CN106520978A - Preparation method for target-DNA-enriching probe - Google Patents
Preparation method for target-DNA-enriching probe Download PDFInfo
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Abstract
The invention discloses a preparation method for a target-DNA-enriching probe. The preparation method for the target-DNA-enriching probe comprises the following steps: preparing N sub-probes and connecting the N sub-probes so as to obtain a long-strand DNA and/or circular DNA; and subjecting the long-strand DNA and/or circular DNA to amplification so as to obtain the target-DNA-enriching probe; wherein the N sub-probes satisfy the condition that the N sub-probes can cover all the sequences of a target DNA; any two adjacent sub-probes on the target DNA satisfy the condition that the downstream of an upstream sub-probe has one or more nucleotides overlapped with the upstream of a downstream sub-probe; and each of the N sub-probes has a length of 50 to 150 bp. Experimental results show that the target-DNA-enriching probe prepared by using the method realizes 100% coverage in capturing of the target DNA, has a capturing rate of 100%, is good in integral homogeneity and in stability and can be used for capturing of the target DNA.
Description
Technical field
The present invention relates in biological technical field, target DNA is enriched with the preparation method of probe.
Background technology
With completing that more and more biological genomes are sequenced, biomedical research has been enter into the genome times afterwards comprehensively.With
The cost of secondary sequencing is constantly reduced, and early in 2014, illumina companies of the U.S. released brand-new high-flux sequence instrument
Device, can sequencing cost be reduced to 1000 dollars, the popularization of gene test and accelerate therewith, existed using high throughput sequencing technologies
In scientific research and Clinical detection.
At present, although for genome sequencing cost has arrived at 1000 dollars, for need superelevation depth survey
In clinical or Technological research field, the particularly lesion detection of sequence, sequencing cost and high flux number that high depth sequencing faces
According to the cost that brings of process, it is impossible to be widely used in Clinical detection and correlational study, so just generating gene trap skill
Art, so-called gene trap, concern required for being aiming at or the purpose region studied design corresponding capture probe, using hybridization
Technology, purpose fragment from full-length genome is enriched with out, and these fragments are carried out high-flux sequence again, can meet research need
Depth requirements are wanted, while can also realize not increasing cost, current gene trapping is in clone, discovery new gene and announcement
There is in terms of gene function uniqueness, become the powerful of functional genome's age study, in biomedical research
The application of every field is increasingly extensive.The probe used in gene trap at present is cDNA single-stranded probes and rna probe.
The content of the invention
The technical problem to be solved is how to prepare the probe that can capture target DNA.
To solve above-mentioned technical problem, present invention firstly provides target DNA is enriched with the preparation method of probe.
Target DNA provided by the present invention is enriched with the preparation method of probe, including：N number of sub- probe is prepared, is connected described N number of
Sub- probe, obtains target DNA enrichment probe；
N number of sub- probe meets the full sequence that N number of sub- probe can cover 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
Ratio of the length (bp) of the target DNA with the length (bp) of the sub- probe is designated as n, when n is by the length determination of sub- probe
During non-integer, the integer part+1 of N=n；When n is integer, N=n.If there is the complete phase of sequence in N number of sub- probe
With probe, the sub- probe of repetition can also be removed in actual applications, as long as making the quantity of the sub- probe meet and cover
Cover the full sequence of the target DNA.In one embodiment of the invention, by calculated N values be 208, but
In this 208 sub- probes, the 207th is identical with the 208th sub- probe, in specifically being tested, can only preparation the
1-207 bars 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 can cover the implication 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 be single-stranded with the two of the target DNA
DNA or one single stranded DNA combines to form double-stranded DNA；When N number of sub- probe single stranded DNA knot only with the target DNA
During conjunction, there is no the site that can not be combined with the sub- probe in N number of sub- probe in the single stranded DNA of the target DNA；When described
When N number of sub- probe is combined with two single stranded DNAs of the target DNA, there is a chain energy in any one site of the target DNA
Combined 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
Two single stranded DNAs or a single stranded DNA complementation of the probe with the target DNA；When N number of sub- probe and the target DNA
Chain DNA is complementary and another chain DNA with the target DNA not mutual added time, and described in N number of sub- probes complementary
Do not exist in that single stranded DNA of target DNA not with N number of sub- probe in sub- probes complementary site；When N number of sub- spy
Pin can with the DNA fragmentation mutual added time in the two of the target DNA chains, any one site of the target DNA have a 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 be single-stranded with the two of the target DNA
DNA combines to form double-stranded DNA, two single stranded DNAs of the target DNA do not exist can not with N number of sub- probe in it is arbitrary
The site that bar single stranded DNA is combined；That is, when N number of sub- probe is double-stranded DNA, in N number of sub- probe each
Two of sub- probe it is single-stranded respectively with two single stranded DNAs of the target DNA in same site complementary, two lists of the target DNA
There is no site not with N number of sub- probes complementary in chain DNA.
