CN107937522B - One group of circRNA marker and its application for children acute lymphoblastic leukaemia diagnosis - Google Patents

One group of circRNA marker and its application for children acute lymphoblastic leukaemia diagnosis Download PDF

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CN107937522B
CN107937522B CN201711246097.4A CN201711246097A CN107937522B CN 107937522 B CN107937522 B CN 107937522B CN 201711246097 A CN201711246097 A CN 201711246097A CN 107937522 B CN107937522 B CN 107937522B
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children
circ
primer
diagnosis
circrna
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CN107937522A (en
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方拥军
薛瑶
康美云
王娅萍
戎留成
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Nanjing Childrens Hospital of Nanjing Medical University
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Nanjing Childrens Hospital of Nanjing Medical University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Abstract

One group of circRNA marker and its application, the marker for children acute lymphoblastic leukaemia diagnosis includes two kinds of circRNAs of has_circ_0005720 shown in has_circ_0117909 and SEQ ID No:2 shown in SEQ ID No:1.Children ALL diagnosis circRNA biomarker provided by the present invention, can be used for the diagnosis of children ALL, the AUC for distinguishing children ALL marrow and normal control marrow can reach 0.962, and sensitivity reaches 90.9%, and specificity reaches 95%.

Description

One group for children acute lymphoblastic leukaemia diagnosis circRNA marker and It is applied
Technical field
The invention belongs to field of biotechnology, are related to one group for children acute lymphoblastic leukaemia diagnosis CircRNA marker and its application.
Background technique
Leukaemia is common hemopoietic system malignant proliferative disease, is candidate stem cell Mr. Yu's single order in atomization There is a kind of malignant proliferation disease blocked in section.Children acute lymphoblastic leukaemia (acute lymphoblastic Leukemia, ALL) it is the Childhood most common malignant tumour, account for about the 25-30% of 15 years old or less children malignant tumors.? In world wide, annual morbidity is about 3-4/10 ten thousand populations.The annual new cases in China are more than 10000, and are had year by year The trend increased.Although as constantly improve for children's ALL Clinics and Practices scheme, a variety of treated with combined medication, supportive treatment And the application of hematopoietic stem cell transplantation, the prognosis of children ALL has more in the past to be significantly improved, and survival rate reaches 80-90% within 5 years, As a kind of malignant tumour of resectability, but still there is about 15% infant to die of chemotherapy side effect and palindromia.Therefore, deep Enter to study the pathogenesis of children ALL, new diagnosis basis can be provided for children ALL, or find new biology Mark and new targeted therapy site provide solid theoretical basis.
With going deep into for oncomolecularbiology research, sight is gradually turned to circular rna (circular by scientist RNA, circRNA), become the new heat of tumor research because of the latent effect that it is manifested in carcinogenic and tumor suppression approach Point.CircRNA is the RNA family newcomer for being different from conventional linear RNA, does not have 5 ' end caps and 3 ' end poly (A) Tail, and with the non-coding RNA molecule of covalent bond formation ring structure.It recent studies have shown that, circRNA has closed hoop knot Structure is mainly generated by the processing of atypia variable sheer, is widely present in various biological cells, has stable structure, it is difficult to Good by conservative between RNA enzyme degradation, gene expression abundance height, species, expression has many characteristics, such as tissue and Space-time speciality, these features So that circRNA has broad prospects in the development and application of disease diagnosis and therapy novel method.At present in oesophagus The malignant tumours such as cancer, gastric cancer, liver cancer summarize has found the circRNA with early diagnosis value in succession, but leads in children ALL There is not yet the report of correlation circRNA in domain.
Summary of the invention
The technical issues of solution: the present invention provides one group of circRNA for children acute lymphoblastic leukaemia diagnosis Marker and its application, the diagnosis for children ALL provide reference frame.CircRNA biomarker pair provided by the present invention There are very high sensitivity and specificity in children ALL, can be used as detection of the novel biomarker for children ALL.
Technical solution: one group of circRNA marker for children acute lymphoblastic leukaemia diagnosis, marker include Two kinds of has_circ_0005720 shown in has_circ_0117909 and SEQ ID No:2 shown in SEQ ID No:1 circRNAs。
Above-mentioned primer combination includes: the specific primer for has_circ_0117909, for has_circ_ 0005720 specific primer.
The above-mentioned specific primer for has_circ_0117909 includes the upstream primer as shown in SEQ ID No:3, The downstream primer as shown in SEQ ID No:4；The specific primer for has_circ_0005720 includes such as SEQ ID Upstream primer shown in No:5, the downstream primer as shown in SEQ ID No:6.
Application of the above-mentioned marker in preparation diagnosis children acute lymphoblastic leukaemia kit.
Application of the above-mentioned primer combination in preparation diagnosis children acute lymphoblastic leukaemia kit.
The application in children acute lymphoblastic leukaemia targeted drug is being screened or is being prepared in above-mentioned primer combination.
A kind of diagnostic kit of children acute lymphoblastic leukaemia is combined including above-mentioned primer.
The utility model has the advantages that (1) this method uses detection of the real-time fluorescence quantitative RT-PCR to one group of circRNA, to children ALL Discovery and diagnosis provide reference value.(2) this method high specificity, sensibility is high, as a result stable, and there is extensive clinic to answer Use prospect.Children ALL diagnosis circRNA biomarker provided by the present invention, can be used for the diagnosis of children ALL, area The AUC of children ALL marrow and normal control marrow is divided to can reach 0.962, sensitivity reaches 90.9%, and specificity reaches 95%.
Detailed description of the invention
Fig. 1 is expression figure of the has_circ_0117909 in children ALL and normal control marrow；
Fig. 2 is expression figure of the has_circ_0005720 in children ALL and normal control marrow；
Fig. 3 is the ROC curve figure that has_circ_0117909 is used for children ALL diagnosis；
Fig. 4 is the ROC curve figure that has_circ_0005720 is used for children ALL diagnosis；
Fig. 5 is that has_circ_0117909 and has_circ_0005720 merges the ROC curve diagnosed for children ALL Figure.
Specific embodiment
Illustrate embodiments of the present invention below by way of specific specific example.
Embodiment 1
1, clinical sample is collected
Collect attached children's hospital, Nanjing Medical University new children ALL marrow specimen in 2016 40 and normal child Control 30, marrow.All participants sign informed consent form.
2, circRNA RT-PCR is tested
2.1 RNA extracting and quantitative PCR
Using Trizol from sample of bone marrow extracted total RNA, RNA is quantified with visible spectrophotometer.
2.2 primer
One group of circRNA provided by the present invention includes has_circ_0117909 and has_circ_0005720.They It is to be screened from the result that children's ALL patient's circRNA chip detects, using GAPDH as reference gene (SEQ ID No:7), primer sequence is as shown in table 1:
The primer sequence that 1 RT-PCR of table is used
2.3 reverse transcription reaction
Reverse transcription is carried out to total serum IgE using Tiangeng FastQuant RT kit, RNA initial amount is 1ug.The kit packet Contained gDNase, can simultaneously digested genomic dna.
2.3.1 Genomic DNA digestion
According to table 2 prepare digestion system, 42 DEG C 3 minutes, cooled on ice.
Table 2
Ingredient Dosage
Starting RNA Sample 1μg
5×gDNA beffer 2μL
Total Volume 10μL
2.3.2 reverse transcription reaction
Be formulated as follows reaction system according to table 3, the postdigestive product of genome be added, totally 20 μ L system, 42 DEG C 15 minutes, 95 DEG C 3 minutes, cooled on ice.
Table 3
Ingredient Dosage
10×Fast-RT Buffer 2μL
FQ-RT Primer Mix 2μL
RT Enzyme Mix 1μL
RNase-Free Water 5μL
Volume of Mix 10μL
2.3 real-time fluorescence quantitative PCR
Real-time fluorescence quantitative PCR is tried using the SYBR Premix Ex Taq (Tli RnaseH Plus) of TaKaRa company Agent box, quantitative instrument are the Q3 type real time PCR instrument of ABI company.Reaction system is as shown in table 4, reaction condition be 50 DEG C 2 minutes； 95 DEG C 3 minutes；95 DEG C 30 seconds, 60 DEG C 1 minute, 40 circulation；Three repetitions are done in each real-time quantitative PCR reaction respectively.
Table 4
Ingredient Dosage
SYBR Premix Ex Taq 5μL
Upstream primer 0.4μL
Downstream primer 0.4μL
ROX 0.2μL
CDNA template 2μL
ddH2O 2μL
Total volume 10μL
3, circRNA expression is analyzed
It is expressed using the circRNA in the method analysis children ALL marrow and normal control marrow of non-matching sample t-test Difference, bilateral P < 0.05 are considered to have statistical difference.
4, circRNA value of diagnosis
Draw area AUC under ROC curve and calculated curve, come evaluate the two circRNA diagnose respectively children ALL and Merge the sensibility and specificity of diagnosis children ALL.When AUC < 0.5, indicate that diagnosis is meaningless；When AUC=0.5-0.7, indicate Diagnostic accuracy is lower；When AUC=0.7-0.9, indicate that diagnostic accuracy is medium；When AUC > 0.9, indicate that diagnostic accuracy is high.
