CN106755506A - Kit for detecting genetic mutation in tumour FFPE samples - Google Patents
Kit for detecting genetic mutation in tumour FFPE samples Download PDFInfo
- Publication number
- CN106755506A CN106755506A CN201710067030.8A CN201710067030A CN106755506A CN 106755506 A CN106755506 A CN 106755506A CN 201710067030 A CN201710067030 A CN 201710067030A CN 106755506 A CN106755506 A CN 106755506A
- Authority
- CN
- China
- Prior art keywords
- probe
- kit
- gene
- gene trap
- design
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- Genetics & Genomics (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Hospice & Palliative Care (AREA)
- Oncology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention provides a kind of kit for detecting genetic mutation in tumour FFPE samples, the kit includes the probe for gene trap, the probe for capturing includes the probe for the design of SNV regions, the probe for the design of CNV regions, the probe for the design of FUSION regions, consensus primer such as SEQ ID NO:Nucleotide sequence primer shown in 1, and Tag primer such as SEQ ID NO:2~SEQ ID NO:Nucleotide sequence primer shown in 5.According to the present invention, in the tumour FFPE samples in the detection method of genetic mutation, it is capable of detection gene SNV, CNV and FUSION of Simultaneous Stabilization.
Description
Technical field
The invention belongs to molecular in vitro diagnosis field, and in particular to be particularly well-suited to genetic mutation in tumour FFPE samples
Kit.
Background technology
FFPE (formalin-fixed paraffin-embedded, formalin fix paraffin are used tumor tissues more
Embedding) form of sample preserved.The filing FFPE samples of enormous amount are retrospective study, illustrate disease mechanisms, are found
Therapeutic targets and instruction prognosis provide valuable resource.But the medical sample of the great challenge of FFPE sample studies, first FFPE
This is very precious, and each sample total is very limited and irreplaceable.Secondly FFPE samples during manufacture, formalin
Fixation can make nucleic acid in tissue that different degrees of degraded and intermolecular crosslinking occurs, it is further that paraffin high temperature gos deep into process
Accelerate the degraded of nucleic acid, the time of preservation and environment also have tremendous influence to the nucleic acid in sample.Therefore how from limited and
It is susceptible in the FFPE samples of nucleolysis successfully detect the variation situation of nucleic acid, is for molecular level retrospective study
It is crucial.
There is research to show, the generation of tumour is generally along with the variation of nucleic acid.Common variation type has SNV (single
Nucleotide variant, single nucleotide variations), CNV (Copy Number Variation, copy number variation) and
FUSION (fusion) etc..
With the fast development of new-generation sequencing technology (next generation sequencing, NGS), greatly carry
Sequencing speed high, while cost is sequenced significantly reducing.Based on new-generation sequencing technology platform, clinically most widely used method
It is to add multipair primer in same PCR reaction systems, can simultaneously expands the multiplex PCR sequencing technologies of multiple DNA fragmentations.
At present, many companies are proposed the kit in multiple PCR method enrichment tumor-related gene region, and such as Qiagen is public
The TruSight Tumor 15 of GeneRead DNAseq Colorectal Cancer Panel, Illumina companies of department,
TruSeq Amplicon Cancer Panel of Illumina companies etc., but these kits can only detect SNV, for
CNV and FUSION, such product can not be detected accurately, therefore, the applicability of multiple PCR method is limited by larger.
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 one kind for detecting tumour FFPE
The kit of genetic mutation in sample, genetic test is carried out using the kit, and is sequenced by high-flux sequence platform,
It is capable of detection gene SNV, CNV and FUSION of Simultaneous Stabilization.
The present inventor has made intensive studies to solve the above problems, and as a result finds：In tumour FFPE sample bases
Because in the detection method that makes a variation, by using the probe of gene trap, i.e., probe for the design of SNV regions, for CNV regions
The probe of design, the probe for the design of FUSION regions, consensus primer such as SEQ ID NO:Shown in 1, Tag primer such as SEQ
ID NO:2~SEQ ID NO:Nucleotide sequence shown in 5.Can be with detection gene SNV, CNV and FUSION of Simultaneous Stabilization.
