CN107794293A - A kind of gene trap kit - Google Patents
A kind of gene trap kit Download PDFInfo
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- CN107794293A CN107794293A CN201610800089.9A CN201610800089A CN107794293A CN 107794293 A CN107794293 A CN 107794293A CN 201610800089 A CN201610800089 A CN 201610800089A CN 107794293 A CN107794293 A CN 107794293A
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Abstract
The present invention provides a kind of gene trap kit.The gene trap kit of the present invention includes the probe for gene trap, and the probe for gene trap includes the probe for target gene and the probe for reference gene.According to the present invention, in the CNV detections being sequenced based on gene trap, even in the later stage Data processing without using background database in the case of, can also obtain good testing result.
Description
Technical field
The invention belongs to molecular in vitro diagnosis field, and in particular to be diagnosed in vitro especially suitable for specific structure variation
Gene trap kit.
Background technology
The copy number variation (Copy Number Variation, CNV) of gene is a kind of clinically very important knot
Structure makes a variation, the prognosis with kinds of tumors, and the sensitiveness of targeted drug is related.Reliable CNV testing results can be clinical application
And condition assessment etc. provides highly important foundation.
Current clinically used CNV detection techniques be mostly the laboratory facilities of PCR-based or SABC (such as
FISH, IHC etc.).Such method can only carry out CNV detections to common known, and single detection can only cover a base
Cause.
CNV detections based on new-generation sequencing (Next-Generation Sequencing, NGS) platform, can protected
The CNV testing results of multiple genes are disposably provided on the premise of card detection performance, are had more simultaneously for low-purity tumor sample
Good Detection results.
Traditional detections of the CNV based on NGS platforms use full-length genome low depth sequencing data.With NGS technologies not
Disconnected progress, the high depth sequencing technologies based on target area capture gradually show advantage under the application scenarios of clinical detection.
, it is necessary to which the Data processing in the later stage introduces background database for being detected based on the CNV of NGS platforms.This
Following shortcomings be present in the data processing method that kind introduces background database：(1) background database is typically derived from healthy normal person
Target gene sequencing data, it is necessary to individually establish, cost is high；(2) experiment condition, sequencing when background database is established are flat
The influence of platform is big, and the experiment condition, microarray dataset when how to ensure to obtain target gene sequencing data are with establishing background database
When experiment condition, the uniformity of microarray dataset be a problem；(3) for this Preference phase of full-length genome high-flux sequence
For less data, the foundation of context vault is excessively cumbersome.
The content of the invention
In view of above-mentioned the deficiencies in the prior art, it is an object of the invention to provide a kind of new gene trap reagent
Box, if in the specific structure variation detection being sequenced based on gene trap, gene is carried out using the gene trap kit
Capture, then even in the later stage Data processing without using background database in the case of, still be able to obtain good detection knot
Fruit.
The present inventor has made intensive studies to solve the above problems, and as a result finds：By being sequenced based on gene trap
Specific structure variation detection in, using the gene trap kit comprising the probe for reference gene, even in the later stage
Data processing without using background database in the case of, good testing result can be also obtained, so as to complete this hair
It is bright.It should be noted that in this manual, specific gene structure variation refers to that gene copy number variation, large fragment are reset
Or sections in series repeats.
That is, the present invention includes：
1. a kind of gene trap kit, it includes the probe for gene trap, the probe for gene trap
Including：
For the probe of target gene；And
For the probe of reference gene.
2. the gene trap kit according to item 1, wherein, the probe for gene trap is biotinylation
's.
3. the gene trap kit according to item 1 or 2, wherein, it is affine that the gene trap kit also includes strepto-
The magnetic bead of element mark.
4. the gene trap kit according to any one of item 1~3, wherein, the gene trap kit also includes
For blocking the DNA fragmentation of non-specific hybridization.
5. the gene trap kit according to any one of item 1~4, wherein, the gene trap kit also includes
For the buffer solution for the ionic environment for providing hybrid capture reaction.
