CN106676095A - Complete set reagent for developing genetic markers and method for developing genetic markers through high-throughput sequencing - Google Patents
Complete set reagent for developing genetic markers and method for developing genetic markers through high-throughput sequencing Download PDFInfo
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- CN106676095A CN106676095A CN201510756142.5A CN201510756142A CN106676095A CN 106676095 A CN106676095 A CN 106676095A CN 201510756142 A CN201510756142 A CN 201510756142A CN 106676095 A CN106676095 A CN 106676095A
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Abstract
The invention discloses a complete set reagent for developing genetic markers and a method for developing genetic markers through high-throughput sequencing. The complete set reagent for developing genetic markers provided by the invention is composed of a linker A, a linker B and a restriction enzyme BcoDI, wherein the linker A is composed of single-strand DNA shown as a sequence 1 and single-strand DNA shown as a sequence 2; the linker B is composed of single-strand DNA shown as a sequence 3 and single-strand DNA shown as a sequence 4. Experiments prove that the linker A and the linker B in the complete set reagent for high-throughput sequencing provided by the invention can be connected to two ends of DNA fragments. By using the complete set reagent for high-throughput sequencing and a database construction method in the invention, a DNA library is successfully constructed and successfully applied to a sequencing platform of next generation sequencing in Illumina Company for sequencing, and lots of genetic marker loci are obtained.
Description
Technical field
The present invention relates to be used to develop the reagent set and high-flux sequence exploitation heredity of genetic marker in biological technical field
The method of labelling.
Background technology
RAD (restriction association site DNA) is the DNA sequence tag near restriction enzyme site.
RAD can be applied to gene mapping, collection of illustrative plates drafting etc. as a kind of molecular marker.The density of RAD labellings can by
Obtain using different restriction endonucleases during detached.The perfect adaptation of RAD labelling techniques and second filial generation sequencing technologies,
Can it is quick and also it is high-throughout realize marker development, collection of illustrative plates is drawn etc..RAD labelling techniques can reduce genome
Complexity, is not limited by genome sequence whether there is, easy to operate, can quickly develop substantial amounts of molecular marker,
So as to be widely used in the genetic research of colony.
The restriction enzyme cleavage recognition site that common RAD technologies are used, the sample given for, its
The restriction enzyme site number contained in nucleotide sequence is molecule mark that is certain, being obtained in that by common RAD technologies
The number of note is fixed, it is impossible to changed.And the labelling to obtaining usually is needed in actual scientific research and application
Number can unrestricted choice, a kind of number of labelling that can be to obtaining is badly in need of at present and be capable of the technology of unrestricted choice.
The content of the invention
The technical problem to be solved is to provide the reagent set and method of exploitation genetic marker.
To solve above-mentioned technical problem, present invention firstly provides the reagent set for developing genetic marker.
Reagent set for developing genetic marker provided by the present invention, by joint A, joint B and restriction enzyme
Enzyme BcoDI is constituted；
The joint that the joint A is made up of positive chain and reverse strand, the positive chain of the joint A is shown in sequence 1
Single stranded DNA, being reversely linked as of the joint A add in 5 ' ends of single stranded DNA shown in the 8-26 positions of sequence 2
Plus the single stranded DNA that 1-10 nucleotide is obtained, the 8-26 positions of sequence 2 and the 5-23 positions reverse complemental of sequence 1；
The joint that the joint B is made up of positive chain and reverse strand, the positive chain of the joint B is shown in sequence 3
Single stranded DNA, being reversely linked as of the joint B add in 3 ' ends of single stranded DNA shown in the 1-18 positions of sequence 4
Plus the single stranded DNA that 1-10 nucleotide is obtained, the 1-18 positions of sequence 4 and the 40-57 positions reverse mutual of sequence 3
Mend.
Wherein, sequence 1,2,3 and 4 from the direction of the 1st to last 1 be corresponding single stranded DNA from 5 '
Hold to the direction at 3 ' ends.
In the above-mentioned reagent set for developing genetic marker, the sequence for reversely connecting of the joint A is sequence 2；Institute
The sequence for stating the reverse strand of joint B is sequence 4.