I.e. the present invention includes：
1. a kind of kit for detecting genetic mutation in tumour FFPE samples, it includes the spy for gene trap
Pin, the probe of the gene trap includes：For SNV regions design probe, for CNV regions design probe, be directed to
The probe of FUSION regions design, consensus primer such as SEQ ID NO:Shown in 1, and Tag primer such as SEQ ID NO:2~SEQ
ID NO:Nucleotide sequence shown in 5.
2. the kit according to item 1, wherein, the tumour be stomach cancer, lung cancer, breast cancer, colorectal cancer, liver cancer and
One or more in oophoroma.
3. the kit according to item 1, wherein, the probe of the gene trap is biotinylated.
4. the kit according to any one of item 1~3, wherein, the gene trap kit also includes strepto- parent
With the magnetic bead of element mark.
5. the kit according to item 1, wherein, the kit also includes the ion for providing hybrid capture reaction
The buffer solution of environment.
6. the kit according to item 1, wherein, the kit is also included for eluting physical absorption or non-specificity
The cleaning fluid of hybridization.
7. the kit according to item 1, wherein, the kit is used to be sequenced in the gene trap of tumor-related gene
In carry out gene trap；Preferably, the gene trap be sequenced SNV, CNV for detecting tumor-related gene and
FUSION。
8. in a kind of tumour FFPE samples genetic mutation detection method, wherein, use any one of item 1~7 try
Agent box carries out genetic test.
9. it is a kind of for detect genetic mutation in tumour FFPE samples two generations be sequenced DNA library construction method, wherein,
The step of including carrying out gene trap to the library for building, wherein the gene trap uses the examination any one of item 1~7
Agent box is carried out.
10. a kind of capture probe, wherein, the probe of the gene trap includes probe for the design of SNV regions, is directed to
The probe, and/or the probe for the design of FUSION regions of CNV regions design.
Beneficial effect：Relative to existing method, kit of the invention can simultaneously detect more regions, while detection
Go out gene SNV, CNV and FUSION.
Brief description of the drawings
Fig. 1 is the schematic diagram of the CNV testing results of sample 1 in embodiment 1.
The specific 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.
On the one hand, the present invention provides a kind of kit for detecting genetic mutation in tumour FFPE samples, and it includes using
In the probe of gene trap, the probe of the gene trap includes：For SNV regions design probe, for CNV regions design
Probe, for FUSION regions design probe, consensus primer such as SEQ ID NO:Nucleotide sequence primer shown in 1 is (as follows
Table), and Tag primer such as SEQ ID NO：Nucleotide sequence primer (such as following table) shown in 2~SEQ ID NO 3.
Tumour described herein be in stomach cancer, lung cancer, breast cancer, colorectal cancer, liver cancer and oophoroma etc. one or two
More than.
Preferably, described is biotinylated for capture probe.So, it is possible to use the solid phase of marked by streptavidin
Carrier will capture the probe separates after target gene out, and the solid phase carrier is preferably magnetic bead.Preferably, base of the invention
Because of the capture agent box also magnetic bead including marked by streptavidin.
Preferably, kit of the invention also includes the buffer solution of the ionic environment for hybrid capture reaction.
Preferably, kit of the invention also includes the cleaning fluid for eluting absorption or non-specific hybridization in room, it
Specificity for improving gene trap.
Preferably, kit of the invention also includes the primer for being expanded for the target gene for capturing.
Preferably, kit of the invention is used to carry out gene trap in genetic mutation detection in tumour FFPE samples,
It is highly preferred that the gene trap is sequenced SNV, CNV and FUSION for detecting related gene in tumour FFPE samples.
Each component in kit of the invention is preferably independently packaged in container, but in the premise for not influenceing to use
Under, it is also possible to some of which component is merged and is packed.
In this manual, SNV (single nucleotide variant, single nucleotide variations) is a kind of body cell
Single nucleotide mutation；CNV (Copy Number Variation copy number variation) refers to rearrangement is occurred by genome and is caused
, referring generally to the copy number of the genome large fragment that length is more than 1kb increases or reduces；FUSION (fusion) refers to two
Or code area or the noncoding region fracture of multiple genes, join end to end again, it is placed in same set of regulating and controlling sequence.