5, result
Expression of 5.1 has_circ_0117909 and has_circ_0005720 in children ALL and normal control marrow It is horizontal
It is compareed in marrow specimen in 40 children ALL marrow specimens and 30 normal children, has_circ_0117909 table It is significantly raised in children's ALL marrow up to level, P value is respectively < 0.0001, sees (Fig. 1)；Has_circ_0005720 expression Level is significantly lowered in children's ALL marrow, and P value is 0.0009, sees (Fig. 2).
5.2 has_circ_0117909 and has_circ_0005720 are individually to the diagnostic value of children ALL
The analysis result of ROC curve is as follows: the area under the curve of (Fig. 3) has_circ_0117909 is 0.897 (95%CI=0.816-0.977), sensitivity 86.3%, specificity 85%；The curve of (Fig. 4) has_circ_0005720 Lower area is 0.748 (95%CI=0.621-0.875), sensitivity 77.2%, specificity 75%.
Both 5.3 has_circ_0117909 and has_circ_0005720 combine the diagnostic value to children ALL
The analysis result of ROC curve is as follows: (Fig. 5) combined analysis has_circ_0117909 and has_circ_ 0005720, area under the curve is 0.962 (95%CI=0.915-1.010), sensitivity 90.9%, specificity 95%.
To sum up, has_circ_0117909 and has_circ_0005720 has certain diagnostic value to children ALL, AUC, sensitivity and the specificity of Combining diagnosis are superior to individual event circRNA measurement, and AUC area under the curve is maximum, sensitivity and Specificity all reaches 90% or more, is the reliable specific biomarkers for diagnosing children ALL.
It should be understood that those skilled in the art can carry out the present invention various after having read above content of the invention Modification and polishing, however be that SNP site of the present invention is related or the various forms of of the site are answered to detect With these modifications or polishing all belong to the scope of the present invention.
Sequence table
<110>Nanjing Children's Hospital
<120>one groups of circRNA markers and its application for children acute lymphoblastic leukaemia diagnosis
<160> 8
<170> SIPOSequenceListing 1.0
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<211> 32953
<212> DNA/RNA
<213>artificial sequence (Homo sapiens)
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gggaaaagct gagagtctgg aactagccta tcttccgagg acttagagac aacagtatgg 60
gaatttcaac gagacgtttt tactttcttt tgaccaagat tcaaattctt tattccagcc 120
cttgataagt aaataagaag gtaaaggact atttatttgt aaaaagtttt tcatgatttt 180
gtgatggcac cttgttccat atcatctcag ataaatcaga ataatttgtg aaaattactc 240
ggtgatttcc acattagata ttttaaacct aatgttattt ctaaaacaaa aaccaaccag 300
gagaatccaa ttaagtaaaa tgtatgtatt aatataaatt agctattccc atctggaaaa 360
gggcagccat ttctgtgttg aggtgcctca atgatactga ggctgagaca ggttagatga 420
tacaggcata ccattagcag cagactcaat actaacccag atctcccatt tcaattgcag 480
tgtgtttttc attcattgtc aataaagaca tcaaaattag ttcaaaacca aatcctcaaa 540
tcaacctttt tttttttctc ctctaagaaa gcgaccgtgt ttcatagaat ttccttaaga 600
catgtttgtc atgctgtttc attttcattc agcactggag aagagccaca gccattcagt 660
tcatggcagg tgatatcaga cagatggatt acacatcttt aggtacttac ggcttgcaga 720
aaatgctcaa actctttctg cactcaaaag cataaaagct gtgattattc cacacagctt 780
agagttggat tgatgatgtg atcatcaaaa ttgacataag atggctaaaa acattagttt 840
tattttgtgc tgcctcccgt aaatttagaa aacacaaaat aaatgatcaa ctgacttttg 900
agtaaaacag aatttttctc aaacctttcg attcatgtac tatacaacct gatggagaaa 960
aaaaaatgtg ctgcccaact ataaaacaga agttctgttc aaaattagta gagaaaaata 1020
acataacttt tgacaggcct gctattttat tttataactt atatagaata taccttcata 1080
gtagcaaaag atataatgac aatgttatat cataaatact aactcaaaga aaagacatat 1140
cttacgttaa aaacactttt tgggcccagt gcattggctc accgctgtaa tcctagcact 1200
ttgggaggcc gaggcgggcg gatggcttga gcccaagcgt ttgagatcag cctgggcaaa 1260
atggcgactg aaacctcatc tctacaaaga tacaaaaatg atccaggcat ggtggagggc 1320
acttgtagtc ctagctactc aggaggctga ggtgggagga tcacctgagc ccgggagttc 1380
aaggctgcag tgagccgtga ttgtgccact gcactccagc ctgggtgaca gagtgagacc 1440
ctgtctcaaa aaaataataa tattataata taataagtaa tgttatagaa acactttttt 1500
gtcattggaa tagatcaggg aactagtaat taagatataa gcaaaaaaac ccccacacac 1560
ataataagct atattgtttt tcttatgtta ttaatagatc cagagcttct gattgtaagc 1620
tcaaatatat gtagtgagct gccaagaatc agagggccat ttagggataa actactacgg 1680
agcctagaat acatgtgggg gcaaagggga atggtaggaa tggggaaaat gtcacagatg 1740
actttgatca tgactttggt gccatagtgg taggttcttt tataagatga ctgatgtact 1800
tattttattt ggtttgtcat tgtttctata ttctatctat cttgattttt taaatgtgat 1860
ttttggcata atgcaaaata aaataactca tatttcgaaa tttacatagc tgatttcttg 1920
tggttttgat agttctatta tgtatcctgc aaatagattt ttgttccctt ccctgagaaa 1980
ttgagcaggg ccactccatc agtctgcctt cccttctttc tcatccaggt gatcggcatg 2040
tgtgagtgtg cctctataca ctgcacttct cagttcattc ctttaattcc gtagtcacaa 2100
ttgtggttcc agcacagcaa gaacttgttt aaagagtggt ttgaattgtg aggatgcctg 2160
aaaccataaa agagggaaaa tagctaggga aactgaagat aatttaatta tgatagttta 2220
caataatata gtgcttacta attccaaaaa ctattctaga cacaacataa tcaactcatt 2280
tattccttat gataacccag gaagtcagtt tccctactac cctcatttta tagatgaatt 2340
caagcaataa ggggttactt tgtccacaat cacacagctc ataagcggca gagccagggt 2400
ttagaacgga cagtgtagca cctgagtcca tgcttttaac cactatacta aatggaagat 2460
tcagggaaaa cgtcaaaatt gtcttggaat aattgaaggg cagcatattg aaataaggag 2520
taaacatatt ctccattgcc tcagaaggaa acagagtctg gcttaataaa agaaaggaaa 2580
tactaaagat aggtagattt ttctggcagc agtggcttcc atgtccctgc tggttggtga 2640
tcagatcttt tctggagcag cagttgtcaa tgatgctgac aacgagaaca gtggttgggt 2700
ttggcaaaac ttcattttct atcctcattt gtatgagtca gggatgtatt acaataattg 2760
tattgttacc agtacttctt tatttgggga gttacaaagt tatgaaaagg cggggggcag 2820
gatgcagaat aattaagcaa ttttattgac aaactttact ggcattactc ttttgctgaa 2880
agtatactat attttggctt acagtgtcaa aacagaattt ttttaaatgc ttttaaaaaa 2940
tggacaaaat tatagatatt cttgaggtaa aatataatgt ttatatatta tatatactgt 3000
acattgtaga atggctaaat caaactaatt aacattaagt acagactttt gatagattta 3060
tgaacttggc ttattgagaa tgaggttgaa tgatgatgtt ttcaagttca aatgtgtagt 3120
gcagtactaa aagcatgact taatgtttat agctttaaaa agttactaaa aaatgacatt 3180
ttggttgatg ttcttatgcc caatcgcttg ctttcctaac tcttgtgcaa tttttctttt 3240
attgcaggta attcgtatgc aagaagctac acgtaattaa atgtgcagga tgaaaagatg 3300
gcacaggcac tgttggtacc cccaggacct gaaagcttcc gcctttttac tagagaatct 3360
cttgctgcta tcgaaaaacg tgctgcagaa gagaaagcca agaagcccaa aaaggaacaa 3420