In the above-mentioned reagent set for developing genetic marker, the positive chain of the joint A is from 5 ' ends to 3 ' ends
It is made up of following part successively：With the microarray dataset sequencing sequence P7 identical oligonucleotide fragments of Illumina companies
And the viscosity restriction enzyme site sequence complementary with the 3 ' ends of DNA to be measured；The reverse strand of the joint A and positive chain except
Outside prominent sticky end, remaining sequence is complementary with positive chain.The structure of the joint A is as shown in Figure 1.It is described to connect
The microarray dataset sequencing sequence p7 identical oligonucleotide fragments with Illumina companies in head A, in being sequence table
The 5-68 positions of sequence 1；The viscosity restriction enzyme site sequence complementary with the 3 ' ends of DNA to be measured in the joint A,
For the 1-4 positions of sequence in sequence table 1.
In the above-mentioned reagent set for developing genetic marker, phosphorus can be carried out in the positive end of chain 5 ' of the joint A
Acidifying modification, concretely carries out phosphorylation modification by the 1st deoxyribonucleotide of sequence in sequence table 1.
In the above-mentioned reagent set for developing genetic marker, the positive chain of the joint B is from 5 ' ends to 3 ' ends
It is made up of following part successively：With the microarray dataset sequencing sequence P5 identical oligonucleotide fragments of Illumina companies
And a deoxyribonucleotide protruding terminus complementary with the 3 ' ends of DNA to be measured；The reverse strand of the joint B with
A part of sequence of positive chain is complementary and carries out phosphorylation modification in 5 ' ends.The structure of joint B is as shown in Figure 2.
The microarray dataset sequencing sequence P5 identical oligonucleotide fragments with Illumina companies of the joint B, are sequence
The 1-57 positions of sequence 3 in table；A dezyribonucleoside complementary with the 3 ' ends of DNA to be measured of the joint B
Acid is the 58th of sequence 3 in sequence table.
In the above-mentioned reagent set for developing genetic marker, phosphorus can be carried out in the end of reverse strand 5 ' of the joint B
Acidifying modification, concretely carries out phosphorylation modification by the deoxyribonucleotide of the 1st of sequence in sequence table 4.
In the above-mentioned reagent set for developing genetic marker, restricted enzyme BcoDI, the joint A and described
Joint B can independent packaging.The ratio of the joint A and the joint B can be 1:1.
For solve above-mentioned technical problem, present invention also offers for develop genetic marker into bell and spigot joint.
It is provided by the present invention for high-flux sequence into bell and spigot joint, be made up of the joint A and the joint B.
To solve above-mentioned technical problem, present invention also offers the joint A.
Wherein, in the positive chain of the joint A, the 1-4 positions of sequence 1 can not be aatt.
To solve above-mentioned technical problem, present invention also offers the system for developing genetic marker.
System for developing genetic marker provided by the present invention, including the reagent set and for developing hereditary mark
Other reagents and/or instrument of note.
In said system, described other reagents and instrument for developing genetic marker can be to carry out restricted enzyme enzyme
Cut, carry out DNA connections, the DNA fragmentation containing sticky end is carried out end-filling, to DNA fragmentation plus dATP,
Enter performing PCR amplification, carry out reagent needed for DNA purification, to smash DNA, DNA quantitative and/or carry out high-flux sequence
Required reagent and instrument.
In said system, described other reagents for developing genetic marker can be single stranded DNA by shown in sequence 5 and
The primer pair of the single stranded DNA composition shown in sequence 6.
In said system, the instrument for developing genetic marker can be Illumina microarray datasets, such as Illumina
The secondary microarray dataset or Illumina Hiseq2000 microarray datasets of company.
To solve above-mentioned technical problem, present invention also offers the method that high-flux sequence develops genetic marker.
The method that high-flux sequence provided by the present invention develops genetic marker, including：By genomic DNA with restricted
Restriction endonuclease BcoD I are carried out after enzyme action, and the joint A and joint B, constructed dna library are connected step by step；To institute
Stating DNA library carries out high-flux sequence exploitation genetic marker.