In this manual, tumor-related gene refers to that can aid in being used as diagnosing tumour, instructs tumour medication and/or prediction
The gene of tumor prognosis.Generally, it is considered that in suffering from the patient of tumour, gene has specific variation result, for example, the gene
Variation can be one or two or three kinds in SNV, CNV and FUSION.
In this manual, the probe of the gene trap can also be included for other related genes of target disease
Probe.Other genes related to disease refer to：The gene of all kinds mutation may occur in target disease, it can be used as
Diagnose target disease, instruct the gene of target disease medication and/or prediction prognosis, but suffering from the Patient cells' of target disease
In genome, the specific structure variation of the gene is not common or do not possess clinical meaning.
The kit of genetic mutation of the invention can be used to detect the two generations sequencing DNA of genetic mutation in tumour FFPE samples
The structure (banking process of the invention) in library, the two generations sequencing DNA library of structure can be used for gene in tumour FFPE samples and become
Different detection.Therefore, on the other hand, the present invention provides a kind of detection method (this hair of genetic mutation in tumour FFPE samples
Bright method), including to build library carry out gene trap the step of, wherein, gene trap is entered using kit of the invention
OK.Specifically include step as follows：
Step A：The genomic DNA of FFPE samples is extracted, genomic DNA is obtained；
Step B：The genomic DNA of extraction is entered into Break Row, the DNA fragmentation after being interrupted；
Step C：DNA fragmentation after interrupting carries out end reparation, obtains flat end DNA；
Step D：Flat terminal DNA fragments are carried out into 3' ends plus A, the DNA fragmentation of 3' ends plus A is obtained；
Step E：The DNA fragmentation that 3' ends add A is carried out into adjunction head, the DNA fragmentation of adjunction head is obtained；
Step F：The DNA fragmentation of adjunction head is entered into performing PCR, library is obtained；
Step G：Hybridized using capture probe, obtained hybrid product；
Step H：Hybrid product is entered into performing PCR amplification, the PCR primer after being hybridized；
Step I：PCR primer after hybridization is sequenced.
The structure in library, gene trap can be operated using method known to those skilled in the art.
The library of capture can carry out upper machine sequencing after quality inspection, so as to obtain sequencing data.Microarray dataset can be example
Such as Illumina HiSeq 2500,4000 platforms or NextSeq 550AR platforms (inventor's confirmation).
Preferably, step C and step D, step D and step E, step E and step F, step F and step G, step G and step
Rapid H, and/or there is purification step between step H and step I, its purification step preferably uses the magnetic bead of marked by streptavidin.
Finally, the present invention provides a kind of capture probe, and the probe of the gene trap includes the spy for the design of SNV regions
Pin, probe, and/or the probe for the design of FUSION regions for the design of CNV regions.Preferably, the gene trap
Probe is biotinylated.
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
1.1 oncogenes detect the design and synthesis of liquid chip
According to the literature and multiple database (NCBI-PDB, PubMed, COSMIC, Clinvar, HGMD,
GeneCards, zj-LOVD etc.) information, have selected the target gene for blood disease detection, or specific target areas carry out
Probe is designed.The databases such as the probe of design and NCBI carry out Blast and compare, and to ensure the base number of probe 60~
Between 80nt, TM values it is moderate, without the special constructions such as palindromic sequence and sequence homology between 50%~80%, by these
Numerical value is ensuring the specificity of probe.
The probe sequence that will be designed synthesizes oligonucleotide probe, tumor-related gene capture is obtained after PCR checkings and is visited
Pin clone bank.
The amplimer or joint component determined using biotin labeling quantity and position are prepared to contain and are conducive to target fragments
The capture probe library of crawl, the capture probe collection for finally giving known biotin labeling quantity and position carries biotin mark
The capture probe storehouse of note.
1.2DNA is extracted
Sample takes from the FFPE samples (sample 1) of the non-small cell lung cancer for preserving half a year and the FFPE of the lung cancer of preservation half a year
Sample (sample 2), FFPE sample DNAs, specific step are extracted using GeneRead DNA FFPE Kit kits (Qiagen companies)
Rapid reference Qiagen DNA extraction kit steps.
1.2.1 interrupt
Instrument being interrupted using Biorupter and entering Break Row, setting interrupts 30 circulations of condition, and 30s ON/30s OFF will
FFPE sample DNAs are broken into the fragment of 200bp or so, the DNA fragmentation after being interrupted.