6. the gene trap kit according to any one of item 1~5, wherein, the gene trap kit also includes
For eluting the cleaning fluid of physical absorption or non-specific hybridization.
7. the gene trap kit according to any one of item 1~6, wherein, the gene trap kit also includes
The primer expanded for the target gene to capturing and/or reference gene.
8. the gene trap kit according to any one of item 1~7, wherein, the gene trap kit is used for
Gene trap is carried out in gene trap sequencing.
9. the gene trap kit according to any one of item 1~8, wherein, the gene trap is sequenced for examining
Cls gene copy number variation, large fragment are reset or sections in series repeats.
A kind of 10. two generations sequencing DNA texts that detection is repeated for gene copy number variation, large fragment rearrangement or sections in series
The construction method in storehouse, it includes：The step of gene trap is carried out to the target gene in DNA library and reference gene；Wherein, institute
Gene trap is stated to carry out using the gene trap kit any one of item 1~9.
11. a kind of gene trap method, the gene trap kit any one of its usage right requirement 1~9 enters
OK.
According to the present invention, in the specific structure variation detection being sequenced based on gene trap, even in the data in later stage
In processing without using background database in the case of, can also obtain good testing result.
Brief description of the drawings
Fig. 1 is shown in the data processing step of embodiment 1, using the figure of the data processed result of A schemes.
Fig. 2 is shown in the data processing step of embodiment 1, using the figure of the data processed result of B schemes.
The embodiment of invention
The scientific and technical terminology referred in this specification has the implication identical implication being generally understood that with those skilled in the art,
It is defined if any definition of the conflict in this specification.
First, in an aspect, the present invention provides a kind of gene trap kit (gene trap reagent of the invention
Box), it includes the probe for gene trap, and the probe for gene trap includes：Probe for target gene, with
And the probe for reference gene.
In this manual, gene trap refers to：Based on the complementary pairing principle between nucleotide sequence, using with mark
The oligonucleotide probe of note (such as biotin) transfers one or more target gene, and by Streptavidin MagneSphere by purpose
Gene is separated from gene library.In this manual, oligonucleotides, polynucleotides and nucleic acid three can exchange and make
With the elementary cell for forming above-mentioned three is nucleotides (including ribonucleotide and/or deoxyribonucleotide).
In this manual, target gene refers to：Target disease state can be reflected, instruct target disease medication and/or pre-
Survey the gene of prognosis.Generally, it is considered that in the patient of target disease is suffered from, the gene has specific structure variation.For example,
In the case that the target disease is cancer, the target gene can be that specific structure variation may occur in cancer
Gene.
It is different because of different target diseases as target gene, it is typically different.The target disease can be example
Such as：Blood disease (such as leukaemia) or solid carcinoma (such as stomach cancer, mammary gland, colorectal cancer, lung cancer etc.).
Specifically for example, in the case of the target disease is hemopathic, the target gene can be such as MLL bases
Cause.
For another example the missing of the large fragment of genome will cause the copy number variation of DNA sequence dna with resetting, and occur new
Arrangement mode.In patient with breast cancer, the exon region of BRCA1/2 genes occurs large fragment deletion and can behave as with rearrangement
More than 200 kinds of hypotype.The common variant form of BRCA1/2 genes includes point mutation, insertion and missing, large fragment deletion is reset.
The tumour such as BRCA1/2 gene unconventionalities and breast cancer, oophoroma, prostate cancer has substantial connection.BRCA1/2 detections are faced
Bed meaning is to assess breast cancer-ovarian cancer syndrome (HBOC) onset risk, prediction platinum medicine sensitiveness, prompts tumour pre-
Afterwards.
In another example in the case of cancer listed during the target disease is following table, the target gene can be
In following table more than one or both of listed gene corresponding with the cancer or all.
