CN108950691A - Probe compositions, kit and the application of genetic disease construction of gene library based on exon trapping - Google Patents
Probe compositions, kit and the application of genetic disease construction of gene library based on exon trapping Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
- C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
Abstract
Probe compositions, kit and the application of the present invention relates to a kind of genetic disease construction of gene library based on exon trapping.The present invention by the study found that design capture probe directly against exon or mRNA, often also can only capture portion exon genes segment, have certain omission, thus a library constructed and not perfect.Therefore, the probe compositions of genetic disease construction of gene library based on exon trapping of the invention are selected using genomic DNA as template, creative classifies to intron sequences, part capture probe is designed in the introne conservative region for closing on exon, the exon trapping region contained than commercial reagent box more includes subregion, and multiple capture probes are set for the conservative region of specific gene, ensure the success rate and covering homogeneity of target area capture by probe density adjustment, to guarantee that gene data that library is included can really reflect all kinds of mutation types of exon level.
Description
Technical field
The present invention relates to genetic test fields, more particularly, to a kind of genetic disease gene text based on exon trapping
Primer composition, kit and the application of library building.
Background technique
Sequencing of extron group technology, which refers to, is caught whole exon 1 domain dnas of genome using target sequence capture technique
The technology of high-flux sequence is carried out after obtaining, the enrichment of target sequence is the key that exon sequencing technologies.Presently commercially available is main
Capture platform have the NimbleGen gene trap chip of Roche, Agilent SureSelect targeting sequence capturing system and
TruSeq and Nextera Rapid sequence capturing system of Illumina etc., main flow be all by DNA library with design
Probe hybridized, by collect hybridized fragment achieve the purpose that be enriched with target gene.
Above-mentioned platform has been widely used, and relevant parameter is also fairly perfect, and has more paper comparative analyses therebetween
Difference, as the core technology of capture, the design of capture probe is the most key.Speculated according to sequencing data, above-mentioned platform uses
Capture probe be commonly the upstream and downstream conservative region of exon transcription site.Building side of the country for exon group library
Method uses in patent of invention 201510419231.0 " a kind of construction method of full-length genome exon sequence capture probe "
It is to be enriched with after extracting total serum IgE reverse transcription using general capture probe；" principle of exon trapping quick separating new gene and
Method " in (Progress in Biochemistry and Biophysics .1997.24 (3) 207-211) using the conservative splicing sequence in 5 ' or 3 ' ends into
Row capture, the target of capture is equally cDNA.Since the expression of mRNA has very strong specificity, what is finally constructed is outer aobvious
Sublibrary is the library of an expression library rather than exon group.Capture probe based on exon or mRNA, often also only
Can capture portion exon genes segment, have certain omission, thus a library constructed and not perfect.
A protein coding gene about more than 20,000 in human genome, protein coding sequence account for whole gene group alkali
Radix amount is less than 1.5%.When abnormal performance occurs for one or more genes, some corresponding phenotype may just produced
Raw clinical symptoms.Hereditary disorder includes gene mutation and numerical abnormalities of chromosomes, if abnormal gene can be from parental generation heredity
To filial generation, that will become a kind of genetic disease.The genetic disease being currently known has more than 7,000 kinds, however known is related to
The gene of inherited genetic only has more than 4,000.Therefore, for genetic disease, if with the entire exon of human genome or
All mRNA of person construct library, then sequencing data is too big, and cost is very high, and there are the unknown genes of a large amount of function, can shadow
Ring genetic analysis as a result, be easy to causeing mistaken diagnosis or failing to pinpoint a disease in diagnosis.
Summary of the invention
Based on this, it is necessary to provide a kind of probe groups of genetic disease construction of gene library based on exon trapping
Object, kit and application are closed, to analyze for the gene studies of a variety of genetic diseases of the mankind.
A kind of probe compositions of the genetic disease construction of gene library based on exon trapping, including sequence difference
With the completely the same a plurality of oligonucleotide probe of the sequence between corresponding site on each chromosome in table 1, each oligonucleotides
5 ' ends of probe are connected with the first linker.
