CN106520978B - The preparation method of target DNA enrichment probe - Google Patents
The preparation method of target DNA enrichment probe Download PDFInfo
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Abstract
The invention discloses the preparation methods of target DNA enrichment probe.The preparation method of target DNA disclosed in this invention enrichment probe include: include: prepare N number of sub- probe, connect N number of Asia probe, obtain long chain DNA and/or cyclic DNA；The long chain DNA and/or the cyclic DNA are expanded, target DNA enrichment probe is obtained；N number of Asia probe, which meets N number of sub- probe, can cover the full sequence of the target DNA；The downstream that any two adjacent sub- probes are all satisfied upstream Asia probe on target DNA has one or more nucleotide Chong Die with the upstream of downstream Asia probe；The length of N number of Asia probe is 50-150bp.It is demonstrated experimentally that the target DNA enrichment probe prepared using method of the invention has reached 100% covering to the capture of target DNA, capture rate reaches 100%, and the whole homogeneity of probe is very good, and stability is good, can be used for the capture of target DNA.
Description
Technical field
The present invention relates in field of biotechnology, target DNA is enriched with the preparation method of probe.
Background technique
With the completion that more and more biological genomes are sequenced, biomedical research has entered the genome times afterwards comprehensively.With
The two generations cost of sequencing constantly reduces, and early in 2014, illumina company, the U.S. released completely new high-flux sequence instrument
Sequencing cost, can be reduced to 1000 dollars by device, genetic test universal and be accelerated therewith, be existed using high throughput sequencing technologies
In scientific research and clinical detection.
Although currently, 1000 dollars are had arrived at for genome sequencing cost, for needing superelevation depth to survey
In the clinic or Technological research field, especially lesion detection of sequence, sequencing cost and high-throughput number that high depth sequencing faces
According to processing bring cost, cannot be widely used in clinical detection and correlative study, so just producing gene trap skill
Art, so-called gene trap design corresponding capture probe aiming at the destination region of required concern or research, utilize hybridization
Target fragment is enriched with from full-length genome and comes out by technology, these segments are carried out high-flux sequence again, and being both able to satisfy research needs
Depth requirements are wanted, while being also able to achieve and not increasing cost, gene trapping is in clone, discovery new gene and announcement at present
Have the advantages that uniqueness in terms of gene function, the powerful of functional genome's age study is had become, in biomedical research
The application of every field is increasingly extensive.The probe used in gene trap at present is cDNA single-stranded probe and rna probe.
Summary of the invention
It can be with the probe of acquisition target DNA the technical problem to be solved by the present invention is to how to prepare.
In order to solve the above technical problems, present invention firstly provides the preparation methods of target DNA enrichment probe.
The preparation method of target DNA enrichment probe provided by the present invention, comprising: prepare N number of sub- probe, connect described N number of
Sub- probe obtains target DNA enrichment probe；
N number of sub- probe, which meets N number of sub- probe, can cover the full sequence of the target DNA.
Wherein, N is a natural number more than or equal to 2.The numerical value of N specifically can be according to the length of the target DNA and described
The length of sub- probe determines, the ratio of the length (bp) of the target DNA and the length (bp) of the sub- probe is denoted as n, when n is
When non-integer, the integer part+1 of N=n；When n is integer, N=n.If in N number of sub- probe, there are the complete phases of sequence
Same probe can also will remove duplicate sub- probe in practical applications, cover as long as being able to satisfy the quantity of the sub- probe
Cover the full sequence of the target DNA.In one embodiment of the invention, N value obtained by calculation is 208, but
In this 208 sub- probes, the 207th identical with the 208th sub- probe, in carrying out specifically experiment, can only prepare the
1-207 item Asia probe.
N number of sub- probe is single stranded DNA or double-stranded DNA.
It is following H1 or H2 that N number of sub- probe, which can cover the meaning of the full sequence of the target DNA:
H1, when N number of sub- probe is single stranded DNA, N number of sub- probe can with two of the target DNA it is single-stranded
DNA or single stranded DNA, which combines, forms double-stranded DNA；When N number of sub- probe only single stranded DNA knot with the target DNA
When conjunction, there is no cannot be with the site in conjunction with the sub- probe in N number of sub- probe for the single stranded DNA of the target DNA；When described
When N number of Asia probe is in conjunction with two single stranded DNAs of the target DNA, there is a chain energy in any one site of the target DNA
In conjunction with the sub- probe in N number of sub- probe；That is, when N number of sub- probe is single stranded DNA, N number of Asia
Probe is complementary with two single stranded DNAs of the target DNA or a single stranded DNA；When N number of sub- probe and the target DNA
A chain DNA it is complementary and with another chain DNA of the target DNA not mutual added time, and described in N number of sub- probes complementary
There is no the sites not with the sub- probes complementary in N number of sub- probe in that single stranded DNA of target DNA；When N number of sub- spy
Needle can have with the DNA fragmentation mutual added time in two chains of the target DNA, any one site of the target DNA chain with
Sub- probes complementary in N number of sub- probe；
H2, when N number of sub- probe is double-stranded DNA, N number of sub- probe can with two of the target DNA it is single-stranded
DNA, which is combined, forms double-stranded DNA, and two single stranded DNAs of the target DNA are not present cannot be with any in N number of sub- probe
The site that single stranded DNA combines；That is, when N number of sub- probe is double-stranded DNA, it is each in N number of sub- probe
Two of sub- probe are single-stranded complementary in same site with two single stranded DNAs of the target DNA respectively, two lists of the target DNA
The not site with N number of sub- probes complementary is not present in chain DNA.
It may also include before connecting N number of sub- probe and elute N number of sub- probe on the chip.Elution
N number of sub- probe on the chip can be carried out using ammonium hydroxide.The molar concentration of the ammonium hydroxide can be 35%.From described
The N number of sub- probe eluted on chip can be dissolved using ultrapure water.
It is described prepare N number of sub- probe can on chip N number of Asia probe described in fabricated in situ.
In the preparation method of above-mentioned target DNA enrichment probe, any two adjacent sub- probes can expire on the target DNA
The downstream of sufficient upstream Asia probe has one or more nucleotide to be overlapped (i.e. sequence is identical) with the upstream of downstream Asia probe.
In the preparation method of above-mentioned target DNA enrichment probe, the quantity of one or more of nucleotide can be according to specific feelings
Condition determines, the quantity of one or more of nucleotide makes the length of N number of sub- probe meet the requirement of specific experiment.At this
In one embodiment of invention, the quantity of one or more of nucleotide makes the length of N number of sub- probe be 108bp.
In the above method, the sequence of one or more of nucleotide is the sequence on the target DNA, it is one or
The sequence of multiple nucleotide is the sequence that the sub- probe extends on the target DNA.
In the preparation method of above-mentioned target DNA enrichment probe, the length of N number of sub- probe can be 50-150bp.The N
The length of a Asia probe specifically can be 108bp.
In the preparation method of above-mentioned target DNA enrichment probe, connection N number of sub- probe may include following A 1) and
A2):
A1 N number of sub- probe) is connected, long chain DNA and/or cyclic DNA are obtained；
A2) the long chain DNA and/or the cyclic DNA are expanded, obtain target DNA enrichment probe.
The target DNA enrichment probe is double-stranded DNA.
In the preparation method of above-mentioned target DNA enrichment probe, it may also include before connecting N number of sub- probe to described N number of
Sub- probe carries out phosphorylation modification.
Phosphorylation modification is carried out to N number of sub- probe and concretely carries out phosphorylation at 5 ' ends of N number of sub- probe
Modification.The phosphorylation modification can be carried out using the T4 polynueleotide kinase kit of T4 polynueleotide kinase or NEB.Benefit
It can with the reaction system that the T4 polynueleotide kinase kit of NEB carries out the phosphorylation modification are as follows: N number of sub- probe 10
μ l, 10 × reaction buffer, 5 μ l, 5 μ l of 10mM ATP, T4 polynueleotide kinase 2.5 μ l and H20 27.5μl.The reaction
The concentration of N number of Asia probe can be 5ng/ μ l described in system.10 × reaction buffer, ATP and T4 poly in the reaction system
Nucleoside monophosphate kinase is the T4 polynueleotide kinase seminal plasma fructose detection kit of NEB.The reaction condition of the phosphorylation modification can be
37℃30min。
Connection N number of sub- probe can utilize ssDNA ligase or the ssDNA connection kit of epicentre
(CircLigaseTMII ssDNA Ligase, epicentre) it carries out.The reaction system of the connection can are as follows: N number of Asia
The Phosphorylated products of probe or N number of sub- 20 μ l of probe, 10 × Reaction of CircLigase II Buffer, 5 μ l,
50mM MnCl22.5 μ l, 10 μ l of 5M Betaine, CircLigase II ssDNA Ligase (100U) 2.5 μ l and H2O 10
μl.The reaction condition of the connection can be 60 DEG C of 60min.
The preparation method of above-mentioned target DNA enrichment probe obtains step A2) in, it is carrying out the long chain DNA or the ring-type
It may also include when DNA is expanded and amplified production be marked using biotin.
Carrying out the amplification can be used random primer progress.The random primer can be for by this four nucleotide of A, C, G and T
The length formed at random is the 4 of 6bp6Single stranded DNA is formed by mixture.All single stranded DNAs in the random primer
Molal quantity can be identical.
The long chain DNA and/or the cyclic DNA are subjected to amplification and amplified production is marked using biotin
It is carried out using the whole genome amplification kit of Qiagen company.Using Qiagen company whole genome amplification kit into
The reaction system of row reaction can are as follows: 10 μ l of the long chain DNA and/or the cyclic DNA, 2 × reaction buffer, 25 μ l, Bio-
5 μ l of 16-dUTP, 1 Replig_Enzyme μ l and H2O 9μl.Wherein, the random primer is contained in 2 × reaction buffer.
The reaction temperature reacted using the whole genome amplification kit of Qiagen company can be 37 DEG C.Utilize Qiagen company
The time that whole genome amplification kit is reacted can be not less than 16 hours.
In order to solve the above technical problems, the present invention also provides the methods of acquisition target DNA.
The method of acquisition target DNA provided by the present invention, the target of the preparation method preparation including target DNA enrichment probe
DNA is enriched with probe acquisition target DNA.
In order to solve the above technical problems, the present invention also provides the methods of target DNA sequencing.
The method of target DNA sequencing provided by the present invention, including the use of the method acquisition target DNA of the acquisition target DNA, so
The target DNA of capture is sequenced afterwards.
In the method for above-mentioned target DNA sequencing, the target DNA of described pair of capture, which is sequenced, can utilize sequencing in the prior art
Method carries out, as long as can achieve the purpose that target DNA is sequenced.
In order to solve the above technical problems, the present invention also provides can any application in following X1-X5:
X1, the target DNA are enriched with application of the preparation method of probe in acquisition target DNA；
The preparation method that X2, the target DNA are enriched with probe is preparing the application in acquisition target DNA product；
X3, the target DNA are enriched with application of the preparation method of probe in target DNA sequencing；
The preparation method that X4, the target DNA are enriched with probe is preparing the application in target DNA sequencing products；
X5, the acquisition target DNA method target DNA sequencing in application.
In above-mentioned application, the progress of two generation sequencing approaches is can be used in the target DNA sequencing.
It is demonstrated experimentally that the full base of capture human mitochondrial of the preparation method preparation using target DNA enrichment probe of the invention
The enrichment probe of cause has reached 100% covering to the capture of homo mitochondrion gene, and capture rate is all larger than 50%, from least
It covers from the point of view of the parameter of 4X, 10X and 20X, reaches 100%, the whole homogeneity of probe is very good, and stability is good.
Show that the preparation method of target DNA enrichment probe of the invention can be used for preparing target DNA enrichment probe, and further to target DNA
It is sequenced.The probe of the preparation method preparation of target DNA enrichment probe of the invention is double chain DNA probe, and there are following advantages:
1, double chain DNA probe is more stable, not degradable；
2, double chain DNA probe after the completion of connection, directly carries out whole genome amplification, the homogeneity of probe in the preparation
It is good；
3, double chain DNA probe, in enrichment, capture double-strand more accurately can exclude the base that single-stranded middle PCR is brought into
Mispairing.
Detailed description of the invention
Fig. 1 is connection product testing result.
Specific embodiment
The present invention is further described in detail With reference to embodiment, and the embodiment provided is only for explaining
The bright present invention, the range being not intended to be limiting of the invention.
Experimental method in following embodiments is unless otherwise specified conventional method.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
The preparation method of target DNA enrichment probe provided by the present invention, comprising: prepare N number of sub- probe, connect N number of sub- spy
Needle obtains long chain DNA and cyclic DNA；Long chain DNA and cyclic DNA are expanded, target DNA enrichment probe is obtained.
Below by taking human mitochondrial full genome is target DNA as an example, how target DNA enrichment of the invention is utilized to be specifically described
The preparation method preparation of probe can capture the enrichment probe of human mitochondrial full genome.
Embodiment 1, the preparation for being enriched with probe that human mitochondrial full genome can be captured
1, the sequence design and preparation of single-stranded sub- probe
The sequence of human mitochondrial full genome as target DNA is as shown in sequence 1 in sequence table.The length of target DNA is
Target DNA is divided into 208 segments by 16569bp, wherein the length of the 1-207 segment is 80bp, the length of the 208th segment
Degree is 9bp, and every two adjacent segment meets the last bit of fragment upstream and is connected with the first place of segments downstream, that is, the 1st segment
Sequence be 1-80 of sequence 1, the sequence of the 2nd segment is 81-160 of sequence 1 ... ..., the 207th segment
Sequence is 16481-16560 of sequence 1, and the sequence of the 208th segment is 16561-16569 of sequence 1.
The single-stranded sub- probe that full target DNA can be covered according to this 208 segment designs of target DNA, by the sequence of this 208 segments
Column extend to its upstream and downstream respectively, and making finally obtained sequence length is 108bp, specific as follows: by the sequence of the 1st segment
Extend 14bp, by the 207th to both ends respectively to extension 28bp downstream, by the sequence of each segment of the 2-206 segment
The sequence of segment to its upstream extend 19bp to extend 9bp (because downstream less than 14bp) downstream, by the 208th segment
Sequence extends 99bp to its upstream, obtains the sequence that 208 length are 108bp.Wherein, the 1st article of sequence is the 1- of sequence 1
108, the 2nd article of sequence is 67-174 of sequence 1, and the 3rd article of sequence is 147-254 of sequence 1, and nth sequence is
Position to position (80n+14) (80 (n-1) -13) of sequence 1, n are any one natural number in 4-205, and the 206th article of sequence is
16387-16494 of sequence 1, the 207th article of sequence are 16462-16569 of sequence 1, and the 208th article of sequence is sequence 1
16462-16569, the 207th article of sequence is identical with the 208th article of sequence.
It is respectively above-mentioned the 1-207 articles sequence in chip synthetic oligonucleotide according to sequence using the method for fabricated in situ
Probe, i.e., 207 single-stranded sub- probes.
Using 35% ammonium hydroxide, be fixed on chip 207 single-stranded sub- probes are eluted, centrifuge tube is collected into
In, after vacuum concentration instrument concentration, the dissolution of 500 μ l ultrapure waters is added, becomes the mixed of the single-stranded sub- probe of target DNA 207
Pond；Concentration is measured, and is diluted to 25ng/ μ l.
2, the preparation of the enrichment probe of human mitochondrial full genome can be captured
1) it 5 ' phosphorylation modifications of single-stranded sub- probe: is carried out using the T4 polynueleotide kinase kit of NEB.5 ' phosphoric acid
The reaction system for changing modification is as follows:
| Component | Volume (μ l) |
| The mixed pond (50ng/ μ l) of 207 single-stranded sub- probes | 10 |
| 10 × reaction buffer (Reaction Buffer) | 5 |
| 10mM ATP | 5 |
| T4 Polynucleotide Kinase (T4 polynueleotide kinase) | 2.5 |
| H20 | 27.5 |
50 μ l of total reaction volume, mixed pond and H in the reaction system, except 207 single-stranded sub- probes2Reagent outside O is
T4 polynueleotide kinase seminal plasma fructose detection kit.After mixing, 37 DEG C of constant-temperature incubations in constent temperature heater or PCR instrument
30min.After reaction, product purification recycling, elution are carried out according to its operating process using trace P CR product QIAquick Gel Extraction Kit
20 μ l of volume, obtains Phosphorylated products；
2) connection reaction: the Phosphorylated products that step 1) is obtained utilize the ssDNA connection kit of business
(CircLigaseTMII ssDNA Ligase, epicentre) it connects, coupled reaction system is as follows:
| Component | Volume (μ l) |
| The Phosphorylated products that step 1) obtains | 20 |
| CircLigase II 10×Reaction Buffer | 5 |
| 50mM MnCl2 | 2.5 |
| 5M Betaine | 10 |
| CircLigase II ssDNA Ligase(100U) | 2.5 |
| H2O | 10 |
Total reaction volume is 50 μ l, in the reaction system, the Phosphorylated products and H that are obtained except step 1)2Reagent outside O is equal
For ssDNA connection seminal plasma fructose detection kit.After mixing, it reacts 1 hour for 60 DEG C.Reaction product utilizes minim DNA reclaim reagent
Box carries out product purification recycling according to its operating process, and recovery product obtains connection product (long chain DNA and cyclic DNA).Pass through
Gel electrophoresis identifies connection product segment, and as a result as shown in Figure 1, No. 1 swimming lane is DNA molecular amount standard in Fig. 1, No. 2 are swimming lane
For connection product, the results show that the connection product being successfully connected.
3) the commercialization whole genome amplification kit of Qiagen company, reaction system label biotin and amplification: are utilized
It is as follows:
| Component | Volume (μ l) |
| The connection product that step 2) obtains | 10 |
| 2 × reaction buffer (2 × Reaction Buffer) | 25 |
| Bio-16-dUTP | 5 |
| Replig_Enzyme | 1 |
| H2O | 9 |
50 μ l of total volume, in the reaction system, the connection product and H that are obtained except step 2)2Reagent outside O is full genome
Reagent in group amplification kit, wherein contain random primer in 2 × reaction buffer, random primer be by A, C, G and T this four
The length that a nucleotide forms at random is the 4 of 6bp6Single stranded DNA is formed by mixture, all single-stranded in the mixture
The molal quantity of DNA is all the same.After mixing, 37 degree constant-temperature incubation 16 hours or more.Reaction product uses DNA reclaim reagent
Box carries out DNA purifying according to the Standard Operating Procedure of kit, obtains target DNA enrichment probe.Using Nanodrop2000 into
Row is quantitative, and target DNA is enriched with probe dilution to 150ng/ μ l, forms final target DNA enrichment probe solution.
3, the quality evaluation of target DNA enrichment probe
In triplicate, repeating test every time, specific step is as follows for experiment:
1) test experiments scheme: the full-length genome library sample of 10 people of selection, each sample are divided into two groups, two components
Confirmatory experiment is not carried out by different batches.
The target DNA in above-mentioned 10 samples is captured using target DNA enrichment probe:
Target DNA enrichment probe is mixed with DNA full-length genome library, the chondriogen in sample is just hybridised to probe
On, absorption is combined by biotin and Streptavidin MagneSphere, removes the DNA fragmentation of nontarget area through elution processing, from
And it is enriched with human mitochondrial full genome, the measurement of 2X150bp high pass is carried out using new-generation sequencing instrument Illumina Nexseq500
Sequence.Specific step is as follows:
A1 it) prepares following mixed system: taking 500ng DNA full-length genome library (20 μ l, 25ng/ μ l), 10 μ l Buffer
BL, the target DNA that 5 μ l steps 2 obtain are enriched with probe solution, 6 μ l Blocking Oligo Mix；
It a2) will be in the mixed system PCR instrument of step a1): 95 DEG C, 7min, 65 DEG C later, 2min；
A3 the 23 μ l of Buffer HY of 65 DEG C of preheatings) is taken to be added to step a2) it reacts in the system terminated, then in PCR
Hybridize 22 hours for 65 DEG C on instrument.
A4 MyOne beads (Invitrogen)) is taken out, whirlpool concussion makes magnetic bead sufficiently suspend, and of short duration centrifugation makes pipe lid
On beads be centrifuged to bottom of the tube.
A5 in the centrifuge tube for) taking 50 μ l MyOne beads to new 1.5ml, whirlpool shakes at least 5s, keeps magnetic bead abundant
It suspends, is put into after of short duration centrifugation on magnetic frame one minute.
A6) centrifuge tube remain stationary (should not rotating centrifugal pipe) on magnetic frame, and careful inhale abandons supernatant.
A7 centrifuge tube) is removed, the 1X Binding Buffer (binding buffer) of 50 μ l is added, the concussion of rotation nest is at least
5s is put on magnetic frame static one minute after of short duration centrifugation, and careful inhale abandons supernatant.
A8 step a6) is repeated) and a7) twice (altogether three times).
A9 centrifuge tube) is removed, 100 μ l 2XBinding Buffer (binding buffer) are added, the concussion of rotation nest is at least
5s is transferred to completely in a new centrifuge tube after of short duration centrifugation.
A10) be transferred to the product of step a3) completely in the centrifuge tube of beads (about 200 μ l of total volume, while handling multiple
Several lower mixings are flicked when sample, after mixing), it is small to be placed in room temperature rotation 1 on Rotator by rotation nest concussion at least 5s (not having to centrifugation)
When.
A11) after above-mentioned steps, primary using WB1buffer room temperature cleaning beads, then 15minutes exists
It is washed in WB3buffer 3 times, 65 DEG C, 15minutes/ times.
A12) DNA bound is eluted using Buffer Elute.The DNA of elution finally expands 15 circulations, utilizes following journey
Sequence: 98 DEG C, 30s (1cycle)；98 DEG C, 25s, 65 DEG C, 30s, 72 DEG C, 30s (15cycles)；72 DEG C, 5min (1cycle) is right
The DNA of elution carries out PCR amplification.
A13) PCR product is purified using SPRI beads (Beckman Coulter) according to laboratory manual, obtains enrichment line
Mitochondrial genes frag-ment libraries.
Wherein, the formula of each reagent is as follows:
It is sequenced using Nextseq500 (illumina) microarray dataset, verifies the repeatability and stability of its probe.
2) analysis of sequencing data: firstly, utilizing the low-quality sequence of Trim-Galore program filters；Then, it uses
Cutadapt program in Trim-Galore removes the universal primer sequence at 3 ' ends and 5 ' ends, retains reading quality and is greater than 20bp
It is greater than the sequence of 80bp with reading length.Then the sequence of filtering uses GATK soft on BWA procedure match to human genome
Part packet calibrates mass value, is then matched on reference sequences again.Repeat reading sequence alignment/matching tool
(SAMtools) 3 remove after, the reading sequence of only unique match is used for subsequent analysis.Matter finally is carried out to the data of sequencing
Amount control, data are shown in Table 1.
Table 1, sequencing data
Parameter declaration in table 1:
A. sequencing data amount refers to the total sequencing data generated when the sample for completing target DNA capture is sequenced；
B. the base number of mesh: refer to the total bases of the probe design destination region to be covered；
C. it covers purpose base number: referring to after enrichment sequencing, can successfully compare the design base area to be covered
The base number in domain；
D. coverage rate: covering purpose base number/purpose base number；
E. capture rate (valid data amount): covering purpose base number/sequencing data amount
F. mean depth is sequenced: when referring to destination region sequencing, average read number；
G. it averagely covers 4X ratio: referring to the minimum base number accounting for reading 4 times or more；
H. it averagely covers 10X ratio: referring to the minimum base number accounting for reading 10 times or more；
I. it averagely covers 20X ratio: referring to the minimum base number accounting for reading 20 times or more；
Probe mass assessment:
Target DNA is enriched with probe and has reached 100% covering to the capture of homo mitochondrion gene, and capture rate is all larger than
50%, from the point of view of at least covering the parameter of 4X, 10X and 20X, reach 100%, the whole homogeneity of probe is very good；It is logical
It crosses and repeats experiment and data the results show that probe has good stability.Show target DNA enrichment probe of the invention
Preparation method can be used for preparing target DNA enrichment probe, and further target DNA is sequenced.
<110>Beijing steps rich permanent industry science and technology limited Company
<120>preparation method of target DNA enrichment probe
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 16569
<212> DNA
<213>people (Homo sapiens)
<220>
<221> misc_feature
<222> (3107)..(3107)
<223>n is a, c, g or t
<400> 1
gatcacaggt ctatcaccct attaaccact cacgggagct ctccatgcat ttggtatttt 60
cgtctggggg gtatgcacgc gatagcattg cgagacgctg gagccggagc accctatgtc 120
gcagtatctg tctttgattc ctgcctcatc ctattattta tcgcacctac gttcaatatt 180
acaggcgaac atacttacta aagtgtgtta attaattaat gcttgtagga cataataata 240
acaattgaat gtctgcacag ccactttcca cacagacatc ataacaaaaa atttccacca 300
aaccccccct cccccgcttc tggccacagc acttaaacac atctctgcca aaccccaaaa 360
acaaagaacc ctaacaccag cctaaccaga tttcaaattt tatcttttgg cggtatgcac 420
ttttaacagt caccccccaa ctaacacatt attttcccct cccactccca tactactaat 480
ctcatcaata caacccccgc ccatcctacc cagcacacac acaccgctgc taaccccata 540
ccccgaacca accaaacccc aaagacaccc cccacagttt atgtagctta cctcctcaaa 600
gcaatacact gaaaatgttt agacgggctc acatcacccc ataaacaaat aggtttggtc 660
ctagcctttc tattagctct tagtaagatt acacatgcaa gcatccccgt tccagtgagt 720
tcaccctcta aatcaccacg atcaaaagga acaagcatca agcacgcagc aatgcagctc 780
aaaacgctta gcctagccac acccccacgg gaaacagcag tgattaacct ttagcaataa 840
acgaaagttt aactaagcta tactaacccc agggttggtc aatttcgtgc cagccaccgc 900
ggtcacacga ttaacccaag tcaatagaag ccggcgtaaa gagtgtttta gatcaccccc 960
tccccaataa agctaaaact cacctgagtt gtaaaaaact ccagttgaca caaaatagac 1020
tacgaaagtg gctttaacat atctgaacac acaatagcta agacccaaac tgggattaga 1080
taccccacta tgcttagccc taaacctcaa cagttaaatc aacaaaactg ctcgccagaa 1140
cactacgagc cacagcttaa aactcaaagg acctggcggt gcttcatatc cctctagagg 1200
agcctgttct gtaatcgata aaccccgatc aacctcacca cctcttgctc agcctatata 1260
ccgccatctt cagcaaaccc tgatgaaggc tacaaagtaa gcgcaagtac ccacgtaaag 1320
acgttaggtc aaggtgtagc ccatgaggtg gcaagaaatg ggctacattt tctaccccag 1380
aaaactacga tagcccttat gaaacttaag ggtcgaaggt ggatttagca gtaaactaag 1440
agtagagtgc ttagttgaac agggccctga agcgcgtaca caccgcccgt caccctcctc 1500
aagtatactt caaaggacat ttaactaaaa cccctacgca tttatataga ggagacaagt 1560
cgtaacatgg taagtgtact ggaaagtgca cttggacgaa ccagagtgta gcttaacaca 1620
aagcacccaa cttacactta ggagatttca acttaacttg accgctctga gctaaaccta 1680
gccccaaacc cactccacct tactaccaga caaccttagc caaaccattt acccaaataa 1740
agtataggcg atagaaattg aaacctggcg caatagatat agtaccgcaa gggaaagatg 1800
aaaaattata accaagcata atatagcaag gactaacccc tataccttct gcataatgaa 1860
ttaactagaa ataactttgc aaggagagcc aaagctaaga cccccgaaac cagacgagct 1920
acctaagaac agctaaaaga gcacacccgt ctatgtagca aaatagtggg aagatttata 1980
ggtagaggcg acaaacctac cgagcctggt gatagctggt tgtccaagat agaatcttag 2040
ttcaacttta aatttgccca cagaaccctc taaatcccct tgtaaattta actgttagtc 2100
caaagaggaa cagctctttg gacactagga aaaaaccttg tagagagagt aaaaaattta 2160
acacccatag taggcctaaa agcagccacc aattaagaaa gcgttcaagc tcaacaccca 2220
ctacctaaaa aatcccaaac atataactga actcctcaca cccaattgga ccaatctatc 2280
accctataga agaactaatg ttagtataag taacatgaaa acattctcct ccgcataagc 2340
ctgcgtcaga ttaaaacact gaactgacaa ttaacagccc aatatctaca atcaaccaac 2400
aagtcattat taccctcact gtcaacccaa cacaggcatg ctcataagga aaggttaaaa 2460
aaagtaaaag gaactcggca aatcttaccc cgcctgttta ccaaaaacat cacctctagc 2520
atcaccagta ttagaggcac cgcctgccca gtgacacatg tttaacggcc gcggtaccct 2580
aaccgtgcaa aggtagcata atcacttgtt ccttaaatag ggacctgtat gaatggctcc 2640
acgagggttc agctgtctct tacttttaac cagtgaaatt gacctgcccg tgaagaggcg 2700
ggcataacac agcaagacga gaagacccta tggagcttta atttattaat gcaaacagta 2760
cctaacaaac ccacaggtcc taaactacca aacctgcatt aaaaatttcg gttggggcga 2820
cctcggagca gaacccaacc tccgagcagt acatgctaag acttcaccag tcaaagcgaa 2880
ctactatact caattgatcc aataacttga ccaacggaac aagttaccct agggataaca 2940
gcgcaatcct attctagagt ccatatcaac aatagggttt acgacctcga tgttggatca 3000
ggacatcccg atggtgcagc cgctattaaa ggttcgtttg ttcaacgatt aaagtcctac 3060
gtgatctgag ttcagaccgg agtaatccag gtcggtttct atctacnttc aaattcctcc 3120
ctgtacgaaa ggacaagaga aataaggcct acttcacaaa gcgccttccc ccgtaaatga 3180
tatcatctca acttagtatt atacccacac ccacccaaga acagggtttg ttaagatggc 3240
agagcccggt aatcgcataa aacttaaaac tttacagtca gaggttcaat tcctcttctt 3300
aacaacatac ccatggccaa cctcctactc ctcattgtac ccattctaat cgcaatggca 3360
ttcctaatgc ttaccgaacg aaaaattcta ggctatatac aactacgcaa aggccccaac 3420
gttgtaggcc cctacgggct actacaaccc ttcgctgacg ccataaaact cttcaccaaa 3480
gagcccctaa aacccgccac atctaccatc accctctaca tcaccgcccc gaccttagct 3540
ctcaccatcg ctcttctact atgaaccccc ctccccatac ccaaccccct ggtcaacctc 3600
aacctaggcc tcctatttat tctagccacc tctagcctag ccgtttactc aatcctctga 3660
tcagggtgag catcaaactc aaactacgcc ctgatcggcg cactgcgagc agtagcccaa 3720
acaatctcat atgaagtcac cctagccatc attctactat caacattact aataagtggc 3780
tcctttaacc tctccaccct tatcacaaca caagaacacc tctgattact cctgccatca 3840
tgacccttgg ccataatatg atttatctcc acactagcag agaccaaccg aacccccttc 3900
gaccttgccg aaggggagtc cgaactagtc tcaggcttca acatcgaata cgccgcaggc 3960
cccttcgccc tattcttcat agccgaatac acaaacatta ttataataaa caccctcacc 4020
actacaatct tcctaggaac aacatatgac gcactctccc ctgaactcta cacaacatat 4080
tttgtcacca agaccctact tctaacctcc ctgttcttat gaattcgaac agcatacccc 4140
cgattccgct acgaccaact catacacctc ctatgaaaaa acttcctacc actcacccta 4200
gcattactta tatgatatgt ctccataccc attacaatct ccagcattcc ccctcaaacc 4260
taagaaatat gtctgataaa agagttactt tgatagagta aataatagga gcttaaaccc 4320
ccttatttct aggactatga gaatcgaacc catccctgag aatccaaaat tctccgtgcc 4380
acctatcaca ccccatccta aagtaaggtc agctaaataa gctatcgggc ccataccccg 4440
aaaatgttgg ttataccctt cccgtactaa ttaatcccct ggcccaaccc gtcatctact 4500
ctaccatctt tgcaggcaca ctcatcacag cgctaagctc gcactgattt tttacctgag 4560
taggcctaga aataaacatg ctagctttta ttccagttct aaccaaaaaa ataaaccctc 4620
gttccacaga agctgccatc aagtatttcc tcacgcaagc aaccgcatcc ataatccttc 4680
taatagctat cctcttcaac aatatactct ccggacaatg aaccataacc aatactacca 4740
atcaatactc atcattaata atcataatag ctatagcaat aaaactagga atagccccct 4800
ttcacttctg agtcccagag gttacccaag gcacccctct gacatccggc ctgcttcttc 4860
tcacatgaca aaaactagcc cccatctcaa tcatatacca aatctctccc tcactaaacg 4920
taagccttct cctcactctc tcaatcttat ccatcatagc aggcagttga ggtggattaa 4980
accaaaccca gctacgcaaa atcttagcat actcctcaat tacccacata ggatgaataa 5040
tagcagttct accgtacaac cctaacataa ccattcttaa tttaactatt tatattatcc 5100
taactactac cgcattccta ctactcaact taaactccag caccacgacc ctactactat 5160
ctcgcacctg aaacaagcta acatgactaa cacccttaat tccatccacc ctcctctccc 5220
taggaggcct gcccccgcta accggctttt tgcccaaatg ggccattatc gaagaattca 5280
caaaaaacaa tagcctcatc atccccacca tcatagccac catcaccctc cttaacctct 5340
acttctacct acgcctaatc tactccacct caatcacact actccccata tctaacaacg 5400
taaaaataaa atgacagttt gaacatacaa aacccacccc attcctcccc acactcatcg 5460
cccttaccac gctactccta cctatctccc cttttatact aataatctta tagaaattta 5520
ggttaaatac agaccaagag ccttcaaagc cctcagtaag ttgcaatact taatttctgt 5580
aacagctaag gactgcaaaa ccccactctg catcaactga acgcaaatca gccactttaa 5640
ttaagctaag cccttactag accaatggga cttaaaccca caaacactta gttaacagct 5700
aagcacccta atcaactggc ttcaatctac ttctcccgcc gccgggaaaa aaggcgggag 5760
aagccccggc aggtttgaag ctgcttcttc gaatttgcaa ttcaatatga aaatcacctc 5820
ggagctggta aaaagaggcc taacccctgt ctttagattt acagtccaat gcttcactca 5880
gccattttac ctcaccccca ctgatgttcg ccgaccgttg actattctct acaaaccaca 5940
aagacattgg aacactatac ctattattcg gcgcatgagc tggagtccta ggcacagctc 6000
taagcctcct tattcgagcc gagctgggcc agccaggcaa ccttctaggt aacgaccaca 6060
tctacaacgt tatcgtcaca gcccatgcat ttgtaataat cttcttcata gtaataccca 6120
tcataatcgg aggctttggc aactgactag ttcccctaat aatcggtgcc cccgatatgg 6180
cgtttccccg cataaacaac ataagcttct gactcttacc tccctctctc ctactcctgc 6240
tcgcatctgc tatagtggag gccggagcag gaacaggttg aacagtctac cctcccttag 6300
cagggaacta ctcccaccct ggagcctccg tagacctaac catcttctcc ttacacctag 6360
caggtgtctc ctctatctta ggggccatca atttcatcac aacaattatc aatataaaac 6420
cccctgccat aacccaatac caaacgcccc tcttcgtctg atccgtccta atcacagcag 6480
tcctacttct cctatctctc ccagtcctag ctgctggcat cactatacta ctaacagacc 6540
gcaacctcaa caccaccttc ttcgaccccg ccggaggagg agaccccatt ctataccaac 6600
acctattctg atttttcggt caccctgaag tttatattct tatcctacca ggcttcggaa 6660
taatctccca tattgtaact tactactccg gaaaaaaaga accatttgga tacataggta 6720
tggtctgagc tatgatatca attggcttcc tagggtttat cgtgtgagca caccatatat 6780
ttacagtagg aatagacgta gacacacgag catatttcac ctccgctacc ataatcatcg 6840
ctatccccac cggcgtcaaa gtatttagct gactcgccac actccacgga agcaatatga 6900
aatgatctgc tgcagtgctc tgagccctag gattcatctt tcttttcacc gtaggtggcc 6960
tgactggcat tgtattagca aactcatcac tagacatcgt actacacgac acgtactacg 7020
ttgtagccca cttccactat gtcctatcaa taggagctgt atttgccatc ataggaggct 7080
tcattcactg atttccccta ttctcaggct acaccctaga ccaaacctac gccaaaatcc 7140
atttcactat catattcatc ggcgtaaatc taactttctt cccacaacac tttctcggcc 7200
tatccggaat gccccgacgt tactcggact accccgatgc atacaccaca tgaaacatcc 7260
tatcatctgt aggctcattc atttctctaa cagcagtaat attaataatt ttcatgattt 7320
gagaagcctt cgcttcgaag cgaaaagtcc taatagtaga agaaccctcc ataaacctgg 7380
agtgactata tggatgcccc ccaccctacc acacattcga agaacccgta tacataaaat 7440
ctagacaaaa aaggaaggaa tcgaaccccc caaagctggt ttcaagccaa ccccatggcc 7500
tccatgactt tttcaaaaag gtattagaaa aaccatttca taactttgtc aaagttaaat 7560
tataggctaa atcctatata tcttaatggc acatgcagcg caagtaggtc tacaagacgc 7620
tacttcccct atcatagaag agcttatcac ctttcatgat cacgccctca taatcatttt 7680
ccttatctgc ttcctagtcc tgtatgccct tttcctaaca ctcacaacaa aactaactaa 7740
tactaacatc tcagacgctc aggaaataga aaccgtctga actatcctgc ccgccatcat 7800
cctagtcctc atcgccctcc catccctacg catcctttac ataacagacg aggtcaacga 7860
tccctccctt accatcaaat caattggcca ccaatggtac tgaacctacg agtacaccga 7920
ctacggcgga ctaatcttca actcctacat acttccccca ttattcctag aaccaggcga 7980
cctgcgactc cttgacgttg acaatcgagt agtactcccg attgaagccc ccattcgtat 8040
aataattaca tcacaagacg tcttgcactc atgagctgtc cccacattag gcttaaaaac 8100
agatgcaatt cccggacgtc taaaccaaac cactttcacc gctacacgac cgggggtata 8160
ctacggtcaa tgctctgaaa tctgtggagc aaaccacagt ttcatgccca tcgtcctaga 8220
attaattccc ctaaaaatct ttgaaatagg gcccgtattt accctatagc accccctcta 8280
ccccctctag agcccactgt aaagctaact tagcattaac cttttaagtt aaagattaag 8340
agaaccaaca cctctttaca gtgaaatgcc ccaactaaat actaccgtat ggcccaccat 8400
aattaccccc atactcctta cactattcct catcacccaa ctaaaaatat taaacacaaa 8460
ctaccaccta cctccctcac caaagcccat aaaaataaaa aattataaca aaccctgaga 8520
accaaaatga acgaaaatct gttcgcttca ttcattgccc ccacaatcct aggcctaccc 8580
gccgcagtac tgatcattct atttccccct ctattgatcc ccacctccaa atatctcatc 8640
aacaaccgac taatcaccac ccaacaatga ctaatcaaac taacctcaaa acaaatgata 8700
accatacaca acactaaagg acgaacctga tctcttatac tagtatcctt aatcattttt 8760
attgccacaa ctaacctcct cggactcctg cctcactcat ttacaccaac cacccaacta 8820
tctataaacc tagccatggc catcccctta tgagcgggca cagtgattat aggctttcgc 8880
tctaagatta aaaatgccct agcccacttc ttaccacaag gcacacctac accccttatc 8940
cccatactag ttattatcga aaccatcagc ctactcattc aaccaatagc cctggccgta 9000
cgcctaaccg ctaacattac tgcaggccac ctactcatgc acctaattgg aagcgccacc 9060
ctagcaatat caaccattaa ccttccctct acacttatca tcttcacaat tctaattcta 9120
ctgactatcc tagaaatcgc tgtcgcctta atccaagcct acgttttcac acttctagta 9180
agcctctacc tgcacgacaa cacataatga cccaccaatc acatgcctat catatagtaa 9240
aacccagccc atgaccccta acaggggccc tctcagccct cctaatgacc tccggcctag 9300
ccatgtgatt tcacttccac tccataacgc tcctcatact aggcctacta accaacacac 9360
taaccatata ccaatgatgg cgcgatgtaa cacgagaaag cacataccaa ggccaccaca 9420
caccacctgt ccaaaaaggc cttcgatacg ggataatcct atttattacc tcagaagttt 9480
ttttcttcgc aggatttttc tgagcctttt accactccag cctagcccct accccccaat 9540
taggagggca ctggccccca acaggcatca ccccgctaaa tcccctagaa gtcccactcc 9600
taaacacatc cgtattactc gcatcaggag tatcaatcac ctgagctcac catagtctaa 9660
tagaaaacaa ccgaaaccaa ataattcaag cactgcttat tacaatttta ctgggtctct 9720
attttaccct cctacaagcc tcagagtact tcgagtctcc cttcaccatt tccgacggca 9780
tctacggctc aacatttttt gtagccacag gcttccacgg acttcacgtc attattggct 9840
caactttcct cactatctgc ttcatccgcc aactaatatt tcactttaca tccaaacatc 9900
actttggctt cgaagccgcc gcctgatact ggcattttgt agatgtggtt tgactatttc 9960
tgtatgtctc catctattga tgagggtctt actcttttag tataaatagt accgttaact 10020
tccaattaac tagttttgac aacattcaaa aaagagtaat aaacttcgcc ttaattttaa 10080
taatcaacac cctcctagcc ttactactaa taattattac attttgacta ccacaactca 10140
acggctacat agaaaaatcc accccttacg agtgcggctt cgaccctata tcccccgccc 10200
gcgtcccttt ctccataaaa ttcttcttag tagctattac cttcttatta tttgatctag 10260
aaattgccct ccttttaccc ctaccatgag ccctacaaac aactaacctg ccactaatag 10320
ttatgtcatc cctcttatta atcatcatcc tagccctaag tctggcctat gagtgactac 10380
aaaaaggatt agactgaacc gaattggtat atagtttaaa caaaacgaat gatttcgact 10440
cattaaatta tgataatcat atttaccaaa tgcccctcat ttacataaat attatactag 10500
catttaccat ctcacttcta ggaatactag tatatcgctc acacctcata tcctccctac 10560
tatgcctaga aggaataata ctatcgctgt tcattatagc tactctcata accctcaaca 10620
cccactccct cttagccaat attgtgccta ttgccatact agtctttgcc gcctgcgaag 10680
cagcggtggg cctagcccta ctagtctcaa tctccaacac atatggccta gactacgtac 10740
ataacctaaa cctactccaa tgctaaaact aatcgtccca acaattatat tactaccact 10800
gacatgactt tccaaaaaac acataatttg aatcaacaca accacccaca gcctaattat 10860
tagcatcatc cctctactat tttttaacca aatcaacaac aacctattta gctgttcccc 10920
aaccttttcc tccgaccccc taacaacccc cctcctaata ctaactacct gactcctacc 10980
cctcacaatc atggcaagcc aacgccactt atccagtgaa ccactatcac gaaaaaaact 11040
ctacctctct atactaatct ccctacaaat ctccttaatt ataacattca cagccacaga 11100
actaatcata ttttatatct tcttcgaaac cacacttatc cccaccttgg ctatcatcac 11160
ccgatgaggc aaccagccag aacgcctgaa cgcaggcaca tacttcctat tctacaccct 11220
agtaggctcc cttcccctac tcatcgcact aatttacact cacaacaccc taggctcact 11280
aaacattcta ctactcactc tcactgccca agaactatca aactcctgag ccaacaactt 11340
aatatgacta gcttacacaa tagcttttat agtaaagata cctctttacg gactccactt 11400
atgactccct aaagcccatg tcgaagcccc catcgctggg tcaatagtac ttgccgcagt 11460
actcttaaaa ctaggcggct atggtataat acgcctcaca ctcattctca accccctgac 11520
aaaacacata gcctacccct tccttgtact atccctatga ggcataatta taacaagctc 11580
catctgccta cgacaaacag acctaaaatc gctcattgca tactcttcaa tcagccacat 11640
agccctcgta gtaacagcca ttctcatcca aaccccctga agcttcaccg gcgcagtcat 11700
tctcataatc gcccacgggc ttacatcctc attactattc tgcctagcaa actcaaacta 11760
cgaacgcact cacagtcgca tcataatcct ctctcaagga cttcaaactc tactcccact 11820
aatagctttt tgatgacttc tagcaagcct cgctaacctc gccttacccc ccactattaa 11880
cctactggga gaactctctg tgctagtaac cacgttctcc tgatcaaata tcactctcct 11940
acttacagga ctcaacatac tagtcacagc cctatactcc ctctacatat ttaccacaac 12000
acaatggggc tcactcaccc accacattaa caacataaaa ccctcattca cacgagaaaa 12060
caccctcatg ttcatacacc tatcccccat tctcctccta tccctcaacc ccgacatcat 12120
taccgggttt tcctcttgta aatatagttt aaccaaaaca tcagattgtg aatctgacaa 12180
cagaggctta cgacccctta tttaccgaga aagctcacaa gaactgctaa ctcatgcccc 12240
catgtctaac aacatggctt tctcaacttt taaaggataa cagctatcca ttggtcttag 12300
gccccaaaaa ttttggtgca actccaaata aaagtaataa ccatgcacac tactataacc 12360
accctaaccc tgacttccct aattcccccc atccttacca ccctcgttaa ccctaacaaa 12420
aaaaactcat acccccatta tgtaaaatcc attgtcgcat ccacctttat tatcagtctc 12480
ttccccacaa caatattcat gtgcctagac caagaagtta ttatctcgaa ctgacactga 12540
gccacaaccc aaacaaccca gctctcccta agcttcaaac tagactactt ctccataata 12600
ttcatccctg tagcattgtt cgttacatgg tccatcatag aattctcact gtgatatata 12660
aactcagacc caaacattaa tcagttcttc aaatatctac tcatcttcct aattaccata 12720
ctaatcttag ttaccgctaa caacctattc caactgttca tcggctgaga gggcgtagga 12780
attatatcct tcttgctcat cagttgatga tacgcccgag cagatgccaa cacagcagcc 12840
attcaagcaa tcctatacaa ccgtatcggc gatatcggtt tcatcctcgc cttagcatga 12900
tttatcctac actccaactc atgagaccca caacaaatag cccttctaaa cgctaatcca 12960
agcctcaccc cactactagg cctcctccta gcagcagcag gcaaatcagc ccaattaggt 13020
ctccacccct gactcccctc agccatagaa ggccccaccc cagtctcagc cctactccac 13080
tcaagcacta tagttgtagc aggaatcttc ttactcatcc gcttccaccc cctagcagaa 13140
aatagcccac taatccaaac tctaacacta tgcttaggcg ctatcaccac tctgttcgca 13200
gcagtctgcg cccttacaca aaatgacatc aaaaaaatcg tagccttctc cacttcaagt 13260
caactaggac tcataatagt tacaatcggc atcaaccaac cacacctagc attcctgcac 13320
atctgtaccc acgccttctt caaagccata ctatttatgt gctccgggtc catcatccac 13380
aaccttaaca atgaacaaga tattcgaaaa ataggaggac tactcaaaac catacctctc 13440
acttcaacct ccctcaccat tggcagccta gcattagcag gaataccttt cctcacaggt 13500
ttctactcca aagaccacat catcgaaacc gcaaacatat catacacaaa cgcctgagcc 13560
ctatctatta ctctcatcgc tacctccctg acaagcgcct atagcactcg aataattctt 13620
ctcaccctaa caggtcaacc tcgcttcccc acccttacta acattaacga aaataacccc 13680
accctactaa accccattaa acgcctggca gccggaagcc tattcgcagg atttctcatt 13740
actaacaaca tttcccccgc atcccccttc caaacaacaa tccccctcta cctaaaactc 13800
acagccctcg ctgtcacttt cctaggactt ctaacagccc tagacctcaa ctacctaacc 13860
aacaaactta aaataaaatc cccactatgc acattttatt tctccaacat actcggattc 13920
taccctagca tcacacaccg cacaatcccc tatctaggcc ttcttacgag ccaaaacctg 13980
cccctactcc tcctagacct aacctgacta gaaaagctat tacctaaaac aatttcacag 14040
caccaaatct ccacctccat catcacctca acccaaaaag gcataattaa actttacttc 14100
ctctctttct tcttcccact catcctaacc ctactcctaa tcacataacc tattcccccg 14160
agcaatctca attacaatat atacaccaac aaacaatgtt caaccagtaa ctactactaa 14220
tcaacgccca taatcataca aagcccccgc accaatagga tcctcccgaa tcaaccctga 14280
cccctctcct tcataaatta ttcagcttcc tacactatta aagtttacca caaccaccac 14340
cccatcatac tctttcaccc acagcaccaa tcctacctcc atcgctaacc ccactaaaac 14400
actcaccaag acctcaaccc ctgaccccca tgcctcagga tactcctcaa tagccatcgc 14460
tgtagtatat ccaaagacaa ccatcattcc ccctaaataa attaaaaaaa ctattaaacc 14520
catataacct cccccaaaat tcagaataat aacacacccg accacaccgc taacaatcaa 14580
tactaaaccc ccataaatag gagaaggctt agaagaaaac cccacaaacc ccattactaa 14640
acccacactc aacagaaaca aagcatacat cattattctc gcacggacta caaccacgac 14700
caatgatatg aaaaaccatc gttgtatttc aactacaaga acaccaatga ccccaatacg 14760
caaaactaac cccctaataa aattaattaa ccactcattc atcgacctcc ccaccccatc 14820
caacatctcc gcatgatgaa acttcggctc actccttggc gcctgcctga tcctccaaat 14880
caccacagga ctattcctag ccatgcacta ctcaccagac gcctcaaccg ccttttcatc 14940
aatcgcccac atcactcgag acgtaaatta tggctgaatc atccgctacc ttcacgccaa 15000
tggcgcctca atattcttta tctgcctctt cctacacatc gggcgaggcc tatattacgg 15060
atcatttctc tactcagaaa cctgaaacat cggcattatc ctcctgcttg caactatagc 15120
aacagccttc ataggctatg tcctcccgtg aggccaaata tcattctgag gggccacagt 15180
aattacaaac ttactatccg ccatcccata cattgggaca gacctagttc aatgaatctg 15240
aggaggctac tcagtagaca gtcccaccct cacacgattc tttacctttc acttcatctt 15300
gcccttcatt attgcagccc tagcaacact ccacctccta ttcttgcacg aaacgggatc 15360
aaacaacccc ctaggaatca cctcccattc cgataaaatc accttccacc cttactacac 15420
aatcaaagac gccctcggct tacttctctt ccttctctcc ttaatgacat taacactatt 15480
ctcaccagac ctcctaggcg acccagacaa ttatacccta gccaacccct taaacacccc 15540
tccccacatc aagcccgaat gatatttcct attcgcctac acaattctcc gatccgtccc 15600
taacaaacta ggaggcgtcc ttgccctatt actatccatc ctcatcctag caataatccc 15660
catcctccat atatccaaac aacaaagcat aatatttcgc ccactaagcc aatcacttta 15720
ttgactccta gccgcagacc tcctcattct aacctgaatc ggaggacaac cagtaagcta 15780
cccttttacc atcattggac aagtagcatc cgtactatac ttcacaacaa tcctaatcct 15840
aataccaact atctccctaa ttgaaaacaa aatactcaaa tgggcctgtc cttgtagtat 15900
aaactaatac accagtcttg taaaccggag atgaaaacct ttttccaagg acaaatcaga 15960
gaaaaagtct ttaactccac cattagcacc caaagctaag attctaattt aaactattct 16020
ctgttctttc atggggaagc agatttgggt accacccaag tattgactca cccatcaaca 16080
accgctatgt atttcgtaca ttactgccag ccaccatgaa tattgtacgg taccataaat 16140
acttgaccac ctgtagtaca taaaaaccca atccacatca aaaccccctc cccatgctta 16200
caagcaagta cagcaatcaa ccctcaacta tcacacatca actgcaactc caaagccacc 16260
cctcacccac taggatacca acaaacctac ccacccttaa cagtacatag tacataaagc 16320
catttaccgt acatagcaca ttacagtcaa atcccttctc gtccccatgg atgacccccc 16380
tcagataggg gtcccttgac caccatcctc cgtgaaatca atatcccgca caagagtgct 16440
actctcctcg ctccgggccc ataacacttg ggggtagcta aagtgaactg tatccgacat 16500
ctggttccta cttcagggtc ataaagccta aatagcccac acgttcccct taaataagac 16560
atcacgatg 16569
Claims (8)
1. the preparation method of target DNA enrichment probe, comprising: prepare N number of sub- probe, connect N number of sub- probe, obtain target DNA
It is enriched with probe；The length of N number of sub- probe is 50-150bp；
Connection N number of sub- probe includes following A 1) and A2):
A1 N number of sub- probe) is connected, long chain DNA and/or cyclic DNA are obtained；
A2) the long chain DNA and/or the cyclic DNA are expanded, obtain target DNA enrichment probe；
N is a natural number more than or equal to 2；
N number of sub- probe, which meets N number of sub- probe, can cover the full sequence of the target DNA.
2. according to the method described in claim 1, it is characterized by: any two adjacent sub- probes are equal on the target DNA
The downstream for meeting upstream Asia probe has one or more nucleotide Chong Die with the upstream of downstream Asia probe.
3. according to the method described in claim 1, it is characterized by: the length of N number of sub- probe is 108bp.
4. method according to claim 1 to 3, it is characterised in that: the method is connecting N number of sub- probe
Before further include that phosphorylation modification is carried out to N number of sub- probe.
5. method according to claim 1 or 2, it is characterised in that: step A2) in, it is carrying out the long chain DNA or institute
State when cyclic DNA is expanded further includes that amplified production is marked using biotin.
6. the method for acquisition target DNA is caught including the use of the target DNA enrichment probe of the method any in claim 1-5 preparation
Obtain target DNA.
7. the method for target DNA sequencing, including the use of method of claim 6 acquisition target DNA, then to the target DNA of capture
It is sequenced.
8. any application in following X1-X5:
Application of any the method in acquisition target DNA in X1, claim 1-5；
Any the method is preparing the application in acquisition target DNA product in X2, claim 1-5；
Application of any the method in target DNA sequencing in X3, claim 1-5；
Any the method is preparing the application in target DNA sequencing products in X4, claim 1-5；
The application of X5, claim 6 the method in target DNA sequencing.
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| CN102102122A (en) * | 2009-12-22 | 2011-06-22 | 中山大学达安基因股份有限公司 | Design method of oligonucleotide probe and application thereof |
| US20120058474A1 (en) * | 2006-08-15 | 2012-03-08 | Genetag Technology, Inc. | Probe-antiprobe compositions and methods for dna or rna detection |
| CN102586229A (en) * | 2012-02-28 | 2012-07-18 | 盛司潼 | Method for preparing capture probe and application of capture probe |
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| US20120058474A1 (en) * | 2006-08-15 | 2012-03-08 | Genetag Technology, Inc. | Probe-antiprobe compositions and methods for dna or rna detection |
| CN102102122A (en) * | 2009-12-22 | 2011-06-22 | 中山大学达安基因股份有限公司 | Design method of oligonucleotide probe and application thereof |
| CN102586229A (en) * | 2012-02-28 | 2012-07-18 | 盛司潼 | Method for preparing capture probe and application of capture probe |
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