplified productions of three pairs of primers, interrupt the fragment into about 200bp by ultrasonic apparatus;
The step of supersound process and condition are:
The PCR primer (product mixtures of three pairs of primers) reclaimed is diluted to 10ng/L;
Getting 100L after dilution adds in EP pipe, and then use new sesame 98-II cup type ultrasonic apparatus in Ningbo to carry out supersound process, adjustment output rating is 1000Hz, 15 seconds on, 15 seconds off, 90 secondary responses;
In EP pipe, add the Ampure bead of 0.9 times of volume (90L) Beckman Ku Erte after supersound process, fully after mixing, leave standstill 5min;
Then slightly centrifugal (use for laboratory palm whizzer, without rotating speed and time requirement, as long as can the liquid be bonded on tube wall and lid be thrown at the bottom of pipe, this operational term is spin-down), after centrifugal, EP pipe is placed in magnet stand, after magnetic bead (Ampure bead) is fully adsorbed, inhale and abandon supernatant;
Then add 700L 70% washing with alcohol magnetic bead (Ampure bead) twice (EP pipe does not take off from magnet stand), after washing, dry magnetic bead;
Then in EP pipe, 16L water is added, abundant resuspended magnetic bead (Ampure bead);
Then EP pipe is placed in magnet stand, after magnetic bead (Ampure bead) is fully adsorbed, transfer supernatant (size of the DNA fragmentation contained is about 200bp) is preserved to new PCR pipe;
3. the fragment of having no progeny of fighting each other carries out biotin labeling;
With reference to Roche High Prime products instruction.Article No. 1585649.
Size in PCR pipe is about the DNA fragmentation of 200bp after 95 DEG C of sex change 5min, inserts on ice rapidly; Completely after cooling, add 4L Biotin-high prime, fully mix, 37 DEG C of overnight incubation.
4. with the successful DNA fragmentation of Beads enrichment biotin labeling of 100L avidin bag quilt; (now biotin labeled DNA fragmentation is combined on the magnetic bead of avidin bag quilt).
5. be combined with the magnetic bead of the avidin bag quilt of DNA fragmentation with 1.25mM aqueous sodium hydroxide solution 50L washing, remove unlabelled strand; (what the magnetic bead of now avidin bag quilt combined is biotin labeled DNA single chain)
6., with the resuspended magnetic bead of deionized formamide 100L, 95 DEG C of heating 10min, make vitamin H be separated with avidin (being namely marked with the DNA single chain of vitamin H from desorption the magnetic bead of avidin bag quilt);
DNA in system described in 7.Qiagen minElute kits 6, products therefrom is biotin labeled DNA probe, about 3g, i.e. capture probe.
8. from people's genome sequencing library, catch human mitochondrial DNA library with biotin labeled DNA probe, specific as follows:
This probe to catch object be the high-throughput sequencing library built;
The probe prepared (2 ~ 2.5% of sequencing library quality) and 100 ~ 500ng sequencing library are mixed, under PCR reaction buffer condition, hybridizes 24h;
Add the magnetic bead of avidin bag quilt, avidin is fully combined with vitamin H;
The library of non-specific binding is washed off with wash buffer;
Add the 1.25mM NaOH aqueous solution, the library double-strand be captured is separated, and reclaims the strand library under washing;
Pcr amplification is carried out to strand library.Capture effect as shown in Figure 1.See Fig. 1, probe capture effect prepared by the inventive method is compared with probe capture effect prepared by traditional method, all reaches the covering in object region 100% and coverage is more homogeneous, and 1% of the not enough traditional method of cost.
The maximum advantage of the present invention under guarantee capture rate and specific prerequisite, greatly can reduce probe preparation cost.Be published in one section of academic paper on Science translational medicine for 2012, in literary composition, the target synthesis region of synthesising probing needle is about 4.1Mbp.Describe according in literary composition, the cost of rough calculation only synthesising probing needle will more than 1,200 ten thousand yuans.And use method of the present invention, prepare less than 100,000 yuans needed for same probe, and capture effect is suitable with it.