A kind of kit of the genetic disease construction of gene library based on exon trapping, including capture magnetic bead and upper
Probe compositions are stated, is fixed on the capture magnetic bead and can connect mark with the second of the first linker reaction bonded
Remember object.
Above-mentioned probe compositions or mentioned reagent box are in genetic disease gene library of the building based on exon trapping
Application.
It, often also can only be outside capture portion by the study found that design capture probe directly against exon or mRNA
Aobvious mrna exon fragment has certain omission, thus the library constructed and not perfect.Therefore, of the invention based on exon trapping
The probe compositions of genetic disease construction of gene library select using genomic DNA as template, it is creative to introne
Sequence is classified, and part capture probe is designed in the introne conservative region for closing on exon, contains and compares commercial reagent
The exon trapping region of box more includes subregion, meanwhile, multiple captures are set for the conservative region of specific gene
Probe ensures the success rate and covering homogeneity of target area capture, by probe density adjustment to guarantee what library was included
Gene data can really reflect the mutation types such as point mutation and the copy numerical mutation of exon level.
The probe compositions and kit of the genetic disease construction of gene library based on exon trapping can be used
In genetic disease gene library of the building based on exon trapping, for the gene studies of a variety of genetic diseases of the mankind point
Analysis, advantageously reduces cost, and reduction is failed to pinpoint a disease in diagnosis, and improves Positive rate, is conducive to clinical expansion.
Detailed description of the invention
Fig. 1 is the electrophoretogram for the DNA fragmentation smashed；
Fig. 2 is the electrophoretogram before capturing after LM-PCR product purification；
Fig. 3 is the electrophoretogram after capturing after LM-PCR product purification.
Specific embodiment
The present invention is from the databases such as NCBI, OMIM, UniProt, HGMD, HUGO and Refseq and the mankind being currently known
Selected in Disease-causing gene multiple Disease-causing genes as target spot and by all assortments of genes together, it is creative to related gene
Intron sequences are classified, and part capture probe is designed in the introne conservative region for closing on exon, contains and compares city
The exon trapping region for selling kit more includes subregion, meanwhile, it is more for the conservative region setting of specific gene
A capture probe ensures the success rate and covering homogeneity of target area capture, by probe density adjustment to guarantee library institute
The gene data for including can really reflect the mutation types such as point mutation and the copy numerical mutation of exon level.
Specifically, the probe compositions packet of the genetic disease construction of gene library of the invention based on exon trapping
The sequence a plurality of oligonucleotide probe completely the same with the sequence between corresponding site on chromosome each in the following table 1 respectively is included, respectively
5 ' ends of the oligonucleotide probe are connected with the first linker.
Table 1
Note: reference sequences hg19；" chr " and subsequent digital (1~22) or alphabetical (" X " or " Y ") indicate specific
Chromosome, the base positions that the subsequent digital representation of chromosome dyes, " | " are used as project and separate symbol.With " chr3 |
48682875 | for 48683082 ", indicate that the 48682875th bit base is between the 48683082nd base on No. 3 chromosomes
Base sequence.
The kit of the present invention also provides a kind of genetic disease construction of gene library based on exon trapping,
Including capture magnetic bead and above-mentioned probe compositions.Being fixed on capture magnetic bead can be with the of the first linker reaction bonded
Two linkers.In a specific example, the first linker is biotin, and the second linker is Avidin
Or Streptavidin.
Further, which further includes that reagent plus A reagent plus linker reagents, LM-PCR reagent, miscellaneous are repaired in the end DNA
Hand at least one of reaction reagent and magnetic capture reagent.
The probe compositions and kit of the above-mentioned genetic disease construction of gene library based on exon trapping can be with
For constructing the genetic disease gene library based on exon trapping, with the gene studies for a variety of genetic diseases of the mankind
Analysis, advantageously reduces cost, and reduction is failed to pinpoint a disease in diagnosis, and improves Positive rate, is conducive to clinical expansion.