Gene cancer
In this manual, reference gene refers to：It cannot act as target gene but can be used as reference, background data is provided
The chromosomal DNA region of information.Reference gene is the metastable gene of copy number or region on human genome, and it is one section
DNA sequence dna highly conserved and without specific structure variation.Generally, it is considered that either in healthy normal person, or
In the patient of target disease, the gene does not all have specific structure variation.
For different target diseases, reference gene can be with identical, can also be different.The inventors discovered that it is selected from the group
In one or more or whole reference genes can be with general in the detection of different target diseases.
In addition to for the probe of target gene and for the probe of reference gene, the probe for gene trap
The probe for the other genes related to target disease can also be included.The other genes related to target disease refer to：
May occur in target disease all kinds mutation gene, its can be used as diagnose target disease, instruct target disease medication and/
Or the gene of prediction prognosis, but in the genome of the diseased cells of patient of target disease is suffered from, the specific knot of the gene
Structure variation is not common or do not possess clinical meaning.
Preferably, the probe for gene trap is biotinylated.So, marked by streptavidin can be used
Solid phase carrier the probe separates after capturing target gene are come out, the solid phase carrier is preferably magnetic bead.It is therefore preferable that
Ground, gene trap kit of the invention also include the magnetic bead of marked by streptavidin.
In the generation of gene trap two is sequenced, the process object of gene trap carries mark generally by what PCR expanded to obtain
The DNA library of (Index) is signed, the end of the DNA molecular in the DNA library generally carries joint sequence.Carrying out gene trap
When, it usually needs the joint sequence is closed.It is used to seal it is therefore preferred that also including in the gene trap kit of the present invention
The oligonucleotides of the joint sequence is closed, it generally can be that the upstream and downstream that PCR expands to use during the obtained DNA library is drawn
Thing.
Preferably, the DNA fragmentation for being used for blocking non-specific hybridization is also included in gene trap kit of the invention, its
For the repeat region in block gene group.The DNA fragmentation for blocking non-specific hybridization can be placenta source
DNA, its length are mainly 50-300bp, and rich in the DNA sequence dna repeated, are generally used for blocking non-spy in array screening
Cutcross, moreover it can be used to suppress reiterated DNA sequences, can use such as Roche COT human DNA or
The Human Cot-I DNA of ThermoFisher companies.
Preferably, also included in gene trap kit of the invention and be used to provide the ionic environment that hybrid capture reacts
Buffer solution.
Preferably, also included in gene trap kit of the invention and be used to elute physical absorption or non-specific hybridization
Cleaning fluid, they are used for the specificity for improving gene trap.
Preferably, also also include being used for capture comprising the gene trap kit in gene trap kit of the invention
To target gene and/or the reference gene primer and/or PCR reaction solutions that are expanded.
Preferably, reference substance is also included in gene trap kit of the invention, it is used as positive control and/or feminine gender is right
According to.
Each component in the gene trap kit of the present invention is preferably independently packaged in container, but is not influenceing to make
On the premise of, some of which component can also be merged and packed.
The gene trap kit of the present invention, it can be used for carrying out gene trap in gene trap is sequenced.Preferably, institute
Gene trap sequencing is stated to can be used for detecting specific structure variation.
The gene trap kit of the present invention can be used for the structure generation of gene trap two sequencing DNA library, the gene trap two
Generation sequencing DNA library detects available for specific structure variation.Therefore, in another aspect, the present invention provides a kind of for spy
The construction method (construction method of the invention) of the two generations sequencing DNA library of fixed structure variation detection, it includes：To DNA texts
The step of target gene and reference gene in storehouse carry out gene trap；Wherein, the gene trap uses gene of the invention
Capture agent box is carried out.When being used to build the generation of gene trap two sequencing DNA library by the gene trap kit of the present invention, gene
Capture, the operation in structure library can use operation known in the art.