Said method specifically may include following 1) -5)：
1) will be digested with restricted enzyme BcoDI for the genomic DNA of marker development, obtain digestion product,
The joint A is connected with the digestion product, the connection product containing joint A is obtained；
2) by step 1) connection product that obtains smashes DNA fragmentation for 300-700bp, obtains smashing product；
3) to step 2) product of smashing that obtains carries out successively end-filling, plus dATP and connects the joint B, obtain
To the connection product containing joint B；
4) to step 3) connection product that obtains enters performing PCR amplification, obtains amplified production, it is sequenced with Illumina
Platform is sequenced to the amplified production；
5) carry out after analysis of biological information, determine genetic marker (labelling such as restriction enzyme site SNP or INDEL).
The flow chart of said method is as shown in Figure 3.
In said method, step 2) may also include purification is carried out to the product of smashing.
In said method, step 3) may also include purification is carried out to the connection product containing joint B.
In said method, step 4) may also include purification is carried out to the amplified production.
Said method step 1) in, it is described to include the enzyme action system of Genomic DNA digestion：Enzyme cutting buffering liquid, limit
Property restriction endonuclease processed, the genomic DNA of 0.1-1 μ g, concentration of the restricted enzyme in enzyme action system is 0.5
U/μl；Described linked system includes：The digestion product, the joint A, ATP, ligase, the joint A
Concentration be 100nM, the concentration of described ATP is 1mM, and concentration of the ligase in the linked system is
0.5U/μl。
Said method step 2) in, it is described smash available covaris and smash instrument carry out, the condition smashed can
It is the corresponding parameter that instrument adjustment is smashed using covaris.
Said method step 3) in, it is to be entered using PCR primer QIAquick Gel Extraction Kit that the method for product is smashed in described recovery
Row is reclaimed；Described end-filling system is to be carried out using the Quick Blunting kit of NEB companies；Described
Plus the system of dATP includes：DATP, plus A buffer, plus the polymerase of dATP, the concentration of described dATP is 5
MM, the concentration of described polymerase in described reaction system is 0.5U/ μ l；The linked system of described joint B
Including：Joint B, ATP and ligase, the concentration of described joint B is 10 μM, and the concentration of described ATP is
1mM, concentration of the ligase in the linked system is 0.5U/ μ l.
Said method step 4) in, described PCR amplification system includes：The primer pair, polymerase mixture,
And step 3) in the connection product that obtains.The concentration of every primer of the primer pair is 0.2 μM, the polymerase
Mixture in the PCR system shared 50% volume.
In said method, the genomic DNA is the DNA of any monoploid or diploid material, such as animal, plant,
Microorganism etc..The plant concretely arabidopsiss.
To solve above-mentioned technical problem, present invention also offers the application of following X1 or X2：
X1, the reagent set, it is described into bell and spigot joint, the joint A or described systems exploitation genetic marker in
Using；
X2, the reagent set, described educate in molecule into bell and spigot joint, the joint A, the system or methods described
Application in kind.
In above-mentioned application, the exploitation genetic marker can be the genetic marker of exploitation animal, plant or microorganism；It is described
Molecular breeding can be animal or plant molecular breeding.
In the present invention, the restricted enzyme BcoDI concretely NEB products.
The present invention is proposed based on SRAD (the selective restriction for using dystopy to cut restricted enzyme
Association site DNA) technology.The experiment proves that, provided by the present invention for high-flux sequence
Joint A and joint B in reagent set, is connectable to the two ends of DNA fragmentation, with the present invention for high pass
The reagent set and banking process of sequence are measured, DNA library is successfully constructed, and is successfully used in Illumina
The microarray dataset of the secondary sequencing of company is sequenced, and obtains substantial amounts of genetic marker site；Simultaneously, it is possible to achieve many
Storehouse is built in the mixing of individual sample, and the number of the genetic marker of acquisition is adjusted as needed.Provided by the present invention for high flux
The reagent set and banking process of sequencing is in researchs such as population heredity, collection of illustrative plates drafting, germ plasm evaluation and identifications and applies
Field has a wide range of applications.
Description of the drawings
Fig. 1 is the structural representation of joint A.
Fig. 2 is the structural representation of joint B.
Fig. 3 is Library development flow schematic diagram.
Fig. 4 is the agarose gel electrophoresis figure of genomic DNA digestion products.Wherein, swimming lane CK is the gene before enzyme action
Group DNA, swimming lane BcoDI are the product after enzyme action.
Fig. 5 is to the agarose gel electrophoresis figure for smashing product after enzyme action genomic DNA product plus top connection A.
