CN102061335B - Asymmetric deoxyribose nucleic acid (DNA) artificial adapters by using second-generation high-throughput sequencing technology and application thereof - Google Patents
Asymmetric deoxyribose nucleic acid (DNA) artificial adapters by using second-generation high-throughput sequencing technology and application thereof Download PDFInfo
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Abstract
The invention provides asymmetric deoxyribose nucleic acid (DNA) artificial adapters by using a second-generation high-throughput sequencing technology, which is composed of two DNA oligonucleotide single strands. The preparation method comprises the following steps: dissolving the two DNA oligonucleotide single strands into a quenching solution; regulating the final concentration to 2mM and volume to 20microlitre; carrying out a quenching reaction to form the DNA artificial adapters which are locally and mutually complemented at a temperature of 95 DEG C for 5 minutes; reducing the temperature of 95 DEG C to 12 DEG C at the speed of 0.1 DEG C per second; keeping the temperature of 12 DEG C, and diluting an adapter solution after quenching to 500 mu M at a ratio of 1:4; and storing at the temperature of minus 20 DEG C. An application of the asymmetric DNA artificial adapters by using the second-generation high-throughput sequencing technology is as follows: A, asymmetric DNA adapters are in ligation with DNA samples; B, non-connected DNA artificial adapters are purified and isolated by using electrophoresis tapping; C, judgment is carried out; and D, a polymerase chain reaction (PCR) amplification reaction is performed based on an asymmetric sequence. The asymmetric DNA artificial adapters by using the second-generation high-throughput sequencing technology has the obvious advantages that: 1) the usage of original DNA samples for establishing a library is reduced to 50 nanogram and the sensitivity of the original DNA samples is improved by 100 times; and 2) 100% of effective sequencing samples are produced in the process of adapter connection for establishing the library.
Description
Technical field:
The present invention relates to a kind of two generation high-flux sequence the double-stranded joint of asymmetric D NA and application thereof.
Background technology:
In the end of the year 2005,454 Corp. has released ultra-high throughput gene order-checking system Genome Sequencer 20 System based on tetra-sodium sequencing of novelty.Within 2007, released again the more excellent s-generation gene order-checking system of performance: Genome Sequencer FLX System.454 high throughput sequencing technologies, when order-checking, has been used flat board (PTP) of a kind of being called " Pico TiterPlate ", and it contains more than 160 ten thousand holes that are comprised of optical fiber, is loaded with chemiluminescence reaction required various enzymes and substrate in hole.When order-checking starts, be placed on four kinds of bases in four independent reagent bottles, according to the order of T, A, C, G, circulate successively and enter PTP plate, only enter a base at every turn.If generation base pairing, will discharge a tetra-sodium.This tetra-sodium is under the effect of various enzymes, and through a building-up reactions and a chemiluminescence reaction, fluorescein is oxidized to oxyluciferin the most at last, discharges optical signal simultaneously.The optical signal that this reaction discharges is captured by the high-sensitive CCD of instrument configuration in real time.There are a base and sequencing template to match, will capture the optical signal of a part; Corresponding one by one thus, just can determine accurately and rapidly the base sequence of template to be measured.
The committed step of order-checking experiment flow is the library preparation of DNA sample.Standard method prepared by the library of 454 platforms is: by genomic dna or transcribe the fragment that cDNA that group reverse transcription becomes interrupted and selected 400-800bp.After end is repaired, (B joint carries out tack (blunt-end) with vitamin H (Biotin) and is connected (ligation) the joint A different from two kinds, and result forms the fragment of three kinds of different splice combinations: AA, AB, BB with B.To connecting (ligation) product through the magnetic bead adhesion with streptavidin (Streptavidin), wash-out, alkaline denaturation, the treatment step such as wash-out again, finally being recovered to two ends is single stranded DNAs (sstDNA) of A and B joint sequence, for next step latex PCR (emulsion-PCR) and tetra-sodium sequencing reaction.