In general, the construction method of the present invention can comprise the steps：
Step A：The DNA fragmentation being desired with the sample of sequencing is subjected to end reparation, obtains multigroup flat end DNA pieces
Section；
Step B：The flat terminal DNA fragments are subjected to 3' ends and add A, obtain the DNA fragmentation that 3' ends add A；
Step C：The DNA fragmentation that the 3' ends add A is subjected to adjunction head, obtains adjunction head DNA fragmentation；
Step D：Described plus linker DNA is entered into performing PCR amplification, obtains amplified production (such as DNA library with label)；
Step E：Using the amplified production as object, gene trap is carried out using the gene trap kit of the present invention,
And the gene to capturing (including target gene and reference gene, can also optionally include related to target disease other
Gene) enter performing PCR amplification, obtain capturing gene library.
The sample can be selected from blood, cerebrospinal fluid, Pleural effusions, tissue or FFPE samples.DNA fragmentation in the sample
Such as can be Circulating tumor DNA (ctDNA).The sample can be one or multiple, such as more than 2, then example
Such as more than 5, then such as more than 10.
The capture gene library upper machine can be sequenced after quality inspection, so as to obtain sequencing data.Microarray dataset can be
Such as with Illumina HiSeq 2500,4000 platforms or NextSeq 550AR platforms.
During data processing is carried out to the sequencing data, traditionally need to introduce background database.But such as
During the fruit structure generation of gene trap two sequencing DNA library, use the gene trap kit of the present invention to carry out gene and caught
Obtain, then (can certainly, introduce the back of the body without introducing background database using the sequencing data of the reference gene of acquisition as background
Scape database can also).The advantages of this mode, includes：(1) without individually establishing background database, cost has been saved；(2) obtain
Reference gene sequencing data when experiment condition, microarray dataset with obtain target gene sequencing data when experiment bar
Part, microarray dataset are completely the same.
Therefore, according to the present invention, in the specific structure variation detection being sequenced based on gene trap, even in the later stage
In the case that Data processing is without using background database (it is of course also possible to using background database), it can also obtain good
Testing result.In this manual, " testing result is good " refers to：In the specific structure variation being sequenced based on gene trap
In detection, in the case where carrying out gene trap using the gene trap kit of the present invention, by by the reference gene of acquisition
Sequencing data it is consistent with the testing result by introducing background database to obtain as background, gained testing result.
Embodiment
More specific description is carried out to the present invention by the following examples.It should be appreciated that embodiment described herein is
It is of the invention not for limiting for explaining the present invention.
Embodiment 1
The design and synthesis of 1.1 blood disease genetic test liquid chips
According to the literature and multiple databases (NCBI-PDB, PubMed, COSMIC, Clinvar, HGMD,
GeneCards, zj-LOVD etc.) information, have selected for blood disease detection target gene, wherein, mll gene is in blood
There may be sections in series to repeat in patient, it is located at o.11 chromosome.Probe is for the outer of specified disease Disease-causing gene
Aobvious son (or extron rear and front end 200bp) or specific target areas are designed.The database such as designed probe and NCBI
Carry out Blast comparisons, and to ensure the base number of probe between 60~80nt, TM values it is moderate, without spies such as palindromic sequences
Different structure and sequence homology are between 50%~80%, by these numerical value to ensure the specificity of probe.
The probe sequence designed is synthesized into oligonucleotide probe, obtained after PCR checkings leucocythemia related because of capture
Probing pin clone storehouse.
In addition, using following genes as reference gene：ABL1, CUX1, DNAH9, FBXW7, GNAS, NTRK2, SMAD4 and
SYK, specific region and the position in human genome refer to the description in specification.Set for the probe of reference gene
Meter and synthesis are identical with the design and synthesis of the probe for target gene.
The amplimer or joint component determined using biotin labeling quantity and position, which prepares to contain, is advantageous to target fragments
The capture probe library of crawl, the capture probe collection for finally giving known biotin labeling quantity and position carry biotin mark
The capture probe storehouse of note.