The genetic disease construction of gene library of the invention based on exon trapping is used below in conjunction with specific embodiment
Probe compositions and kit and application be described in further detail.
The present embodiment constructs genetic disease gene library to 18 samples, and specific step is as follows.
1, genomic DNA is extracted, and is smashed, DNA fragmentation is obtained
DNA is extracted from 400 μ l whole bloods of each sample according to conventional DNA extraction method, 2 μ l DNA samples is taken to exist
Concentration mensuration is carried out on NanoDrop.DNA concentration requirement OD260/OD280 ratio is measured between 1.8~2.0, OD260/
For OD230 ratio between 1.8~2.2, two above ratio may determine that extracted DNA purity.If exceeding above range,
It is believed that the DNA purity extracted is undesirable, need to extract again or repurity.According to the concentration of measurement, is dissolved and buffered with DNA
Liquid is diluted further to final concentration of 25ng/ μ l.
The DNA of extraction is interrupted with Q800R sonicator, the specific steps are as follows:
Pure water is added into cup type probe cup, and did not had titanium alloy probe.40 μ l are transferred in the centrifuge tube of 0.5ml
The DNA of 25ng/ μ l, is fixed on sample holder, and gently bullet walks the bubble of bottom.Sample holder is put into cup
In formula probe, then into cup type probe pure water is added, until pure water liquid level is concordant with DNA liquid level in pipe.
Temperature is arranged at 3 DEG C in the circulating condensing water system for opening sonicator in advance.It sets and interrupts on host
Program: Pulse on20sec；Pulse off 30sec；Time 20min；Amplitude 40%.Setting temperature is dropped to temperature
When spending 3 DEG C, it can open and interrupt program.After the completion of program operation, the sample in centrifuge tube is placed in -20 DEG C of preservations.All samples
After this completion interrupts, turn off circulating condensing water system, closes engine.
Every sample take 2 μ l to smash after DNA fragmentation, detected with 1.5% agarose gel electrophoresis, final every sample
Fragment length should in 500bp hereinafter, peak value 350bp be qualification.The agarose gel electrophoresis results of 18 samples such as Fig. 1 institute
Show.Wherein, the 1st swimming lane is marker, the 2nd~6 swimming lane difference sample interrupt after DNA fragmentation, clip size about 500bp with
Under, subsequent step purified pool peak value about 350bp or so segment.
2, end is repaired
Every sample takes 50 μ l DNA fragmentations, and 20 ends μ l are added and repair reaction mixture, 70 μ l of total volume.In thermal cycle
In 20 DEG C of incubation 30min on instrument, after purified immediately.
The specific method is as follows for purifying: the Agencourt of 120 μ l being added into every sampleR AMPureR XP
Reagent, 190 μ l of total volume.It is inhaled and is beaten repeatedly up and down with liquid-transfering gun, mix well solution.Incubation at room temperature 10 minutes, make DNA with
Magnetic bead sufficiently combines.Centrifuge tube is placed on magnetic frame, until solution turned clear, carefully discards supernatant.Centrifuge tube is continued
It stays on magnetic frame, 200 μ l, 80% alcohol is added.Incubation at room temperature 30 seconds or more, carefully siphon away alcohol.Centrifuge tube is continued to stay
On magnetic frame, 200 μ l, 80% alcohol is added.Incubation at room temperature 30 seconds or more, alcohol is carefully siphoned away, this step should be as far as possible by wine
Essence blots only, but should be noted that the magnetic bead that do not siphon away bottom.Room temperature is sufficiently dry.Centrifuge tube is taken out from magnetic frame.
3, add A
Contain be added 50 μ l in DNA plerosis-magnetic bead pipe plus A reaction mixture reaction solution, 50 μ l of total volume to each.
Liquid-transfering gun is inhaled repeatedly up and down to be beaten, and sample is mixed well.In 30 DEG C of incubation 30min on thermal cycler, after carry out immediately it is pure
Change.