Repair (End Repair) in 1.3 ends:
(1) reagent needed for being taken out from -20 DEG C of kits of preservation in advance, is placed on ice chest and thaws and shake mixed
It is even.Single sample amount of preparation can in proportion prepare Mix referring to table 1, multisample.
Table 1
(2) reaction is repaired in end：To 25 μ L Mix are dispensed in the centrifuge tube of 1.5mL, packing completes and confirms to add nothing
After by mistake, add DNA sample that 1.5mL centrifuge tubes are placed in Thermomixer into 20 DEG C of warm bath 30 minutes.Reaction is used after terminating
DNA in 1.8 × nucleic acid purification magnetic bead recovery purifying reaction system, is dissolved in 32 μ L EB.
1.4 ends add " A "
(1) reagent needed for being taken out from -20 DEG C of kits of preservation in advance, is placed on ice chest and thaws and shake mixed
It is even.Single sample amount of preparation can in proportion prepare Mix referring to table 2, multisample：
Table 2
(2) end adds " A " to react：To 18 μ L Mix are dispensed in the centrifuge tube of 1.5mL, after confirmation is errorless, add on 32 μ L
The DNA that the purifying of one step is reclaimed.Concussion is mixed and is 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 of 1.5 joints
(1) reagent needed for being taken out from -20 DEG C of kits of preservation in advance, is placed on ice chest and thaws and shake mixed
It is even.Single sample amount of preparation can in proportion prepare Mix referring to table 3, multisample：
Table 3
(2) coupled reaction of joint：To 27 μ L Mix are dispensed in the centrifuge tube of 1.5mL, after confirmation is errorless, add on 18 μ L
The DNA that the purifying of one step is reclaimed.Concussion is mixed and is centrifuged after adding, and sample tube is placed in into 20 DEG C of 15 points of warm bath in Thermomixer
Clock.Using the DNA in 1.8 × nucleic acid purification magnetic bead recovery purifying reaction system, it is dissolved in the EB of 30 μ L.
1.6PCR reacts
(1) reagent needed for being taken out from -20 DEG C of kits of preservation, the sequence label primer that wherein sample 1 is used is mark
The nucleotide sequence shown in primer I n1 is signed, the sequence label primer that sample 2 is used is the nucleotides sequence shown in Tag primer In2
Row.It is placed on ice chest and thaws and shake mixing.PCR reaction systems are prepared in the PCR pipe of 0.2mL, multisample can be in proportion
Prepare Mix：
Table 4
(2) PCR programs are set, the program setting of PCR reactions is as follows：
Reaction terminates timely take out sample and is put into 4 DEG C of Refrigerator stores and exits on request or close instrument.
(3) with the DNA in 0.9 × nucleic acid purification magnetic bead recovery purifying reaction system, library after purification is dissolved in 20 μ L's
ddH2In O.Label is posted, storehouse completion is built in small fragment library.Qubit detections are carried out to library, each library concentration size is recorded.
By library censorship Agilent 2100, peak figure result is preserved.
Consensus primer sequence (SEQ ID NO:1)：
5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3'
Tag primer In1 primer sequences (SEQ ID NO:2)：
5'-CAAGCAGAAGACGGCATACGAGATTCGTAAGCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
-3'
Tag primer In2 primer sequences (SEQ ID NO:3)：
5'-CAAGCAGAAGACGGCATACGAGATTAGGAATAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
-3'
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 commercially obtained.
(2) Hybrid Library is prepared：By DNA library to be hybridized in thawed on ice, the μ g of gross mass 1 are taken (in subsequent operation step
This DNA library is referred to as sample library in rapid).
(3) Ann primers Pool is prepared：By the corresponding Tag primer In1/In2 of sample library Index (100 μM) and public
Primer (100 μM) is placed in thawed on ice, respectively takes 1000pmol mixing and (this mixture is referred to as into Ann in subsequent process steps to draw
Thing pool).
(4) preparation of sample is hybridized：To adding 5 μ L COT DNA (Human Cot-1DNA, Life in 1.5mL EP pipes
Technologies, 1mg/mL) and 1 μ g samples library.To addition Ann primers pool in mixture.Sealed with sealed membrane and made
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
The EP pipes of primer pool are placed in vacuum plant until being completely dried.