1.2DNA extraction
Embrane method was used to extract poba gene group DNA, specific steps are with reference to Tiangeng blood extracts kit step.
Repair (End Repair) in 1.3 ends:
(1) in advance from -20 from the kit of preservation take out needed for reagent, be placed on ice chest thaw and shake it is mixed
It is even.
Single sample amount of preparation can prepare Mix in proportion referring to table 2, multisample.
Table 2
(2) reaction is repaired in end：25 μ L Mix are dispensed into 1.5mL centrifuge tube, packing is completed and confirms to add nothing
After by mistake, add DNA sample and 1.5mL centrifuge tubes are placed in Thermomixer 20 DEG C of warm bath 30 minutes.Reaction uses after terminating
DNA in 1.8 × nucleic acid purification magnetic bead recovery purifying reaction system, it is dissolved in 32 μ L EB.
1.4 ends add " A " (A-Tailing)
(1) in advance from -20 DEG C preservation kit in take out needed for reagent, be placed on ice chest thaw and shake it is mixed
It is even.Single sample amount of preparation can prepare Mix in proportion referring to table 3, multisample：
Table 3
(2) end adds " A " to react：18 μ L Mix are dispensed into 1.5mL centrifuge tube, after confirmation is errorless, are added on 32 μ L
The DNA of one step purifying recovery.Concussion is mixed and centrifuged after adding, and 1.5mL centrifuge tubes are placed in into 37 DEG C of warm bath in Thermomixer
30 minutes.Using the DNA in 1.8 × nucleic acid purification magnetic bead recovery purifying reaction system, it is dissolved in 18 μ L EB.
The connection (Adapter Ligation) of 1.5 joints
(1) in advance from -20 DEG C preservation kit in take out needed for reagent, be placed on ice chest thaw and shake it is mixed
It is even.Single sample amount of preparation can prepare Mix in proportion referring to table 4, multisample：
Table 4
(2) coupled reaction of joint：27 μ L Mix are dispensed into 1.5mL centrifuge tube, after confirmation is errorless, are added on 18 μ L
The DNA of one step purifying recovery.Concussion is mixed and centrifuged after adding, and sample tube is placed in into 20 DEG C of warm bath 15 in Thermomixer divides
Clock.Using the DNA in 1.8 × nucleic acid purification magnetic bead recovery purifying reaction system, it is dissolved in 30 μ L EB.
1.6PCR reaction
(1) reagent needed for being taken out from the kit of -20 DEG C of preservations, is placed on ice chest and thaws and shake mixing.
PCR reaction systems are prepared in 0.2mL PCR pipe, multisample can prepare Mix in proportion：
Table 5
(2) PCR programs are set, the program setting of PCR reactions is as follows：
Reaction, which terminates in time to take out sample, to be put into 4 DEG C of refrigerators preservations and exits or close instrument on request.
(3) it is dissolved in 20 μ L's with the DNA in 0.9 × nucleic acid purification magnetic bead recovery purifying reaction system, library after purification
ddH2In O.Label is posted, storehouse completion is built in small fragment library.Qubit detections are carried out to library, record each library concentration size.
By library censorship Agilent 2100, peak figure result is preserved.
1.7 prepare Hybrid Library
(1) in this experiment, for provide hybrid capture reaction ionic environment buffer solution and for elute physics inhale
Attached or non-specific hybridization cleaning fluid, rinsing liquid are purchased from Roche companies.
(2) Hybrid Library is prepared：DNA library to be hybridized is melted on ice, takes the μ g of gross mass 1 (to be walked in subsequent operation
This DNA library is referred to as sample library in rapid).
