CN108018344A - The lesion detection approach of high-flux sequence based on MLPA+Seq - Google Patents
The lesion detection approach of high-flux sequence based on MLPA+Seq Download PDFInfo
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Abstract
The present invention provides a kind of lesion detection approach of the high-flux sequence based on MLPA+Seq, include the following steps：(1) genomic DNA is prepared；(2) probe is designed；(3) probe is hybridized with genomic DNA, and is attached using DNA ligase；(4) product of step (3) and primer are subjected to PCR reactions；(5) PCR product is sequenced after purification, the copy number and nucleotide variation of DNA is determined by bioinformatics method, calculate the base combination frequency of heterozygote.The present processes can detect multiple sites at the same time, and cost when building storehouse is low, and easy to operate, accuracy is high, high specificity.
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of lesion detection of the high-flux sequence based on MLPA+Seq
Method.
Background technology
The multiple join dependency probe amplification technology (multiplex of MRC-Holland b.v. companies exploitation
Ligation-dependent probe amplification, MLPA), i.e. MLPA technologies, utilize simple hybridization
(hydriation), (ligation) and PCR amplification (PCR amplication) reaction are connected, can be at the same time in a reaction
Detect 45 kinds of gene copy number variations (Copy number variations, CNV), single nucleotide polymorphism (Single
Nucleotide Polymorphisms, SNP), it may also be used for DNA methylation assay and the mRNA analysis of gene.MLPA detection techniques
Based on Capillary Electrophoresis, to distinguish each probe, each probe fragment size need at least be differed 4bp, which limits multiple effect
Fruit can only be 45 weights, and simply the change of the detection nucleotide sequence of sxemiquantitative, its sensitivity be not high.SNP refers in genome
The variation of upper single nucleotide acid, including conversion, transversion, missing and insertion, SNP are a strong work in terms of medical diagnosis on disease
Tool, research find that SNP is distributed quite extensively in genome, and SNP variations and various diseases, the generation such as tumour is related, therefore
It is significant that SNP variations relevant with tumour are screened in disorder in screening.
Capitalbio Corporation Co., Ltd. provides a kind of detection method of the SNP based on high-flux sequence, it is substantially former
Reason is first to design probe and the primer pair expanded in advance, and 5 ' ends of pre- amplimer mark biotin, first amplify default
Section, is exactly to hybridize probe library and pre- amplified fragments afterwards, and with magnetic capture hybrid product, hybridization production is connected with DNA ligase
Thing, it is high-temperature denatured, double-strand is dissociated, purpose fragment is expanded with the universal primer of belt lacing.Though the method can be reduced in pre- amplification
Non-specific amplification, but after adding this wheel PCR, then carry out being possible to cause the random mutation between template during PCR again.
Since the detection of the prior art is based on Capillary Electrophoresis, so each probe fragment size need at least be differed 4bp, this
It can only be 45 weights to limit multiple effect, and by Capillary Electrophoresis can only sxemiquantitative detection nucleotide sequence change,
Its sensitivity is not high.The flow of existing two generations high-flux sequence includes the extraction of DNA, the fragmentation of DNA, fragment screening and text
Storehouse structure, the sequencing of upper machine, data analysis, the template initial amount needed in the process is high, i.e., higher to the quality requirement of DNA.
Therefore, the cost of the prior art is of a relatively high.
The content of the invention
The present invention provides a kind of lesion detection approach of the high-flux sequence based on MLPA+Seq, with the method for MLPA into
Row builds storehouse, only probe need to be allowed to hybridize completely with template when building storehouse, after being attached with DNA ligase, then with archaeal dna polymerase, use
General weight primer plays the effect of multiplex amplification to build library, and the final method with the sequencing of two generations carries out upper machine.
The purpose of the present invention is realized by following scheme：
A kind of lesion detection approach of the high-flux sequence based on MLPA+Seq, includes the following steps：
(1) genomic DNA is prepared；
(2) probe is designed；
(3) probe is hybridized with genomic DNA, and is attached using DNA ligase；
(4) product of step (3) and primer are subjected to PCR reactions；
(5) PCR product is sequenced after purification, determines that the copy number of DNA and base become by bioinformatics method
It is different, calculate the base combination frequency of heterozygote；
Probe in the step (3) includes left probe and right probe；The left probe include 5 ' universal linker sequences and
3 ' specific hybridization sequences, right probe include 5 ' the specific hybridization sequences and 3 ' universal linker sequences of phosphorylated modification.