The specific method is as follows for purifying: the PEG/NaclSPRI of 90 μ l being added into every sampleRSolution, it is overall
140 μ l of product.It is inhaled and is beaten repeatedly up and down with liquid-transfering gun, mix well solution.Incubation at room temperature 10 minutes, combines DNA sufficiently with magnetic bead.
Centrifuge tube is placed on magnetic frame, until solution turned clear, carefully discards supernatant.Centrifuge tube is continued to stay on magnetic frame,
200 μ l, 80% alcohol is added.Incubation at room temperature 30 seconds or more, carefully siphon away alcohol.Centrifuge tube is continued to stay on magnetic frame, is added
Enter 200 μ l, 80% alcohol.Incubation at room temperature 30 seconds or more, alcohol is carefully siphoned away, this step should as far as possible blot alcohol only, but answer
It is careful not to siphon away the magnetic bead of bottom.Room temperature is sufficiently dry.Centrifuge tube is taken out from magnetic frame.
4, adjunction head
2 μ l water, 45 μ l connection mixture (Ligation Master are added into each plus A DNA- magnetic bead centrifuge tube
Mix), 3 μ l linker fragment, 50 μ l of total volume.
It is inhaled and is beaten repeatedly up and down with liquid-transfering gun, mix well sample.In 20 DEG C of incubation 15min on thermal cycler, after
Following purification steps are carried out immediately.The specific method is as follows for purifying:
The PEG/NaclSPRI of 50 μ l is added into every sampleRSolution, 100 μ l of total volume.Above and below liquid-transfering gun
It inhales and beats repeatedly, mix well solution.Incubation at room temperature 10 minutes, combines DNA sufficiently with magnetic bead.Centrifuge tube is placed in magnetic frame
On, until solution turned clear, carefully discards supernatant.Centrifuge tube is continued to stay on magnetic frame, 200 μ l, 80% alcohol is added.Room
Temperature incubation 30 seconds or more, carefully siphon away alcohol.Centrifuge tube is continued to stay on magnetic frame, 200 μ l, 80% alcohol is added.Room temperature
Be incubated for 30 seconds or more, alcohol is carefully siphoned away, this step should as far as possible blot alcohol only, but should be noted that the magnetic that do not siphon away bottom
Pearl.Room temperature is sufficiently dry.Centrifuge tube is taken out from magnetic frame.The 10mM Tris-HCl (pH 8.0) of 50 μ l is added, room temperature is incubated
It educates 2 minutes, so that DNA is separated from magnetic bead.Supernatant is fully transferred in a new centrifuge tube, subsequent behaviour is continued
Make.
5, LM-PCR before capturing
Reaction system are as follows: 2 × KAPA HiFi Hot Start Ready Mix, 25 μ l, the Pre-LM- that concentration is 5 μM
The 20 μ l of DNA fragmentation sample of each 2.5 μ l of PCR Oligos 1 and 2, adjunction head, 50 μ l of total volume.
Reaction condition are as follows: 98 DEG C 45 seconds；98 DEG C 15 seconds, 60 DEG C 30 seconds, 72 DEG C 30 seconds, totally 9 circulation；72 DEG C 1 minute.
Sample saves 72 hours at 4 DEG C or -20 DEG C, or directly carries out subsequent purification experiment.
Purifying method particularly includes:
The Agencourt of 50 μ l is added in every sampleR AMPureRXP reagent, 100 μ l of total volume.Use liquid-transfering gun
It inhales and beats repeatedly up and down, mix well solution.Incubation at room temperature 10 minutes, combines DNA sufficiently with magnetic bead.Centrifuge tube is placed in magnetic
On power frame, until solution turned clear, carefully discards supernatant.Centrifuge tube is continued to stay on magnetic frame, 200 μ l, 80% wine is added
Essence.Incubation at room temperature 30 seconds or more, carefully siphon away alcohol.Centrifuge tube is continued to stay on magnetic frame, 200 μ l, 80% alcohol is added.