The library preparation of 454 sequenced dna samples, larger to the sample size requirement of DNA/cDNA, at least 5 microgram samples.Meanwhile, the efficiency of technology prepared by the library of DNA sample is lower, low as the efficiency of tack (blunt-end) connection own; 50% connection (ligation) product (AA, BB) is invalid; And step is more loaded down with trivial details, need to carry out the detection of a plurality of steps to DNA amount, not only need expensive instrument, the 2100Bioanalyzer (instrument name) as Agilent (u s company), also will consume a lot of consumptive materials and reagent.
Summary of the invention:
In order to solve the problems of the technologies described above, the invention provides a kind of two generation high-flux sequence the double-stranded joint of asymmetric D NA and application thereof.
The technical solution adopted for the present invention to solve the technical problems is: a kind of two generation high-flux sequence the double-stranded joint of asymmetric D NA, described two generation high-flux sequence the double-stranded joint of asymmetric D NA by two DNA oligonucleotide strands, formed, its preparation method is as follows:
Described DNA oligonucleotide strand is dissolved in quenching solution, adjusts ultimate density and reach 2mM, volume 20 microlitres, then utilize the reaction of quenching of following condition, form the DNA double chain joint of part complementation: in 95 ℃ 5 minutes; 95 ℃ drop to 12 ℃, 0.1 ℃ per second; Remain on 12 ℃, the joint solution after annealing is diluted to 500 μ M at 1: 4, and-20 ℃ of preservations.
Above-mentioned two DNA oligonucleotide strands can adopt following flag sequence, thereby form 3 different, local complementary DNA double chain joints, Y1-ACGAGTGCGT, Y2-AGCGTCGACT, Y3-TGACGCACCT.
Meanwhile, the present invention also provides the banking process of the double-stranded joint of asymmetric D NA of a kind of application this high-flux sequence in two generation, and it adopts following flow process:
The ligation of A, asymmetric D NA joint and DNA sample:
After DNA sample fragment is used atomising method to interrupt, after Qiagen MinElute PCR purification kit purifying, after end reparation 5 ' phosphorylation, utilize Klenow archaeal dna polymerase and dATP to add 3 ' A-tail end, then 3 ' the A-tail end DNA sample forming is connected with the double-stranded joint of aforesaid asymmetric D NA;
The DNA double chain joint that B, electrophoresis rubber tapping purifies and separates do not connect:
To the DNA sample after ligation, first after Qiagen MinElute PCR purification kit purifying, with 2.0% agaropectin, carry out electrophoretic separation again, extract the agar blob of viscose that comprises DNA of 400-800bp position, utilize QIAquick Gel Extraction Kit to reclaim DNA;
C, judgement
As the DNA reclaiming is quantitatively greater than 10 nanograms, can directly enters 454 emPCR reactions steps, and then check order; If the DNA quantity not sufficient reclaiming is in the emPCR step that directly enters 454, also can carry out PCR amplification by following step D, then electrophoresis rubber tapping purifying is removed the unreacted DNA primer of PCR, finally enters 454 emPCR reactions steps;
D, the PCR iodine based on asymmetric sequence:
With the recovery DNA of B step, be template, with following two oligonucleotide strand primers
PA：5’-CCATCTCATCCCTGCGTGTCTC-3’70C
PB：5’-CCTATCCCCTGTGTGCCTTGGCA-3’66C
DNA profiling is carried out to 10-30 cycle P CR iodine.
Further:
In above-mentioned flow process A, 3 ' A-tail end DNA sample with the condition that the double-stranded joint of asymmetric D NA is connected is: 0.1 μ g DNA/ μ l; The asymmetric joint of 20 μ M; 2mM ATP; 1/20 T4 DNA ligase; Insulation at 14 ° over 6 hours.
In above-mentioned flow process D, the condition of the solution that described PCR iodine adopts is: 2 μ M PA primers; 2 μ M PB primers; 1x Phusion Master Mix.