(3) Ann primers Pool is prepared：By Tag primer In1 (100 μM) and consensus primer corresponding to the Index of sample library
(1000 μM) are placed on ice to melt, and respectively take 1000pmol mixing (this mixture to be referred to as into Ann primers in subsequent process steps
pool)。
(4) preparation of sample is hybridized：5 μ L COT DNA (Human Cot-1DNA, Life are added into 1.5mL EP pipes
Technologies, 1mg/mL) and 1 μ g samples library.Ann primers pool is added into mixture.Sealed and made with sealed membrane
The hybridization sample EP pipes got ready, the temperature of regulation vacuum drying instrument will fill sample library/COT DNA/Ann to 50~60 DEG C
Primer pool EP pipes are placed in vacuum plant until being completely dried.
(5) solution of sample is hybridized：Added into sample library/COT DNA/Ann primers pool dry powder：
7.5 μ 2 × hybridization buffers of L
3 μ L hybridization components A
So far, following components is contained in EP pipes：
1.8 prepare Hybrid Library
(1) said mixture for adding hybridization buffer is vortexed and shaken 10 seconds, maximum (top) speed centrifugation 10 after fully mixing
Second.
(2) said mixture is placed on preprepared 95 DEG C of heating modules and be denatured 10 minutes.After removing denaturation
Mixture, at room temperature maximum revolution centrifuge 10 seconds.
(3) said mixture is transferred in the 0.2mL flat cover PCR pipes that chip is captured containing 4.5 μ L.Be vortexed concussion 3 seconds,
Maximum revolution centrifuges 10 seconds after fully mixing.So far, following component is contained in Hybridization samples mixture：
(4) Hybridization samples mixture is placed on 47 DEG C of heating modules 16 hours.The hot lid temperature of heating module needs to set
For 57 DEG C, product need to carry out subsequently eluting reclaimer operation after hybridization.
(5) by 10 × cleaning fluid (I, II and III), 10 × rinsing liquid and 2.5 × magnetic bead cleaning fluid be configured to 1 × working solution.
Table 8
(6) following reagent is preheated at 47 DEG C in heating module：
400 μ 1 × rinsing liquids of L
100 μ 1 × cleaning fluids of L I
1.9 prepare affine absorption magnetic bead
(1) before experiment starts, by Streptavidin MagneSphere (Dynabeads M-280Streptavidin, hereinafter referred to as magnetic
Pearl) balance is after 30 minutes at room temperature, and magnetic bead is fully vortexed mixing 15 seconds.
(2) 100 μ L magnetic beads are dispensed into 1.5mL centrifuge tubes, 1 1.5mL centrifuge tube, which at most can be used to prepare 6 hybridization, catches
Obtain required magnetic bead.
(3) centrifuge tube for filling magnetic bead is placed on magnetic frame, careful inhale abandons supernatant after about 5 minutes, by magnetic bead stay in from
In heart pipe, add 1 × magnetic bead cleaning fluid of twice magnetic bead initial volume.Centrifuge tube is removed from magnetic frame, is vortexed and mixes 10
Second.The centrifuge tube for filling magnetic bead is put back into magnetic frame, adsorbs magnetic bead.Treat that solution is clarified, supernatant is abandoned in suction.Time step is repeated, is washed altogether
Wash twice.
(4) inhaled after washing and abandon magnetic bead cleaning fluid, be vortexed with 1 × magnetic bead cleaning fluid of magnetic bead initial volume and magnetic is resuspended
Pearl.100 μ L magnetic beads of resuspension are transferred in 0.2mL PCR pipe.PCR pipe is placed on magnetic frame and adsorbs magnetic bead, after solution clarification
Supernatant is abandoned in suction.So far, the affine absorption magnetic bead needed for capture dna is prepared and finished, and should carry out lower step association reaction immediately.
1.10DNA and affine absorption magnetic bead combination and rinsing
(1) the sample library of hybridization is transferred in the 0.2mL PCR pipes for filling affine absorption magnetic bead, suction is made a call to 10 times, by two
Person mixes.
(2) 0.2mL PCR pipes are placed in 47 DEG C of heating modules 45 minutes, were vortexed and are mixed once every 15 minutes, make DNA with
Magnetic bead combines.