Incubation at room temperature 30 seconds or more, alcohol is carefully siphoned away, this step should as far as possible blot alcohol only, but it should be noted that not siphon away bottom
Magnetic bead.Room temperature is sufficiently dry.Centrifuge tube is taken out from magnetic frame.52 μ l ddH are added2O is incubated at room temperature 2 minutes, so that DNA
It is separated from magnetic bead.Centrifuge tube is put back on magnetic frame until solution turned clear.50 μ l supernatants are taken to be transferred to one newly
Pipe in, continue subsequent operation.With 1.5% agarose gel electrophoresis, Qubit and qPCR measurement amplification after DNA concentration and
Clip size.For agarose gel electrophoresis results as shown in Fig. 2, the 1st swimming lane is marker, other swimming lanes are to carry out to different samples
LM-PCR before capturing, clip size is about in 400bp or so.DNA concentration result after Qubit measurement amplification see the table below 2.
Table 2
6, hybridize and capture
(1) reagent and instrument prepare
1. hybridization prepares
A) PCR instrument is opened, setting temperature is 95 DEG C and keeps the equilibrium temperature.
B) 1 middle probe composition of table, 4.5 μ l is taken, thaw at RT is placed.
2. the preparation of the HE Universal Oligo and HE Index Oligo of 5 samples.
3 HEUniversal Oligo and HE IndexOligo of table is prepared
| Ingredient | Content |
| SeqCap HE Universal Oligo | 4500pmol(9μl of 500μM) |
| SeqCap HE Index 1Oligo | 250pmol(0.5μl of 500μM) |
| SeqCap HE Index 2Oligo | 250pmol(0.5μl of 500μM) |
| SeqCap HE Index 3Oligo | 250pmol(0.5μl of 500μM) |
| SeqCap HE Index 4Oligo | 250pmol(0.5μl of 500μM) |
| SeqCap HE Index 5Oligo | 250pmol(0.5μl of 500μM) |
| SeqCap HE Index 6Oligo | 250pmol(0.5μl of 500μM) |
| SeqCap HE Index 7Oligo | 250pmol(0.5μl of 500μM) |
| SeqCap HE Index 8Oligo | 250pmol(0.5μl of 500μM) |
| SeqCap HE Index 9Oligo | 250pmol(0.5μl of 500μM) |
| SeqCap HE Index 10Oligo | 250pmol(0.5μl of 500μM) |
| SeqCap HE Index 11Oligo | 250pmol(0.5μl of 500μM) |
| SeqCap HE Index 12Oligo | 250pmol(0.5μl of 500μM) |
| SeqCap HE Index 13Oligo | 250pmol(0.5μl of 500μM) |
| SeqCap HE Index 14Oligo | 250pmol(0.5μl of 500μM) |
| SeqCap HE Index 15Oligo | 250pmol(0.5μl of 500μM) |
| SeqCap HE Index 16Oligo | 250pmol(0.5μl of 500μM) |
| SeqCap HE Index 17Oligo | 250pmol(0.5μl of 500μM) |
| SeqCap HE Index 18Oligo | 250pmol(0.5μl of 500μM) |
| Ultimate density | 9,000pmol(18μl of 500μM) |
3. the preparation of DNA sample mixed liquor
According to the concentration of specimens that Nanodrop is measured, every sample takes 300ng to be mixed in a 1.5ml centrifuge tube, often
Six samples are mixed into a library, and mixing library sample is made, finally takes the mixing library sample of 1.8 μ g for hybridizing.
4. the preparation of probe capture and magnetic bead eluent
By in Nimble Gen Seq Cap EZ Hybridization and Wash kit 10 × eluent (I,
II, III and Stringent) and 2.5 × Bead eluent be diluted to 1 × working solution, prepare 1 capture unit quantity work
The eluent dosage of liquid is as shown in table 4.