(5) solution of sample is hybridized：Added in the dry powder of sample library/COT DNA/Ann primers pool：
7.5 μ 2 × hybridization buffers of L
3 μ L hybridization components A
So far, following components is contained in EP pipes：
Table 5
1.8 prepare Hybrid Library
(1) said mixture of hybridization buffer will be added to be vortexed to shake 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 is denatured 10 minutes.After removing denaturation
Mixture, at room temperature maximum revolution is centrifuged 10 seconds.
(3) said mixture is transferred in the 0.2mL flat cover PCR pipes containing 4.5 μ L capture chips.Be vortexed concussion 3 seconds,
Maximum revolution is centrifuged 10 seconds after fully mixing.So far, following component is contained in Hybridization samples mixture：
Table 6
(4) Hybridization samples mixture is placed in 47 DEG C of heating module upper 16 hours.The hot lid temperature of heating module needs setting
It it is 57 DEG C, product need to subsequently be eluted 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 7
(6) following reagent is preheated in heating module at 47 DEG C：
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) at room temperature balance 30 minutes after, by magnetic bead fully be vortexed mixing 15 seconds.
(2) to dispensing 100 μ L magnetic beads in 1.5mL centrifuge tubes, 1 1.5mL centrifuge tube at most can be used to prepare 6 hybridization catches
Obtain required magnetic bead.
(3) centrifuge tube that will fill magnetic bead is placed on magnetic frame, and careful suction abandons supernatant after about 5 minutes, by magnetic bead stay in from
In heart pipe, plus twice magnetic bead initial volume 1 × magnetic bead cleaning fluid.Centrifuge tube is removed from magnetic frame, is vortexed and is mixed 10
Second.The centrifuge tube that magnetic bead will be filled puts back to 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 is finished and abandon magnetic bead cleaning fluid, with 1 × magnetic bead cleaning fluid resuspended magnetic of vortex of magnetic bead initial volume
Pearl.100 resuspended μ L magnetic beads are transferred in the PCR pipe of 0.2mL.PCR pipe is placed in magnetic bead is adsorbed on magnetic frame, 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 immediately carry out lower step association reaction.
The combination and rinsing of 1.10DNA and affine absorption magnetic bead
(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 every 15 minutes and mixed once, make DNA with
Magnetic bead is combined.
After (3) 45 minutes are incubated, to 47 DEG C of μ L of 1 × cleaning fluid I 100 of preheating of addition in the DNA sample that 15 μ L are captured.
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, supernatant is abandoned.
(4) 1.5mL centrifuge tubes are removed from magnetic frame, the 1 × rinsing liquid for adding 200 μ L to 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 5 points on 47 DEG C of heating modules after mixing
Clock.This step is repeated, is washed twice altogether with 47 DEG C of 1 × rinsing liquid.The centrifuge tube of 1.5mL is placed on magnetic frame, magnetic is adsorbed
Pearl, abandons supernatant.
(5) to 1 × cleaning fluid I that 200 μ L room temperatures are added in above-mentioned 1.5mL centrifuge tubes, it is vortexed and mixes 2 minutes.Will centrifugation
Pipe is placed on magnetic frame, adsorbs magnetic bead, abandons supernatant.To 1 × cleaning fluid II that 200 μ L room temperatures are added in above-mentioned 1.5mL centrifuge tubes,
It is vortexed and mixes 1 minute.Centrifuge tube is placed on magnetic frame, magnetic bead is adsorbed, supernatant is abandoned.To adding 200 in above-mentioned 1.5mL centrifuge tubes
1 × the cleaning fluid III of μ L room temperatures, is vortexed and mixes 30 seconds.Centrifuge tube is placed on magnetic frame, magnetic bead is adsorbed, supernatant is abandoned.
(6) 1.5mL centrifuge tubes are removed from magnetic frame, add 45 μ L PCR water, dissolving wash-out 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 is mixed.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) the amplification program setting of magnetic bead adsorption of DNA PCR 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 × magnetic bead, and library after purification is dissolved in the ddH of 30 μ L2In O.Post label, hybrid capture library
Build storehouse completion.