To cut before glue reclaim, right figure is to cut after glue reclaim to left figure.
Fig. 6 is the electrophoretogram that pair connection product for adding top connection B carries out pcr amplification product.
Specific embodiment
The present invention is further described in detail with reference to specific embodiment, the embodiment for being given only for
The present invention is illustrated, rather than in order to limit the scope of the present invention.
Experimental technique in following embodiments, if no special instructions, is conventional method.
Material used, reagent etc. in following embodiments, if no special instructions, commercially obtain.
Nucleotide sequence used is synthesized by Shanghai Invitrogen biotech firms in following embodiments.Agents useful for same for example without
Special instruction is the product of NEB companies, and water used is ultra-pure water.
Embodiment 1, for developing the application of the reagent set of genetic marker in exploitation arabidopsiss genetic marker
For developing the reagent set of genetic marker, it is made up of joint A, joint B and restricted enzyme BcoDI,
Single stranded DNAs of the joint A shown in sequence 1 and the single stranded DNA shown in sequence 2 are constituted, and the joint B is by sequence
Single stranded DNA composition shown in single stranded DNA and sequence 4 shown in row 3.
1st, the connection of the enzyme action of genomic DNA and joint A
1) sample concentration is determined
Arabidopsis thaliana genomic dna is extracted, using Qubit (U.S.) to sample DNA concentration mensuration, is carried out precisely quantitatively,
And using 0.8% agarose gel, 120v voltages, electrophoresis 1 hour, detection sample quality, it is ensured that arabidopsis gene
Group DNA sample is completely without degraded.
2) with step 1) detection arabidopsis thaliana genomic dna 100ng-1 μ g carry out restricted enzyme action as template.
Reaction system and reaction condition are as follows：Sample DNA (150-250ng/ μ l) 0.1-1 μ g, Restriction
The μ l of Enzyme (BcoDI) 1,10 × NEB Buffer 25 μ l, add H2O to cumulative volume is 50 μ l, will be upper
State 37 DEG C of mixed liquor to process after 15min-2h, 65 DEG C of process 20min.Taking out 10 μ l carries out electrophoresis detection, knot
Fruit shows that enzyme action fully, obtains the band of even dispersion, such as accompanying drawing 4, and swimming lane M is trans15k marker,
CK is that, used as control, BcoDI is the swimming lane of digestion products without digested DNA.
3) connection of joint A
In step 2) in the digestion product that obtains, ATP, T4 ligases, connection buffer and joint A are added,
Room temperature connects.Specific reaction system and reaction condition are as follows：Step 2) in digestion product 50 μ l, 100mM ATP
The μ l of 1 μ l, 10 × NEB Buffer 21, (1000u) the μ l of T4DNA Ligase 0.5, plus H2The μ l of O 5,
100nM Adaptor12.5 μ l, altogether 60 μ l, under room temperature (or 16 DEG C) 2h is connected；65 DEG C 20 of connection product
Min, connection enzymatic activity inactivation.
2nd, connection product is smashed at random, filling-in, plus dATP
1) connection product obtained in step 1 is crushed to into 300-700bp using covaris (U.S.), is used
Qiagen PCR kits are reclaimed and smash product, and concrete steps are carried out according to Qiagen test kits operating instruction.
2) end-filling is carried out to above-mentioned recovery product.Reaction system and reaction condition are as follows：The μ l of product DNA 30 are smashed,
The μ l of 10 × Blunting Buffer 5,1mM dNTP 10 μ l, Quick Blunting kit Enzyme Mix, 1
μ l, plus H2The μ l of O 9, the μ l of total system 50；Reaction condition：30 DEG C of incubation 30min.
3) to step 2) filling-in product carry out purification：Above-mentioned product is carried out into glue reclaim, purification purpose fragment.1.0%
Agarose gel electrophoresiies, cut the band of glue reclaim 300-700bp scope.Whole removal process is according to Qiagen glue reclaims
Test kit operating instruction is carried out.Such as accompanying drawing 5.Swimming lane M is trans100bpladder marker, and swimming lane 1 is benefit
Show no increases in output the track of thing.