In above-mentioned flow process D, PCR iodine the temperature condition that adopts as follows: the 1st step: in 98 ℃ 30 seconds; The 2nd step: in 98 ℃ 10 seconds; The 3rd step: in 65 ℃ 45 seconds; The 4th step: in 72 ℃ 30 seconds; The 5th step: repeat 2-4 step 10-30 time; The 6th step: in 72 ℃ 5 seconds; The 7th step: remain in 4 ℃.
The present invention is with respect to building storehouse for 454 platforms of high-flux sequence and have several significant advantages when the first two: 1) build storehouse initiate dna amount of samples and be low to moderate 50 nanograms, sensitivity and improve and reach 100 times, 454 platforms are built the initiate dna sample that storehouse needs 3-5 microgram before this; 2) the joint connection procedure of building storehouse can produce effective order-checking sample of 100%, and 454 platforms are built the storehouse sample that effectively checks order and only accounted for connection and connected 50% of product before this; 3) while building storehouse, the joint efficiency of joint is higher, in the art of this patent, adopt be 3 ' A-tail end DNA with the outstanding joint of 5 ' T, stick end and be connected, and 454 platforms adopt flat end to connect before this; 4) simplified and built storehouse flow process, shortened the stability of building the storehouse time, having improved technology, 454 platforms are built storehouse and need be adopted various step to process DNA sample before this, finally to obtain effective single-stranded DNA banks; 4) the DNA library building is stable, easy long-time preservation, and utilizing the DNA library that the art of this patent finally builds is two strands, and before this 454 platforms be strand.
Accompanying drawing explanation:
Fig. 1 is that DNA sample fragment interrupts working drawing by atomization.
Fig. 2 is that DNA sample is built the quality monitoring figure in the process of storehouse.
Fig. 3 is DNA staple diagram.
In figure: 1, blank; The standard DNA sample of 2, Agilent; 3, the DNA sample of smashing with atomization; 4, connect the DNA library of 3 ' A-tail end before double-stranded joint; 5, connect the DNA library after double-stranded joint; 6, use the DNA sample of ultrasonic grinding.
Embodiment:
Below in conjunction with drawings and Examples, the present invention is further described.
Two generation high-flux sequence the double-stranded joint of asymmetric D NA, by two DNA oligonucleotide strands, formed, its preparation method is as follows:
Described DNA oligonucleotide strand is dissolved in quenching solution, adjusts ultimate density and reach 2mM, volume 20 microlitres, then utilize following condition to carry out double-stranded joint annealing: 5min in 95 ℃; 95 ℃ drop to 12 ℃, 0.1 ℃ per second; Remain on 12 ℃, the double-stranded joint solution after annealing is diluted to 500 μ M at 1: 4, and-20 ℃ of preservations.
Described two DNA oligonucleotide strands can adopt following flag sequence:
Be the banking process of the double-stranded joint of asymmetric D NA of application this high-flux sequence below, it adopts following flow process in two generation: (Fig. 2 is that DNA sample is built the quality monitoring figure in the process of storehouse, and in conjunction with shown in Fig. 1)
1) the interrupting of long segment gDNA or cDNA (atomization):
Prepare atomization damping fluid: 10ml (total amount)
Glycerine 5.31ml
1M?Tris-HCl(pH?7.5)0.37ml
0.5M?EDTA(pH?8.0)0.11ml
The super hydrostatic of VWR (product of the VWR of u s company) 4.21ml
DNA sample (0.05-1 μ g) is dissolved in to atomization damping fluid (200 μ l) and put into the cup of spraying gun, pass into nitrogen, pressure 30psi, time 1min.Reclaim the DNA sample that atomization is smashed.((to DNA, also can use hyperacoustic method, but the method that the rate of recovery is smashed not as atomization, with reference to figure 3).