The dilution scale of 4 eluent of table
| Reagent dosage | The PCR grade water of addition | Reaction volume (1 ×) |
| 10×Stringent Wash Buffer(vial 4)–40μl | 360μl | 400μl |
| 10×Wash Buffer I(vial 1)–30μl | 270μl | 300μl |
| 10×Wash Buffer II(vial 2)–20μl | 180μl | 200μl |
| 10×Wash Buffer III(vial 3)–20μl | 180μl | 200μl |
| 2.5×Bead Wash Buffer(vial 7)–200μl | 300μl | 500μl |
Working solution can be reserved for 2 weeks at room temperature.Buffers was placed in 47 DEG C of water-baths in 2 hours and is carried out in advance before elution
Balance.
5. capturing magnetic bead (Capture Beads) pretreatment
Capture magnetic bead was restored to room temperature in 30 minutes before use premise.Magnetic bead sufficient vortex is mixed 15 seconds.It is caught with each
Obtaining unit adds 100 μ l magnetic beads in 1.5ml centrifuge tube.Each centrifuge tube can at most add the magnetic bead amount of 6 capture units.It will centrifugation
Pipe is placed on magnetic frame, when liquid, which becomes, to be clarified (this process is no more than 5 minutes), is carefully discarded supernatant, is careful not to siphon away bottom
The magnetic bead in portion, residual liquid will be removed in subsequent elution step.
Add 1 × Bead Wash Buffer of 2 times of magnetic bead volumes in the centrifuge tube of magnetic frame.By centrifuge tube from magnetic force
It takes out and is vortexed 10 seconds in frame.Centrifuge tube is put back in magnetic frame to combine magnetic bead.Once liquid becomes clarification, can discard
Clearly.It is primary to repeat this step.
Magnetic bead is resuspended with isometric 1 × Bead Wash Buffer.Take 100 μ l be resuspended magnetic bead be added new 0.2ml from
In heart pipe.Centrifuge tube is put back in magnetic frame to combine magnetic bead.Once liquid becomes clarification, supernatant can be discarded.At this point, capture magnetic
Pearl pretreatment is completed, can be in conjunction with the DNA of capture.Subsequent operation should be carried out after this step immediately, avoids capture magnetic bead
Dehydration dries out.A small amount of magnetic bead eluent will not noise capture magnetic bead in conjunction with DNA.
(2) hybridize
The 1mg/ml COT DNA and 1.5 μ g mixing library samples of 5 μ l are added into new 1.5ml centrifuge tube, is mixed
Close object 1.500 μM of the 500 μM of Seq Cap HE Universal Oligo and 3 μ l of 3 μ l are separately added into mixture 1
SeqCap HE Index Oligo (by taking 5 samples as an example) obtains (the sample library/COT DNA/ of mixture 2
SeqCap HE Universal Oligo-SeqCap HE Index Oligo mixture), the composition of mixture 2 such as 5 institute of table
Show.
The composition of 5 mixture 2 of table
| Ingredient | Content |
| COT DNA | 5μg |
| Mix library sample | 1.5μg≤50ul |
| 500μM Seq Cap HE Universal Oligo | 3μl |
| 500μM Seq Cap HE Index Oligos | 3μl |
| Total volume | Volume≤57 μ l |
The centrifuge tube lid of mixture 2 is opened, 60 DEG C is put into DNA vacuum concentration instrument, 15min is concentrated.With addition liquid
The volume of body increases, and concentration time need to be appropriately extended, until liquid is evaporated.
It is added into the mixture 2 being evaporated: 7.5 μ l 2 × Hybridization of of Buffer (vial 5) and 3 μ l
Of Hybridization Component A (vial 6), obtains mixture 3.The composition of mixture 3 is as shown in table 6.