gataatgatg atgagaacaa accaaagcca aatagtgact tggaagctgg aaagaacctt 3480
ccatttattt atggagacat tcctccagag atggtgtcag agcccctgga ggacctggat 3540
ccctactata tcaataagaa agtgagtatt gattttagac ttctaataaa tctttaatga 3600
aactcttaac tgtaatatac ttttctgggc cttatataca gcatcacaat ttttcttctg 3660
ttaaagattt tataatactc ttcactgtca cttattttta tcacaatata ataaaacaaa 3720
catttataag aaatgaagtc aagagttggt tacagtcagg aaatatgaat agatgaatga 3780
tttctacaat ttcacagtga taattcagat agtcaaaatt aaaattaata tcaacaaagt 3840
gaaatatctg ttatggctga catattcaag cacagggtag actacagaat aatacctgtg 3900
cagaaaagaa ttatgagcat atgttgtact taattatttt ttagttcatg actgacttga 3960
tgtaccttca caataattta tattctagag gacaataaat aatctgctta gcatcctcct 4020
gtagccagac aatacatgaa gaagcatgtt cacccagaga ttcctttccc ccccagtaac 4080
ctagaatatg attaggatct tctcttaacc ttatatagtt tgtggcatgc tagattctgg 4140
atcatgtata ccattaaaac atcaccctga agaaaaaaat gtcaaaattg gaagaccaaa 4200
aagaaagaac tttcaaattt tctccatcca tacatgattt aaattagtgg ttatgactgt 4260
gctttcctaa tgctaaaata aatgcataat tgtatcagaa ttagctggtt tctatagtta 4320
aactagctgg gtgatatttt atagaaaata taaaaaatag gtggtatact ctatgctttc 4380
taattggata ttaaggcatt ttataaccac gatttatatt ttacacttaa aagcgtaaaa 4440
atagctcaat gtagaaacta aagataaatt agtatcacca tgagttgatg acttatagat 4500
tatggaagat attaaacacc tttattagtc aaagcacatc cttttaaaag tttataattt 4560
ataaaatcaa tgatggctaa aggcaatgac aaccattcag acatggaatt agtcctgttg 4620
ttctaaataa gtacttctca aacttgcaca tccatacaga ttacctcaag atgttttaat 4680
ttgcagattc tgaatccata ggtctgggct agggccagag atcctgaatt tcttacaaat 4740
catcaggctg ttacatggcc acactttgaa tagcaagctt taatgtttta tttaaagtca 4800
taagactaca tgtaaatagc taaacagagt ctcctttttc cccttttata gctagtttca 4860
taaaaggtga catttaatac attcatgtac ttctaaaaat aacttatttc aacaatttac 4920
tatagaattg ataaagaaaa atttgaaatg tagttatgat gattacaata agtcaccccc 4980
caagaaattg gacaaaatat tatccggtga aaactgttgc tctgaaggat atgtttgtaa 5040
agatatcttt aagaaaggag gcatgattta ctctgctatg attaaaagta tttgctactt 5100
aaagaaagga agtgtcctta aaaaagagaa tcaatcaggc tggtaagatt aagggggaaa 5160
caaaaggaga acaacagagg caaacgttac tattttccaa gggagacaga actttagtag 5220
tagcactgaa agcattttct tcccaatggc caggccacct cattgtggag gggctacact 5280
gaactaccta acacatgcta ttaaacattc cacatcattt gattaggtca gctggcttgg 5340
gctccatatt cagcaagaaa ccttctgact acaatttgtt ttcccctaaa cctgcttgaa 5400
gaaaagaacg tgctcattac atcactacaa ttatctcagt ccatatgaaa gtaatgtatc 5460
tcattgtaaa taaatacact ggaggccagg catgatggcc cacacctgta atcctagggc 5520
ttcgagaggc caaggcagga ggattgcttg aggtcaggag tttgaggctg cagtgagcta 5580
tgattgtgct actgcactct agcctaggtg acagatcgag gtcctgtcta aataaataaa 5640
taggttggca ttcttatttt atatatcagg ttttcaagtt tggttaatgt gagtctgcca 5700
ctaaaaaaca gaattgtcct ctgtttttga caaagtcaat ttttattgcc tccatggctg 5760
agagccagac ttagaccctg atttattcat agtggctgtg aaccatcttg cgagacatct 5820
agatagtctt tacggttggc tgcactgctt tcgtttatga agtatccata aagatgaaga 5880
ttgaggtgct attaaacaag gaggtaattt gtcattctta gcatagaagg agtaaagtaa 5940
gaagctaatg gaggaagttt tagaatttaa aaatatgtta tgaagctgtg ttaattatat 6000
ggaaaaatac aagtaaaagg catttaaaag ctttaaagca ttatgtaact ctttaccatc 6060
attaattagt gatacatttt gaatacctaa tttaattaaa tgagtaatat cacaatgtat 6120
tttaatggca tttaatttga ccagggtgct attaataata gtgacagtga taatcataac 6180
taacttttct tgtgctcata ctgtgtcaag cactgctaag ttcttgaact catttactat 6240
atagtgaacc agtgaggcag gtcctattgt tagctcagtt tgtaaatggg gaagtgaagc 6300
acagagagat gaggtaactt gctcaaggcc acactcctta agtggtgaat ccaggatctg 6360
agtccaggca ttcgactcca gagccagagc tcttaaccac taagtgaaat tatatactat 6420
gcttattaac ataataaaat aaaagggaaa gttaaaaaat tggctgcatc attttttgag 6480
ttctctgatt gatgtggtcc taatcattga gatgtaaatc ttattcttgc cacaaagtag 6540
taaaactttt agtaaactta taagatgtgc atctaagtaa cattctagga aaaagaagca 6600
agtaacattc cagagtacaa aatagctggt ttaacatact tccagctgta gcaatgacga 6660
tctatctctc ccctattcat ctgtcatttt ctcaagtaat tttcagttta ggtatatctc 6720
ttcatatcat cagagcccca taatttcaaa ttcttgcaat tctttcacta tgcaggacat 6780
tagtatttta ttctgcattt ctcagaactc agggttctct ttgtgtcagg tattgttcct 6840
gagacataaa ctgggattgt agtcttaagg cctgctctgt tttagaacct ataaattcaa 6900
cgaagatgct cttagcatgt cagctgtaat tccgctagta ctacgaaggc taatcttgca 6960
ggaggagcaa agggttccat gttcaaaggg ataatttatt gctgttcact ctgtttttgg 7020
agatagttca aaatgaaact ctctcctaat gtcgcaagca gaaattgctt tttcagggaa 7080
attgtttcaa aagagcatga gtacctgaac cttaaattac ggtgagcttt ttcaagttat 7140
gctgttgtgt tgctgaatgc ctaaatggat cttgactgtt caactgtaat attaaataat 7200
ctaatcttgt gactccaagg caacccttta atgcttctgt gcttcaacaa cagtctttcc 7260
aaggtccact gtcctttaca cacagtccta ctgctagtaa agaatagttt gcttgcccgc 7320
ttcataaaac tattcacatt tatagtgtgc agtttctgga aaattgttag ttaagtaatg 7380
gttggctcta tggtcttaaa tacaaaaagg caatgaacag agaaactgtg tagtttctga 7440
aaaatggtta gttaaataat ggttggcttg atggtcttaa acacaaaggg ataatgaaca 7500
gaaaaactgt tcagtttctg gaaaatggtt agttaagtaa tggttggcac catggtctta 7560
aatacaaaag agcaatgaac agagaaattg ttttccctgc cctttatact gacaatataa 7620
atatttttgt ctacctactc aaacaaagca tgttgtcagt gaacataaat gtatggcata 7680
tagcccctag ttaaaggaga gaaacatatt attaagtaaa tggatataca gaatagatgt 7740
cttaaaggta aatagtatct ttaaggtaaa cccacattta attaaaacaa gtgtttattt 7800
gcaaaccagg ctattaaata ctgtgaaaaa aatggagaaa aagtagatta caagctcttt 7860
atgctcaatg gtttataaca atttctaacc tagttctcac tggaatgaac actgtgtaaa 7920
ttgctacttg actataatta caagcaacat gtaaaagagt acaaattaat gtactaagac 7980
ttactggtaa agagaaacaa ttagggagtg atcaggaggt agtgtgattt atttggaagg 8040
acatgattaa ggaggtgatt gatagctgag aaaattccca gacagaaggg attttcaacc 8100
ttgattgcat attggaatcg cctcaggaaa gttttaaaaa tacccaaatc tgtgcccatc 8160
cctcagaaat tttcattcaa ttggcaattt taaaatcttt acaggacagc ctaatattca 8220
ggcaggatta agaacctctg acataatagg gggtcactgt aggtggaact atattgtaat 8280
gtgttgtgga gacatttccc tgaaatgaag ggagtttgca ttaaatgacc tttcagttgc 8340
caccttcttc taggagtcta tgacattcat attcagagtc atagggagag tttgactagt 8400
tgtgagtgag gctgacaact gaattgaatt ggccatagca aagatgactc tagtgggaaa 8460
aaaataaaag atgattctat aggaacatgg taaataatca cttgtagcat agctaatact 8520
aagtggatga ttgtagcttt cagatcatag aattttaatt gtggaatgcc ctttgaaatg 8580
gtgtatttca gactaaattt caatgcatcc atctctcttc agtattagct gagaagttga 8640
acccttctca cacatctata gtcagcaaaa ccagattatt caacctgttt gaacatttta 8700
aatctacagg gacaatcaga agccatcata aactttgaag ttaaaagatt tgatatgatt 8760
aaaatactta taaaagatta ttttagtagc aatgtgtaca gagtaatctg aaaagttacc 8820