May also include before N number of sub- probe is connected and the described N number of sub- probe on the chip is eluted.Eluting
Described N number of sub- probe on the chip can be carried out using ammonia.The molar concentration of the ammonia can be 35%.From described
The described N number of sub- probe eluted on chip can utilize ultrapure water dissolution.
It is described prepare N number of sub- probe can be in N number of sub- probe described in fabricated in situ on chip.
In the preparation method of above-mentioned target DNA enrichment probe, on the target DNA, the adjacent sub- probe of any two can be expired
The downstream of sufficient upstream Asia probe has one or more nucleotide and the upstream of downstream Asia probe to overlap (i.e. sequence is identical).
In the preparation method of above-mentioned target DNA enrichment probe, the quantity of one or more of nucleotide can be according to concrete feelings
Condition determines that 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 said 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 individual sub- probe can be specifically 108bp.
In the preparation method of above-mentioned target DNA enrichment probe, the connection N number of sub- probe may include following A 1) and
A2)：
A1) connect N number of sub- probe, obtain long chain DNA and/or cyclic DNA；
A2) the long chain DNA and/or the cyclic DNA are expanded, target DNA enrichment probe is obtained.
The target DNA enrichment probe is double-stranded DNA.
In the preparation method of above-mentioned target DNA enrichment probe, may also include to described N number of before N number of sub- probe is connected
Sub- probe carries out phosphorylation modification.
Phosphorylation modification is carried out to N number of sub- probe concretely carries out phosphorylation at 5 ' ends of N number of sub- probe
Modification.The phosphorylation modification can utilize T4 polynueleotide kinases or the T4 polynueleotide kinases test kit of NEB to carry out.Profit
The reaction system that the phosphorylation modification is carried out with the T4 polynueleotide kinases test kit of NEB can be：N number of sub- probe 10
μ l, 10 × reaction buffer, 5 μ l, 5 μ l, T4 polynueleotide kinases of 10mM ATP 2.5 μ l and H20 27.5μl.The reaction
The concentration of N number of sub- probe described in system can be 5ng/ μ l.10 × reaction buffer, ATP and T4 polies in the reaction system
Nucleoside monophosphate kinase is the T4 polynueleotide kinase seminal plasma fructose detection kits of NEB.The reaction condition of the phosphorylation modification can be
37℃30min。
It is described to connect the ssDNA connection test kits that N number of sub- probe utilize ssDNA ligases or epicentre
(CircLigaseTMII ssDNA Ligase, epicentre) carry out.The reaction system of the connection can be：N number of Asia
The Phosphorylated products of probe or N number of 20 μ l of sub- 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, entering to be about to the long chain DNA or the ring-type
May also include when DNA is expanded and amplified production is marked using biotin.
Carrying out the amplification can be carried out using random primer.The random primer can be by this four nucleotide of A, C, G and T
The length of random composition is the 4 of 6bp6The mixture formed by bar single stranded DNA.All single stranded DNAs in the random primer
Molal quantity can be identical.
The long chain DNA and/or the cyclic DNA are expanded and amplified production be marked using biotin
Carry out using the whole genome amplification test kit of Qiagen companies.Entered using the whole genome amplification test kit of Qiagen companies
Row reaction reaction system can be：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, Replig_Enzyme 1 μ l and H2O 9μl.Wherein, contain the random primer in 2 × reaction buffer.
The reaction temperature reacted using the whole genome amplification test kit of Qiagen companies can be 37 DEG C.Using Qiagen companies
The time reacted by whole genome amplification test kit can be not less than 16 hours.