4) to step 3) in purified product carry out plus dATP, reaction system and condition are as follows：Step 3) in benefit
Show no increases in output the μ l of thing 22, the μ l of 100mM dATP 1,10 × NEB Buffer 23 μ l, Klenow exo-(NEB) (50,
000units/ml) 0.3 μ l, H2The μ l of O 3.7, the μ l of cumulative volume 30；Reaction condition：Fully mix, 37 DEG C incubate
Educate 30min, 75 DEG C of 20min heat inactivations.
3rd, the connection of joint B
1) by the connection product in step 2 4), joint B is added, reaction system and condition are specific as follows:Step 2
In the μ l of connection product 30, the μ l of NEB Buffer 22 4), 10 μM of joint B 2.5 μ l, (1000u) T4DNA
The μ l of Ligase 0.5, plus H2The μ l of O 15, the μ l of cumulative volume 50, connect 2 hours under room temperature (or 16 DEG C), and connection is produced
65 DEG C of 20min of thing, connection enzymatic activity inactivation.
2) by step 1) in connection product equal-volume AMPure XP Beads purification 1 time.Purge process is strictly pressed
Carry out according to operation instructions.
3) by step 2) in recovery product to carry out Qubit quantitative, for the reaction of next step PCR.
4th, PCR amplifications purpose fragment
1) product that will be reclaimed in step 3, precisely quantitatively 10ng reacts for PCR, the PCR primer for obtaining, i.e.,
For constructed library.Reaction system and condition are as follows：The μ l of DNA sample 3, PCR Primer 11 μ l,
The μ l of PCR Primer 21,2 × Phusion PCR Master Mix 25 μ l, H2The μ l of O 20, altogether 50 μ l,
The condition is answered to be：First 98 DEG C of denaturations 1min；Then 98 DEG C of 10s, 60 DEG C of 30s, 72 DEG C of 30s, totally 10
Circulation；Last 72 DEG C of extensions 5min.The sequence of Primer 1 is 5 '-AATGATACGGCGACCACCGA-3 ' (sequences
Sequence 5 in list)；The sequence of Primer 2 is 5 '-CAAGCAGAAGACGGCATACGA-3 ' (sequences in sequence table
Row 6).
2) by step 1) in PCR primer equal-volume AMPure XP Beads purification twice.Purge process is strictly pressed
Carry out according to operation instructions.Take out 1 μ l and enter row agarose gel electrophoresis detection, as a result show, obtain 300-700
The band of enrichment, such as accompanying drawing 6, swimming lane M is trans100bpladder marker, and swimming lane 1 is the electricity of PCR primer
Swimming swimming lane.
3) by step 2) in purified product to carry out Q μ bit precisely quantitative.
5th, the inspection of library storehouse and upper machine
1) purified product of gained in step 4 is diluted to into 1ng/ μ l, taking out 1 μ l is used for Agilent2100 (U.S.s
Agilent companies of state) detection, 1 μ l are taken again in addition for QPCR (Biorad companies) detections, according to detection knot
Really, machine concentration in decision.
2) according to step 1) obtained by concentration, by library be diluted to it is upper it is confidential ask after, the two of Illumina companies
It is sequenced for microarray dataset.
6th, the statistical analysiss of sequencing result
1) BcoDI restriction enzyme site analyses are carried out to arabidopsis gene group data, totally 192,332；
2) lower machine data 100M are filtered out with the reads of restriction enzyme site, then PEreads is compared to plan with BWA
On southern mustard genome, total number of all data containing restriction enzyme site is 191,640, you can with the genetic marker for obtaining
Number be 191,640, the restriction enzyme site for having 99.6% is covered to.
Claims (10)
1. it is used to develop the reagent set of genetic marker, is made up of joint A, joint B and restricted enzyme BcoDI；
The joint that the joint A is made up of positive chain and reverse strand, the positive chain of the joint A is shown in sequence 1
Single stranded DNA, being reversely linked as of the joint A add in 5 ' ends of single stranded DNA shown in the 8-26 positions of sequence 2
Plus the single stranded DNA that 1-10 nucleotide is obtained, the 8-25 positions of sequence 2 and the 5-22 positions reverse complemental of sequence 1；
The joint that the joint B is made up of positive chain and reverse strand, the positive chain of the joint B is shown in sequence 3
Single stranded DNA, being reversely linked as of the joint B add in 3 ' ends of single stranded DNA shown in the 1-18 positions of sequence 4
Plus the single stranded DNA that 1-10 nucleotide is obtained, the 1-18 positions of sequence 4 and the 40-57 positions reverse mutual of sequence 3
Mend.