1.12 libraries quantify
2100Bio Analyzer (Agilent)/LabChip GX (Caliper) and QPCR detections, note are carried out to library
Record library concentration.
Machine sequencing on 1.13 libraries
The library for building is sequenced with NextSeq 550AR.
1.14 data processing and inversions
Comparative example
It is same to be tried using the TruSeq Amplicon Cancer Panel of Illumina companies using sample 1 and sample 2
Agent box is hybridized, and the specification referring in particular to kit is carried out.
Embodiment is by the analysis to sequencing result, and concrete outcome is as follows：
In sample 1 there is SNV in EGFR gene：When the EGFR gene of sample 1 is with NM_005228 as reference sequences.Code area
2573 bit bases sport G by T, cause the 13rd amino acids of albumen coded by EGFR to sport arginine by leucine (L)
(R), the frequency of mutation is dealt into 70.74%.
In sample 1 there is CNV in ERBB2：As shown in Figure 1, transverse axis behaviour reference gene group (hg19) coordinate, middle coordinate be through
Cross the depth signal value of homogenization treatment, it is considered that it is normal that the signal value fluctuates near 0.The area of ERBB2 is marked in Fig. 1
Domain is region to be detected：It can be seen that the depth signal value in region to be detected has deviated considerably from the region near 0, therefore the result
It is considered as ERBB2 positive, the sample has CNV.
In sample 2 there is fusion in EML4 genes and ALK gene：EML4 genes are located at the 29446588th alkali of No. 2 chromosome
Base is broken, and same ALK gene is located at the 42552712nd bit base on No. 2 chromosomes and is broken, EML4 and ALK gene weight
New connection, forms new EML4-ALK fusions.
In comparative example, found after data analysis, there is EGFR gene SNV in sample 1, and the position that occurs and prominent
Change situation is consistent with result in embodiment, but ERBB2 has CNV in not finding sample 1 by the method for comparative example.In sample 2
In do not find in EML4 genes and ALK gene there is fusion.
In order to further prove the accuracy of result of the present invention, inventor is using generation sequence measurement to the SNV feelings of sample
Condition is verified, and uses the united method validation CNV of RT-PCR and qPCR, while using the method validation of FISH
FUSION, as a result shows that the sample has SNV, CNV and FUSION situation, and as a result the result with present invention detection is consistent, explanation
The method of the present invention and kit are capable of detecting when SNV, CNV and FUSION of sample.
Industrial applicibility
In accordance with the invention it is possible to provide a kind of kit for detecting genetic mutation in tumour FFPE samples.
Sequence table
<110>Peace promise is excellent up to Gene science（Beijing）Co., Ltd
<120>Kit for detecting genetic mutation in tumour FFPE samples
<130> 1624-2TGCN
<160> 5
<170> PatentIn version 3.3
<210> 1
<211> 58
<212> DNA
<213>Artificial sequence
<400>Consensus primer
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT 58
<210> 2
<211> 66
<212> DNA
<213>Artificial sequence
<400>Tag primer In1
CAAGCAGAAGACGGCATACGAGATTCGTAAGCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT 66
<210> 3
<211> 66
<212> DNA
<213>Artificial sequence
<400>Tag primer In2
CAAGCAGAAGACGGCATACGAGATTAGGAATAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT 66
<210> 4
<211> 66
<212> DNA
<213>Artificial sequence
<400>Tag primer In3
CAAGCAGAAGACGGCATACGAGATCCTCAGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT 66
<210> 5
<211> 66
<212> DNA
<213>Artificial sequence
<400>Tag primer In4
CAAGCAGAAGACGGCATACGAGATAACTCGTAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT 66
Claims (10)
1. a kind of kit for detecting genetic mutation in tumour FFPE samples, it includes the probe for gene trap, institute
The probe for stating gene trap includes：For SNV regions design probe, for CNV regions design probe, for FUSION areas
The probe of domain design, consensus primer such as SEQ ID NO:Nucleotide sequence primer shown in 1, and Tag primer such as SEQ ID NO:
2~SEQ ID NO:Nucleotide sequence primer shown in 5.
2. kit according to claim 1, it is characterised in that the tumour is stomach cancer, lung cancer, breast cancer, Colon and rectum
One or more in cancer, liver cancer and oophoroma.