To solve above-mentioned technical problem, present invention also offers the method for capture target DNA.
The method of capture target DNA provided by the present invention, including target prepared by the preparation method of target DNA enrichment probe
DNA enrichment probe capture target DNAs.
To solve above-mentioned technical problem, present invention also offers the method for target DNA sequencing.
The method of target DNA sequencing provided by the present invention, captures target DNA including using the method for the capture target DNA, so
Target DNA to capturing is sequenced afterwards.
In the method for above-mentioned target DNA sequencing, the target DNA of described pair of capture carries out sequencing and can utilize sequencing of the prior art
Method is carried out, as long as the purpose to target DNA sequencing can be reached.
To solve above-mentioned technical problem, the present invention also provides arbitrary application that can be in following X1-X5：
X1, the target DNA are enriched with application of the preparation method of probe in capture target DNA；
X2, the preparation method of target DNA enrichment probe are preparing the application captured in target DNA product；
X3, the target DNA are enriched with application of the preparation method of probe in target DNA sequencing；
X4, the target DNA are enriched with application of the preparation method of probe in target DNA sequencing products are prepared；
X5, the capture target DNA method target DNA sequencing in application.
In above-mentioned application, the target DNA sequencing can be carried out using secondary sequence measurement.
It is demonstrated experimentally that the full base of capture human mitochondrial prepared using the preparation method of the target DNA enrichment probe of the present invention
The enrichment capture of the probe to homo mitochondrion gene of cause has reached 100% covering, and its capture rate is all higher than 50%, from least
From the point of view of covering the parameter of 4X, 10X and 20X, 100% is reached, the overall homogeneity of probe is very good, and good stability.
Show, the preparation method of the target DNA enrichment probe of the present invention can be used for preparing target DNA enrichment probe, and further to target DNA
It is sequenced.Probe prepared by the preparation method of the target DNA enrichment probe of the present invention is double chain DNA probe, there is following advantage：
1st, double chain DNA probe is more stable, not degradable；
2nd, double chain DNA probe, in the preparation, after the completion of connection, directly carries out whole genome amplification, the homogeneity of probe
It is good；
3rd, double chain DNA probe, in enrichment, captures double-strand, more accurately, can exclude the base that single-stranded middle PCR brings into
Mispairing.
Description of the drawings
Fig. 1 is connection product testing result.
Specific embodiment
The present invention is further described in detail with reference to specific embodiment, the embodiment for being given is only for explaining
The bright present invention, rather than in order to limit the scope of the present invention.
Experimental technique in following embodiments, if no special instructions, is conventional method.
In following embodiments, material used, reagent etc., if no special instructions, commercially obtain.
Target DNA provided by the present invention is enriched with the preparation method of probe, including：N number of sub- probe is prepared, connects N number of sub- spy
Pin, obtains long chain DNA and cyclic DNA；Long chain DNA and cyclic DNA are expanded, target DNA enrichment probe is obtained.
Below so that human mitochondrial full genome is as target DNA as an example, it is specifically described and how is enriched with using the target DNA of the present invention
The preparation method of probe prepares the enrichment probe that can capture human mitochondrial full genome.
Embodiment 1, the preparation for being enriched with probe that human mitochondrial full genome can be captured
1st, the sequential design of single-stranded sub- probe and preparation
As shown in sequence 1 in the sequence such as sequence table of the human mitochondrial full genome of target DNA.The length of target DNA is
Target DNA is divided into 208 fragments by 16569bp, wherein, the length of 1-207 fragment is 80bp, the length of the 208th fragment
Spend for 9bp, the adjacent fragment of each two is met the last position of fragment upstream and is connected with the first place of segments downstream, i.e. the 1st fragment
Sequence for sequence 1 1-80 positions, the 81-160 positions ... ... of the sequence of the 2nd fragment for sequence 1, the 207th fragment
16481-16560 position of the sequence for sequence 1, the sequence of the 208th fragment are the 16561-16569 positions of sequence 1.