Claims (9)
1. a preparation method for DNA capture probe, is characterized in that: comprise the following steps:
(1) design special amplimer for area-of-interest in genome, utilize the amplimer described area-of-interest that increases to obtain amplified production;
(2) method of supersound process is adopted to interrupt as DNA fragmentation by described amplified production;
(3) biotin labeling is carried out to DNA fragmentation;
(4) after step (3), be separated from described DNA fragmentation and obtain the DNA single chain being marked with vitamin H, obtain DNA capture probe.
2. the preparation method of a kind of DNA capture probe according to claim 1, is characterized in that: described area-of-interest is human mitochondrial DNA.
3. the preparation method of a kind of DNA capture probe according to claim 1, it is characterized in that: described step (2) specifically comprises the following steps: amplified production is diluted to 10ng/ μ L, then the amplified production got after 100 μ L dilutions is put into ultrasonic apparatus and is carried out supersound process, the output rating of ultrasonic apparatus is 1000Hz, supersound process number of times is 90 times, the single sonication time is 15 seconds, 15 seconds, interval between twice supersound process.
4. the preparation method of a kind of DNA capture probe according to claim 1, it is characterized in that: before carrying out biotin labeling, according to clip size, purifying is carried out to the DNA fragmentation that step (2) obtains, then biotin labeling is carried out, the method of described purifying comprises the following steps: mix add nucleic acid purification magnetic bead in the 100 μ L 10ng/ μ L amplified productions processed through step (2) after, then leaves standstill 5min; Leaving standstill is placed on magnet stand, after nucleic acid purification magnetic bead is adsorbed, inhales and abandons supernatant; Then use 70% washing with alcohol nucleic acid purification magnetic bead, dry nucleic acid purification magnetic bead after washing, the nucleic acid purification magnetic bead after drying with water is resuspended is placed on magnet stand, and after nucleic acid purification magnetic bead is adsorbed, transfer supernatant, containing the DNA fragmentation after purifying in this supernatant.
5. the preparation method of a kind of DNA capture probe according to claim 4, is characterized in that: the add-on of described nucleic acid purification magnetic bead is 90 μ L, and the consumption of 70% ethanol is each washing use 500 ~ 750 μ L.
6. the preparation method of a kind of DNA capture probe according to claim 1, is characterized in that: described step (4) specifically comprises the following steps:
A () adsorbs the successful DNA fragmentation of biotin labeling with the magnetic bead of avidin bag quilt;
B (), after step (a), washs the magnetic bead of described avidin bag quilt with aqueous sodium hydroxide solution, removed by the magnetic bead of the DNA single chain of vitamin H unmarked in described DNA fragmentation from described avidin bag quilt;
C (), after step (b), in 95 DEG C of heating 10min, makes described vitamin H be separated with described avidin with the magnetic bead of the resuspended described avidin bag quilt of deionized formamide, is then separated and obtains the DNA single chain being marked with vitamin H.
7. the preparation method of a kind of DNA capture probe according to claim 6, is characterized in that: the concentration of described aqueous sodium hydroxide solution is 1.25mM.
8. the DNA capture probe prepared of the preparation method of a DNA capture probe is as claimed in claim 1 in the application of catching human mitochondrial DNA library from people's genome sequencing library.
9. the DNA capture probe prepared of a kind of preparation method of DNA capture probe, in the application of catching human mitochondrial DNA library from people's genome sequencing library, is characterized in that: the consumption of DNA capture probe is 2 ~ 2.5% of described sequencing library quality according to claim 8.
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| 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 |
| 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 |
| CN106591285A (en) * | 2015-10-19 | 2017-04-26 | 安诺优达基因科技(北京)有限公司 | Method for constructing high available data rate Hi-C library |
| 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 |
| CN107236727A (en) * | 2017-05-31 | 2017-10-10 | 江苏为真生物医药技术股份有限公司 | The single-stranded probe preparation method of polygenes capture sequencing |
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| 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 |
| 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 |
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| CN106591285A (en) * | 2015-10-19 | 2017-04-26 | 安诺优达基因科技(北京)有限公司 | Method for constructing high available data rate Hi-C library |
| 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 |
| 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 |
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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 |
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| 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 |
| 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 |
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