Preferably, the method that genomic DNA is prepared in the step (1) is that the base of blood is extracted using the paramagnetic particle method of Tiangeng
Because of a group DNA；Comprise the following steps that：
1.+200 μ l lysate BL+20 μ l Proteinase Ks of 200 μ l blood samples, vibration, 70 DEG C of incubation 15min, the vibration one per 7min
It is secondary；
2. adding the μ l magnetic beads of absolute ethyl alcohol+30 of 450 μ l, overturn 10min or stand 10min, vibrated once per 3min；
3. being put in magnetic frame, supernatant is abandoned, 700 μ l cleaning solution BW1 is added, overturns repeatedly, abandon supernatant；
4. adding 800 μ l cleaning solutions, overturn repeatedly, abandon supernatant；
5. adding 80% ethanol of 800 μ l, overturn repeatedly, abandon supernatant；
6. drying at room temperature 5-10min；
7. add 60 μ l eluents CE, 56 DEG C of incubation 10min, 5min vibrations；
8. supernatant is drawn in the EP pipes of new 1.5ml.
Preferably, the detecting probe information in the step (2) is as shown in table 1：
The sequence information of 19 pairs of probes of table
Preferably, the hybridization temperature of the step (3) is Gradient annealing from 60 DEG C to 25 DEG C, and 1 DEG C is dropped per 4min；DNA connects
Connect enzyme and be selected from appointing for T4DNA Ligase, T3DNA Ligase, Taq DNA Ligase, T7DNA ligase and Ligase-65
One kind, after 37 DEG C connect 1h, uses 0.8 × VAHTS TM DNA Clean Beads to purify magnetic beads for purifying.
Preferably, the universal linker sequence and universal primer included in the universal primer in the step (4) is as follows：
5 ' end universal joints：CCTACACGACGCTCTTCCGATCT；
3 ' end universal joints：TGGAATTCTCGGGTGCCAAGGAAC；
Universal primer：
P5 ends universal primer：
AATGATACGGCGACCACCGAGATCTACAC[i5barcode]
ACACTCTTTCCCTACACGACGCTCTTCCGATCT；
P7 ends universal primer：
CAAGCAGAAGACGGCATACGAGAT(i7barcode)GTGATGGAGTTCCTTGGCACCCGAGAATTCCA。
Preferably, the PCR system is as follows：The whole quality for adding gDNA templates is 150ng, and the amount for adding probe mix is 2
μ l, the amount for adding Taq DNA Ligase Buffer are 2 μ l, this system is mended to 10 μ l with water, i.e., first carries out the denaturation of template,
The hybridization of probe and template is carried out again, adds Taq DNA Ligase 0.2 μ l, 9.8 μ of water after the completion of hybridization in this system again
L, cumulative volume are 20 μ l；37 DEG C of connection 1h；0.8 × VAHTS TM DNA Clean Beads purifying connections are used after the completion of connection
Product, comprises the following steps that：
1. magnetic bead liquid is shifted to an earlier date 30min makes its equalized temperature to room temperature from 2-8 DEG C of taking-up, standing；
2. the vibration of reverse or vortex makes magnetic bead liquid fully mix, draw 16 μ l magnetic beads liquid and add DNA sample
In, gently inhaled using pipettor and play 10 fully mixings；
3. being incubated at room temperature 10min, DNA is set to be attached on magnetic bead；
4. sample is placed on magnetic frame, after solution clarification (about 5min), supernatant is carefully removed；
5. keeping sample all the time on magnetic frame, the 80% ethanol rinsing magnetic bead of 200 μ l Fresh is added, room temperature is incubated
30s is educated, carefully removes supernatant；
5. 6. repeat step once, it is secondary to amount to rinsing；
7. keeping sample all the time on magnetic frame, uncap dry magnetic bead about 5-10min at room temperature；
8. sample is taken out from magnetic frame, 12 μ l H are added2O, vortex oscillation or use pipettor piping and druming fully mix,
It is stored at room temperature 2min；5min is stood on magnetic frame, it is careful to draw the new EP pipes of supernatant 10 μ l to one after solution clarification
In；
The amplification system of Q5 enzymes is as follows：Q5Hot Start High-Fidelity 2×Master Mix：10μl；
0.5 μ l of purified product 9.5 μ l, primer mix.