The single-stranded sub- probe of full target DNA can be covered according to this 208 fragment designs of target DNA, by the sequence of this 208 fragments
Row extend to its upstream and downstream respectively, make the sequence length for finally giving be 108bp, specific as follows：By the sequence of the 1st fragment
To downstream extend 28bp, by the sequence of each fragment of 2-206 fragment respectively to two ends extension 14bp, by the 207th
The sequence of fragment extends 19bp to extending 9bp (because downstream less than 14bp) downstream, by the 208th fragment to its upstream
Sequence extends 99bp to its upstream, obtains the sequence that 208 length are 108bp.Wherein, 1- of the 1st article of sequence for sequence 1
108, the 67-174 positions of the 2nd article of sequence for sequence 1,147-254 position of the 3rd article of sequence for sequence 1, nth bar sequence is
(80 (n-1) -13) position to (80n+14) position of sequence 1, n are any one natural number in 4-205, and the 206th article of sequence is
The 16387-16494 positions of sequence 1,16462-16569 position of the 207th article of sequence for sequence 1, the 208th article of sequence are sequence 1
16462-16569 positions, the 207th article of sequence be identical with the 208th article of sequence.
Using the method for fabricated in situ, above-mentioned the 1-207 article sequence is respectively in chip synthetic oligonucleotide according to sequence
The single-stranded sub- probe of probe, i.e., 207.
Using 35% ammonia, be fixed on chip 207 single-stranded sub- probes are eluted, centrifuge tube is collected
In, after instrument concentrated in vacuo concentration, the ultrapure water dissolutioies of 500 μ l of addition become the mixed of the single-stranded sub- probe of a target DNA 207
Pond；Concentration is determined, and is diluted to 25ng/ μ l.
2nd, the preparation of the enrichment probe of human mitochondrial full genome can be captured
1) 5 ' phosphorylation modifications of single-stranded sub- probe：Carried out using the T4 polynueleotide kinases test 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 kinases) | 2.5 |
| H20 | 27.5 |
50 μ l of total reaction volume, in the reaction system, except mixed pond and the H of 207 single-stranded sub- probes2Reagent outside O is
T4 polynueleotide kinase seminal plasma fructose detection kits.After mix homogeneously, 37 DEG C of constant-temperature incubations on thermostatic heater or PCR instrument device
30min.After reaction terminates, product purification recovery, eluting are carried out according to its operating process using trace P CR product QIAquick Gel Extraction Kit
20 μ l of volume, obtain Phosphorylated products；
2) coupled reaction：By step 1) Phosphorylated products that obtain, using the ssDNA connection test kits of business
(CircLigaseTMII ssDNA Ligase, epicentre) connect, coupled reaction system is as follows：
| Component | Volume (μ l) |
| Step 1) Phosphorylated products that obtain | 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, except step 1) Phosphorylated products that obtain and H2Reagent outside O is equal
Connect seminal plasma fructose detection kit for ssDNA.After mix homogeneously, 60 DEG C are reacted 1 hour.Product utilizes minim DNA reclaim reagent
Box carries out product purification recovery according to its operating process, and recovery product obtains connection product (long chain DNA and cyclic DNA).Pass through
Gel electrophoresiss, differentiate connection product fragment, and as a result as shown in figure 1, No. 1 swimming lane is DNA molecular amount standard in Fig. 1, No. 2 is swimming lane
For connection product, as a result show, obtained the connection product being successfully connected.
3) labelling biotin and amplification：Using the commercialization whole genome amplification test kit of Qiagen companies, reaction system
It is as follows：
| Component | Volume (μ l) |
| Step 2) connection product that obtains | 10 |
| 2 × reaction buffer (2 × Reaction Buffer) | 25 |
| Bio-16-dUTP | 5 |
| Replig_Enzyme | 1 |
| H2O | 9 |
50 μ l of cumulative volume, in the reaction system, except step 2) connection product that obtains and H2Reagent 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 individual nucleotide is constituted at random is the 4 of 6bp6The mixture formed by bar single stranded DNA, it is all single-stranded in the mixture
The molal quantity all same of DNA.After mix homogeneously, 37 degree of constant-temperature incubations more than 16 hours.Product adopts DNA reclaim reagents
Box, according to the Standard Operating Procedure of test kit, carries out DNA purification, obtains target DNA enrichment probe.Entered using Nanodrop2000
Row is quantitative, and target DNA is enriched with probe dilution to 150ng/ μ l, forms final target DNA enrichment probe solution.