2. reagent set according to claim 1, it is characterised in that：The sequence for reversely connecting of the joint A is
Sequence 2；The sequence of the reverse strand of the joint B is sequence 4.
3. be used for develop genetic marker into bell and spigot joint, by joint A described in claim 1 or 2 and the joint B
Composition.
4. joint A described in claim 1 or 2.
5. the system for developing genetic marker, including reagent set described in claim 1 or 2 and for developing heredity are used for
Other reagents of labelling and/or instrument.
6. system according to claim 5, it is characterised in that：Described other reagents for developing genetic marker
It is primer pair that the single stranded DNA shown in the single stranded DNA and sequence 6 shown in sequence 5 is constituted；It is described for high flux
The instrument of sequencing is Illumina microarray datasets.
7. the method that high-flux sequence develops genetic marker, including：By genomic DNA restricted enzyme BcoD I
After carrying out enzyme action, the joint A and joint B described in substep connection claim 1 or 2, constructed dna library；
High-flux sequence exploitation genetic marker is carried out to the DNA library.
8. the method that high-flux sequence develops genetic marker, including following 1) -5)：
1) genomic DNA is digested with restricted enzyme BcoDI, digestion product is obtained, by claim 1 or 2
Described in joint A be connected with the digestion product, obtain the connection product containing joint A；
2) by step 1) connection product that obtains smashes DNA fragmentation for 300-700bp, obtains smashing product；
3) to step 2) obtain smash product carry out successively end-filling, plus dATP and connection claim 1 or 2
Described in joint B, obtain the connection product containing joint B；
4) to step 3) connection product that obtains enters performing PCR amplification, obtains amplified production, it is sequenced with Illumina
Platform is sequenced to the amplified production；
5) carry out after analysis of biological information, determine genetic marker.
9. the method according to claim 7 or 8, it is characterised in that：The genomic DNA is plant, animal
Or microbial DNA.
10. the application of following X1 or X2：
Described in reagent set described in X1, claim 1 or 2 or the joint A or claim 3 into bell and spigot joint,
Or application of the system described in claim 5 or 6 in exploitation genetic marker；
Described in reagent set described in X2, claim 1 or 2 or the joint A or claim 3 into bell and spigot joint,
Or appoint in system described in joint A described in claim 1 or 2 or claim 5 or 6 or claim 7-9
Application of one methods described in molecular breeding.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107841544A (en) * | 2017-12-14 | 2018-03-27 | 浙江海洋大学 | A kind of method for obtaining Japanese eel high density SNP marker |
| CN108004303A (en) * | 2017-12-14 | 2018-05-08 | 浙江海洋大学 | The screening technique of Japanese eel adaptability SNP site |
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| CN102978205A (en) * | 2012-11-19 | 2013-03-20 | 北京诺禾致源生物信息科技有限公司 | High-throughput sequencing junction applied to marker development and application method thereof |
| CN102978206A (en) * | 2012-11-27 | 2013-03-20 | 北京诺禾致源生物信息科技有限公司 | High-throughput sequencing joint applied to hybrid library building and library building method thereof |
| CN103937899A (en) * | 2005-12-22 | 2014-07-23 | 凯津公司 | Method for high-throughput AFLP-based polymorphism detection |
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| CN103937899A (en) * | 2005-12-22 | 2014-07-23 | 凯津公司 | Method for high-throughput AFLP-based polymorphism detection |
| CN102978205A (en) * | 2012-11-19 | 2013-03-20 | 北京诺禾致源生物信息科技有限公司 | High-throughput sequencing junction applied to marker development and application method thereof |
| CN102978206A (en) * | 2012-11-27 | 2013-03-20 | 北京诺禾致源生物信息科技有限公司 | High-throughput sequencing joint applied to hybrid library building and library building method thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107841544A (en) * | 2017-12-14 | 2018-03-27 | 浙江海洋大学 | A kind of method for obtaining Japanese eel high density SNP marker |
| CN108004303A (en) * | 2017-12-14 | 2018-05-08 | 浙江海洋大学 | The screening technique of Japanese eel adaptability SNP site |
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