The composition of 6 mixture 3 of table
| Ingredient | Content |
| COT DNA | 5μg |
| Mix library sample | 1.8μg≤50ul |
| 500μMSeqCap HE Universal Oligo | 3μl |
| 500μMSeqCap HE Index Oligos | 3μl |
| 2×Hybridization Buffer(vial 5) | 7.5μl |
| Hybridization Component A(vial 6) | 3μl |
| Final volume | 10.5μl |
Mixture 3 is mixed 10 seconds, and with maximum (top) speed centrifugation 10 seconds under the model centrifuge of concentrating instrument.Mixture is set
In heating 10mins on 95 DEG C of heating modules with denatured DNA.At room temperature with maximum (top) speed centrifugation 10 seconds.The mixture of denaturation is added
In the 0.2ml centrifuge tube for entering 4.5 μ l, 4000 human disease's target gene specific Probe Library.It is vortexed and mixes 3 seconds and be centrifuged 10
Second.Above mixture is fully transferred in 0.2ml centrifuge tube, and covers pipe lid, obtains mixture 4.Mixture 4 is followed in heat
47 DEG C of thermal modules (57 DEG C of hot lid) are incubated for 24 hours on ring instrument.The composition of mixture 4 is as shown in table 7.
The composition of 7 mixture 4 of table
(3) combination of magnetic bead and DNA is captured
The sample of hybridization is added in the capture magnetic bead pre-processed.It is mixed well 10 times, is placed in up and down with pipettor
(57 DEG C of hot lid) combines 45 minutes 47 DEG C in thermal cycler.Sample was vortexed in every 15 minutes and is mixed 3 seconds, to ensure that magnetic bead uniformly hangs
It is floating.
(4) magnetic bead-DNA conjugate elutes
1 × Wash Buffer I of 47 DEG C of 100 μ l is added into the magnetic bead-DNA conjugate of 15 μ l.It is vortexed and mixes 10
Second.Content in 0.2ml centrifuge tube is fully transferred in the centrifuge tube of 0.5ml by brief centrifugation.
Centrifuge tube is put back in magnetic frame to combine magnetic bead.Once liquid becomes clarification, supernatant can be discarded.By centrifuge tube from
It is taken out in magnetic frame, 1 × Stringent Wash Buffer of 47 DEG C of 200 μ l is added, liquid-transfering gun is inhaled up and down plays 10 mixings.
47 DEG C of constant temperature fast operatings of this process are to ensure that temperature does not decline.47 DEG C are incubated for 5 minutes.It is primary to repeat this step.
Centrifuge tube is put back in magnetic frame to combine magnetic bead.Once liquid becomes clarification, supernatant can be discarded.Add 200 rooms μ l
Temperature 1 × Wash Buffer I is vortexed and mixes 2 minutes.If pipe has covered liquid residual, continue gently to beat before next step from
Heart pipe collecting pipe covers liquid.Centrifuge tube is put back in magnetic frame to combine magnetic bead.Once liquid becomes clarification, can discard
Clearly.Add 200 μ l room temperature 1 × Wash Buffer II, is vortexed and mixes 1 minute.Centrifuge tube is put back in magnetic frame to combine magnetic bead.
Once liquid becomes clarification, supernatant can be discarded.Add 200 μ l room temperature 1 × Wash Buffer III, is vortexed and mixes 30 seconds.It will centrifugation
Pipe is put back in magnetic frame to combine magnetic bead.Once liquid becomes clarification, supernatant can be discarded.Centrifuge tube is taken out from magnetic frame,
Every pipe beads-DNA combines capture sample to add 50 μ l ddH2O.The sample of magnetic capture is saved in -15 DEG C to -25 DEG C.DNA
Without being separated magnetic bead again, the sample of magnetic capture will be used for the LM-PCR after next capture as template.
7, LM-PCR after capturing
Reaction system are as follows: 25 μ l of KAPA HiFi Hot Start Ready Mix, the Post- that concentration is 5 μM
The 20 μ l of DNA sample of each 2.5 μ l of captureLM-PCR Oligos 1 and 2, magnetic capture, 50 μ l of total volume.
Reaction condition are as follows: 98 DEG C 45 seconds；98 DEG C 15 seconds, 60 DEG C 30 seconds, 72 DEG C 30 seconds, totally 14 circulation；72 DEG C 1 minute.
Sample saves 72 hours at 4 DEG C or -20 DEG C, or directly carries out subsequent purification experiment.