3rd, target DNA is enriched with the quality evaluation of probe
Experiment repeats comprising the following steps that for test in triplicate, every time：
1) test experiments scheme：The full-length genome library sample of 10 people, each sample is selected to be divided into two groups, two components
Confirmatory experiment is not carried out by different batches.
Probe is enriched with using target DNA and captures the target DNA in above-mentioned 10 samples：
Target DNA enrichment probe is mixed with DNA full-length genomes library, the mitochondrial gene in sample is just hybridised to probe
On, absorption is combined by biotin and Streptavidin MagneSphere, the DNA fragmentation of nontarget area is removed by the process of Jing eluting, from
And human mitochondrial full genome is enriched with, 2X150bp high pass measurements are carried out using new-generation sequencing instrument Illumina Nexseq500
Sequence.Comprise the following steps that：
A1) prepare following mixed system：Take 500ng DNA full-length genomes library (20 μ l, 25ng/ μ l), 10 μ l Buffer
BL, the target DNA enrichment probe solution that 5 μ l steps 2 are obtained, 6 μ l Blocking Oligo Mix；
A2) by step a1) mixed system PCR instrument on：95 DEG C, 7min, 65 DEG C afterwards, 2min；
A3 the 23 μ l of Buffer HY for) taking 65 DEG C of preheatings are added to step a2) react in the system for terminating, then in PCR
On instrument, 65 DEG C hybridize 22 hours.
A4 MyOne beads (Invitrogen)) are taken out, whirlpool concussion makes magnetic bead fully suspend, and of short duration centrifugation makes lid
On beads be centrifuged to bottom of the tube.
A5) take in the centrifuge tube of 50 μ l MyOne beads to new 1.5ml, whirlpool concussion at least 5s makes magnetic bead abundant
Suspend, after of short duration centrifugation, be put into magnetic frame upper one minute.
A6) centrifuge tube remains stationary (should not rotating centrifugal pipe) on magnetic frame, careful suction abandon supernatant.
A7 centrifuge tube) is removed, adds the 1X Binding Buffer (binding buffer) of 50 μ l, rotation nest to shake at least
5s, is put into after of short duration centrifugation static one minute on magnetic frame, and careful suction abandons supernatant.
A8) repeat step a6) and a7) twice (altogether three times).
A9 centrifuge tube) is removed, adds 100 μ l 2XBinding Buffer (binding buffer), rotation nest to shake at least
5s, is proceeded to after of short duration centrifugation in a new centrifuge tube completely.
A10) by step a3) product proceed to (about 200 μ l of cumulative volume, while processing multiple in the centrifuge tube of beads completely
During sample, several lower mixings after mixing, are flicked), rotation nest concussion at least 5s (without centrifugation) is placed in room temperature rotation 1 on Rotator little
When.
A11) after above-mentioned steps, using WB1buffer room temperatures cleaning beads once, 15minutes, Ran Hou
3 times, 65 DEG C, 15minutes/ time are washed in WB3buffer.
A12 the DNA for) binding utilizes Buffer Elute eluting.Finally amplification 15 is circulated the DNA of eluting, using 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 eluting enters performing PCR amplification.
A13) PCR primer obtains being enriched with line using SPRI beads (Beckman Coulter) according to laboratory manual purification
Mitochondrial genes frag-ment libraries.
Wherein, the formula of each reagent is as follows：
It is sequenced using Nextseq500 (illumina) microarray dataset, is verified the repeatability and stability of its probe.
2) analysis of sequencing data：First, using the low-quality sequence of Trim-Galore program filters；Then, use
Cutadapt programs in Trim-Galore remove the universal primer sequence at 3 ' ends and 5 ' ends, retain reading quality and are more than 20bp
It is more than the sequence of 80bp with reading length.The sequence of filtration is with BWA procedure match to human genome, then soft using GATK
Part bag is calibrated to mass value, is then matched again on reference sequences.Repeat reading sequence alignment/matching tool
(SAMtools) 3 remove after, the reading sequence of only unique match is used for subsequent analysis.Finally the data to being sequenced carry out matter
Amount control, data are shown in Table 1.