After (3) 45 minutes are incubated, the μ L of 1 × cleaning fluid I 100 of 47 DEG C of preheatings are added in the DNA sample captured to 15 μ L.
It is vortexed and mixes 10 seconds.Whole components in 0.2mL PCR pipes are transferred in 1.5mL centrifuge tubes.1.5mL centrifuge tubes are placed in magnetic force
Magnetic bead is adsorbed on frame, abandons supernatant.
(4) 1.5mL centrifuge tubes are removed from magnetic frame, adds 1 × rinsing liquid that 200 μ L preheat 47 DEG C.Mixing is played in suction
10 times (need to operate rapidly, prevent reagent, sample temperature to be less than 47 DEG C).Sample is placed in 47 and mixed in thermal modules 5 minutes after mixing.
This step is repeated, is washed twice altogether with 47 DEG C of 1 × rinsing liquids.1.5mL centrifuge tube is placed on magnetic frame, adsorbs magnetic bead,
Abandon supernatant.
(5) 1 × cleaning fluid I of 200 μ L room temperatures is added into above-mentioned 1.5mL centrifuge tubes, is vortexed and mixes 2 minutes.Will centrifugation
Pipe is placed on magnetic frame, is adsorbed magnetic bead, is abandoned supernatant.1 × cleaning fluid II of 200 μ L room temperatures is added into above-mentioned 1.5mL centrifuge tubes,
It is vortexed and mixes 1 minute.Centrifuge tube is placed on magnetic frame, magnetic bead is adsorbed, abandons supernatant.200 are added into above-mentioned 1.5mL centrifuge tubes
1 × cleaning fluid III of μ L room temperatures, it is vortexed and mixes 30 seconds.Centrifuge tube is placed on magnetic frame, magnetic bead is adsorbed, abandons supernatant.
(6) 1.5mL centrifuge tubes are removed from magnetic frame, add 45 μ L PCR water, dissolving elution magnetic capture sample.By magnetic
Pearl-sample mixtures are placed on -20 DEG C of preservations.
The PCR amplifications of 1.11 capture dnas
(1) according to the form below prepares PCR mix after capture, and the concussion that is vortexed after preparing mixes.Remaining magnetic bead adsorption of DNA is put
In -20 DEG C of preservations.Enriching primer F and enriching primer R are purchased from Invitrogen Corp..
(2) magnetic bead adsorption of DNA PCR amplification program setting is as follows：
(3) recovery purifying of hybrid capture DNA PCR primers：With in nucleic acid purification magnetic bead recovery purifying reaction system
DNA, magnetic bead usage amount is 0.9 ×, library after purification is dissolved in 30 μ L ddH2In O.Post label, hybrid capture library Jian Ku
Complete.
1.12 library quantifies
2100Bioanalyzer (Agilent)/LabChip GX (Caliper) and QPCR detections, record are carried out to library
Library concentration.
Machine is sequenced on 1.13 libraries.
Claims (10)
1. a kind of gene trap kit, it includes the probe for gene trap, and the probe for gene trap includes：
For the probe of target gene；And
For the probe of reference gene.
2. gene trap kit according to claim 1, wherein, the probe for gene trap is biotinylation
's.
3. gene trap kit according to claim 2, wherein, the gene trap kit also includes Streptavidin
The magnetic bead of mark.
4. gene trap kit according to claim 1, wherein, the gene trap kit also includes non-for blocking
The DNA fragmentation of specific hybrid.
5. gene trap kit according to claim 1, wherein, the gene trap kit also includes miscellaneous for providing
Hand over the buffer solution of the ionic environment of capture reaction.
6. gene trap kit according to claim 1, wherein, the gene trap kit also includes being used for eluate
Reason absorption or the cleaning fluid of non-specific hybridization.
7. gene trap kit according to claim 1, wherein, the gene trap kit also includes being used for capture
To the primer that is expanded of target gene and/or reference gene.