Preferably, the PCR conditions are as follows：First 98 DEG C be denatured templet gene group 10min, then from 60 DEG C of Gradient annealings to
25 DEG C, condition is that 1 DEG C is dropped per 4min, drops to 25 DEG C and adds Taq DNA Ligase and water, and 1h is connected at 37 DEG C；
Program during using Q5 enzymatic amplifications is as follows：
98℃ 30s；
98℃ 10s；55℃ 30s；72 DEG C of 15s, the wheel of circulation 30；
72 DEG C of 5min,
4℃ +∞。
Preferably, the archaeal dna polymerase in PCR reaction include Q5Hot Start High-Fidelity 2 ×
Master Mix, 2 × KAPA HiFi HotStar Ready Mix and Pfusion polymerase.
Preferably, the purifying in the step (5) is carried out pure using 1.5 × VAHTS TM DNA Clean Beads magnetic beads
Change.
Preferably, the microarray dataset in the step (5) includes illumina HiSeq, MiSeq, NextSeq sequencing are put down
Platform, Life Ion microarray datasets, Illumina and Affymetrix chip platforms.
The present invention is directed to the probe of specific nucleic acid-templated design different loci, and by different probe combinations one
Rise, the nucleotide change of different loci is detected in same pipe.In same single nucleic acid strands template, which includes left probe
With right two fragments of probe, as shown in Figure 1.Left probe includes 5 ' universal linker sequences and 3 ' specific hybridization sequences, right probe bag
5 ' the specific hybridization sequences and 3 ' universal linker sequences of phosphorylated modification are included, close adjacent no base notch is answered between probe,
It can hybridize at the same time on template strand, adjacent left and right probe can be catalyzed 5 ' P and 3 ' OH by nucleic acid ligase and form di(2-ethylhexyl)phosphate fat
Key and connect into same nucleic acid chains；In the present invention used 5 ' universal sequence and 3 ' universal sequences be can be with sequenator
Or the joint sequence of biochip compatibility, include but is not limited to illumina HiSeq, MiSeq, NextSeq microarray dataset,
Life Ion microarray datasets, Illumina or Affymetrix chip platforms；Universal linker sequence can select that P5's and P7 is complete
Portion or partial sequence.
Brief description of the drawings
Fig. 1 is the MLPA technical schematic diagrams of the present invention.
Embodiment
With reference to embodiment, detailed explanation is carried out to the present invention.
A kind of lesion detection approach of the high-flux sequence based on MLPA+Seq, includes the following steps：
(1) genomic DNA is prepared；
(2) probe is designed；
(3) probe is hybridized with genomic DNA, and is attached using DNA ligase；
(4) product of step (3) and primer are subjected to PCR reactions；
(5) PCR product is sequenced after purification, determines that the copy number of DNA and base become by bioinformatics method
It is different, calculate the base combination frequency of heterozygote；
Probe in the step (3) includes left probe and right probe；The left probe include 5 ' universal linker sequences and
3 ' specific hybridization sequences, right probe include 5 ' the specific hybridization sequences and 3 ' universal linker sequences of phosphorylated modification.
The method that genomic DNA is prepared in the step (1) is that the genome of blood is extracted using the paramagnetic particle method of Tiangeng
DNA；Comprise the following steps that：
1.+200 μ l lysate BL+20 μ l Proteinase Ks of 200 μ l blood samples, vibration, 70 DEG C of incubation 15min, the vibration one per 7min
It is secondary；
2. adding the μ l magnetic beads of absolute ethyl alcohol+30 of 450 μ l, overturn 10min or stand 10min, vibrated once per 3min；
3. being put in magnetic frame, supernatant is abandoned, 700 μ l cleaning solution BW1 is added, overturns repeatedly, abandon supernatant；
4. adding 800 μ l cleaning solutions, overturn repeatedly, abandon supernatant；
5. adding 80% ethanol of 800 μ l, overturn repeatedly, abandon supernatant；
6. drying at room temperature 5-10min；
7. add 60 μ l eluents CE, 56 DEG C of incubation 10min, 5min vibrations；
8. supernatant is drawn in the EP pipes of new 1.5ml.