3. kit according to claim 1, it is characterised in that the probe of the gene trap is biotinylated.
4. the kit according to any one of claims 1 to 3, it is characterised in that the gene trap kit also includes
The magnetic bead of marked by streptavidin.
5. kit according to claim 1, it is characterised in that the kit also includes anti-for providing hybrid capture
The buffer solution of the ionic environment answered.
6. kit according to claim 1, it is characterised in that the kit also include for elute physical absorption or
The cleaning fluid of non-specific hybridization.
7. kit according to claim 1, it is characterised in that the kit is used for the gene in tumor-related gene
Gene trap is carried out in capture sequencing；Preferably, the gene trap be sequenced for detect tumor-related gene SNV, CNV,
And FUSION.
8. in a kind of tumour FFPE samples genetic mutation detection method, it is characterised in that usage right requirement 1~7 in it is any
The item kit carries out genetic test.
9. a kind of two generations for detecting genetic mutation in tumour FFPE samples the construction method of DNA library is sequenced, its feature exists
In, including to build library carry out gene trap the step of, wherein the gene trap usage right requirement 1~7 in it is any
Kit described in is carried out.
10. a kind of capture probe, it is characterised in that the probe of the gene trap includes probe, the pin for the design of SNV regions
To the probe, and/or the probe for the design of FUSION regions of the design of CNV regions.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611225422 | 2016-12-27 | ||
| CN2016112254224 | 2016-12-27 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106755506A true CN106755506A (en) | 2017-05-31 |
Family
ID=58956101
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710067030.8A Pending CN106755506A (en) | 2016-12-27 | 2017-02-07 | Kit for detecting genetic mutation in tumour FFPE samples |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106755506A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108424955A (en) * | 2018-05-09 | 2018-08-21 | 合肥中科金臻生物医学有限公司 | A kind of high-flux sequence method and its application of a variety of variation type genes of detection |
| CN111662981A (en) * | 2020-06-09 | 2020-09-15 | 俊兮生物科技(上海)有限公司 | Cancer gene detection kit based on second-generation sequencing probe capture method |
| CN111926075A (en) * | 2020-06-09 | 2020-11-13 | 俊兮生物科技(上海)有限公司 | Tumor microsatellite instability detection kit based on second-generation sequencing probe capture technology |
| CN111926058A (en) * | 2020-06-09 | 2020-11-13 | 俊兮生物科技(上海)有限公司 | Kit for constructing second-generation DNA sequencing library based on chemical enzyme cutting method |
| 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 |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN105861710A (en) * | 2016-05-20 | 2016-08-17 | 北京科迅生物技术有限公司 | Sequencing joint and preparation method and application thereof in ultra-low frequency mutation detection |
| US20160244838A1 (en) * | 2013-09-27 | 2016-08-25 | Natera, Inc. | Cell free dna diagnostic testing standards |
| CN105925665A (en) * | 2016-03-30 | 2016-09-07 | 广州精科生物技术有限公司 | Kit, database establishment method, and method and system for detecting area target variation |
| CN105925675A (en) * | 2016-04-26 | 2016-09-07 | 序康医疗科技（苏州）有限公司 | Method for amplifying dna |
-
2017
- 2017-02-07 CN CN201710067030.8A patent/CN106755506A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160244838A1 (en) * | 2013-09-27 | 2016-08-25 | Natera, Inc. | Cell free dna diagnostic testing standards |
| 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 |
| CN105925665A (en) * | 2016-03-30 | 2016-09-07 | 广州精科生物技术有限公司 | Kit, database establishment method, and method and system for detecting area target variation |
| CN105925675A (en) * | 2016-04-26 | 2016-09-07 | 序康医疗科技（苏州）有限公司 | Method for amplifying dna |
| CN105861710A (en) * | 2016-05-20 | 2016-08-17 | 北京科迅生物技术有限公司 | Sequencing joint and preparation method and application thereof in ultra-low frequency mutation detection |
Non-Patent Citations (2)
| Title |
|---|
| CHOI, J ET,AL.: "Comparison of dPCR versus hybridization capture methods for next generation sequencing of ctDNA for a SNV, copy number amplification, and fusion liquid biopsy assay", 《CANCER RESEARCH》 * |