aaagctgaag acagatggta agctttaaat agagttggaa tctgtaacgc tacaattcca 8880
taaaaaaatt tgtgtagaat tgtcagtttg tatcagcaag ttggaacctt agagatagat 8940
taggtaatag taaatagtat ctttatttgt aactatatgt taatttaaac atctaacatg 9000
tttgtagtta tgatatatca actggtttaa acaaacaagt ttgaacaaac aaattctatt 9060
ttttaaaaag ttcttcatgt atgtaagctc tttaaataag cccatgtcta atttagtaat 9120
tttactcgta ttttcttttt cagactttta tagtaatgaa taaaggaaag gcaattttcc 9180
gattcagtgc cacctctgcc ttgtatattt taactccact aaaccctgtt aggaaaattg 9240
ctatcaagat tttggtacat tcatatcctt ttaatgtgaa ttgcctaaat gctatttcta 9300
acagttgatt ttaaagaaaa tgtcagttat attttcaagt atctgtaaaa tttctttgag 9360
attaatggta acattgttag tttaattcat ttatttgcat tggttttcca ctatttataa 9420
tgagagtatt atagaatgac tacagcatta tagaagaaaa tatgtaacca accatgatag 9480
tgaatgtgca aaggaaacac atagtaggaa aaagagcaaa tctccttaca cctttatcag 9540
cttcaatgag gtcatatata accattgcta aagattagat tatggttagt attactattt 9600
gcattaccaa gtgttcaaac ttttacgcct taattgtttc tgtatcttca gcgcctggat 9660
ttatccttct ttgtgtcctt ccttccttct ttctgtcctt cctgtcttct tcccttcctt 9720
cattcttccc ttttcccttc cgtcttattc ccttcttttt ttcagtaaat atttatcaga 9780
ctcttactat gtgctcatgt acctggttga gaatatgtat caaaaagtcg aacttgaaat 9840
tatttacaat gttagtcaaa aagcatatgt aaaagggcag aatatgtttg gatttcaaga 9900
aaattctcaa gtaatgataa aatagaatgg tgattttctg aggcagtgtt caagaatggg 9960
aattttgtat aggcagattc aaagcaggct tctatataaa cttaaagact gattcaccta 10020
gagacagggc aaatgaaact gataaccttt tgaggtatga attctctcat tctatggctt 10080
gatgttggaa agtatttata ttctaaaata aaatatttat ctcaaaataa tcataaactt 10140
tcttacaggg aagtctggac ttagatccca agagaacgta ctacagctaa ttctcagaag 10200
agcatcagaa tagtgctatc tgtttaggta taatgtggac tagatacaag agtatagcca 10260
gatggtctgt tggggtccgc cacaatctaa agtgtaaata aaaagctaga aaataacatt 10320
accttatcag cacaaaattg gatctccctc ttttatgcta taaattgtaa aggagtaagt 10380
tacatcacta caaacctact atttatgata gaattctcaa tcaatgtaat ctttgattat 10440
actgggctct acattctcac actttttaca ttgcattcgt taacgtttta taagtcagtg 10500
aggatgaata tatctgaagt acacctttta cacttatggc atactataaa cttactaagg 10560
gatggtgagt tcttcttcca gggcaatttc acacaactga ttggaatgca aaataacttg 10620
catttgcttc aaatagagat catttttcta ttaaattcag ttttgtgagt atatggtata 10680
aaatgaaaaa aagagtgcac caaggccata tcacaggctt tgaagtttct tattatttta 10740
tcattgtttt aaaacaaata atattaattt cacagttttt gcatcgataa acttttttgt 10800
gtgttttgga tcatttataa atggccatgg taacctacta acatttattc cttaactata 10860
atctacttta ttcagcatgc ttatcatgtg cactattttg accaactgtg tatttatgac 10920
cttgagcaac cctcctgact ggacaaagaa tgtagagtaa gtaggaataa cttctgggaa 10980
tgagaaatgc acactcaaat tctctagcaa tctccttgtg ggtatagcct gacttatggt 11040
ttccacttct gtctaagaaa aagttatttt cataatatga aagaagataa gggaagttct 11100
ttgggggagc tattcttcta cgaggtaagt atttttccaa caaaatcatg acaatatctg 11160
ttaaagagag gttataaatt agtatatgct ttctggtaaa ctaaaccaat cacctagcct 11220
aaaattaggt ctcaatcatg gttaatcatt tgagataaaa tacagtgatc agttcagtaa 11280
tttgatgaat taaattattt tattccagta tatagtgatt attgttatat tcgaatagaa 11340
tagatttagg tttttagctc taaattaagt agatgtctta attatacatc taagatgtct 11400
aagttatatt tagatatgta agttttttat acacaacatg tgtgctgaaa cagcttagat 11460
gtgttctgta aaagaaataa aatgtcttcc ataaaactgt gttcataaaa taacagggct 11520
taggggaaac atgattgggg gagagtaaaa cttatggaac tttgtaacta gaatacaaca 11580
tagaaaccat aataatccca ataaaaagac acttgcccag ttgattcttt tctaatgttt 11640
gtacctaaaa tcatagccat tgagttcttt gtggcctcat atcccaggcc tcatcctttc 11700
tcccttgaaa tggagactgg ggatatttgt atgatttcat caaaataaaa tgcttcattc 11760
tgtattttaa tacatggaga aaggtctaag cacattagtt gttcaggatg ttaaaccagg 11820
cccttgttgc actaaaagaa aacacttctg tggatgttgt gtattagtat caagatcttt 11880
ttattattat tattgcaagg gattttccat ttaagtgtga gaaaaaatgc ccttcattgt 11940
ttcaaaacat caatatgctg gggaaaactg tgcatgaact aacacaattt ctttatcgta 12000
acaacattat ttcattttct tatttttcag tcacatatga ggttatttgc atcaaagaaa 12060
catagttttt agtgaaacaa agtggttata taggctactt atatatagta taatacaata 12120
atcaggtatc tgactggtta agtaaaaaat gcagtatgct ctttttattg ttatattttt 12180
aaaagtaatg tacaatcgga gataagatag tgtaacaaat actgccactt ttagttccgt 12240
ttttatagat ttctctgagg atttactgta ccactctaag ccattatttg tcagttattt 12300
aacatgttat ttaaaaaaaa atagtagctt caaaatgctg cctagaaatt attgtaatag 12360
agaatccagc tcccatattg aggaaagcca tttaacttag aactctcatt tactatacat 12420
gattttggag ttaacaaaat ggacactcct atgtaagact actctgtatg tggttttccc 12480
agcagaaatt attgaagcat ttaaacatgt atcaatttca tttatttgta taacattatt 12540
aaaagatagt taaagaaggc agttggagtt tgtgaccagc ctggccaaca tggtgaaacc 12600
ccatctctac taaaaataca aacaatgagc caggtgtggt ggcaagcacc tgtagtccca 12660
gctactcagg aggctgagac aagagaatcg cttgaaccca ggaggtggag gttgcagtga 12720
gccaagatcc caccactgca ttccagcctg gacgacagag cgaaactctg tttcaaaaca 12780
aaaaaaaaag gcaattgaat atacaggcct ctctttctaa agaagcagta ataacttaga 12840
gatgattata cccattatga aggcagtctt agctttagaa agtttttgat ttgcatcatt 12900
cagctggaaa actttgctca ggttttctga tcaattgtct tcagtagctt gaaagcaagc 12960
cagcaggaaa aaagcaaagt agtagtggtt actaccccaa accctgggtt cagtgaaaca 13020
gaagataact tagttctgag atgaaaactg aagtcaccga tatttgaaac agtccctgta 13080
caaagatgtg gcatacacaa aaccatatta tatagtgagg ggaatgttat ataaggcaag 13140
gcacttctat agactgcatt ttatttaatt ttatttatac tgcattttat ttaaaccaat 13200
atacttaaaa tatgaaaatt caacaattaa gattagccaa atttcaaggt tttaaaacac 13260
caaagttaca ggaacataga cagaattgac agcttgatta tgagcctagt gtgggcatag 13320
ccacgaaaga taaaggtccc ctgactccca cttacctgcc caacctagtt actaataacc 13380
atttttgaat acctattctg tgtagagctc tgtctcagta ggctctagga ctctcttcca 13440
tggacagcca tgaacacttc ccatacattc caaatgtacc cacatatttt cttaatttcc 13500
ccaaatcaag aacaaaagta gggactttaa tcttattcct agtaactcta tagataagac 13560
agaattgcct cagcataaac cacagggcac taaccactaa tacaagaaac agaacataca 13620
tgtgtggtgt ttctaggcct ttaaaaggcc agctatctct acagacagaa atatcacggt 13680
gattatgtgt gtcacacaac gatccaaact acaacacagt aaacataagc ttgatattgt 13740
aaaatatatc tcttcctaat agtcagctac ttcatataag taaagatagg aattattgct 13800
tatgagatca attgaattgt tgggggagaa aaggaaggga atgtacttaa gaaaactaga 13860
cttgaagata tctgcataaa ttattcttga tgtctttgtt ccctcatatg aaagaaactt 13920
gtgcagaaaa catttcaaaa taatgatcct aacatttttc atatataaat actggtaggc 13980
tgaaaaatct aatgaattta atatgagtaa atcaaatctt tgtggcttgg gtgaagtgct 14040
ctgaaaaata aacttaaatt gtgcttttaa agaacacaat gttgaaaaac aagtactctc 14100