Detecting probe information in the step (2) is as shown in table 1：
The sequence information of 19 pairs of probes of table
The hybridization temperature of the step (3) is Gradient annealing from 60 DEG C to 25 DEG C, and 1 DEG C is dropped per 4min；DNA ligase is selected from
T4DNA Ligase, T3DNA Ligase, Taq DNA Ligase, T7 DNA ligase's and Ligase-65 is any, and 37
DEG C connection 1h after, use 0.8 × VAHTS TM DNA Clean Beads purifying magnetic beads for purifying.
The universal linker sequence and universal primer included in universal primer in the step (4) is as follows：
5 ' end universal joints：CCTACACGACGCTCTTCCGATCT；
3 ' end universal joints：TGGAATTCTCGGGTGCCAAGGAAC；
Universal primer：
P5 ends universal primer：
AATGATACGGCGACCACCGAGATCTACAC[i5barcode]ACACTCTTTCCCTACACGACGCTCTTCCG
ATCT；
P7 ends universal primer：
CAAGCAGAAGACGGCATACGAGAT(i7barcode)GTGATGGAGTTCCTTGGCACCCGAGAATTCCA。
The PCR system is as follows：The whole quality for adding gDNA templates is 150ng, and the amount for adding probe mix is 2 μ l, is added
The amount of Taq DNA Ligase Buffer is 2 μ l, this system is mended to 10 μ l with water, i.e., first carries out the denaturation of template, then visited
The hybridization of pin and template, adds 0.2 μ l of Taq DNA Ligase, 9.8 μ l of water, cumulative volume in this system again after the completion of hybridization
For 20 μ l；37 DEG C of connection 1h；0.8 × VAHTS TM DNA Clean Beads purifying connection products, tool are used after the completion of connection
Body step is as follows：
1. magnetic bead liquid is shifted to an earlier date 30min makes its equalized temperature to room temperature from 2-8 DEG C of taking-up, standing；
2. the vibration of reverse or vortex makes magnetic bead liquid fully mix, draw 16 μ l magnetic beads liquid and add in DNA sample, use liquid relief
Device, which is gently inhaled, plays 10 fully mixings；
3. being incubated at room temperature 10min, DNA is set to be attached on magnetic bead；
4. sample is placed on magnetic frame, after solution clarification (about 5min), supernatant is carefully removed；
5. keeping sample all the time on magnetic frame, the 80% ethanol rinsing magnetic bead of 200 μ l Fresh is added, room temperature is incubated
30s is educated, carefully removes supernatant；
5. 6. repeat step once, it is secondary to amount to rinsing；
7. keeping sample all the time on magnetic frame, uncap dry magnetic bead about 5-10min at room temperature；
8. sample is taken out from magnetic frame, 12 μ l H are added2O, vortex oscillation or use pipettor piping and druming fully mix,
It is stored at room temperature 2min；5min is stood on magnetic frame, it is careful to draw the new EP pipes of supernatant 10 μ l to one after solution clarification
In；
The amplification system of Q5 enzymes is as follows：Q5Hot Start High-Fidelity 2×Master Mix：10μl；
0.5 μ l of purified product 9.5 μ l, primer mix.
The PCR conditions are as follows：Templet gene group 10min are first denatured at 98 DEG C, then from 60 DEG C of Gradient annealings to 25 DEG C, bar
Part is that 1 DEG C is dropped per 4min, drops to 25 DEG C and adds Taq DNA Ligase and water, and 1h is connected at 37 DEG C；
Program during using Q5 enzymatic amplifications is as follows：
98℃ 30s；
98℃ 10s；55℃ 30s；72 DEG C of 15s, the wheel of circulation 30；
72 DEG C of 5min,
4℃ +∞.Archaeal dna polymerase in PCR reaction include Q5Hot Start High-Fidelity 2 ×
Master Mix, 2 × KAPA HiFi HotStar Ready Mix and Pfusion polymerase.In the step (5)
Purifying is purified using 1.5 × VAHTS TM DNA Clean Beads magnetic beads.Microarray dataset in the step (5) includes
Illumina HiSeq, MiSeq, NextSeq microarray dataset, Life Ion microarray datasets, Illumina and Affymetrix cores
Piece platform.