2) DNA fills and clears up:
Prepare dna filling-in liquid: 20 μ l (total amount)
Polysaccharase holds liquid (10X NEB T4 DNA: the product of U.S. New England Biolab company) 6 μ l
The super hydrostatic of VWR (product of the VWR of u s company) 4.5 μ l
The bovine serum albumin 0.5 μ l of 100X U.S. New England Biolab company
25mM dNTP mother liquor 2.5 μ l
100mM ATP (mononucleotide) 0.5 μ l
3U/ μ l T4 DNA Polymerase (archaeal dna polymerase) 3 μ l
10U/ μ l T4 Polynucleotide Kinase (oligonucleotide kinases) 3 μ l
DNA sample 40 μ l after adding atomization to smash during DNA filling-in, follow these steps to react:
In 12 ℃ 20 minutes
In 25 ℃ 20 minutes
In 75 ℃ 20 minutes
Reacted rear with QIAquick PCR Purification Kit (U.S. Qiagen company product) cleaning and recovery DNA (32 μ l).
3) add 3 ' A tail end to DNA fragmentation:
Prepare dna adds the reaction solution of 3 ' A-tail end: 50 μ l (total amount)
DNA sample 32 μ L
Klenow solution 5 μ L
dATP?10μL
Klenow excision enzyme (the circumscribed shortage of 3 ' to 5 ') 3 μ L
Then reaction solution is processed 30 minutes at 37 ℃.Reacted rear with Qiagen MinElute PCR Purification kit (U.S. Qiagen company product) cleaning and recovery DNA (10 μ L).
4) DNA fragmentation of 3 ' A-tail end connects the double-stranded joint of asymmetric D NA:
Prepare dna ligation liquid: 50 μ l (total amount)
DNA sample 10 μ L
Nuclease free water (Nuclease-free water) 4 μ L
Ligase enzyme solution (DNA ligase buffer) 25 μ L
The double-stranded joint of asymmetric D NA: 6 μ L
Ligase enzyme (DNA ligase) 5 μ L
Reaction solution insulation is made to have reacted at 14 ° for about 6 hours.Reacted rear with QIAquick PCR Purification Kit (U.S. Qiagen company product) cleaning and recovery DNA (20 μ l).
5) electrophoresis rubber tapping purification connects the DNA fragmentation of double-stranded joint
With TAE damping fluid, prepare 2.0% Agarose glue and pack electrophoresis chamber into, packing DNA sample fragment and low molecular dna mark into.Run adhesive tape part: the about 45min of 120V cuts out the adhesive tape of 400-800bp position under UV light with sterile blade.Utilize Qiagen Gel Extraction Kit (U.S. Qiagen company product) to reclaim DNA sample.The DNA reclaiming can directly enter 454 emPCR reactions steps, and then check order (if directly carry out emPCR after this step, need to carry out effectively the quantitative of (being the DNA fragmentation that two ends all connect joint) to the DNA reclaiming, with the PCR amplification primer in this programme, carry out qPCR and can carry out accurate quantitative analysis).
6) based on asymmetric sequence, DNA sample is carried out to PCR iodine
(can carry out this step working the lower situation of storehouse sample size of establishing, 500ng as not enough in sample size can consider)
The DNA that step 4 reclaims can directly enter 454 emPCR reactions steps.If the DNA quantity not sufficient reclaiming, can carry out PCR amplification by this step, reclaim after enough DNA, in the emPCR reactions steps that enters 454.
By step 4, reclaiming DNA is template, by two sequences that the following describes, is primer,
PA：5’-CCATCTCATCCCTGCGTGTCTC
PB：5’-CCTATCCCCTGTGTGCCTTGGCA
According to the amount of template, carry out 10-30 cycle P CR iodine.The final condition of the solution that PCR iodine adopts: μ M PA primer; 2 μ M PB primers; 1x Phusion Master Mix (product of U.S. New England Biolab company) [see handbook: < < Phusion High Fidelity Master Mix User ' s Guide (Finnzymes) > >]; The DNA that connects double-stranded joint.
The temperature condition that PCR iodine adopts is as follows:
The 1st step: in 98 ℃ 30 seconds
The 2nd step: in 98 ℃ 10 seconds
The 3rd step: in 65 ℃ 45 seconds
The 4th step: in 72 ℃ 30 seconds
The 5th step: repeat 2-4 step, 10-30 time
The 6th step: in 72 ℃ 5 minutes
The 7th step: keep in 4 ℃
After PCR iodine, utilize the method identical with step B, electrophoresis rubber tapping purifying is removed the unreacted DNA primer of PCR.The DNA reclaiming can directly enter 454 emPCR reactions steps, and then checks order.
Claims (5)
- Two generation high-flux sequence the double-stranded joint of asymmetric D NA, it is characterized in that: described two generation high-flux sequence the double-stranded joint of asymmetric D NA by two DNA oligonucleotide strands, formed, its preparation method is as follows:Described DNA oligonucleotide strand is dissolved in quenching solution, adjusts ultimate density and reach 2mM, volume 20 microlitres, then utilize the reaction of quenching of following condition, form the DNA double chain joint of part complementation: in 95 ℃ 5 minutes; 95 ℃ drop to 12 ℃, 0.1 ℃ per second; Remain on 12 ℃, by the joint solution dilution after annealing, to 500 μ M, and-20 ℃ of preservations, the double-stranded joint sequence of described two asymmetric D NA is as follows:Above-mentioned two DNA oligonucleotide strands adopt following flag sequence, thereby form 3 DNA double chain joint different, local complementation: Y1-ACGAGTGCGT, Y2-AGCGTCGACT, and Y3-TGACGCACCT:5’CCATCTCATCCCTGCGTGTCTCCGAC?TCAG?ACGAGTGCGT3’3’GATAGGGGACACACGGAACCGTCAG?AGTC?TGCTCACGC-5’PO 4Y1-ACGAGTGCGT whereinOr5’CCATCTCATCCCTGCGTGTCTCCGAC?TCAG?AGCGTCGACT3’3’GATAGGGGACACACGGAACCGTCAG?AGTC?TCGCAGCTG-5’PO 4Y2-AGCGTCGACT whereinOr5’CCATCTCATCCCTGCGTGTCTCCGAC?TCAG?TGACGCACCT3’3’GATAGGGGACACACGGAACCGTCAG?AGTC?ACTGCGTGG-5’PO 4Y3-TGACGCACCT wherein.
- Application rights require described in 1 two generation high-flux sequence the banking process of the double-stranded joint of asymmetric D NA, it is characterized in that: described banking process adopts following flow process:The ligation of A, asymmetric D NA joint and DNA sample:After DNA sample fragment is used atomising method to interrupt, after Qiagen MinElute PCR purification kit purifying, after end reparation 5 ' phosphorylation, utilize Klenow archaeal dna polymerase and dATP to add 3 ' A-tail end, then 3 ' the A-tail end DNA sample forming is connected with the double-stranded joint of the asymmetric D NA described in claim 1;The DNA double chain joint that B, electrophoresis rubber tapping purifies and separates do not connect:To the DNA sample after ligation, first after Qiagen MinElute PCR purification kit purifying, with 2.0% agaropectin, carry out electrophoretic separation again, extract the agar blob of viscose that comprises DNA of 400-800bp position, utilize QIAquick Gel Extraction Kit to reclaim DNA;C, judgementAs the DNA reclaiming is quantitatively greater than 10 nanograms, can directly enters 454 emPCR reactions steps, and then check order; If the DNA quantity not sufficient reclaiming is in the emPCR step that directly enters 454, also can carry out PCR amplification by following step D, then electrophoresis rubber tapping purifying is removed the unreacted DNA primer of PCR, finally enters 454 emPCR reactions steps;D, the PCR iodine based on asymmetric sequence:With the recovery DNA of B step, be template, with following two oligonucleotide strand primersPA：5，-CCATCTCATCCCTGCGTGTCTC-3，PB：5’-CCTATCCCCTGTGTGCCTTGGCA-3’DNA profiling is carried out to 10-30 cycle P CR iodine.
- 3. the banking process of the double-stranded joint of asymmetric D NA of a kind of application two generations high-flux sequence according to claim 2, it is characterized in that: in described flow process A, 3 ' A-tail end DNA sample with the condition that the double-stranded joint of asymmetric D NA is connected is: 0.1 μ g DNA/ μ l; The asymmetric joint of 20 μ M; 2mM ATP; 1/20 T4DNA ligase enzyme; Insulation at 14 ℃ over 6 hours.
- 4. the banking process of the double-stranded joint of asymmetric D NA of a kind of application two generations high-flux sequence according to claim 2, it is characterized in that: in described flow process D, the solution that described PCR iodine adopts is: 2 μ M PA primers, 2 μ M PB primers, 1x Phusion Master Mix.
- 5. the banking process of the double-stranded joint of asymmetric D NA of a kind of application two generations high-flux sequence according to claim 2, is characterized in that: in described flow process D, PCR temperature condition that iodine adopts is as follows: the 1st step: in 98 ℃ 30 seconds; The 2nd step: in 98 ℃ 10 seconds; The 3rd step: in 65 ℃ 45 seconds; The 4th step: in 72 ℃ 30 seconds; The 5th step: repeat 2-4 step 10-30 time; The 6th step: in 72 ℃ 5 seconds; The 7th step: remain in 4 ℃.
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| CN102296065B (en) * | 2011-08-04 | 2013-05-15 | 盛司潼 | System and method for constructing sequencing library |
| CN102978205B (en) * | 2012-11-19 | 2014-08-20 | 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 |
| WO2015117040A1 (en) * | 2014-01-31 | 2015-08-06 | Swift Biosciences, Inc. | Improved methods for processing dna substrates |
| CN104862302B (en) * | 2015-05-05 | 2020-12-15 | 华南师范大学 | DNA fragmentation method and device for implementing same |
| CN105154444A (en) * | 2015-10-15 | 2015-12-16 | 南京普东兴生物科技有限公司 | Asymmetric high-throughput sequencing linkers capable of effectively improving library construction efficiency, and application of linkers |
| CN105567681B (en) * | 2015-12-31 | 2018-08-31 | 广州赛哲生物科技股份有限公司 | A kind of method and label connector based on the noninvasive biopsy virus of high-throughput gene sequencing |
| CN105950612B (en) * | 2016-07-08 | 2019-06-21 | 北京全式金生物技术有限公司 | A kind of efficient DNA connector connecting method |
| CN108300716B (en) * | 2018-01-05 | 2020-06-30 | 武汉康测科技有限公司 | Linker element, application thereof and method for constructing targeted sequencing library based on asymmetric multiplex PCR |
| CN108034705A (en) * | 2018-01-15 | 2018-05-15 | 武汉爱基百客生物科技有限公司 | A kind of full-length genome methylates high-flux sequence method |
| CN108148899A (en) * | 2018-01-15 | 2018-06-12 | 武汉爱基百客生物科技有限公司 | A kind of genome simplifies and two generations sequencing SNP compound detections system and detection method |
| CN110791813B (en) * | 2018-08-01 | 2023-06-16 | 广州华大基因医学检验所有限公司 | Method for processing single-stranded DNA and application thereof |
| CN110872615A (en) * | 2018-08-31 | 2020-03-10 | 成都先导药物开发股份有限公司 | High-throughput next-generation sequencing method for sequencing nucleotide double strands |
| CN111041069B (en) * | 2019-12-26 | 2021-01-19 | 人和未来生物科技（长沙）有限公司 | High-throughput sequencing library construction method for low-initial-quantity DNA sample and application thereof |
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