aactattttg ggaaaaaaac gaaaacagca ataacttata ttgtcaagac tcaaagaaaa 14160
gagggctaga aatacatgac attaaacagc aaatagaaaa gtcttccacc attttggagg 14220
ctgaaaattg aatttaagca aggtttgaat gatttagccc aattgcatag agatagtagc 14280
tgatgcaagg tttgatttaa gaattttgtg tatccgtgta ttttggcatt tgtgcaaaca 14340
cacacatata aaatactagt gaactggaac atttcatatg ataatatata cacacacaca 14400
cacatctcta tacagcatgc tcatgcacac acacacacat acactcaggg tgggaggaag 14460
gagggcagag agagagaaag aagagagaga aatggggggc agtttataac tctacatgat 14520
tcttaaaaat tgaaaaaaaa gaaatatatt ttctatggta gacatgtaaa ctagccctca 14580
aaatgagtct actcattaag aaatttgaat aaaatatttt attatttaag taaatgaatc 14640
caagctatgt tgcctctaaa gttgtactgt ctgatgagga agccactagc cacatatggc 14700
tatggaacct tgaaatttgg ctaatattaa ttgttgaatt ttcatatttt agatatattg 14760
gtttaaataa aatgcagtat aaaaaatacg ttgacttgct tcttctcatc ttttcgcttg 14820
ctactaggaa tttgaaaatt acatttgtgg cttccattac atttctatca gatactgctg 14880
cgctagaagg tcaaactaga tgattatcca taagatacat gaaactatta ttctaaaacc 14940
caaatagtta aaccagatta gattcctaaa gaatatattt tctcttcagt ttaactcttt 15000
gctcaggctt gtaaaactaa ctaaatgaat agattatttg gtaaatagaa gtaaggaaca 15060
atattttaat gaattgaaaa accacaaaag gataggattt gctatgattg aaaacattta 15120
ttttaacagt tcaagcaaaa ttgttaattt tggcttggat gtttttccta ggtacacatt 15180
cactggaatc tatacctttg agtcacttat aaaaatcttg gcaagagggt tttgcttaga 15240
agattttacg tttcttcgtg atccatggaa ctggctggat ttcagtgtca ttgtgatggc 15300
gtgagtaact ttgaaaattt gataagcgca aaggagtgaa aatagtcata gtacaaacaa 15360
ggtctttgtg tcatatatta aatgtagagc tttcttgtta gtcaagttaa ctatatggat 15420
tgtgtatttt aagaatacat attagaatac atattgcaat gtaaatatat ccagtaaatg 15480
atcaataaat ggggttatct tcatgtcata tagtctttct cttcatcaaa atgaagttat 15540
tcattgacta caatttaata gcaacaactg tacttggatt agttttgcaa acaaatgtgt 15600
tcatattagt gaaaattaca ttgaaataaa gcaacacgat tttgtttctt aaaagcaacc 15660
tttagcatat ggcagcctga ggtctatagc aattccattt gttaaactca cagggctcta 15720
tgtgccaaac ccagcattaa gtccttattt agtataaact ttgccaaaac tatcagtaac 15780
tctgatttaa ttctgcaggt atgtaacaga atttgtaagc ctaggcaatg tttcagccct 15840
tcgaactttc agagtcttga gagctctgaa aactatttct gtaattccag gtaagaagaa 15900
actggtgtaa ggtagtaggc cccttatatc tccaactttt cttgtgtgtt attgtgtttg 15960
tgtgtgaact cccctattac agatatgtga cagagtttgt ggacctgggc aatgtctcag 16020
cgttgagaac attcagagtt ctccgagcac tgaaaacaat ttcagtcatt ccaggtgaga 16080
gctaggttaa acaccgaggt tgactttaat tattgagttt gaaatcaatt tatatgactt 16140
acagcattag ccttgttgct tattattaca gttcatcccg ttaaataatg ccaaatgatg 16200
tttcaatgtc agtttagctc ctaaaatttt ataaattaca tgcgtattta taaagtcagc 16260
ctttgagttt aacagaaaat tgcatgagac atcttcaaaa aatgctaatt tgggcctctt 16320
gcgctctctc tctctctttt tcactaccat ggctttacta acagatttgg attttaccat 16380
tcgctgcaga tgtagttcaa aaatggatct ccttttacaa aggagaaaat attattgttg 16440
aagagcatgt atatggcatg agacatgaga cttacattat tttaaaattc ttttctcctc 16500
taaattacta ctgcttggag gctagagtaa agaatggatt ggtggaaaat ggctgcatga 16560
aacacaaaat ctgaaatgaa aaataactcc aagttttttt gtatgtttgt tcagcatgta 16620
aaaccacaca aacaaaactt cctgactaga tatttaaacc ttcatattga atttccagca 16680
agcacactgt tcatgtgtaa aatctgctgt tcatctattt cccaaatcat caggctatcc 16740
atacagcttt ggtgtctaaa tagtcaagca atcatttatg ggggaaagag aatgtgtgtg 16800
actattaaga aatcatgatt tctggcactc ttcctcaggt aacctatagt tctctctctg 16860
caggtttaaa gaccattgtg ggggccctga tccagtcggt aaagaagctt tctgatgtga 16920
tgatcctgac tgtgttctgt ctgagcgtgt ttgctctcat tgggctgcag ctgttcatgg 16980
gcaatctgag gaataaatgt ttgcagtggc ccccaagcga ttctgctttt gaaaccaaca 17040
ccacttccta ctttaatggc acaatggatt caaatgggac atttgttaat gtaacaatga 17100
gcacatttaa ctggaaggat tacattggag atgacagtaa gaagtattac attatgttaa 17160
ccttagtgtt gctgaatgaa ttttcaacta taaatagttg agactgtggg tgtacagcca 17220
cctttgtaaa taactgaaat agtccaactc tgatttatta ctaatactaa tgtgaatagg 17280
attaatatga aataaaatgg gttttttttt gtattaacag gtcactttta tgttttggat 17340
gggcaaaaag accctttact ctgtggaaat ggctcagatg cagggtaaga aacataatat 17400
attttttaag atatagaact ctttgcgaaa aaaaaaagta ggtaggaaaa caactacatg 17460
gttatatgtg tagccttacc atgtatgcaa taaagagcag tgctgctccc ctaggaagtg 17520
ccttgtctgc cttaccggat tgccactggt cctaaactca cagcaattaa aaattatccc 17580
tttgtgaaga cctttcccca aaatttcaca gttaagatgt tcttaaattg atgctccaat 17640
gtgtgaaggc ccagagtctg tctttgctgt acatctatca gagctgttag gaaacacagg 17700
aacacaagat gggccccttt ttattggtgt acatatgtat caatgggatg agttttgtcg 17760
tgactggaaa agacccagac tataatgtgg tcttcagggt ggaagcacac ttggggaagt 17820
gctttgaaat atttaagtat tcttgaagga gcatgtattt ctgtatacaa agaagccaag 17880
ggtgcatgtg tgccaaaggc atgtccaagt atcagttgac ataatgaata gttcttttag 17940
gccacacgtt acaacaaaag atgccataat atcttagtca tgctgttatt ctctaaccct 18000
taaatatttt cctaagagta ttgtaagtta attttaaatc aaaataggga attttttaag 18060
agctgagaga tagcaatagt aagtgactct taggacacgg aacttaaata tattttgagt 18120
catcctttaa aaagggaaac tggtgtagcc ataaaaatct aagagagtta atgaaaatgt 18180
atactagtca tataaataga gctttgggga attttttttc ctgacagttt tcctagcctt 18240
attatgaatg tagggggtaa gagcataggt tgtagagttc atgtatacca gggttcaaat 18300
cctaacttca ccacttgtaa gctctttgac cttggaaaag tgtcctaagc tctctgtacc 18360
ttagtttact catcgttaaa atggagagag taatattctg tccctttaat agttgtgatg 18420
attaatgatt taatacacat aaagtatttg aactatcaca tggacaattg taagaattta 18480
ttgtatgtac cagcctaagc aacaaagcaa gaccctacct ctacaaaaca aaaaaaaaaa 18540
aaaaaaaaaa aaaagaaaga aagaaaaaaa agaaaagaaa agaagaagaa aaaaagaaaa 18600
acttagccag gcattgtgat gcacacgtgt agtcctaact tctcaggggg ctaaagtaga 18660
aggattgctt gagcctagga attccaagcc acagtgagct atgatcatgc cactgcactc 18720
cagcctaggt gataggctgt ctctgaaaaa tggctgggag cggtggctca cacctgtaat 18780
cccagcactt tgggaagcag aggcaggggg attacttgag gccaggagat ctagaccagc 18840
ctggccaaca tggcgaaacc ttgtctctac tacaaaatgc aaaaattagc tgggtgtggt 18900
ggggcacacc tgtatctcag ctactctgga ggctgaggca caataatctc ttaaacgtga 18960
aagatggagg tagcagtgag ctgagatggc accactacac tccagcctgg atgacaaagc 19020
gagaccctgt ccccccaaaa aattttttta ataaatttaa aaaaaaagca cactctaaaa 19080
aaagaaaaga atttattgcc tattaactat tatttattat gatgatgatt aacataaaag 19140
cccagataaa attttggata aatttggtta atacatactt tattcctatt aaaccataat 19200
aatctattcc ccttaatgaa ttcctattat ggcttaagta atttacatca acagtcccca 19260
gcctttttgg caccagggac tgttttgtgg aagacaattt ttccaccgac caagggtggt 19320
ggtgggaaat gatttcagga tgattcaagc acgttacatt tattgtgtac attatttata 19380
ttattagcat actgtaatat ataatgaaat aattgtacaa gtcaccataa tgtagaatca 19440
gtgggagcct gatctcaaat gattcacctg cctcggcctc ccaaagtgct ggggattaca 19500
ggcatgagcc actgcgcctg gccattggtg atgagatttt tgtttttgtt tttgtttttt 19560
tttgagacgg agtttagctc tgtcgcccag gctggagtgc agtggcgcga tctcggctca 19620
ctgcaagttc cgcctcccgg gttcaagcta ttctcctgcc tcagcctccc gagtagctgg 19680
gaccacaggc gcccgccacc acgcccggct aattttgtat tcttaataga taccaggttt 19740
caccatgtta gccaggatgg tctcaatctc ctgacctcgt gatccgcccg cctcggcctc 19800
ccaaagtgct gggattacag gagtgagcca ggtgatgagt tttaaataca aaaccatttt 19860
gtatttaagc ggcacattcc attaaagaaa taaatgattt gtattttatt aaaattaaaa 19920
gctgctgttt tacaaagata ttcttaagag aatgaaaaga caaaccagca actggaagac 19980
aatatttgca aagcacatat ctgatttaaa aaaaaaaaaa aaaagactgg ggctaggtga 20040
agtagctcat tcctgtaatc cagcactttg ggaagctgag aggggaagat catttgagcc 20100
caagagtttg agactggcct gggcagcata gggagacact gtttctccaa aaaataaaaa 20160
taaaaaaatt agccaggcat ggtgtagtgt atctgtggta ccagctactc aggaggctga 20220
ggcagaagga ttacttgagc ctgggaggtc gaggctgcag tgagccatga tcagaccact 20280
gcactatagc ctgggcaaca gaattatacc ctgtctcaaa agaaaacaaa acaaaacaaa 20340
acaacaaacc agtgtccaga atatctaaag aactcttaaa actcaacaat aagaaaacaa 20400
atgacccaat ttaaaactgg caaaagatct gaacagatac cccacacgag tatatagtac 20460
aggtggttat taaggaggta aaaaggcact cgacatcatt tgtcattagg gaattgcaaa 20520
ctaaaacaac aaggagttac cactgcacat ctgttagaat gactgaaaaa tgtatctggc 20580
aataccaact tctgatgagg atgcagaata acaggaactc ttgcttattg ctggtgggaa 20640
tgcaaaacta gacaaccgcc ttggaagaca gtttggcggt ttcttacaaa ataaacatag 20700
tcttaccata tgatccaaca actgcacttc taggtattta gctgatcaat ttgaaaactt 20760
atgtcttcac aaaaaacctg catgtgaata tttatagcag ctgtattcat aatgacccca 20820
aactggaagt aaccaagatg cccttcaatg accccaaact ggaagcaacc aagatgtcct 20880
tcaatagttg tatagataaa taaattgtta tgcccataca ataaaatatt actgagtgat 20940
taaaaagaaa tgagctatca agatacaaac aaatattaat gaatattaaa tgcacatttc 21000
taagtgagaa aaagccaacc tcaaaaagct acatactgta tgatttcaat tacctgacat 21060
tctagaaaac acaaagcaac agagatgata agaaaatagt gttttccagg ggttcatggt 21120
gagggattat ctaggtaaag caaaggggat ttttttttag ggtgagaacc tgttctctaa 21180
gattctataa tggtgaatat gaaacttaat gcatttgtta aaactcatag acctttataa 21240
cacaaagagt gaatcttaat atatacaaat taaaaaaaaa atcttttgtg aggtttgggg 21300
atcccaggaa gaaatggaat atgtgacaaa aatatataca tgtattgcaa aatatgaaat 21360
gacctcactg taggtggtgg ggtaataaca tgctaaccta agtaactttg gaaatgagtg 21420
gagtctgtaa gactaaaggt aaaaggaaca ataccctagt tgataaagtt gtttccaatg 21480
gtagtataga ttaacaatcc tgaaaccact gtgcatgtgt accagtactg agcaattaca 21540
aacatgtgta tctccttgtt ctgtcagctg agagggccta gaagcaatga caccccagta 21600
gcaacaaaca cagctagcac tcagatcttg gtttctaata ccattttcca ataaaaagaa 21660
ctagggctcc ttggagaaat ggttgattct aggactgcgg cagaaattat aaaagatgag 21720
cctggagcat ctgatagtac ctaaaactaa gaaagtgtta aaaaacaaaa aaaaatgcaa 21780
cccacaacca tggtgtatgt caaactgtta caggtgtcaa ctgaaagagc ttccagtggc 21840
caaagatgaa gccatttgaa caaataaatg aaatatcatt gaattgtaat ccaaaatata 21900
aaatgactat ccatgactac atgttaattg aaatgattga ataattaaat aaaatatgaa 21960
tggacaaata tcctatgcag aataatttca aataattttg gtatatactt aagatcataa 22020
ctgcgggttc cttaaatgtg gtctgtacac agtgacttta tttcaaagag cacatatgga 22080
aaaggaagaa aagggagtaa atttacagta gagaaaccta acactacctc aagacaggtg 22140
gtgaaagtta acatcaagag tgataagtca tagtgagtga taagtatgta cccttggcat 22200
gatgtgatgt gatgacagtg acattttacc tctgtagtct tccacccaaa aatccataac 22260
ctaaatataa tcatgagaca aacattagac aaatcccaat tgaagggtat tctataaaat 22320
atctgaccag taatcctcaa aactgttgtg gtcatcaaaa acaagggaag tctgagagat 22380
tgtcacagcc aagaggagcc taaaaagaca taactactaa atgcgatgtg tatcctgaat 22440
gggatcctgg aacaaaaaaa ggacattaag aaaaaaactg agggctgggc acggtggctc 22500
tcgcctgtaa tcccagcact ttgggaggcc gaggcaggcg gatcacgagg tcaggagatg 22560
gagaccatcc tggctaacac tgtgaaaccc tgtctccact aaaaatacaa aaaattagct 22620
gggcgtgatg gcaggcacct gtagtcccag ctatttggga ggctgaggca ggagaatggt 22680
gtgaacctgg gaggcggagc ttgcattgag ccgagattgc accactgcac tccagcttgg 22740
gcgacagaac gagactacat ctcaaaacaa aacaaaaaaa aagaaaaaaa ctgatgaaat 22800
ctgaataaaa tagtaacttt aggtaataat aatgtattaa ggctgggtgc ggtggctcgc 22860
acctgtaatc ccagtacttc aggaggccaa ggcaggagat tgcttgagcc cagaagtttc 22920
agaccagcct gggcagcgta gcaagatccc atctcaatta aataataaca atatatcaat 22980
attgaaaatg gcatggaatg tatgaagcct gtactatcct catacttttt ctgtaaataa 23040
aaaattgtta taaatattaa ctttatttta gaaaattcta caaaaccagc taatgaaatg 23100
gttgattccc cactctttgt ctgttttctg gcttttaatt tttttttttt tttttttttt 23160
tttttttttt tttttttttg agtcagagtc tcgctctgtc acccaggctg gagtgcaatg 23220
gcacaatatc ggctcactgc aagctccacc tcccgggttc acgccattct cctgcctcag 23280
cctcccgagt agctgggact acaggcaccc accacaacgc ccagctaatt gttttttttt 23340
tgtattttta gtagagatgg agtttcactg tgttagccag gatagtctcg atctcctgac 23400
cttatgatcc gcccgccttg gcctcccaaa gtgctgggat tacaggcgtg agccactgca 23460
cccggcctta atttttaact attgattctt gccactttgt aaagagtaga aatatggtaa 23520
tgtcagagcc aaaagtgtgt ggttgctagc tttctgccat tctaaatgtc cgtaaatatt 23580
tatttgcatc taaattttct atcggtcttc ctagtgaatt tcatctgata agtttcacgg 23640
tgggcaatca cctaaagtgt tctggaaatt aaagcaagat aattcgtcac agatagcagc 23700
tttgggtttt gaaaattcct ataagtcaaa taaattgaaa ttgctgtaat ttctaaactg 23760
accctacctc catttctctc tcttatagcc agtgtccaga aggatacatc tgtgtgaagg 23820
ctggtcgaaa ccccaactat ggctacacaa gctttgacac ctttagctgg gctttcctgt 23880
ctctatttcg actcatgact caagactact gggaaaatct ttaccagttg gtaaggtcca 23940
aatgagcatg cataacattt atttttatag acatgtatga aatgaaaagc ataggctggg 24000
tgtggtggct catgcctgta accctagcac tttgggaggc agaggcggat ggatcacttg 24060
agtccaggaa ttcgagacca gcctgaacaa catggcaaaa ccccatctct accaaaatac 24120
aaaaattagc ccagcatggt ggcgcccatc tgtagcccta gctacaaggg agtctgaggt 24180
gggaggatca cccaagccca ggaggtggag gttgcagtga gtcgagatca tatcattgca 24240
ctccagccta gatgacagag taagacccca tctcaaaaaa agaaagggaa agggaaaggg 24300
aatgggaaaa ggaaagggaa agggaaaggg agagaaggga atggaaggga agggaaggga 24360
aaggacctgc ttataacact ctggaacaca gaggaaggtc caacataaag caagtgtgcc 24420
aggaatccta attgctcact gagtcagtac ctatatatgt acactgagga cttttttatg 24480
tgaaaggcat tctgctagga acttggagat gctcatgact cttagaagtt gataatccag 24540
ttgttggtat ataaacagca cgttaaaaga ttttaaaaat cagtgatgtt gatgtgaaaa 24600