Embodiment 1
In order to prove that the mononucleotide polymorphic in DNA can be effectively detected for genomic DNA design multiple probe group
Property (Single Nucleotide Polymorphisms, SNP), we select the peripheral blood genomic DNA of normal person as just
Begin nucleic acid-templated, and therefrom select 5 specific regions design probes (table 1) and tested as follows：
(1) extraction of peripheral blood genomic DNA
The blood of people is taken, using the genomic DNA of the paramagnetic particle method extraction blood of Tiangeng.Concentration is measured using Nanodrop,
The genomic DNA of Quality Control qualification is used for ensuing experiment；
(2) the whole quality for adding gDNA templates is 150ng, and 5 pairs of probes are added into the phase, add Taq DNA Ligase
Buffer, 98 DEG C of denaturation 10min, it is single-stranded to be first denatured the gDNA of double-strand, then from 60 DEG C of Gradient annealings to 25 DEG C, is dropped per 4min
1 DEG C so that probe fully hybridizes with template.Taq DNA Ligase, 37 DEG C of connection 1h are added in this system again.
(3) using 0.8 × VAHTS TM DNA Clean Beads purifying magnetic beads for purifying connection products, free spy is removed
Pin, and remove Taq DNA Ligase, and Taq DNA Ligase Buffer.
(4) using the connection product purified in the step 3 of 10 μ l as template, expanded using general PCR primer P5 and P7,
Archaeal dna polymerase uses Q5, and 20 μ l PCR reaction systems are carried out with last storehouse of building and is handled, 1.5 × VAHTS is used after the completion of PCR
TM DNA Clean Beads purify magnetic beads for purifying PCR product.
(5) library Quality Control and it is sequenced using NestSeq 500.
(6) data analysis：
The clean reads that sequencing data will obtain after Quality Control removes low quality reads, then with target sequence into
Row compares and counts read quantity on each target respectively, as shown in table 2；
Table 2
These as shown by data present invention can be used for detecting the base change of specific genome area, and the repetition of technology
Property is also fine.Rs12951053, rs2287498 and rs 2287498 wherein in this 5 sites belongs to the inspection of P53 tumor suppressor genes
The gene in set meal is surveyed, the detection of p53 tumor suppressor genes is one kind of cancer early screening, therefore this technology can be used to detect tumour.
Embodiment 2
In order to prove that such a method can effectively detect single nucleotide polymorphism (the Single Nucleotide in DNA
Polymorphisms, SNP), we equally select the peripheral blood genomic DNA of normal person another as original nucleic acid template, selection
Simultaneously tested as follows with relevant other 4 sites of tumour occurred frequently, specific regions design probe (table 1) outside：
(1) extraction (the same) of peripheral blood genomic DNA
(2) the whole quality for adding gDNA templates is 150ng, and 4 pairs of probes are added into the phase, add Taq DNA Ligase
Buffer, 98 DEG C of denaturation 10min, it is single-stranded to be first denatured the gDNA of double-strand, then from 60 DEG C of Gradient annealings to 25 DEG C, is dropped per 4min
1 DEG C so that probe fully hybridizes with template.Taq DNA Ligase, 37 DEG C of connection 1h are added in this system again.
(3) using 0.8 × VAHTS TM DNA Clean Beads purifying magnetic beads for purifying connection products, free spy is removed
Pin, and remove Taq DNA Ligase, and Taq DNA Ligase Buffer.
(4) using the connection product purified in the step 3 of 10 μ l as template, expanded using general PCR primer P5 and P7,
Archaeal dna polymerase uses Q5, and 20 μ lPCR reaction systems are carried out with last storehouse of building and is handled, 1.5 × VAHTS is used after the completion of PCR
TM DNA Clean Beads purify magnetic beads for purifying PCR product.
(5) library Quality Control and it is sequenced using NestSeq 500.
(6) data analysis：
The clean reads that sequencing data will obtain after Quality Control removes low quality reads, then with target sequence into
Row compares and counts read quantity on each target respectively, as shown in table 3；
Table 3
These as shown by data present invention can be used for detecting the base change of specific genome area, and the repetition of technology
Property is also fine.Rs753955 in this 4 sites is the lung cancer in the main tumour occurred frequently of detection, and rs9275319 is that detection is main
Liver cancer in tumour occurred frequently, rs3781264 are the stomach cancers in the main tumour occurred frequently of detection, and rs10505477 is that detection is main occurred frequently
Colorectal cancer in tumour.