Purification process are as follows:
The Agencourt of 90 μ l is added into each centrifuge tubeR AMPureRXP reagent, 140 μ l of total volume, with shifting
Liquid rifle is inhaled repeatedly up and down to be beaten, and solution is mixed well.It is incubated at room temperature 15min, combines DNA sufficiently with magnetic bead.Centrifuge tube is placed in
On magnetic frame, stand until liquid becomes limpid.Carefully discard supernatant.Centrifuge tube is continued to stay on magnetic frame, 200 μ l are added
80% alcohol incubation at room temperature 30 seconds or more, carefully siphons away alcohol.Centrifuge tube is continued to stay on magnetic frame, 200 μ l are added
80% alcohol incubation at room temperature 30 seconds or more, carefully siphons away alcohol, and room temperature is sufficiently dry.Centrifuge tube is taken out from magnetic frame, is added
Enter the ddH of 52 μ l2O is incubated at room temperature 2min, separates DNA from magnetic bead.Centrifuge tube is put back into magnetic frame until molten
Liquid becomes limpid.Every sample directly shifts 50 μ l into 1.5ml centrifuge tube, continues subsequent operation.
DNA concentration and segment after being expanded with 1.5% agarose gel electrophoresis, Nanodrop, Qubit and qPCR measurement is big
It is small.For agarose gel electrophoresis results as shown in figure 3, the 1st swimming lane is marker, the second swimming lane is different samples, and peak value about exists
400bp or so.The results are shown in Table 8 for DNA concentration after Qubit and qPCR measurement amplification.
DNA concentration result after table 8 Qubit and qPCR measurement amplification
| Sample number | Group 1 | Group 2 | Group 3 |
| Qubit measured value (ng/ μ l) | 29.8 | 49.5 | 49.5 |
| QPCR measured value (nM) | 100.34 | 168.2 | 174.2 |
Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, all should be considered as described in this specification.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention
Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (9)
1. a kind of probe compositions of the genetic disease construction of gene library based on exon trapping, which is characterized in that packet
Include the sequence a plurality of oligonucleotide probe completely the same with the sequence between corresponding site on following each chromosome respectively:
It is hg19 with reference to genome, 5 ' ends of each oligonucleotide probe are connected with the first linker.
2. a kind of kit of the genetic disease construction of gene library based on exon trapping, which is characterized in that including catching
Magnetic bead and probe compositions as described in claim 1 are obtained, mark can be connect with described first by being fixed on the capture magnetic bead
Remember the second linker of object reaction bonded.
3. the kit of the genetic disease construction of gene library based on exon trapping as claimed in claim 2, special
Sign is that first linker is biotin, and second linker is Avidin or Streptavidin.
4. the kit of the genetic disease construction of gene library based on exon trapping as claimed in claim 2 or claim 3,
It is characterized in that, further includes that reagent plus A reagent plus linker reagents, LM-PCR reagent, hybridization reaction reagent and magnetic are repaired in the end DNA
Pearl captures at least one of reagent.
5. probe compositions as described in claim 1 or the kit as described in any one of claim 2~4 are in building base
Application in the genetic disease gene library of exon trapping.
6. application as claimed in claim 5, which is characterized in that the building process in library includes the following steps:
Human DNA sample is extracted, DNA fragmentation is broken into；
End reparation plus A and the processing of adjunction head are successively carried out to the DNA fragmentation smashed；
To the LM-PCR processing that treated before DNA fragmentation captures of adjunction head；
Hybridized using the probe compositions with the target DNA fragment in LM-PCR product, is cooperated using with the probe compositions
The capture magnetic capture target DNA fragment used；
Target DNA fragment is eluted from the capture magnetic bead, and after being captured to the target DNA fragment of capture
LM-PCR processing.
7. application as claimed in claim 6, which is characterized in that the length of the DNA fragmentation is not more than 500bp.
8. the use as claimed in claim 7, which is characterized in that the length of the DNA fragmentation is 340bp~360bp.
9. the application as described in any one of claim 6~8, which is characterized in that described to use the probe compositions and LM-
It further include the addition COT DNA into LM-PCR product before target DNA fragment hybridization in PCR product, for blocking non-spy
The step of specific hybridization.
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