Table 1, sequencing data
Parameter declaration in table 1：
A. sequencing data amount refers to total sequencing data that the sample for completing target DNA capture is produced when being sequenced；
B. the base number of mesh：Refer to that probe designs the total bases of the purpose region for arriving to be covered；
C. cover purpose base number：Refer to after enrichment sequencing, can successfully compare design base area to be covered
The base number in domain；
D. coverage rate：Cover purpose base number/purpose base number；
E. capture rate (valid data amount)：Cover purpose base number/sequencing data amount
F. mean depth is sequenced：When referring to that purpose region is sequenced, the number of times for averagely being read；
G. 4X ratios are averagely covered：Finger at least reads the base number accounting of more than 4 times；
H. 10X ratios are averagely covered：Finger at least reads the base number accounting of more than 10 times；
I. 20X ratios are averagely covered：Finger at least reads the base number accounting of more than 20 times；
Probe mass is assessed：
Target DNA enrichment capture of the probe to homo mitochondrion gene has reached 100% covering, and its capture rate is all higher than
50%, from the point of view of the parameter at least covering 4X, 10X and 20X, 100% is reached, the overall homogeneity of probe is very good；It is logical
Cross and repeat experiment and the result of data shows, probe with good stability.Show, the target DNA enrichment probe of the present invention
Preparation method can be used for preparing target DNA enrichment probe, and further target DNA is sequenced.
<110>Beijing steps rich perseverance industry science and technology limited Company
<120>Target DNA is enriched with the preparation method of 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 (10)
1. target DNA is enriched with the preparation method of probe, including：N number of sub- probe is prepared, is connected N number of sub- probe, is obtained target DNA
Enrichment probe；N is a natural number more than or equal to 2；
N number of sub- probe meets the full sequence that N number of sub- probe can cover the target DNA.
2. method according to claim 1, it is characterised in that：On the target DNA, the adjacent sub- probe of any two is equal
The downstream for meeting upstream Asia probe has one or more nucleotide Chong Die with the upstream of downstream Asia probe.
3. method according to claim 1 and 2, it is characterised in that：The length of N number of sub- probe is 50-150bp.
4. according to arbitrary described method in claim 1-3, it is characterised in that：The length of N number of sub- probe is
108bp。
5. according to arbitrary described method in claim 1-4, it is characterised in that：Under the connection N number of sub- probe includes
State A1) and A2)：
A1) connect N number of sub- probe, obtain long chain DNA and/or cyclic DNA；
A2) the long chain DNA and/or the cyclic DNA are expanded, target DNA enrichment probe is obtained.
6. according to arbitrary described method in claim 1-5, it is characterised in that：Methods described is connecting N number of sub- probe
It is front also to include carrying out phosphorylation modification to N number of sub- probe.
7. the method according to claim 5 or 6, it is characterised in that：Step A2) in, entering to be about to the long chain DNA or institute
State and amplified production is marked using biotin.
8. the method for capturing target DNA, is caught including the target DNA enrichment probe prepared using arbitrary methods described in claim 1-7
Obtain target DNA.
9. the method for target DNA sequencing, captures target DNA including using the method described in claim 8, then the target DNA to capturing
It is sequenced.
10. the arbitrary application in following X1-X5：
Application of arbitrary methods described in capture target DNA in X1, claim 1-7；
Application of arbitrary methods described in capture target DNA product is prepared in X2, claim 1-7；
Application of arbitrary methods described in target DNA sequencing in X3, claim 1-7；
Application of arbitrary methods described in target DNA sequencing products are prepared in X4, claim 1-7；
The application of X5, claim 8 methods described in target DNA sequencing.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106929588A (en) * | 2017-04-12 | 2017-07-07 | 北京迈博恒业科技有限责任公司 | SLC26A4 gene traps probe and its application in SLC26A4 detection in Gene Mutation |
| CN108546739A (en) * | 2018-04-20 | 2018-09-18 | 曹顺 | A method of the nucleic acid target sequence enrichment for NGS sequencings |
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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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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106929588A (en) * | 2017-04-12 | 2017-07-07 | 北京迈博恒业科技有限责任公司 | SLC26A4 gene traps probe and its application in SLC26A4 detection in Gene Mutation |
| CN108546739A (en) * | 2018-04-20 | 2018-09-18 | 曹顺 | A method of the nucleic acid target sequence enrichment for NGS sequencings |
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