This method is directly connected to 5 ' P and 3 ' OH by DNA ligase and forms phosphodiester bond, and single-stranded one is connected into by two
Bar chain.Probe is so only needed to can be achieved with template hybridization completely, it is easy to operate, then gone with the universal primer of stand-by universal joint
Amplification, plays the effect that a weight primer reaches multiplex amplification, and cost is more much lower.
MLPA+Seq probes design in this patent is two-part, it can also be designed to three-stage or four-part form,
In addition to leftmost probe, by 5 ' the equal phosphorylations in end of every section of probe, the later stage connects single-stranded nucleotide into one using DNA ligase
Bar nucleotide chain, can so increase the specificity of product, and detection SNP is sensitiveer.
The foregoing is only a preferred embodiment of the present invention, but protection scope of the present invention be not limited thereto,
Any one skilled in the art is in the technical scope of present disclosure, the change or replacement that can readily occur in,
It should all be included within the scope of the present invention.Therefore, protection scope of the present invention should be with the protection of claims
Subject to scope.
<110>U.S. is because of healthy science and technology（Beijing）Co., Ltd
<120>The lesion detection approach of high-flux sequence based on MLPA+Seq
<130> GW1172978DF
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cttcactggtcagctcctccccccacatcttcagtggaattctcgggtgccaaggaac
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cctacacgacgctcttccgatctcagggccgcgtggtacaaagagcaggar
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cactgagaaaagtacaaagaatttttatgtgctattgactttattttggaattctcgggtgccaaggaac
Claims (10)
1. a kind of lesion detection approach of the high-flux sequence based on MLPA+Seq, it is characterised in that include the following steps：
(1) genomic DNA is prepared；
(2) probe is designed；
(3) probe is hybridized with genomic DNA, and is attached using DNA ligase；
(4) product of step (3) and primer are subjected to PCR reactions；
(5) PCR product is sequenced after purification, the copy number and nucleotide variation of DNA is determined by bioinformatics method, counted
Calculate the base combination frequency of heterozygote；
Probe in the step (3) includes left probe and right probe；The left probe includes 5 ' universal linker sequences and 3 ' spies
Different hybridization sequences, right probe include 5 ' the specific hybridization sequences and 3 ' universal linker sequences of phosphorylated modification.
2. the lesion detection approach of the high-flux sequence according to claim 1 based on MLPA+Seq, it is characterised in that institute
The method that genomic DNA is prepared in step (1) is stated as using the genomic DNA of the paramagnetic particle method of Tiangeng extraction blood；Specific steps
It is as follows：
1.+200 μ l lysate BL+20 μ l Proteinase Ks of 200 μ l blood samples, vibration, 70 DEG C of incubation 15min, vibrate once per 7min；
2. adding the μ l magnetic beads of absolute ethyl alcohol+30 of 450 μ l, overturn 10min or stand 10min, vibrated once per 3min；
3. being put in magnetic frame, supernatant is abandoned, 700 μ l cleaning solution BW1 is added, overturns repeatedly, abandon supernatant；
4. adding 800 μ l cleaning solutions, overturn repeatedly, abandon supernatant；
5. adding 80% ethanol of 800 μ l, overturn repeatedly, abandon supernatant；
6. drying at room temperature 5-10min；
7. add 60 μ l eluents CE, 56 DEG C of incubation 10min, 5min vibrations；
8. supernatant is drawn in the EP pipes of new 1.5ml.
3. the lesion detection approach of the high-flux sequence according to claim 1 based on MLPA+Seq, it is characterised in that institute
The detecting probe information stated in step (2) is as shown in table 1：
The sequence information of 19 pairs of probes of table
4. the lesion detection approach of the high-flux sequence according to claim 1 based on MLPA+Seq, it is characterised in that institute
The hybridization temperature for stating step (3) is Gradient annealing from 60 DEG C to 25 DEG C, and 1 DEG C is dropped per 4min；DNA ligase is selected from T4DNA
Ligase, T3DNA Ligase, Taq DNA Ligase, T7DNA ligase's and Ligase-65 is any, 37 DEG C of connection 1h
Afterwards, magnetic beads for purifying is purified using 0.8 × VAHTS TM DNA Clean Beads.
5. the lesion detection approach of the high-flux sequence according to claim 1 based on MLPA+Seq, it is characterised in that institute
State the universal linker sequence included in the universal primer in step (4) and universal primer is as follows：
5 ' end universal joints：CCTACACGACGCTCTTCCGATCT；
3 ' end universal joints：TGGAATTCTCGGGTGCCAAGGAAC；
Universal primer：
P5 ends universal primer：
AATGATACGGCGACCACCGAGATCTACAC[i5barcode]ACACTCTTTCCCTACACGACGCTCTTCCGATCT；
P7 ends universal primer：
CAAGCAGAAGACGGCATACGAGAT(i7barcode)GTGATGGAGTTCCTTGGCACCCGAGAATTCCA。
6. the lesion detection approach of the high-flux sequence according to claim 5 based on MLPA+Seq, it is characterised in that institute
It is as follows to state PCR system：The whole quality of gDNA templates in the step of adding embodiment 1-2 (2) is 150ng, adds probe mix's
Measure as 2 μ l, the amount for adding Taq DNA Ligase Buffer is 2 μ l, this system is mended to 10 μ l with water, i.e., first carries out template
Denaturation, then the hybridization of probe and template is carried out, add 0.2 μ l of Taq DNA Ligase, water after the completion of hybridization in this system again
9.8 μ l, cumulative volume are 20 μ l；37 DEG C of connection 1h；Purified after the completion of connection using 0.8 × VAHTS TM DNA Clean Beads
Connection product, comprises the following steps that：
1. magnetic bead liquid is shifted to an earlier date 30min makes its equalized temperature to room temperature from 2-8 DEG C of taking-up, standing；
2. the vibration of reverse or vortex makes magnetic bead liquid fully mix, draw 16 μ l magnetic beads liquid and add in DNA sample, it is light using pipettor
Light inhale plays 10 fully mixings；
3. being incubated at room temperature 10min, DNA is set to be attached on magnetic bead；
4. sample is placed on magnetic frame, after solution clarification, supernatant is carefully removed；
5. keeping sample all the time on magnetic frame, the 80% ethanol rinsing magnetic bead of 200 μ l Fresh, incubation at room temperature are added
30s, carefully removes supernatant；
5. 6. repeat step once, amounts to rinsing twice；
7. keeping sample all the time on magnetic frame, uncap dry magnetic bead about 5-10min at room temperature；
8. sample is taken out from magnetic frame, 12 μ l H are added2O, vortex oscillation or use pipettor piping and druming fully mix, room temperature
Stand 2min；5min is stood on magnetic frame, it is careful to draw in 10 μ l to one new EP pipe of supernatant after solution clarification；
The amplification system of Q5 enzymes is as follows：Q5Hot Start High-Fidelity 2×Master Mix：10μl；Purified product
0.5 μ l of 9.5 μ l, primer mix.
7. the lesion detection approach of the high-flux sequence according to claim 6 based on MLPA+Seq, it is characterised in that institute
It is as follows to state PCR conditions：Templet gene group 10min is first denatured at 98 DEG C, then from 60 DEG C of Gradient annealings to 25 DEG C, condition is per 4min
1 DEG C of drop, drops to 25 DEG C and adds Taq DNA Ligase and water, and 1h is connected at 37 DEG C；
Program during using Q5 enzymatic amplifications is as follows：
98℃30s；
98℃10s；55℃30s；72 DEG C of 15s, the wheel of circulation 30；
72 DEG C of 5min,
4℃+∞。
8. the lesion detection approach of the high-flux sequence according to claim 7 based on MLPA+Seq, it is characterised in that institute
The archaeal dna polymerase stated in PCR reactions includes Q5Hot Start High-Fidelity 2 × Master Mix, 2 × KAPA
HiFi HotStar Ready Mix and Pfusion polymerase.
9. the lesion detection approach of the high-flux sequence according to claim 1 based on MLPA+Seq, it is characterised in that institute
The purifying stated in step (5) is purified using 1.5 × VAHTS TM DNA Clean Beads magnetic beads.
10. the lesion detection approach of the high-flux sequence according to claim 1 based on MLPA+Seq, it is characterised in that
Microarray dataset in the step (5) includes illumina HiSeq, MiSeq, NextSeq microarray dataset, Life Ion sequencings
Platform, Illumina and Affymetrix chip platforms.
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