8. gene trap kit according to claim 1, wherein, the gene trap kit is used to survey in gene trap
Gene trap is carried out in sequence；Preferably, the gene trap is sequenced for detecting gene copy number variation, large fragment is reset or portion
Divide tandem sequence repeats.
9. a kind of gene trap method, the gene trap kit any one of its usage right requirement 1~8 carries out gene
Capture.
10. a kind of two generations sequencing DNA library that detection is repeated for gene copy number variation, large fragment rearrangement or sections in series
Construction method, it includes：The step of gene trap is carried out to the target gene in DNA library and reference gene；Wherein, the base
Because the gene trap kit any one of capture usage right requirement 1~8 is carried out.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020168668A1 (en) * | 2001-03-27 | 2002-11-14 | Millennium Pharmaceuticals, Inc. | 14691, a human glutamate receptor family member and uses therefor |
| CN102358910A (en) * | 2011-11-03 | 2012-02-22 | 东南大学 | Chemiluminescence detection method for copy number polymorphism based on magnetic separation and primer extension |
| WO2013074492A1 (en) * | 2011-11-14 | 2013-05-23 | Five Prime Therapeutics, Inc. | Methods of treating cancer |
| CN103667268A (en) * | 2012-09-18 | 2014-03-26 | 邵阳 | DNA (Deoxyribonucleic Acid) probe library hybridized with BRAF (v-Raf murine sarcoma viral oncogene homolog B1) gene, and method for enriching BRAF gene segments by adopting same |
| CN103667254A (en) * | 2012-09-18 | 2014-03-26 | 邵阳 | Enrichment and detection method of target gene fragment |
| CN103667269A (en) * | 2012-09-18 | 2014-03-26 | 邵阳 | DNA (Deoxyribonucleic Nucleic Acid) probe library for hybridization with BCR or ABL gene and method for enriching BCR-ABL gene segments |
| CN105524981A (en) * | 2014-09-28 | 2016-04-27 | 浙江大学 | Capture kit and method of target gene |
-
2016
- 2016-08-31 CN CN201610800089.9A patent/CN107794293A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020168668A1 (en) * | 2001-03-27 | 2002-11-14 | Millennium Pharmaceuticals, Inc. | 14691, a human glutamate receptor family member and uses therefor |
| CN102358910A (en) * | 2011-11-03 | 2012-02-22 | 东南大学 | Chemiluminescence detection method for copy number polymorphism based on magnetic separation and primer extension |
| WO2013074492A1 (en) * | 2011-11-14 | 2013-05-23 | Five Prime Therapeutics, Inc. | Methods of treating cancer |
| CN103667268A (en) * | 2012-09-18 | 2014-03-26 | 邵阳 | DNA (Deoxyribonucleic Acid) probe library hybridized with BRAF (v-Raf murine sarcoma viral oncogene homolog B1) gene, and method for enriching BRAF gene segments by adopting same |
| CN103667254A (en) * | 2012-09-18 | 2014-03-26 | 邵阳 | Enrichment and detection method of target gene fragment |
| CN103667269A (en) * | 2012-09-18 | 2014-03-26 | 邵阳 | DNA (Deoxyribonucleic Nucleic Acid) probe library for hybridization with BCR or ABL gene and method for enriching BCR-ABL gene segments |
| CN105524981A (en) * | 2014-09-28 | 2016-04-27 | 浙江大学 | Capture kit and method of target gene |
Non-Patent Citations (4)
| Title |
|---|
| SEOANE-ZONJIC P.等: "Establishing gene models from the Pinus pinaster genome using gene capture and BAC sequencing", 《BMC GENOMICS》 * |
| 余元勋等: "《中国分子白血病学》", 30 April 2016, 安徽科学技术出版社 * |
| 刘旭霞等: "目标基因捕获测序技术检测1例肥厚型心肌病致病基因突变", 《临床检验杂志》 * |
| 吴德沛等: "《白血病诊疗临床实践》", 28 February 2013, 科学技术文献出版社 * |
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