tttcaaatta aagctctagc tattaactga gactagaagg ggaggggagc aaaagtcatc 24660
tatagatggt atggagaata gctgcagtgc aaatggaatc ccactgaaag gatttaaagg 24720
ttcagtgctc ttacttgtca tattattgct ctgcatatga actctaataa aagcattata 24780
gtggggctca catatatatc cactatatat gagtttaaaa tgtatttata tccaacaaga 24840
ctgcagtcaa gcctcaaata atctgattct taattttgtg tgttgagcat atcaatacca 24900
aaaaattctt ggtgaaaata aactggatac taaaaagcta attagtctac tgactatcta 24960
actgtggtaa tcagatattt atttggggac attatactaa aatactgatg gaattatccc 25020
ccatttcccc tagacattac gtgctgctgg gaaaacatac atgatatttt ttgtcctggt 25080
cattttcttg ggctcatttt atttggtgaa tttgatcctg gctgtggtgg ccatggccta 25140
tgaggagcag aatcaggcca ccttggaaga agcagaacaa aaagaggccg aatttcagca 25200
gatgctcgaa cagcttaaaa agcaacagga agaagctcag gtactgagtg ataaaagcaa 25260
agatttatca ttattattat tagtttctaa gtagaaatag tgttatacta tagagggtag 25320
attggaactg ctttttcatt ttatatatag gcattgtcat tagaaaaaaa agtattcaaa 25380
tatgaacttg tttctgaagt tagagtctaa caaacttttt agccactgta tataaagaaa 25440
tcagctagcg gatagaaaat acattagaga taaggtttac tatggaattg tctcatagaa 25500
cctaagagag gaatttaatt ttttatttaa tttagaaaag aaataaaact gatatttcca 25560
attattagtg attcacccct aattatgaca tttgagattt tgttctgaag ttaattgatt 25620
ttaaaggttg acaagagact ttatatatac ctatgcttaa caattctttg ggatttctgt 25680
agcaaaaaaa aaaaaaaaaa aaaatgtttg ctatagccca catcaggata tcaggaaggt 25740
atctaaagtt agatgccaac ttgagatttt tattaatatt aaaggctgtg tgagctaggg 25800
gtatagtgaa tgggacatta atgtgttgag gagaagagac ggaaacacag tactgtatta 25860
aaagacagaa aaaaaaatcc atatactgaa aggtgaagac tcacgaatat atagtaaaaa 25920
tctgattcat gccagttaga attataattt ctcaatattt ttgccaacat ttggtatggc 25980
cagtcttttt aattttagcc attctaataa gtatctcatt tctgttttga ttttcatttt 26040
cctaattact aatgttgagc acctttttct gttcttattt tccatctgta tagcttcttt 26100
tatgaagtgg ttgttcaaat ctttaatccc ctcctttttt tttagttagg ttgtttgatt 26160
tcatattatt gagttttgag cattctttga ctctttggga tgtaagtcct atatcacata 26220
tatgcttctc aaatatttaa agtgcagttt atccagagta ttatttctta gcttttttgt 26280
ttaaatcatt cttttggatc taagaaattt ttgccaagtc aacaccatga gtattttctt 26340
ctaaattttc ttcttaagtt agttttatgc tttatattta ggtaaataac ccattttagt 26400
taatttttgt ataacatatg agtgatatgt tgagattcac ttttttaaaa tgagtatcta 26460
attattccaa ctgcataaaa acaaaatatt cattctccac tgcattgcct ttgaactttt 26520
gtcaattatt agttgtccat aggtgagttt gtctatttct gaactctatt ctgtttagtt 26580
tatttgtcat ttttatacca gtaccacact ctcttaattc ctgtaccttt tatgacgagt 26640
cttgaaataa catagtgcag gtccccaact ttttgttctt tttttttttt ttcaaagctg 26700
ttttggctgt tttatgtccc ttgtagttct gtatgagttt tttttctaag tttcatatat 26760
atatattttt tattatactt taagttctag ggtacatgtg cacaacatgc aggtttgtta 26820
catatgcata catgtgccat gttggtgtgc tgcacccatt aactcgtcat ttacattagg 26880
tatacctcct aatgctatcc ttcccacctc ccccacccac cagttagaat ggcgatcatt 26940
aaaaagtcag gaaacaacag gtgctggaga ggatgtggag aaataagaac acttttacac 27000
tgttggtggg actgtaaact agttcaacca ttgtggaaga cagtgtggcg attcctcagg 27060
tatctagaac tagaaatacc atttaaccca gccatcccat tactggatat atactcaaag 27120
gaatataaat catgctgcta taaagacaca tgcacacgta tgtttattgc ggcactattc 27180
acaatagcaa agactctata tgagttttaa actccatttg tcaattgcaa tttgaatgat 27240
ttctattgct gtcttcaagt caattatctt ttcttttgaa catttaatct gccgctaatt 27300
tcatccagtg tatttgtcat ctgaaacatt gtagttttca cctctgcaag cttgatctgt 27360
gtctttttta tatttcatat atctctaatt aacattgtga acatacacaa tacagttata 27420
acaactgttt aagttccctg tctgctaatt tgaacatctg tgtcacttct tggtcagttt 27480
tgattaattt ttctctttat tatgggtcac atattcctgc agttttatgt tcctgacaat 27540
cttcaattga attccagaca ctgtcaattt tacttttttt aagtgctgga tatttttgta 27600
ttcccataaa aattattgat ctttgtttcg ggacatagtt atgttgttta cacagtttga 27660
tccttttgta tctttaaaca tttgaaaatt tgttaggcag gaccagcata ccatttattc 27720
taagactaat tattcctcac tagtgaatca agacccttcc taatactcta cccaatgcgc 27780
atttaaaacg aagtgtttgc cagtgtagga acagacactg ttctggcaca gtgtgattgc 27840
caggtggtgt tctctcttat cctcagcctc aggtagtttt ctaagacaca tgcttggtga 27900
ataatcttct cagtactcaa ggggaacctg agaagatccc tagagttctc tctctgctca 27960
actctctcct ctgtggtacc ctgttttgtg aactatacaa ccaccctgat tttcctaaac 28020
tcagcttcat ctcatccatt catggagtct gcagagatac ttcctctcta catcatagtc 28080
tggaagttcc aggccataag ctaacaaaag catagaactg acttgcttgc ttttccattt 28140
tccaatatct taaaacatcc tgatctgatg tccagtgcct tcaaaactac tgtaacatgt 28200
attttgtctg ttttattggt tgcttcagga gagagcaaat ctggtctctg ttactcaatt 28260
ttggccagaa gcagaaatcc aaataatatt tttatgcact gacttttttg aggatttttt 28320
tcagatgaat ggctccacat atttgaagta ttctttggat gacagtatat aacagaaatt 28380
ggtatattca aaatttaagt ttttaacaat tctgagttac tttgaagatg cacaacaaat 28440
agcattaggt cctttctcaa ggcatttttt ggggtaatgg agatacagca atgagttgtt 28500
tagtgaatta ttgagctcaa ctctaggcat agcttttgct gaacatgcta gcttgaccac 28560
tttcatctta ccattggata cagagtagta atgtggatat accttattga ttttatacag 28620
aaaactataa acaaatttgg tgaaacttga aaatatttta aaatatacct catgcttgcc 28680
agaaagttct acatttgaat tatttaagag ttttattcta tgtcaccata aataaaacac 28740
atttataata taataaaata aaaatagcta tttacagggc aaatacttta tttcaaggtt 28800
ttaaataagt gttataggaa ataatgttca taaattgcat atgattgtat tttaaagcac 28860
tgggtttttc tattatcatt aataaaagaa gacaaattaa tgtgcttgaa aatcctacac 28920
agataatgag tttattctaa aatgcagcca ttttagggta aaatatgaca tcaattaaaa 28980
agagtatagt ctttttgaac ttagaaataa acatgcacta tttctagtag aagtttaaag 29040
aacctatgat tatgatgagt tcggctaaaa aacatcagtt ctattacctt agtagtcagt 29100
tactgaatct tagtgaccaa atcatcacaa tttgattcag ctttagcttc atacatataa 29160
aatttaaatt tatccaaggt aaagtataat actgattgga gtagcacagt gccaaatcca 29220
ttttagttcc ttattgacaa cacaagtaat gatgactagt aaattatcaa tgttgaagcc 29280
aggaggaagt ggaggcaaag acagaattag tgcacctata agatcattta tgctgcagtg 29340
tagcttaagt ctccctaacc acctacgact cttcaaaagt tactatctag ccaatgactt 29400
catgaaggct agcatcactt ccatctttgt ttattattgg ttgaggggcc acctagggct 29460
agtcacggtg taggcatgag aaaataatgt ggagaaacac aaaccctgca aagaaatcaa 29520
tgttttgtcc tagatcattc taattgtgtt tgacatttaa attgtgatat ttaagtgcct 29580
aatgttgccc agcttagtat atcccatagc agtctatatt gctctgatag gaaaaaattt 29640
gggagtaagc tctagaacaa aattcgaaat agtagctctt aagaaatcta atataattgg 29700
ggtagaacag aaataggtat aatgtcttgt agctttgaat gggattttaa ctggattata 29760
tacataaaat gaacaaagta ttagttatgt ggttatattt ttacggacac acagatacag 29820
tgagttacag agcctacttt gaatcacaca aaaatgttat aagaaaatgt aatttcctga 29880
gtaaatatta agtgaaattt cttatttgtt aaaagaggaa ttcagcaagt tggctgtgaa 29940
agtaacacag ttattctcat acgcaggaca ttggtagtgt cacagagttg ggtataaaca 30000
ggtacacaga catgagagat gacaggaaac atctgccttt gagggaactg gcaaatcact 30060
ggaagtggaa caaatgacag atttgagttg atgagtgaca cagaaaatgt gtctgaggtt 30120
acctttgttg ccttagaaat ctgttttggt tttgagttat ttgaactgtt acccagaata 30180
ggtttggaaa taatgtctta tgcaaaacaa agtttgtggc cgggtgcggt ggctcgcgcc 30240
tgtaatccca gcactttggg aggctgaggt gggtgggtca cttgaggcca ggagtttgag 30300
atcagcctgg ccaacatagt gaaaccctgt ctctactaaa aatacaaaaa ttagccaggt 30360
gtggtggctc acgcctgtaa tcctagctac tcagggggct gaggcaggag aatcacttga 30420
acccgggggt ggaggttgca gtgagccgag atcatgccat tgcactccag cctgggcgac 30480
agagtgagac cccatctaaa aaaaaaaaaa aagtttggaa gtgatttaaa tggacataag 30540
ttgtaggatt atggtaaggg actattacca ccacttcctt tgtttgcagg gacaaacaag 30600
tcaataaatg attaacactg tgtgttgtgt gtttgtgtct atataaaata gaaaagagaa 30660
ttaaggactt tatttggata agaaagggta tatatatatt cactgtttca acaaatggtg 30720
tatattatgt acctcatatg tatccagaac tctgtaacta tatttaactt gctatagata 30780
agtgattcat ttattcactt tattttgcat atatagtcag agttataaaa tgtacttaat 30840
atttctcatt ttacatcatg aattatatta aaagtgatac tgatgatcaa taattccatt 30900
aaaaacctgg ctatatgact ctataaaatt ttataaatat ctaaattctg aattactgtt 30960
ttatttaaat gctgggtatt gtcttatatc aagtcttaga tttgagcttc cattatgaat 31020
atagtgctta atataaataa atatgttaca ttttatgtgt tgcttggtca aacacataaa 31080
tttaaaatca tatttagtac taagtttccc tccctgagat cattgtaatc ctagagaatt 31140
tgcagcatag taattttaga tttaaaatct tttttaaatt tttaaacatg aagtttaaaa 31200
atgaaaacta cttaggagag tttgcttctt ctcaagaaag atgaaacata aagcacaaag 31260
tgttgtcaaa gcattataaa taggttttat ctccatttct tcctaaaata tgttgttata 31320
tagactgtta tgacagtctt ttatgatgtc ttgaaaagtc aagtctttta tgatgtcttg 31380
aaaagtcaac ttgctatctg tagcaagtta aatataagtt ctataaagtt cttcagaagc 31440
atcaaagtca aggttaagta tgtcttgttc ttcccccacc cccaccccag tgtctgagac 31500
aattgatagc aaagtacatt tttaaatctt tattcaccaa ttatttactg agccttattt 31560
atgtcaggtg acatactagt cactgagcat acaatgatac aatttctatc tagcttacag 31620
ttaatgggaa agataatata ttagataagt tagatttgtt tgtatttagt gatgacatat 31680
gtaaaaagtt taactgatat tgtcataatt taattatttt cacttctgtg gttacattta 31740
caaatagtaa tatacttaat gagaactcag ctttgatgca caccatattg tttcatatcc 31800
tctgtatgtc attgttagat catttccagt ctaaaaatac tatttgccat gtttcacctc 31860
agagctgact gtgtttcatt tgtaggtaca gaaattcaaa gtgcttaaga atacttagtg 31920
ctgcaatata gaaagaaaat atttgaagca gttctcataa tcctttgaat gaatttatgt 31980
aatgctatgt taatcatttg ttatttataa tattaaataa tctatgaacg tttaacattt 32040
tttaagtgat atgtaataaa ttctctggga gtaacatgat cacagagatg tgaactacag 32100
tcctaaatgt aattcaaagg aaggaagagg ataagctatc taatacattt ccctacttcc 32160
atctggaggt aaaatagtag acacttgaaa tgttttatgt tcacaaaaaa taaaagaaac 32220
agtcttttgt actggattta gaggggaaaa aatacgactc agctgctcag aagtgcacac 32280
aggaggaaac gttttacaga tggaatcaga atggtagtta gataagcaac caccacgaag 32340
aaaacatttg ctttttaaag tatagaagac actttgaaga gaacagttgt caccaacccc 32400
ccccccccaa aaaaaagaca atccagaaga taggcaaaac cagatttttc ttgctttaat 32460
gcatacacta gcatattgct ttatatgatt taaaacataa gggtcattgt taagtaaact 32520
cttgtggtga gacaaaataa aaggactaga ggatagactt ttcacttgta gttaatgagt 32580
cttaaatggc aatattttaa ccatactcaa ctgttttcaa agctctgtgt tctaaatagt 32640
gcctggcttt gttttatgac aggcagttgc ggcagcatca gctgcttcaa gagatttcag 32700
tggaataggt gggttaggag agctgttgga aagttcttca gaagcatcaa agttgagttc 32760
caaaagtgct aaagaatgga ggaaccgaag gaagaaaaga agacagagag agcaccttga 32820
aggaaacaac aaaggagaga gagacagctt tcccaaatcc gaatctgaag acagcgtcaa 32880
aagaagcagc ttccttttct ccatggatgg aaacagactg accagtgaca aaaaattctg 32940
ctcccctcat cag 32953
<210> 2
<211> 484
<212> DNA/RNA
<213>artificial sequence (Homo sapiens)
<400> 2
gttgaagaaa tatggagatc aacacaaaac tgctcatcag tgttacctgt atcagctttt 60
tcacctttca gcttcttttc tactttgtaa gttactggtt ttcagcaaaa gtttctccag 120
gtttcaatag tctcagcttc aaaaagaaga ttgaatggaa ctcaagggta gtatccacat 180
gccattcttt ggtggttggt atttttggcc tgtacatttt cttattcgat gaggctacta 240
aagctgatcc actttggggt ggtccatcac ttgcaaacgt gaatattgct attgcctcag 300
gctacctcat ttctgatttg tccattataa ttttgtattg gaaagtgatt ggtgacaaat 360
tttttataat gcatcattgt gcgtccctgt atgcatacta ccttgtactg aaaaatggag 420
tgctggcata cattgggaat tttcgcctgc ttgcagagct ttccagcccg tttgtgaatc 480
agcg 484
<210> 3
<211> 21
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 3
aattctgctc ccctcatcag g 21
<210> 4
<211> 25
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 4
ccatactgtt gtctctaagt cctcg 25
<210> 5
<211> 19
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 5
cattgggaat tttcgcctg 19
<210> 6
<211> 21
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 6
tccatatttc ttcaaccgct g 21
<210> 7
<211> 19
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 7
gaaggtgaag gtcggagtc 19
<210> 8
<211> 20
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 8
gaagatggtg atgggatttc 20

Claims (5)

1. one group of circRNA marker for children acute lymphoblastic leukaemia diagnosis, it is characterised in that the marker Including two kinds of has_circ_0005720 shown in has_circ_0117909 and SEQ ID No:2 shown in SEQ ID No:1 circRNAs。
2. the primer combination of one group of circRNA marker described in claim 1, it is characterised in that the primer combination includes: needle To the specific primer of has_circ_0117909, for the specific primer of has_circ_0005720.
3. the primer combination of one group of circRNA marker according to claim 2, it is characterised in that described to be directed to has_ The specific primer of circ_0117909 include the upstream primer as shown in SEQ ID No:3, as shown in SEQ ID No:4 under Swim primer；The specific primer for has_circ_0005720 includes the upstream primer as shown in SEQ ID No:5, such as Downstream primer shown in SEQ ID No:6.
4. application of the combination of primer described in Claims 2 or 3 in preparation diagnosis children acute lymphoblastic leukaemia kit.
5. a kind of diagnostic kit of children acute lymphoblastic leukaemia, it is characterised in that draw including as claimed in claim 3 Object combination.
CN201711246097.4A 2017-12-01 2017-12-01 One group of circRNA marker and its application for children acute lymphoblastic leukaemia diagnosis Active CN107937522B (en)

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