| DI SHAO等: "A targeted next-generation sequencing method for identifying clinically relevant mutation profiles in lung adenocarcinoma", 《SCIENTIFIC REPORTS》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108424955A (en) * | 2018-05-09 | 2018-08-21 | 合肥中科金臻生物医学有限公司 | A kind of high-flux sequence method and its application of a variety of variation type genes of detection |
| CN108424955B (en) * | 2018-05-09 | 2022-01-11 | 合肥中科金臻生物医学有限公司 | High-throughput sequencing method for detecting multiple variant genes and application thereof |
| CN111662981A (en) * | 2020-06-09 | 2020-09-15 | 俊兮生物科技(上海)有限公司 | Cancer gene detection kit based on second-generation sequencing probe capture method |
| CN111926075A (en) * | 2020-06-09 | 2020-11-13 | 俊兮生物科技(上海)有限公司 | Tumor microsatellite instability detection kit based on second-generation sequencing probe capture technology |
| CN111926058A (en) * | 2020-06-09 | 2020-11-13 | 俊兮生物科技(上海)有限公司 | Kit for constructing second-generation DNA sequencing library based on chemical enzyme cutting method |
| 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 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107475375B (en) | A kind of DNA probe library, detection method and kit hybridized for microsatellite locus related to microsatellite instability | |
| CN106755506A (en) | Kit for detecting genetic mutation in tumour FFPE samples | |
| CN107190329B (en) | Fusion based on DNA is quantitatively sequenced and builds library, detection method and its application | |
| CN104350152B (en) | Selective kernel acid fragment is reclaimed | |
| CN106650312A (en) | Device for detecting DNA copy number variation of circulating tumor | |
| CN109609635B (en) | Multi-gene enriched probe bank and detection method of multiple genes related to multiple tumor treatments | |
| JP2022191311A (en) | Methods for detecting dna mutations using mitra tip extraction | |
| CN106835291B (en) | The preparation method and kit of DNA library | |
| CN108517360A (en) | A kind of circulating tumor dissociative DNA abrupt climatic change quality-control product and preparation method thereof | |
| JP2020512405A (en) | How to quickly build an amplicon library in one step | |
| CN111712580A (en) | Method and kit for amplifying double-stranded DNA | |
| CN106845154A (en) | A kind of device for the copy number variation detection of FFPE samples | |
| CN111690748B (en) | Probe set for detecting microsatellite instability by using high-throughput sequencing, kit and microsatellite instability detection method | |
| CN109652525A (en) | Pulmonary thromboembolism gene panel kit and its application | |
| CN105986039A (en) | Detection kit for drug-resistant mutation locus and genotype of hepatitis B virus, use method and application thereof | |
| CN109385469A (en) | A kind of high sensitivity double-strand Circulating tumor DNA detection method and kit | |
| CN109337956B (en) | Design method and kit for enriching multi-gene stem-loop probe based on NGS technology | |
| CN110157808A (en) | A kind of application of non-coding RNA relevant to larynx squamous carcinoma occurrence and development | |
| CN106282361B (en) | Gene capturing kit for capturing genes related to blood diseases | |
| CN106755505A (en) | Kit for detecting genetic mutation in blood plasma ctDNA | |
| CN103797130B (en) | For determining that human body has the system and method for abnormality | |
| CN106591486A (en) | Kit for detecting blood disease related gene variation | |
| CN112626214A (en) | Primer group, kit and method for detecting 1p/19q heterozygosity loss | |
| CN106815491A (en) | A kind of device for detecting FFPE sample Gene Fusions | |
| CN106929564A (en) | breast cancer susceptibility gene detection kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20171215 Address after: 100176 Beijing branch of Beijing economic and Technological Development Zone Street 88 Hospital No. 8 Building 2 unit 701 room Applicant after: Annoroad Genetic Technology (Beijing) Co., Ltd. Applicant after: Zhejiang Annuo uni-data Biotechnology Co. Ltd. Applicant after: Annuo uni-data (Yiwu) Medical Inspection Co. Ltd. Address before: 100176 Beijing branch of Daxing District economic and Technological Development Zone Street 88 Hospital No. 8 Building 2 unit 701 room Applicant before: Annoroad Genetic Technology (Beijing) Co., Ltd. |
|
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |