JP2020516280A - 角膜ジストロフィーに関連する対立遺伝子の多重検出のための方法 - Google Patents
角膜ジストロフィーに関連する対立遺伝子の多重検出のための方法 Download PDFInfo
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- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- OVTCUIZCVUGJHS-VQHVLOKHSA-N trans-dipyrrin Chemical compound C=1C=CNC=1/C=C1\C=CC=N1 OVTCUIZCVUGJHS-VQHVLOKHSA-N 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000000472 traumatic effect Effects 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
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Abstract
Description
本開示は、反応混合物の発見に少なくともある程度基づく、角膜ジストロフィーの検出を改善するための反応キットである。
用語「発明」または「本発明」は、本明細書において使用される場合、本発明のいずれか1つの具体的な実施形態への限定を意図しておらず、概して、特許請求の範囲および明細書において記述されている通りの本発明のありとあらゆる実施形態に当てはまる。
一態様において、本開示は、対象において角膜ジストロフィーを検出するための反応混合物であって、TGBIにおける突然変異を検出するための検出プローブを含む、反応混合物を提供する。いくつかの実施形態において、検出プローブは、本明細書において記述されているアミノ酸変異を引き起こすSNPを検出する。一態様において、本開示は、対象において角膜ジストロフィーを検出するための反応混合物であって、配列番号25〜30、36および54からなる群から選択される変異ヌクレオチド配列を含む、反応混合物を提供する。反応混合物は、配列番号19〜24、33および50からなる群から選択される正常ヌクレオチド配列を含む、対応する標識プローブをさらに含んでよい。いくつかの実施形態において、標識プローブは、配列番号25〜30、36および54からなる群から選択される変異ヌクレオチド配列からなり、および/または、対応する標識プローブは、配列番号19〜24、33および50からなる群から選択される正常ヌクレオチド配列からなる。追加の実施形態において、反応混合物は、配列番号33または36のヌクレオチド配列を含む標識TGFBI G623Dプローブ、および配列番号24または30のヌクレオチド配列を含む標識TGFBI M502Vプローブを含む。またさらなる実施形態において、標識TGFBI G623Dプローブは、配列番号36のヌクレオチド配列を含み、標識TGFBI M502Vプローブは、配列番号30のヌクレオチド配列を含む。
一態様において、本明細書において記述されている試薬のいずれかまたはすべては、診断キットにパッケージ化されている。そのようなキットは、本明細書において記述されているプライマー、プローブ、緩衝液および/または他の試薬のいずれかおよび/またはすべてを、任意の組合せで含む。
一態様において、本開示は、角膜ジストロフィーを検出するための方法であって、TGFBI遺伝子において、G623D、M502V、R124S、A546D、H572RおよびH626R変異からなる群から選択される1つ、2つ、3つ、4つ、5つまたは6つのTGFBI変異を検出するステップを含む、方法を提供する。いくつかの実施形態において、方法は、G623D、R124S、H403Q、R124CおよびR124Hからなる群から選択される1つ、2つ、3つ、4つまたは5つのTGFBI変異を検出するステップをさらに含んでよい。
a.リアルタイムPCR
b.リアルタイムPCRサイクル
いくつかの実施形態において、プライマーは、実験室の設定で試験および設計される。いくつかの実施形態において、プライマーは、コンピュータベースのインシリコ法によって設計される。プライマー配列は、アンプリコンの配列または増幅される標的核酸配列に基づく。より短いアンプリコンは、典型的には、より長いアンプリコンと比較して、より効率的に複製し、より効率的な増幅を招く。
当業者によって一般的に用いられる検出プローブは、加水分解プローブ(TAQMAN(登録商標)プローブ、5’ヌクレアーゼプローブまたは二重標識プローブとしても公知)、ハイブリダイゼーションプローブおよびスコーピオンプライマー(プライマーおよび検出プローブを1個の分子中で組み合わせたもの)を含むがこれらに限定されない。いくつかの実施形態において、プローブは、効率的なシグナル生成を促進するために、プライマーよりも高いTmを有するように設計される。Tmは、当該技術分野において公知である様々な方法のいずれかを使用して計算され、当業者ならば、Tmを計算するためのそのような種々の方法を容易に理解するであろうが、そのような方法は、例えば、ワールド・ワイド・ウェブでpromega.com/techserv/tools/biomath/calc11.htmにおいて利用可能な計算機など、オンラインツールにおいて利用可能なものを含む。
実施例1:世界の文献検索
TaqPath ProAmpマスターミックス体積;検査1回あたり2.5μL、
M502V V1プライマーフォワードおよびリバースプライマー、ならびにVICおよびFAMプローブミックス体積;検査1回あたり0.05μL、
G623D 20 pM V1プライマーフォワードおよびリバースプライマー体積:検査1回あたり0.05μL、
G623D 50 pM V1 ABYプローブ体積:検査1回あたり0.025μL、
G623D 50 pM V1 JUNプローブ体積:検査1回あたり0.025μL、
水体積:検査1回あたり2.35μL、
PCR蛍光検出増幅サイクリング数および条件:
サイクル数:40サイクル、
サイクリング条件;
PCR読み出し前(保留状態):60.0℃−01:00分、
保留状態:95.0℃−00:20分、
サイクリング状態:40サイクル、95.0℃−00:30分、
PCR読み出し後(保留状態):60.0℃−01:00分。
1 agagggaaca gaagcatcta ggagagattt ggaaagaaca cctgcaggat cttggtgact
61 gattgcacgt gggggaccag agagcaggga caggcaaaac tgaatgcaag gtttccaacc
121 ttgagcggca ccacaggcaa gaatgaagaa atgaagaagg ggagctggac gaaagagcca
181 agggatttct gcattttgga atgaattgct gctgggtggt gtccatttcc ctgaaggcct
241 ttatcctacg tgcaagaaaa ctcgtgggaa gcagaggaaa ggcatgtgta agccaacaat
301 catctgtggg catccttcca ctaaagtatt tgaggtcagg caactaaagc aacctcaaaa
361 gtgcctctgg attcttctta gatattttag ctgagccaaa tcaatgaaac tctcatgaaa
421 aatcggtttc cctggaaaat gaaattgggt tctaaccaac aagtagcatt tggcaggccc
481 tgattaagaa agccagtgtt tggagaagtt gtgaaaacag ccaagtcatt taagaaacta
541 aacactgggg cctaatgcca ttctagggct gcgacggctg ttctgttccc atcaattgca
601 gagcccgaag cctcaagttt gttttaagtt cctgccatta caaacctgtc gattatccca
661 gcctcccttg cgggctttga aaagagagaa gaatggaagg tgactgtggc caatttcccc
721 tccctgtcca gtgtgtggaa gacactgaat atgcaactac tgaccttgtg cctgggcatc
781 ttgaaggtct tccacaaagt gagctgggcc tcagcggaag atgagagttc ctctgtggtc
841 acttcactgg tacacatttt caggtgtatt tcgtttcttc catgcctaca taaattgaat
901 cctctgttaa ccacctctga gctcatagct atttaacatg accctgtagt cctgtgcata
961 caaatcacct tgggatctgg tgaaaatgca gattcagtgg gtcttgggag gttgggaggt
1021 tataagattc cacgtttctt catgagagct agaaaaaata aataaataaa taaaaaattt
1081 ttaaattttc cacatttcta atgaactctg gggttgtgct gatgatgctg ttttgcagat
1141 cacattttga gtggcaagac tgtggaaaat ccttgagaaa tcaatccaaa atcccctaaa
1201 tggtactaca atcacacctt aatgttagta aactgagatg tttcttacct ttatttgtaa
1261 catggaaaaa acaattactg tatatgaagt accattctaa gttctgtgtg ttacacaagg
1321 gatggcaatt ttccccaaaa tttgattcac atcttttcat ttggatatct cttgccaaaa
1381 ctcacctttt tttctcccta gcaagtcttg gggagctgaa ttttaagagc tctttattta
1441 gctatatggt ggcctctgaa aatgattttg actgtatctt ctgtctccat gtatgcccaa
1501 gcatcaccag gaactttagg gagtaaggaa aaggcaggcc tggtgtcagc tgggctgcag
1561 atgccagctc tcccaccaac aggcccagaa ccagtttctt tcctaggttc ctttgtgaag
1621 aacttgttgg aactactaat ttatcatgat gcataaagct tgttgtcata ccctacagta
1681 ttattttcaa aacctgaatg tttttggtga cctttcatgt gccacaaaat gtaaaagcag
1741 tcatttttta aaaagtgctt gaaaaagtct agtaaagatt cttccaagca agcctcactt
1801 tctcctgttt agattgttta atctggaagg aaaaaattct ttctcaaatg acagggtttc
1861 tggtgctctg tgtttgcctg gttggctctg ggtcatctgg ggatggaggg tccctgctct
1921 tacctccagc agcatcactc ttgtctccaa agaagcagca acctcaggtg ggagaatggt
1981 tatactcaca gcattctgct tttcatgttt gaaagagggg atgggtggtg gggcatggat
2041 gtgggatttt aaaaaaatat ctaaaccata aataaagtat tactgcaatc tctttactga
2101 gctcatggaa aaactcaagt catcgaatgt tagttttgca gactggagaa gtgaggtcca
2161 gtgaacttgc ttgacttgcc ctaaatcttg ctagagagag agctggaacc agatggcagg
2221 gctcctggcc tcttacatac aaggagcatt tttcctagaa actgcaatgc agccaaattc
2281 tactggtctc aggggaaact tgttctggga gtcagcctga gcttgaatcc ctttgggttc
2341 ttcccattat cctatgccaa gcagtcatgc tgaaaccgag aaatgttttg ctttcaataa
2401 atgaaatgag cattttcaga taattatttc tgtagttgct caaaactatc atattgtttc
2461 attgaaccct actatataga acaatgactg gggagaggta ataataataa tagcaatgca
2521 tatttattgg ccattttact tgaattgtat catgtaatct agtttagagt cctgtgaggt
2581 aggttttatt atcctctcta tgaggttgaa taacttgccc aagaccacac agctaggaag
2641 tagaaagact ggtatttgaa cccatcttct ccttttcttc tccttcctcc tcctcctctc
2701 ttccaacacc tgctcccaag gaagctcatc cagtgcatga ctttagctac cacctgctcg
2761 tagtggtgac tcaaatctgc atctccaatc ctcataccta tcctgagctc aagacctttg
2821 aatatagctc cctcctgtcc atccctcctg gaaatgcagg tggcttgttc acacataatg
2881 tgaacacaaa tggagcactc tcctcacaca cccaaatgtg caccttcacc agcgtgccca
2941 gcacaggcat cccttcctgc cagctatgag cctcgaggtt agctctactc cccctcccta
3001 accctgcatg cccaaggggt ttccaagtct aatcaatgct accactaaaa tctcccatac
3061 acctgttccc tcctctccac tagcttgatc actccccatg caggccctca gttgctttat
3121 gctctcagta ggccctcctc cagtgcccac actctctccc ttctccttcc caccttcttt
3181 ctaccagagt tctaacctct ccaagccccg cttgtctttt tctttccctg gctgccatcc
3241 taactcgccc cttcccttct cagacaagct tctacatgct actcatctct ccatcaaacc
3301 accatattcg ggctttggcc atctgctctc cacagccaag tccccagtgg cctctctgct
3361 tctgacacag tgaaagccat tcagatctgt cttgttggca gcattcctca ctttgagcag
3421 cgccctccta ctaggatacc cctccttgac tacaacccca cattctctac ttcctgggct
3481 cttctgtcac tggaggatga ctcccaggtg tgaatcttca tcccgcgtcc ctcactcaag
3541 cccccgatcc tcatatccag ctttatcctc atgggatgct tcaccaggat gagtcataag
3601 cacctcagac tcagggtgtc ccaaaccact catctacctg gcaagcctgc actctgcatg
3661 tgcctcattc tgaacatggc accatcacct gctgcaatgt ccagaccaca aacaccctac
3721 aatatccttg actctccttt ctccccttct ccctgtatac agactccaaa ttctattgag
3781 actattacct cctacacccc tcacatttgc ccagccttcc ccatctctgc ctctaccacc
3841 atagttcaag ctctcccatg gtcccttcct ggttacctgt tcttcttgcc tccttaagcc
3901 tctcatgaca ctggccatgt cacttgcctc cacccatcac ccgctaggct cttagctgga
3961 gtctgggccc tgctaccttc ctccccttct tccctaccct tgactccacc tccctgtgct
4021 tcagccaacc agataacttg agtttcgtga atgcatgcct cagtttacct gattaactca
4081 ttttcatctt tcaggcctca gagcaggtat caccctgtca gggccaggtg cctcttctta
4141 gctcccaaag ccccagctac tcttcatgga acatcattgg cttgggctac ggatcttccc
4201 aaattggagc tttttcacaa agggcttagg tctcactcat tctattaatc catctgtgtc
4261 tccccagggc tagcagtgcc aagtaactga caggtgatta atagatgctt gggtaagtat
4321 cacctcttta ccatgtgaca atttgtttac ctgccttgag ctcctccagg gcaggactct
4381 tgcctttgca gaatctatct ggcaggtact gttgcagaga tgtttactga agaagggaat
4441 gaattagtac caaggtgagg accccaccct tccccacggg ctccaaaagc agcttagagc
4501 ccaacaaaac ctgccccaca tttttggcgt ttctgtggat cacacgattt actcatctgt
4561 ctttcaatga gcatgacagg tggggtgggg gtggagggat tagagattga ggagctgggg
4621 agggtggtca gctcctgggg tgcagaaaca agtctgatgg gccatggtgt tctgggaatc
4681 agcactgcct cccctcaccc ctccctgcag tgttttgtag cctcaagatc agtgagggaa
4741 tcttcgggcc cccagcatgc aggaccgaag cccccgagac agctgtccct cagtcccaag
4801 gtccccattt ggaagcagcc acaggaggcc taagggacct atacccttgg tttgaggaag
4861 actgtggcga gggagagagg gagggagggc tggcagtgag ggcaagggct gggaaaactg
4921 agcacgggca cagtgcggga gcgggtgggt gcccagggca gccaggggcg cacgggttgg
4981 gaggcgccag gcggcccgcc ctccttgcac gggccggccc agcttccccg cccctggcgt
5041 ccgctccctc ccgctcgcag cttacttaac ctggcccggg cggcggaggc gctctcactt
5101 ccctggagcc gcccgcttgc ccgtcggtcg ctagctcgct cggtgcgcgt cgtcccgctc
5161 catggcgctc ttcgtgcggc tgctggctct cgccctggct ctggccctgg gccccgccgc
5221 gaccctggcg ggtcccgcca agtcgcccta ccagctggtg ctgcagcaca gcaggctccg
5281 gggccgccag cacgggtaag ccgagccgcc tggccagggg ctgcggaagg tcaggtagtc
5341 ggggctcgga gcgcaagccg ctgggggcat tgaactgggc tgggggcgca ggggacaaag
5401 cccgaactaa aaaccttgca gcatggagcg ctcggacacc agccctgcac gcggtggaag
5461 gagagaggga gggaggtgga ggaccatgga gggaaagcgg gaggccgccg ctttgtagaa
5521 gggagtgggg aagtggacca gagactttcg acgcaggcca agagcctgag acggacagcg
5581 ctttcagctt ctcctcccag ccactgcaga aagggggaaa tggcaactct ttggccataa
5641 tcaccgtggg agggtgccaa gggcaaagcc cacccagcag tacacctatt ccaacccagc
5701 caggcccccg gccagcgact ccagacaaga acctgggcca cacacggtgg cagcatctaa
5761 ggtgccccag gctcctgtgc tcctggccag gccctgcact cagacactgc tggcacccga
5821 cactgctctc tgggtacagc aagggcaatg tggcacttct tgtcctgccc gatgaagagc
5881 aggagaatgc actgggccct cacacacact gttcaaatgg ggaaactgag tcctgagtgg
5941 ttccactttc ccacagtcct gaagtgtgca ctggagccag gattggagtc tgtcttaaag
6001 taatagctgg gtttgtaaat gtaggacact atcattgcag gaattccttt gagaccctga
6061 agatgtgttg gctttaggag acaaactcaa gcagaaggtc tggtctgata gtggccctaa
6121 tactgaccca ggcagaggca ggcaacattt ctacctcaaa aaccaggcca tacctgcgtc
6181 acaaataccc aggctttgct gcagcttcca gcctacctgg ttgcaccaac ttctttttca
6241 taactaggta aaactatata tgagtagaat cttgtagtga ctcctcagag gaagcctaaa
6301 taccatcggg gtctggcgtt cacacccaca agcaatgccc aaacctccaa gagactgggc
6361 agatctgtgc tcaaatcaaa actcattgtt gggggtgata gagttgactt cacaggccct
6421 gaaagtcttg gctccttgca ctaggagtgc tctgggtacg ggtacaggct gccccttgta
6481 gggcatagtt gctcttgttt cctctacttg tggctttatg gtctaggcct ttcaggagtt
6541 tggggctctg gcggagaggg cctgctggga gcacatctgg ccaccctgca gagtgaaatc
6601 aaaccaggcc tggctgcaac ctcaacaccc tcctggaaag aggagaatac tggggatatc
6661 ctggggtctt tctggaagtg ggagaatcag ctttgacttg ggcagtgtgc agaatagagt
6721 gaggggggat gtcagaaaga tgagagggat atgaggcctc aacatcaaaa tgcaagcacc
6781 tggcattttt attatctctg cccacctctc cgttggtctc tctgcctttc ctgccaatga
6841 attgtgttat gtttgggtgc ctcaatttgc ctaggagggt tctatttctt ctgtatcttc
6901 gccactaagt caggagaaga tccttatagc atgccctgca acagtgtcac ctgtaagggc
6961 atctctctgc acagccacag tgaaggatcc tcaaaggtat tgagggcttt ccatcaagag
7021 ccatctttac agcaaacctc tttcccttca gagcccagaa gagtgctgac cagctggaaa
7081 acagggtttt tttcttaaat gcagatgctc ttgattatga gttccagata ttagatcaac
7141 ttccccacca tacccctgca ggcaaagcct cttaattagc ttcctgcagc acagctggaa
7201 aggcctattg taatctgtga tgggcagagt aatctaagaa gtcacaggag cacccctgtc
7261 ccagtagaat ctggatgcgc aggcacatga accatggcaa aatggttgca ggcacagttg
7321 tatttactct gatctaactg tccctgttaa tgccacaggg ctgcctggcc tggcacacag
7381 ggctgtggcg ccttgtgcaa atggataacg ttgttctagc tccagccttt cattcaaagt
7441 gaaaactgtt agaaagggaa ggaaaacttt gctattttaa ggaattgtag cgtgctgcct
7501 gatatgaagg aagaaataac agctgtgcct tgcttgtgcg cagcactcga ttgccgcttt
7561 tgctttcgac ctcaccacaa cacagtgaga tctactgttc atgttcccat tttacaggag
7621 gtgaaactgc agcttagtga ggtagagagt gacttagttc agacacagaa tgctgttggg
7681 agagtaataa ctatgatatg gtctcttgac tcccagctat atctgtgttg ctatagggaa
7741 ggggaaaaat aatactgaaa gagaagtaaa aatacaatca cacttccaaa catcaaccac
7801 caaaaactga actgaatttc ctgaagcact tggttttcaa atctaagctg aacatcaatg
7861 ctgttattct tgaggcccag aagcaacttg ctcatttcaa ttaagcttca gcatgaactt
7921 cctatgtaca cagcccaccc acactccccg atgtgagaag gagagggtca cagccgcccc
7981 cagcctctgc tgctgccaca aggacagcag cagtggaaac attcagcaaa ggaatgttgg
8041 agccacatcc acaagagact cactgaagat tcgccaaacg cctacggaaa gtggcaggga
8101 attcattgac agtaattgtt tcctgcttga tcagattgaa gagcttctgg gattctgtaa
8161 caataaatag gaccgggggc tggagtatgg ccagcaagga ctcttcaggg gttattcagg
8221 gactgtctaa cctgtgaatc ctaggcagca aacagaaacc aggtattcag aaatctggag
8281 gatttggtca ggcccagcta ggactaggga ggcatgggcc tctgctggct gtggtccctt
8341 ctccagcctt cacttctctt gtccctagat ccttacatgg attcattaat gctcattgtc
8401 cctcctgggc ccactcactt tcacctgttg aacaaaaaac tggccaagag gtgacagtca
8461 tatcaccgca gaagagacag ggcagagaaa tgaaggggca gaatggactc ccacccaaaa
8521 gcctgactct gaatatttga gaattgttca agttcctgca gaggaatcat gatggggaca
8581 gtaggtgtag tttttactgc aatattggtg tcttcttaac aaatacgctg cacatcaagt
8641 gatgtctgtg gatggcattc ttaaagtaac agggaaattg atgttaaaga aatacttcat
8701 cctttgggtg atacctgaag ttctctgagc ttggaggtct tgtgaaagcc ctcagtattg
8761 tttgttttat ttgctttcct ctgacttgtg attcagtcag atgcatgcct gcctctggct
8821 caggaagatc aaccctctcc tgactgacca cgcctctcct gactgaccac gtagcacagc
8881 agcttccttt ccctaggggc tcctaatgaa gctttcacaa tcacctggcc tgagcacagt
8941 ttgggtcagg acttggtata cttgaaaaaa acatgcaaaa ccaaaatcct gtggttctgg
9001 aaaaggcttc ttagcagaac ccccagacat ttacactctg ctttttcaca gggtccctga
9061 ggattctttg gatctgggta gtttggggag cagtattttc aacaagttca tttcgtgctc
9121 cttctacacc ctgcctggat gctaggcccc atctagaatg tgaacaacag aacaaggcag
9181 aacacttgtc ctcaaggttc tgttgagtgt tagatgcaga gaagagacac cccccacctc
9241 cccgcatcac ttacaggaat tctgtttgga acccaacatc aaataaggac cgtatccact
9301 gtcagaggat gggaagcagc atgtcatctg ggacattgga gaaaggctcc tgggggaagt
9361 gggacttgag ctgtgatcta agtaatgaac aactgagagt taaatgggag agcatcccct
9421 atcagggtcc tgagagcaac cagccatggt ttaaaccagc tataaagcct cgggtttata
9481 ggatagacag taacaatggc ttgtctttgg gagccaagca gctggtccag gcatgcagag
9541 catgtctgta tggagagctg cctgagagat gcttttgttt acacttatca attgcccatg
9601 tcaaagaagg atatgtacat gaagttacat cagtatgtaa gagagatttt aacaattttt
9661 gcaggggaag ctttcatggg ggctgatggg aatctaggta aacagaacca aagtctaaac
9721 ccaagatatc cccagtacca agactgaaat gactctctcc tctatctcta gaaagttcca
9781 gtgacccaag gaggcaaaca cgatgggagt cattaaagtg gggtggacgt gctgatcatc
9841 ttcctaattc tgctgctttt gttttcagcc ccaacgtgtg tgctgtgcag aaggttattg
9901 gcactaatag gaagtacttc accaactgca agcagtggta ccaaaggaaa atctgtggca
9961 aatcaacgtg agtatctgta accagccagg agaccaagct gtatgcacgc tggctgcagt
10021 tccccagggc ctgggccagc cttctagaag gtcaggttgc ctaaaaagcc atgaagatgc
10081 atgtgcgaac atgtctggga cctgcgtgct agggagtggc atttttagga agctggccaa
10141 ttttgttttg catttttaag gctgctgaca agacttggag acatttttca gggctggttt
10201 gggtttgcaa gaaacatgaa acactgcgtg tgtgtgtgtg tgtgtgtgtt tctcaatcct
10261 cataaaataa tacagatatg cagtggagaa gccaccagca tgtgactctg gaaaagaaag
10321 cccattggtg aatctgtact aaagaatgcc atccctatct tacagtccta aggtaaacac
10381 cccaaaaaga cttagagcac taaacatatg cagattatga gacagcatag catataatat
10441 ttgcacagac ttcctcattc aaaccctagc tctacctggg ccagtcgatt catctttaga
10501 accctccatt gctttacctg aaaagttcgt ataacaaaag gacccacctt atggggttgt
10561 tacaaggatt gaatgaaata atgtacataa gagactgaat atggtgccca gcatatatca
10621 gtgctcaata aatgctagct actattatta ttatcaccct agatttgcaa atctagacca
10681 cacaagcaga agtaagagtg ccaacggggt gtggaccagt gtggttacaa tagggcttgt
10741 tgatgtctgt ttcagcaagg agggaggcag cttttacccc actgcccagc tccctggtgg
10801 aatcaggtgc atgttctaac aattctgggg aaacctaatc tgttttggca ctgtcaacag
10861 atctcaaagc tggctgtctc ctatagctag gaagatgtgt atgacaaatc tcctgagcca
10921 cttgtgaagg cctgaccttc ctcctgtctc catacataat gggatgatta agaaactcta
10981 agccactctc ttaagcactt ttcaatgtta gggattttta agtttattgt tgtgacattg
11041 cttttgagca gacatctcct ccaatttaat agccaactga aagaagagaa aatgctcttt
11101 ccttaaactg tatgtggaaa taaatattcc aatgtgtgac cctgattatg ttaggcaatt
11161 agcaatccta atatgaattg agggaagttg ggattcatgg cacagctggg gagataccag
11221 cagtccctgg gagcctgtcc agggcaggtc catggcagct tgctccatgc ctgattgaca
11281 gcccagcctg caagctaaaa gttgagtgag ctaggaggac acactgccaa gattcagcta
11341 acagacaccc agcgatattc ttgctgctat gaacaaaagg agactatgca aattatacac
11401 cacccattct tccaggatgc ctgacttaaa aaataagaaa aaagatgggc cgggcacagt
11461 ggctcacgcc tgtaatccca acactttggg aggccgaggt gggcggatca caaggtcagg
11521 agacagagac catcctggct aacatggtga aaccccgtct ctactaaaaa aatacaaaaa
11581 tattagcggg cgtggtggcg ggcacctgta gtcccagcta ctcgggaggc tgaggcagga
11641 gaatggcgtg aacctgggag gcggagcttg cagtgagcca agatcgtgcc actgcagtcc
11701 agcctgggtg acagagtgag acaccgtctc aaaaaaaaaa aaaaaaaaag aaaagaaaac
11761 ctttagtact gattgatttt ttcccatgtg tgtatattat ctactcaaat taacaattaa
11821 ttacttaatt aaacacaaag ccaggcctca cctaattgct tcttggaagg tgaccagagt
11881 gctagtgcca agcaaacaac tcttctatat ctcaagagcc ctgggcttca gagggccatc
11941 ttttttgtta attcaagttt ctctgaaaat ggagacccgt ttatgatgac aagctggcta
12001 cagggtagca tctgccacac tgtttcgggg gtgccgctgg gctgaagcat ttgcccagct
12061 agttaacaat agctcgataa cattccctat cagtgtccag gctgagaata ctgtcagtga
12121 tgagtcgcct tggctcttgt acctgtatct ttgtgtgcca ggacaaggca caagcaacag
12181 agctgtgtgt tgccaaaatg ttcctgatga gcaggtcaac ccctcggggg caggtttgga
12241 tatgataatg tggtgatgtg gtggcgcagc tcccttaccc agtgagcaca aggggagtcc
12301 tctaggaaaa ggaagaaatg tctggatgag gtggggagat ggggttcaga gtggactcag
12361 gcaaagcccg atgcccagtc ccagctgttg gcctagtctc acaaagccag aaggatatga
12421 catttacatt caactcttga atttgtggcc actgctttgg gcaacttcaa agagagaaaa
12481 tgaagataga aaaatattat ttgatataaa acttctagga caagagaggc ccttcctgga
12541 acattacatg tagtattagg aaggtggagc tgccctggaa aagatccaga gaactcagag
12601 agaggaagag gtggaaccca tctctgttct tgtagagagc tcagtaagag tggcttggca
12661 gggctcctgt gtacctgaga ccaagaccag tgaggaggct actgtctgac caccatacgg
12721 tcagaattca gtgccatggg tggtcaggtg ggaaggggag aggactgtgc tggctggagt
12781 tgatgttatc ctggggaaag taggtcccta gatgccttta gttgagtgag gagcagactg
12841 ggaaatggga gcacagtagt ggttggggca aaaaggactg tctctgcatg aggtccatag
12901 gcagttggaa ttttctcagc aagactccag agaaggaggc tggagcagag gtgtatgttg
12961 ggatgaaaag gagtaaagta tcatggggga ggaggcagct caggttgtca agggtcaaga
13021 aaccagaagg agaatttcac cttggaagca gacaacgggt accaagcata caggggaata
13081 ctttgtggtg agaggtcaca cagagataca ggagccgacc tggtgagaca ggagcctgga
13141 gccacctgcc tgcttttgtg aggccccaga ctccactgct atcatcaggt gaagctctgt
13201 tgcctgcaca caaaagcttt tctgcattta caaagagaga agggcctgag tttctggtgc
13261 aatgcgtcaa gctgacatat ggactttatt acaggaagtg gttaccagtg ggtccctatt
13321 tagtggctgt tattgtgaat tttattgttc ggaaattcac tttagcattt atttcagatc
13381 ctaaatagca ccggagtgat acaatggcta atcaaacaaa gagggctgtg gggagcagac
13441 agtcagcatc cccctctgtg atttcaggcc ctggtttgat tagtagccat aaaatttttt
13501 acgtgtggca ctttgagcaa aggtgcagga aattgtggtc aggaagcctg gctgcctctc
13561 gacaggcttc ctttgtgcta gccccaggga gaggaggcct atttaacagc caagtccaag
13621 ttgacatcat gggactggaa tagtcatagc aggagctcag acatcataaa cgtggcatag
13681 ggagggctgg tggaggagct agcgggtatg ggtggcagct attcattcca aaagtcttga
13741 aattgtttca cgagcaacac atttcacaag tgcgaagccc ttctctggag ccaagatgag
13801 ctggcagagc actcctgttt ctctagtagc aagtgttcct ttgcccaggg gcaaaaatat
13861 taatactcct tcagcactgc attaatgctt aaagatttaa cttttaaaga gatcagctgg
13921 tgcatggtcg agcttttcca tcagctggca gggctttttc agtaggtgtc cttctgggca
13981 gggcactggg gacagctgac gtgaaggtga agaagagctg tcgttttcct cccttatatc
14041 ccacaacctt ggtcccaaga ggaaaaaaaa gaagatggtg agaagtcatc caagcagacc
14101 ccagacccat actagtgcct cctttcctgt ttcatatccc tgtgcagcca gctgggatct
14161 cttgaataat ctgctctggg ggcactgaga ttggacatac accaaacagc ggagatcgac
14221 caaacgcctc tgttgggcag tgtttcctga gggttctgtc ccattctgta aactaggagg
14281 ctgactagct gacaaggaat tttattctgt tgggtattta catgaaccta tgtgccacct
14341 ggggtaagac cctgtggtag gtagaaacat gacttcccaa aaatgtccac atcctaatct
14401 ctaattctgt aaatatattc ccttactgga aaaagagact ttgcaggtgt gattaaatta
14461 aggatcataa gagggagaga ttatccagga ttatttgatg agtctaatat aatcatcagg
14521 gtacttaaaa gagggaggca ggctgtgcct ggtggttcac gcctttaatc ccagcacttt
14581 gggagactga ggcgagcggg tcacgaggac aggagttgga gaccagcctg accaacatgg
14641 tgaaactccc cctctagtaa aaaaaaaaat acaaaaatta gccaggcatg gtggtacaca
14701 cctgtaatcc cagctactca ggaggctgag gcgggagaat tgcttgaacc caggaggcag
14761 aggttgtggt gagctgagat cgcaccactg ccctccagcc tgggcaacag agcaagactc
14821 catctcaaaa aaaaaaaaag agggaggcag tgggatcaga gtcagagaag gcaacgtgat
14881 gatgaaagct gacatttgag tgatgcaacc acaagccaag gaatgcaggc agcttctcaa
14941 agctggaaag gacgagcaat ggattcttcc ctacagcctc tgtgaggaat gcagcctttg
15001 attttaaccc cataaggccg atttctgact ctagcctctg gaattgtaag ataatttgca
15061 tgatctcaag ccactaaatt tgtggtaatt tgtcacagaa agcaatggga agccaacaca
15121 ggccttattt gttgacttat agatgcattt ttctttattt caatgtactt ttatcaatgg
15181 tctcatgtag ggtattgctt tcaatgaaga tattaacata gtttcaactt taaggtttat
15241 atctggagtt tctttagaag cttcacaact gaccacttag taaacagtaa gcatctgtta
15301 agtgcttctc atatgtaagt tcattcaatt ctcacaatca cactataaga taaatatgat
15361 tattagccca tttacagatg aggagacagg ctcaaaagac ttttatgcaa cctggtcaaa
15421 gtcattcact ggtaagctga ggaggtctgt ccacttcctt ttgctgcccc cagggggtat
15481 caagcctggc agttagtgtc agcgacttag gaggtgaaca agtgagcagg cctgtaggac
15541 ctggctaaac tgccccaggt ctctgtctac agcctcaaac ctgtggctgt gggtcccaga
15601 gacaaggcct cctcagcatc agagaaggat gcctttgtct cagggtcatc aaccttctcc
15661 aggttgctca ccccctgctg taaaggggat ccccaagacc gctcatcaga caaggagctt
15721 gggaactgag gagacacagt cagcctccag gagtgcccaa aatgccctca catgctgcat
15781 acagattgcc acaaataaag tacatccaca ttctgaagac tctgtcctca tcaccaacca
15841 ggctggcccc tggtgagggc tgtagtggtt gaggcctttg ttggtagaca gtaggttaaa
15901 gcaagccatg attttctatt gggaggcttc agaatcagct cagctgtgtt tccaagacca
15961 ggagggcaga aagcaaacca tcccaggcaa gcagtccatg ggccatgtca gatgtctaga
16021 cgttatgggt ctgtgtttgc tctgccattc ctctcggaaa ctatgatgcc ctgtatggtt
16081 taccttcagt cacaggtgac tggcctacag ggccattcct tgttccaacg acttctcgag
16141 tataattaat ccccaggcat ttacggccag agcagccggc caaatccgtg aagtgcagtg
16201 gttgttttaa attatattaa cttcttggaa acttatttta gggagagaaa actcagtact
16261 tctctctatc caatcttgag taaaaatgtt agaagggact ggtggagagc ctcccagaca
16321 tccctacaca tagactttgg gttgacatta tctctttgca ccttccttga aactttcttc
16381 taaattaggt gccttcccta atttaggcac cttcccagta ctagtctgtg acctgttagg
16441 aaccaggcca cacagcagga gttgagtggc agggagtgag cattattgcc tgagctccgc
16501 ctcctgtcag atcagcagtg gcattagatt ctcatagcag tccgaatact attgtgaact
16561 gtgcgtgtaa gggatctagc ttgtgcattc cttatgagaa tctaatgccc gatggtctga
16621 gatggaagag tttcatacca aaaccacccc ttccccctgc caccatctgg ggaaatattg
16681 tctaccacga aactgatccc tggtgccaaa aaggttgggg accgctgtcc taagggatct
16741 gctttttctg acctgaggtt tttctttatt agactgtatc tggctgagga gaagcctgaa
16801 gcctttaatc ggaacagctt tggctgatga gattagattc agaaaccaac agattggtct
16861 tttctatgca gggaagccta ggaactgggg ggctatggct gggaagcccc ctattgtttc
16921 catcctttcc tatgttcatc ctggaggaat ggcatcagac ccatgcctct gtgattgctc
16981 ccagcccatc caaccacagc atctatgttc tgcctgggac cagggccagg gagcatggca
17041 cactgagctg agtataagga gagtggagca ggccactgcc agcccagaaa attttggtca
17101 aagttgcctg aaatcttctc agccttcgat tcacagctgc tctctgctgc tctggggcca
17161 tgcagaccag ttcagaaaag agttaatttg ttggggcagt tggaggcagg tggactgcca
17221 gctttgacac cttcccagcc cacaggctgc tgcactgggg ctgaaggcgt ggctaacccc
17281 tgcacaccta gagagtgaca gagatgccag actgggcagc aggaaggcaa gaggattaag
17341 agagagcttc ctggctgaaa gccacactcg gttaaccagg aaaaagccct tggcacgaga
17401 agactcagtg gcctgaggga ctgagccttg gttgttgggc atgtgctgca taagccatcc
17461 atgtgtgaca gtagagtgta gtccagccac tgtgggacat gggtgctgaa agaccacatg
17521 gagaggaaca gtgagtgctg acaagggcta gccttgatca ctttggagac accccctgtg
17581 tcttctagat gtcagacttt ccaaatctgt ctgctatcct ccaaacgtgc attttcaaga
17641 gcaatggaaa aaggattgga cttgatggaa tgcagcaaga gtcctaggtc tgttactacc
17701 tacctatgac cttaagaaac tccttcaccc ctcagaaccc ttacagcttt ctttctgatt
17761 ctatcctgag ttactctact ccaagctgag acttttctgc ttagatctat cccttcctcc
17821 taaaccccca acctccattt ctcctggtgt ctttctttac acacccctca gcatacacac
17881 acacctagcc acaggaacca atgagttaat atttgaggag ttggttttct tttgtcctca
17941 atgagatcct ggtgaggcca cttgagctgt tcagctccct tgcggtattt tggggatgga
18001 actcagaagc caacaatata gaaaaagagt ctttggccag ctttcccagg ggctccatgc
18061 catagagagt actgcacccg tgtgcacagg gggccctgac atgaggactt tgaggataac
18121 actattcctc caactctgct tcagcatctc catggatttt cacacagaca ctttaggaaa
18181 gaaactaagt ttggggggac ttgacctaat cccacatcac agccccagta atacagccct
18241 ggaatttatc acagaaagcc tagaatccca tgcatatccc atgcatatgc atccctagtc
18301 ctatgggttc aaggcttgga gctctccctg gatttagctg ggaaaagttg gcagacagtt
18361 cttctctgtc ttctagaaat atggactaga atcgtgagtg tgagattgca agtaactttt
18421 aaaatcatct agtttaactt caccccattt catagaccaa gaaactgaga ccagagagag
18481 aaatggactt tcaagttcac cctgctagtt actgatggat cacaagtcaa atctcctgat
18541 tctagcactg tttctcttac accacaccac ctttgaaagt gtgtcaatca aatcttactt
18601 tagttgcaga ggatgacttt agtttctgaa gataaaattg tgagtcaatc aagatgagtc
18661 ccaagacaat agcctgttta gcccttataa gttcagggat gaaaggttag aaagaaacag
18721 gatggaagga ggactggaga aaaaaacaaa agaggaagga aggaggagga agcaaacagg
18781 aaaaaaaaag aatgtgcata gcttgtcact cctcagtcat ttcctgggag cccatttcta
18841 gcaaagtgac agctgcaact ccctggccac ctgagcatct tagctgatct gtctctgaaa
18901 caccccctgg agaacagatg aatcaggctt catcttcgct taactaagtc ttccctgaga
18961 cgactccatt taaatgaaca agagcaggat ttcctgggca cactgagagc accttccaga
19021 ggcccctcca gagccctaaa gcctgtattt cttccagtcg gcctgtttct ttcctggtga
19081 tgtcattaaa cgccctttga gagtcccaca gtgagcagtt ctgcggtaaa acccgctgca
19141 attaaagtct gagtcctttc ctgtctcaaa gggcatattc atatagaaga aaggaaaagg
19201 aaggactggc tgtttgcatt tggttccagg cctgttgagt agaggtcgtg ctcactccac
19261 cgaaggtaca gggtagcctt cagcagaacc tggggatttg gttttaagca agtctttctt
19321 aggtgtgggc tttcagaaca cttccttcct tgcaatatta tttgaaattc tcagtgtttt
19381 agccgtcccc agaatattgg ttcgttaaag ctgtgtattt cagatctcca gacagtggtc
19441 actgtttgta tattttcaat ttcaaaccag aaaacaaaag ttcttattga ttactttttt
19501 tatttaaaaa ataaaaagta agtatcttcg taagaggagc tttgttttaa ttttaaagtt
19561 taaaatttga ttgtgaagac agagaaaaac ttgatgattg tagatatatt cccctctttg
19621 gctattcaat cagagaacta gaaaatcatg agagatttaa tgaccactgc ctgatacaca
19681 tatgtgtttt acagatgagg aaactgagac ccagagagat gatgaaattg gctgaggatg
19741 gcccagctgg tcagtgaaag actcagagcc agagctggtg cagggctctt tctattcctt
19801 cctgttccct ttcaggaaca ctcaccatcg gctttcctgt gaataatgtt gagataaaat
19861 ccttggtgca ttatgttttc tagtcacaac attgactagg ctgccagagt cctctgttct
19921 cccagttggt tggctgtagg tgttggcagc cgccaggagc attctacaga acagaggagg
19981 agtgagactc tccttgctca ggaaaggcag acctatgact tagcaaataa ctcctaagag
20041 gagagtgttt cacccaccat tcctcttcct tggctgtgga ggcaacttag tggagagggg
20101 ccagatgacc tgtgaggaac agtgaagccc tgcctaacac aatgtatggt tgtcttgtta
20161 cagagtcatc agctacgagt gctgtcctgg atatgaaaag gtccctgggg agaagggctg
20221 tccagcaggt gaatgaatcc tccgggcctt gcctgttggt gtgggtggaa gggaatggtg
20281 ggagagagga gtacccacat aaaaggcagc agagtgtgaa tgggggcagt ggcacaagga
20341 catggcattc tccccacgtg cccactggcc ccaggctcta tgcgaggggc tgaggaatgg
20401 aagctggaaa cagcgcattt cctgagctgc tcctcctggc ctccttacca cactggtgga
20461 gtagactcca actgtggcct gtccatgccc ttcccagcag gcacaggctc aggctcaggc
20521 tcttggcctc tgcctctggc tgggagtgat tctaaacaca tccagcaggg tcagcctgat
20581 agcccatcag tttccgatca gctctgctag agagccgatg ggatgtggga ggagggggtc
20641 actggtgggc tggcaacccc aagccatccc catctccctc tgtgtctaaa cttggccctt
20701 tggagttcgg tagggagaag agccataggc caggtgggct cacccagagt cagcagagag
20761 tcccacaaat ggttgcactg ggcgaaagac agcatggcac ctgtgaattt tattagagct
20821 tttcttttag tgctacacac aagtgactgt acaggggagt tagtattttg ttttaatttt
20881 gaaatagagt catcttttgg tatctgcggg ggattgattc taggacccat tctaggatgc
20941 catatcctca gatgttcaag tccctgatat aaagtggtat agtatttgca tgtaatctat
21001 gcatattctt ccatgtactt taaatcatct caagattact tataatacca aatataatgt
21061 aaatcctatg taagtagttg ttataccctc ttttaaattt ttgtattatc ttttattgta
21121 tttcaaaaaa tatttttggt ccatgtttag ttgaatctgt gggtgaagaa cccacagata
21181 cgaagggcca actgtattgg ctattttttt agttaagaat gtgagactga ggccaggcgc
21241 agtggctcat gcctttgatt ccagcacttt gggaggccaa gaggggacga tcacctgagc
21301 caagaattcg agaccagcag cccgtgcaac atagtgagac cttgtctctt aaagattgtg
21361 agactgggct gggcacggtg gctcacgcct gtaatcctag cactttggga ggccaaggca
21421 ggtggatcaa ctgaggtcag gagtttgaga tcagcctggc taacatagtg aaactctgtc
21481 tctactaaaa atacaaaaaa attagctggg tgtggtggtg ggcgcctata atcccagcta
21541 ctcaggaggc tgaggcagga gaatcgcttg tatccaggag gcggaggttg cagtgagctg
21601 agatagggcc gttgcactcc agcctgggca agaagagcaa aactccatct caaaaataaa
21661 taaataaata aataaataaa tcatgagact gagacataac aggaaggagg gcaatttggt
21721 tggttccaag gttcctagag tatgtgatgg gagaggttgg tgcgggtggg gccatggagg
21781 tactgactca agtggaggga caggtgggga aatgggatgg gaaaagaaga ttgaccttag
21841 aaggggagct caacctctga accctaattt cagacccttc aaaatgaata ttaagctcat
21901 tttggtctaa gaaacaaaaa acaaatgaac atgaaactca ttttggtctt ataaggtctg
21961 agaaacccct tctaaacttc aagctgcttt aagaaataac attttattac ctgcaaatac
22021 acacagtact ttggagattt ataatagtct cttattctaa tagaagccat tagggaacca
22081 gtttcaataa acaggtaaat ctgtaagact agtttgtaat taggatatct gtttccagtg
22141 tccattcctg cctctgttat ctaaatgtct gggaacaaga gctgtgctct gctgtgttta
22201 aaatgattaa aaatcaccaa ttagttgagt tcacgtagac aggcatttga cttattgagt
22261 tgttttaaga agactataac aagccttaag ccccccagaa acagcctgtc tttgggcttt
22321 cccacatgcc tcctcgtcct ctccacctgt agatgtaccg tgctctctgt cagagaaggg
22381 agggtgtggt tgggctggac ccccagaggc catccctcct tctgtcttct gctcctgcag
22441 ccctaccact ctcaaacctt tacgagaccc tgggagtcgt tggatccacc accactcagc
22501 tgtacacgga ccgcacggag aagctgaggc ctgagatgga ggggcccggc agcttcacca
22561 tcttcgcccc tagcaacgag gcctgggcct ccttgccagc tgtgagatga cctccgtctg
22621 cccgggggac tcttatgggg aactgcctta cttccccgag gggtgggcat gatgaatggg
22681 agtctgcagt catttcctac tgtttcagga agctttctcc ttaacccctt agaaaaggct
22741 gtggaacttg agctaaaata tgtcttacca ggttgcgtct aatgcccccc gttccctact
22801 gggcagaaag acttgggtgc ttcctgagga gggatccttg gcagaagaga ggcctgggct
22861 cacgagggct gagaacatgt ttcccagagt tgcaaggacc catctcttaa acacagagtc
22921 tgcagcccct aactgacacc ctgtccttcc tcctaggaag tgctggactc cctggtcagc
22981 aatgtcaaca ttgagctgct caatgccctc cgctaccata tggtgggcag gcgagtcctg
23041 actgatgagc tgaaacacgg catgaccctc acctctatgt accagaattc caacatccag
23101 atccaccact atcctaatgg ggtaggggat ccccagccat actgcatggc ccttggtgca
23161 taatgaaccc atttctgttc catgtgtggg ctggtttctg gggtttaagc tgtagacaac
23221 ccaccctctt tgtgcctgct tctccttggg ccctctattc cacagcttgt ggaacccaca
23281 ttttgctact gtgtttgaaa acactgtttt ctcctcccgg ggctttggga ctatgcctct
23341 gttgtgttga ctgctcatcc ttgctgcttc tctgggcaga ttgtaactgt gaactgtgcc
23401 cggctgctga aagccgacca ccatgcaacc aacggggtgg tgcacctcat cgataaggtc
23461 atctccacca tcaccaacaa catccagcag atcattgaga tcgaggacac ctttgagacc
23521 cttcgggtaa gggactgccc tgggtggagg cccaggcttg ggacacattg cctcccaaga
23581 ggggcctagc aggaactctt ctgcaggaga ggtagaggat ggctcctgta ggggaacata
23641 gagcaggttc ccctgaatgc ccttgaacat ggagaattca ttgaccagac attcagcttg
23701 acctaacctg tgaaattctc catcttcttt ataaagtgtt cccttccttg cctcccctgg
23761 aaaggtcagt ggtgtgtggc tgcagcagca cagtgtcctc tgagccctgg acctgcactg
23821 tggcttccag aggtggcagt tcccacatgg ggtactagaa taaatggcct atcaggctgt
23881 gtgtgctttg ggatcacatg tccccaccct aggaccctgg ttccaaccat acgcatgttc
23941 tcttggagcc cagaacagca gagaagccac cagtgtggac acagaagtca agggtctgat
24001 ttccagcctg gcttctgact gctctggggc cgcaggaata cggttccttc ccccatgccc
24061 agcaggcatt tgtcttacaa ctggagggga aggcatgttc ctcttggcaa ggactgctca
24121 ggaggaagtg gaggcaggct gccctgtcag ggtttttgcc ttgattcaag gagaacttcc
24181 taaccacaaa ggatacaagt gggagtgagg cggaccctcc ctagagatct ccaacacaga
24241 gagacaaaca cgctggggct ggctggcact gacaggcctc gcaggtgtgg atggctgtta
24301 gctgggagct tcgctgtcta agctcctctc ccatgctttt cttctgggtt gctcgaagga
24361 cgggggtctg caagaaaatg atgttcccac atagttggca gcacgtgaac agcaattgat
24421 ccctttgcat cacctcctct tactgtttag atttggtaaa tatttcttcc ttccctcttc
24481 tgaccctcca ttttgccgat ctttccttct tataacacat acttactagg tacctgctac
24541 ttcccgggtg ggcctatgtg ccaggagtat agaggtgaac aaggaaggca aagttctatt
24601 ctcagtagag ctaatactct atctggagag agacaacaaa caaatcaaca aggtagccag
24661 gggctgtgat aatttatgtc aagtgggcag gtaaatcggg agtgacagta gtgcagggag
24721 gattggaaag tcagggagtt ctctctggag gaggtggctt ttgatctgca gcctaaagga
24781 tgagaatggg tccattatac aaaatgctgg ggcaagagca cacccagtag aggggagagt
24841 aatagcaaag gctcagggca ggaagggcaa gggagaggcc agtgggtgag gtcacatgtg
24901 aagggcatac aatgggcaaa gacaaggcca gagtggccag gcccaatcct ccaggacttg
24961 cagacctggg aaagagtgca tctccatcct gggagcagca ggaaaccact caggccttta
25021 gaagatcctt ctggcagctg tgtagagaat gggtggtgtg atccttccat gcatgggctc
25081 atgtacgtga ttaccagtaa ctgtcgagtg acagtgtgag gagggctgca agccatgagt
25141 gtaggcacag cagacagact cacctttgtc tggcggtgag atggggtggg aagtgtgcca
25201 agttgacctc ccaaagaaat gatattttag tggaagaatg aatagaatca gagaagcaaa
25261 gtaagaggga agagcagaga ggacagcagg gacaaggact tgggggcagg aagaggaaag
25321 gcaggttaag gacatgaaag atggccaggc tggctggagc tcaggcccag caaggccccc
25381 tgggggccat ggtcatgggt gagcttgggt ttggcttctg ttttcgtctt gggcttctgt
25441 gaaagcctcg agcccttgcg gggaaccagt gaagctgtgt gtgcatcttc tgtggggagt
25501 gccagagtct tcagggagca ctccatcttc tctcctcccc acaggctgct gtggctgcat
25561 cagggctcaa cacgatgctt gaaggtaacg gccagtacac gcttttggcc ccgaccaatg
25621 aggccttcga gaagatccct agtgagactt tgaaccgtat cctgggcgac ccagaagccc
25681 tgagaggtga gcatcctttg gctcctgctg ctgcctcatt tgtgcagcta gattgagccc
25741 aagacctgct ctggtccaag atgaacatac cacctgccat gaggtgaccc tcaggatatc
25801 cactgcagcc atgggctggg gtcatcctgt cctgttgctt cagctaaccg tgtctctagc
25861 agccacacta ctctgagggc tgactacaga atccagcagc ttttgtctgg gagagctgga
25921 ctgaagagag gcatagctgg agacccatag ctggccctgg ccagaaacag ggagagtgaa
25981 aggctggaat agccaaggcc agagcaaggc taataggtag agcaacagct tacaggtgtg
26041 ggggtggcag atactggcac ccttgaaatg gattcctcat gcccacgctt cactattctt
26101 ctctgtggct aggggattta tggataaacc aaaattacag ttaaaaacca gccataggcc
26161 aggcacagtg actcacgcct ttaatatcag cactttggga ggacaaggtg ggcggatcac
26221 ctgagatctg gaatttgaga ccagcctggc caacatggcg aaaccccatc tctactaaaa
26281 atacaaaaat tagctgggca tggtggtggg cacctgtaat cccagttact caggggctga
26341 ggcaggagaa ccacttgaac ccaggaggtg gaggttgcag tgagccaagc ttgcaccact
26401 gcactccagc ctgggtgaca cagcgacact ccgtctcaag aaaaaaaaaa aaaaaaacag
26461 ttatagtagt caacttttga ctctccattt cagatttcgt catgccctcc tcaatgagct
26521 gctaagttag gcagtgcatt gattattgct gcaggagagg gaaggaagga gctaacgtgt
26581 tttcacatgt tttccttttg gagatgagaa aggaggactc tgccttcccc ctaccctgcc
26641 cctttctact ccaggacctc tgaaaggcca tgagcacaaa gctgctgcct gagtcccctg
26701 aaatgcaggg tacgccccag gtctctgatg taccccacca cacttttcct ctcaaacata
26761 ttccaggatc acttgatttc ttttgaatct atttaaaccc accgtgtcaa tgtgctatat
26821 aaaatgtcta atgcatttca gacaccctat acatctatac atttaaagtg ttctccttct
26881 atctgtgcag ggatgggaaa gggcatattt ctgaaagcac agatgggaag acgggatttg
26941 ttccgtgtcc aggtgattat ggtacctcta tgcgcctggc cggcactggg gacagaggcc
27001 atgaaaatga atacagcaca gcctttgcct ccaagaaact taagacctag tagaaatggc
27061 aggctttaaa acaggttgtt gggatctgat ttggtgagtg caatgacaga gatactcaca
27121 gcacaaaatg gggaatgagg gcgggcattg ggacacacat agccttaagg ggcccaaagg
27181 cttttagaac tgtattccct attaaaacat gatttgcaca gagcacattc tttgctttgg
27241 agacctcaga actccttact ataggccggg catggttata atcccagcac tttgggaagc
27301 caaggcgggc agatcacttg aggctgagag ttcaagacca gcctggccaa catggtaaaa
27361 ccccgtctct actaaaaata caaaaattag ctgggtgtgg tggtggccac ctgtaatccc
27421 agctactcag gaggctgagg taggagaatc acttgaacct gggaggcaga agttgcaata
27481 agcccagatc atgccactgc actccagcct gggcaacaaa gctagactct ctcaaaagaa
27541 aaaaacaaaa caaaacaaaa caaaacaaaa aaaactcctt attataaact gtaagaaaaa
27601 aaaggcccct acttcgtccc ttttgcaaat ctgccttttc ctactcacta accagctggt
27661 tcagagcaag gacactctgt ttggtgccat cgctgcagac tggaaggaag aggtccttgc
27721 cccacaccca acagtctcct gctgttaccg gcaggttggc aggcaggcag gcgagaagca
27781 gccagggctg gtggtgtgtc cagtttgaag actagtttcc agccctggcc ctgctcaccc
27841 tccaagtggc cctggcaggt tcctctacca catcgtggac ttcaccttcc ttctctaaga
27901 agctcaatcc ccaaggcctc attcccatag gccttctcac cctttttctt tccctctggc
27961 tgaatgtggc cagcacgggc ttccaaggcc atcaactcgt ctgcagcagc cccatgcctt
28021 gcagggcctc agagcttcct cctgcctatg acagtgtggt tttggttccc acacttggga
28081 tcagattgaa actcgcctcc gtggtgagaa tatgggacat agagcctcgg tgaccttggt
28141 gagcagcagt ccaggccacc tgctcagcct ggggttgggg ggggctcctc ctccttgact
28201 ggtccttgca tttgcctcca tccagcctgt ctgggctctc cgaggcaatg gagaccagca
28261 ggagtcacga tgggtcagga gccccctttg ggcctcagcc ctgccctgcc ccctaaagta
28321 gcacttggat aagcaaataa attattatac ttactattta tgggtgtggt gaatgggatg
28381 gcaaaggcca agtcttactg atcaccaaac cttaagatat atcctggcag ctagtagacc
28441 cttgggctaa atgaacagaa aactggacaa ataaagtgta cacaaataac tcaaagctgt
28501 catttgtaca cttttcgtct tttcctacta cagtttacat ttttataaag gtgagtagat
28561 ttctaaaatc ccgtggtagg ctctcttgag tttttcttgt atccctgaag ttcagctaca
28621 aataagctaa tcactaacat ttgttgagca tttactctgt tgtcaggccc cgtgccgagt
28681 gctttaggtt cagaatttca tgtcatcccc acagcagccc taggagatga atgcaattct
28741 tatgtccact tgactgataa ggaagttgag gttcaaagag gctaaatgac tctcccaggg
28801 tcccacagct ggaaagtggc cacagggccc cagctggttt tctagggcag caggcagaag
28861 gcgaggagga tctgggccct gtggtgcccc agcctcatct gagggtcctc atctgagaga
28921 acaggatcct cacagcatgg gcaggctgca agtggtccct gaggttatcg tggagtggac
28981 cctgacttga cctgagtctg tttggacccc agacctgctg aacaaccaca tcttgaagtc
29041 agctatgtgt gctgaagcca tcgttgcggg gctgtctgta gagaccctgg agggcacgac
29101 actggaggtg ggctgcagcg gggacatgct cactatcaac gggaaggcga tcatctccaa
29161 taaagacatc ctagccacca acggggtgat ccactacatt gatgagctac tcatcccaga
29221 ctcaggtagg ccaggcctcc gggggccttg gccctgcctg gcccaccatc tcttctgcca
29281 tcctttgtgg cgggggaggg gaaattcaga gatctttggg cgacttccct gcctggaccc
29341 agctcacagc ttctcggcca ctgcaaatgt gtgggttgtg accagactga tgtgtcttga
29401 gcttcaggct tgcaagtgca gtggagaggc agtggggagc tattgaaggg gtctggggac
29461 agactcaatc acagaggcct ttcagaagat ctgcctgctg tgcatgggca aagagggcca
29521 cttgctgacc tcagagcatg tgctttctca gtagtgccca agctgtccca tggtcactga
29581 cccagttaga atgactgaat ggactttggc ttgtgtctca ttaggaatcc tagccccatt
29641 ctagtcttcc agtgagatct gtccatgagt gaaggaatct cacaggaaaa aacaaaatgc
29701 ttctatgggt gtggttgctg gccttatcta caccacagaa gccatcacac agactgtctt
29761 tcttcccatt gttagaatgt gccctgacca agcagcccac agggcctggg acagaggctg
29821 atctctgcct aactgagctc acctctcctc cctctcctcc tgactggtta gattttctag
29881 gtgactgttc ccctgatgac acaagcccgc tgggccccag cagtgtttag aggggttgtt
29941 gactcacgag atgacattcc tgctgatgtg tgtcatgccc tggggtggat gaatgataaa
30001 tgaaaacagc gcttttaact tttgaaccca ctttctcctt ccttgtagcc aagacactat
30061 ttgaattggc tgcagagtct gatgtgtcca cagccattga ccttttcaga caagccggcc
30121 tcggcaatca tctctctgga agtgagcggt tgaccctcct ggctcccctg aattctgtat
30181 tcaaaggtaa catggggaag gcatccctgt tagattgtcc ctggaggcag cttccccacc
30241 cctgtcacct ccacaacact ctccgattta cagcacccca tgggacatta gaacttccac
30301 tcagctcaac caaaagcaga tgtgacttca gcagaaactt cagaggctct gttgtttcat
30361 taggcagtgc agagaatgcc tttggggagc cgttcctcag aactcaagac ttgacatctg
30421 ggaggcagcc gttcctcaga actcaagact tgacatctgg gagagcagag cattcccttg
30481 cctttctatt tgcagggtca cttgccaatg tatagtcaag aggtcagagt gagggtacag
30541 ctgagctgca gccccaggaa ggcagagaag ggggccaagt tgtgtgcgtg cctgcccttc
30601 cctcttaggg caaaactcca aacacccttg attatctgga tcttctttaa ttctccatag
30661 aagataccag atgttaagga atattggcag cttcacttgg tttctcaatc cctgtttcca
30721 aactcaagga gggatgggct ttttcactgt atttatctct catcactctc ttcattgcag
30781 gagcacatct ctctggacct aaccatcacc ctttcttgta gatggaaccc ctccaattga
30841 tgcccataca aggaatttgc ttcggaacca cataattaaa gaccagctgg cctctaagta
30901 tctgtaccat ggacagaccc tggaaactct gggcggcaaa aaactgagag tttttgttta
30961 tcgtaatgta agttctgggt cctaaatcat gctcctggga agctccttac tgtgggactt
31021 gtattagtgt aaaaaaaaat gtcctcaata agcaggagtt tgcatgagaa ctggttgctg
31081 acaaggaagg aaataatttc tggaaaatat agataacaaa atgagatcct gcagaaggat
31141 tggaatctct ttttctggag gcctttgaga ataaaccaca caattatcca acctgtattg
31201 tgaaggaata agtccttctt gaattcagga attaacacct gggaggaggg atggagttca
31261 gactctttct gagcttatga gaagagaagc cccctaaact aaaatacagc cctccttggt
31321 ccaaaaggtg ccttctctct tctgctgtat cttctttgtt ttcaaaccca acagttaccc
31381 tggaaatcaa aaaggaagta caactcaaca tagctcttgc ctgggaccaa ccagcaccat
31441 ttggctaaag atggttatca tctgttaaac aaagaaataa ataaatgggt tcaacgtatt
31501 tatttcaaca ttgtcaatgg acctcatgtg taactgatat tctcattatg ggacctctgt
31561 gtgactttat tggggcctct ctaaccgttc tttccttaag gaagaccatt tattgtttta
31621 tttcctggag aaaatacatc attttatccc agccttaata acccatccca gtgtatactc
31681 cttcatcttc atggataatg accctgctac atgctctgaa caaatcagga ggcccctcgt
31741 ggaagtataa ccagtccttt ctttctctgt ccctcttctg tgcagagcct ctgcattgag
31801 aacagctgca tcgcggccca cgacaagagg gggaggtacg ggaccctgtt cacgatggac
31861 cgggtgctga cccccccaat ggggactgtc atggatgtcc tgaagggaga caatcgcttt
31921 aggtaattag ttccatcccc gggtggagct tctgcccagt ggtcatgctg gagtgggatg
31981 tggggcccca gctatttgtc aagctttctt ctaccttggg gattcaatta acactagcag
32041 tgcactgctg cgaccttcca gacttgggat ggggaaaagg caagggtcgc cttgaaagct
32101 tacattggga agaagggtta cttctaagag tgtaatcttc acatgcatgg gaagcaggga
32161 ggggggacta catttttatg actgaagtgc aaggaaaaca tcaccctctc attgtaaagc
32221 tccaagtgag ccaagagcac atagtttaca gtgcacgatg agcctctcac tctctgcgca
32281 gtatctgttt attgcaactg aagcaccctt gtgagtttgt tttcttgccc ggctatctcc
32341 atttctgact tgctcattca ccttggggtg ctgtcatatt gaatgtttcc ctgtcactga
32401 cttcagccac ctgcacaagg gcttggagac cacacccctc tgccctccca gaatcatatc
32461 cctggaggct cagctagtct ctgggtcagc catacctctg ccctttcttt tccctccttt
32521 ctcctgtggc ctctgacgtc tggccattta acagagctta gcatttttgc tgggtggaga
32581 gagctggagc ctggaatcac tccctctttg tgcatacgga gggcatgaaa accaaggtgt
32641 gtgcattcca gtggcctgga ctctactatc ctcagtggtg aggtatttaa ggaaaatacc
32701 tctcagcgtg gtgaggtatt taaggaaaat acctgttgac aggtgacatt ttctgtgtgt
32761 gtatctacag catgctggta gctgccatcc agtctgcagg actgacggag accctcaacc
32821 gggaaggagt ctacacagtc tttgctccca caaatgaagc cttccgagcc ctgccaccaa
32881 gagaacggag cagactcttg ggtaaagacc aacttaagta cacgtctcca tttttctaaa
32941 gtagtgatcc ctcagggccc cagcagcaaa cagttggcac atcaaggatt gacttgaagg
33001 gattttatga caagactatt agtgaaagag tgggcgggac taaaggaact agcaaaggat
33061 gaggccaacc agggactagc aaccctggga agcctttact acccctaggc ctgggggaat
33121 gggaggatga gagcaggaac cagggaggtc atgagccttg gacaagggca cagaacagca
33181 gccagagcca tgtgcagcca gccactgtca gaaccatgca agggggacca ctcagcgccc
33241 cagcctccct ctcagacagt tgccatctgg gtctcttgtt ggctgatgcg agagcaggag
33301 ggagcccact gatgcagttc atagagctca gcctcctggg caggaaaccg ggcagagagg
33361 agtagaaaag aattaagggt ggctgcgacc agcccagtca ctgaggcacg tttcccactg
33421 gagacctatg agcacagtga taataaagcc agttacctgc actgactatc cctccagaca
33481 aaagctttcc caagaagtta gtcatggctc tgagagatct agttgaggat gtttggcagg
33541 ggatctagtg gttacgggtg gctaagaaaa atgaggaagg taagagtatc ttgcagcctg
33601 tgttgggagg attaaatagg atgccacaca cagggccagg cagacagcct ggtcagtaat
33661 agccatgacg atgggggcgg ggggagcagg aatgggagtt gcagtgttta gctcagatgc
33721 atgcctgtga gagatgcttc cactctcaca gaaagatgag accaaggaaa aggaggagga
33781 agaggaagga ccttgacaaa ccttggggcc cacattgtct acacctccct tcctgctcta
33841 gagcagaata gaaagttcag gttgcaggca gctctaagtt gaattcgtgt cctgtttaat
33901 tttctttatt gctaaatgaa tgcctgtgtc tgtgatgctg acgtatgttc ctaaggagag
33961 gggagaagtt cattctgaac ataaactttt catcctctct ctgtccagca agaatggaat
34021 attccccaag tggcctgagc cagcttggct ttctttttgt tttcaattat gtgggagttg
34081 aggaggggga tgggaaaagc ttcccaaaca caccctcccc caggcctgag gcacccctgg
34141 gggacagaga gtgttagagg ttggtacagg tgttagagat attgaaagga catcccatgc
34201 accccagggg ctggtgtggc tctgtacttc caggcaatat tttgtggaag gggaaccttg
34261 tcagctccag gttgtggatg tttgaaaatc agttggtacc cagtggctcc atcctctggc
34321 aggcatgtgg atttgtcaat aaccaagtga actctccaaa ataagttaaa acttcctccc
34381 ttctcagttt caagatgctg gaaatagctg ttcataagcc ctggggaaat ttagcccttt
34441 ggctggtaat gggagtatcc gagatgagag ggcagctgga aactttcgga atgacctccc
34501 acacttaatt tgggaaatgc ctctgcacct ttatgggcaa ccagatgcct gccccagttg
34561 ctggagacac tgatgtgggc tgaaaggaat gctgagacgt gacgaggaga gatgctgcgg
34621 agggaatatc cccctcagcc ctgacctcat cggctccatg gctcctccac agtacagctg
34681 tctactcttt taagttctcc cttcaggaaa tagccatctc aaacagaatg tgcatttgag
34741 ggcagaatgt gtaaatattg cactactgtg ttataaccgt caggagccat gctgatgatg
34801 aaacgtccca gatgccggtg ctggaaaggt ccctggcttt ccaagcaaat atttatctca
34861 tggaaacatg agtcatactc acagaggagt atggattaac tccttctcag cagccaggga
34921 gcccagcatc ccagacagca tatttaaccc agaggccaac tgactgctgg ggcagatttg
34981 tggtcatgaa catgtgcttt gtgtcctctg accattagac agattgtggg tcacaacgtt
35041 gagtatacag tgggagctta ataagtgctt attccctggg cagggagttc ttcatttcag
35101 gggtgaccac ttacatcttc tcctctgggc cctccttgac caggctaatt accattcttg
35161 ggattaactc tatctccttt tcccgcaacc tgcaggagat gccaaggaac ttgccaacat
35221 cctgaaatac cacattggtg atgaaatcct ggttagcgga ggcatcgggg ccctggtgcg
35281 gctaaagtct ctccaaggtg acaagctgga agtcagcttg gtaagtgtcc tgcaaatcaa
35341 aggctggcta aatttcccca gggcagggct ccaggacata tctcaccccc aggatggaat
35401 tatacacaca caaccttcaa gttgcagccc gaatctctga gtgtaattcg tccaaagaaa
35461 aagagaaaag agaagagggt cttcagggaa atcaagtgag atcatagtta gacatgagta
35521 agaacttcca gatttacaag ggaatagagc atctgatttg gcatctgaga gaggctatta
35581 gatcttcctt ctcttaagga ggttgtaggc aactagttat gtgactgaag agatcagtct
35641 gtactcacac catcccaccc cccaaaccca gggcttcact gagttgtacc atgaaccaga
35701 ccatcccaag aggctttttg agttctgaca cttgctctgt gagccttccc ttgctctgca
35761 cattgatgat ataactttgt aactgcacta agagtgttcc taaagcagat agccagccga
35821 gctccagaaa tctccctggc tgcacctgca gaggccactg acccctctgt ggagggaccg
35881 ctcttcagtg tgtggctggc ttctactctc tgctcctctc tcttggtctt cagccatcca
35941 ttgctcacca gtttctcacg aggagcatag gaagatatgc atgtagggag gtaggcacgg
36001 ggatgacttg tttgacttta gcaggtcatt caagaatctc ctcgcacctg gtttcagatg
36061 ctggggtcct gtctgtcaca ggcttctgtg cctcctaccc ccttgagttt gtcacatggc
36121 ccttcaggaa ggcctgagat agatttgccc tgggtgggcc tcctatgaga aaatcttaag
36181 tgaggcaccc aggcaaaatg gaaagagcct tttgcccaga gcaggaagcc tgtcttccat
36241 ttccagctgt tccacctact tagcttaaaa gaggcacttc gcctgtcttc agtctcagtc
36301 tcagtctcct cttctgtgga atgggacaat aatatctact ctccttatca tacactgctg
36361 tgaggactga gtggatcaca caaaaaagca ttatgtaaat tgcaaagtgc taaatccaca
36421 caggagattt gaattaatcc accacactga aggtctgtca agggcaggga ctgtttcatt
36481 caccagagta tccccagtct aacacaggac ttggcatatg aaaagtgttc agtaggccgg
36541 gtgcagtggc tcatgcctgt aatcccagca ctttgggagg ccaaagtggg cggatcatct
36601 gaggtcagga gttcaagtcc agcctggcca acgtggtgaa accacatctc tactaaaaat
36661 acaaaattag ctgggcgtgg tggcacatgc ctgtaatcac agctactctg gaggctgagg
36721 caggagaatc acttgaaccc aggaggcgga ggttgcagtg agtcgagatc atgccactgc
36781 actccagcct gggcgacaag attgaaactc catctcaaaa acaaagaaca aggaaaaaaa
36841 cgaaaactgt tcagtaaaca cttgctgaat gaataaaata aatatataaa tgtataaata
36901 aatgctctac tttcaaccac tactctgttt ttcttttaga aaaacaatgt ggtgagtgtc
36961 aacaaggagc ctgttgccga gcctgacatc atggccacaa atggcgtggt ccatgtcatc
37021 accaatgttc tgcagcctcc aggtaagtgt cgcatcccca ctgactctgc agccagtcct
37081 tttcttcatg tggcagttgg tggagagaag aaaaactgtt ctaaacaatg atgagaataa
37141 catgtaattg tgatagttaa actgtgccta tgtgactgat tgcagagtga attgggagct
37201 gttggttttg aatgcaccac actaaggaat gtgaggacac attgctcttt gcggagttgc
37261 ccagctatat tagctcccct cggacacagc ccagttttct gtattcgcgt ggatgctgtc
37321 cgcgcgattc ccagcactcc tcttacagca tctcacctca gtgtatgttc cttgcctcca
37381 gtgcagttga acctcagtcc tgcctctcct catgtgtgca ttcacctttc ttggtgctct
37441 ctccccatgg gccaagttct accatgagtt atgaaacatt atggagaaaa catgtctttg
37501 gaaatgtgag ccagaaagcc caccagtgcc cctcagtcac ggttgttatg aatgacatgc
37561 taatggtttc actctggtca aacctgcctt ttctttcctc ttcagccaac agacctcagg
37621 aaagagggga tgaacttgca gactctgcgc ttgagatctt caaacaagca tcagcgtttt
37681 ccagggtaag atgcctgcta ggtttgcgcc tagcctgagc agcctcaggt cctctgtttg
37741 ggccatagag gagcctctcc agcccctgtc ttccttggct gctccccagg gctctcttaa
37801 aacttctccc cactcccact gaggcatcct cagccccagc ctgtgtcaaa ttcagagtaa
37861 agaaccaagg caactccctg gctttcatgg gccaaagcgc aggctttcac accgaggcct
37921 ctgagcctca gatcatgggg aagtcactgc tggagagaac agacatagct ctggaagcca
37981 tctgcccaag agggcagccc atcccaagtt catcttacag tggccaggcc tgccctgagc
38041 cggggcctct gggtcactct tctgctgtcc atggcattgc ccatcctggg tgaggctggg
38101 gctctcctgg gcactgtatg tattctggat acagggatac tgggctcgct atgtgtgtgg
38161 agccatccct tccttgcccc agccccacct ccctctcaaa ccctctctgg ctctttctga
38221 gcttcctttc ctgctcccca gcttgcccag tgctcagtgc cccacttggc tcttttgcta
38281 cttcgggtca ggtggagcct cttgggaatg tgaagtgcct tacagaaaga ttgcacttca
38341 agaggagagg ctgcagggag ccatcctaaa cccagaggcc tggagcttac tgtgtcactt
38401 tacttttgta cacaggggtc tccttagtgc cctcgagaag gattcttggc cctgagcttc
38461 tactcctgag gccacctctg tgcagcccca gctccctcaa ctctaggctg tagtctcagt
38521 gggaaagcct ggcttggggg tctcctagga atgtccacct gaaggcacac ttgatagggg
38581 cttgcacaac ttatgtctgc caaggccacc tgaggaactc cctggtgcct ataagttcca
38641 ccttcccctt cctcttcctc gccccagcat tttttctgag taggggtggc aatgggcaaa
38701 gccattgtca taagcagttg caggtataac tttcactaga aaacctgaca ccttgtgttt
38761 tctttcaggc ttcccagagg tctgtgcgac taggtgagtc tggtctgggt ttgaagtcat
38821 tgcagacctg tttaggcctt acccccaagc aagcccaagc ctgccatctg ctgtatatag
38881 ataagaacat catggtgcag taaaagaagc ctggcctttg gagtcagaac agcagggtga
38941 cttggggtca gacccagagc accccatttc cttctctgta agatgaggat aataagagta
39001 acaacctttt agggttaagg tgagttttca gcttaggaag tctgggaata ttgcaaaggg
39061 cttggcagga acccatggtg aggatctagt tccaagttga taggtacaga aaaccagaac
39121 atcgggcctt gagtaaagag tgaagtttca caaaccacaa agcacctgct atgtgcagga
39181 gagcatggca gaaggaggct gcttggccct ggtccttgag attctgacag tgtcctagac
39241 agacatgggg agatctgcac ctatttgacg ttaccaactt ctctttttca gcccctgtct
39301 atcaaaagtt attagagagg atgaagcatt agcttgaagc actacaggag gaatgcacca
39361 cggcagctct ccgccaattt ctctcagatt tccacagaga ctgtttgaat gttttcaaaa
39421 ccaagtatca cactttaatg tacatgggcc gcaccataat gagatgtgag ccttgtgcat
39481 gtgggggagg agggagagag atgtactttt taaatcatgt tccccctaaa catggctgtt
39541 aacccactgc atgcagaaac ttggatgtca ctgcctgaca ttcacttcca gagaggacct
39601 atcccaaatg tggaattgac tgcctatgcc aagtccctgg aaaaggagct tcagtattgt
39661 ggggctcata aaacatgaat caagcaatcc agcctcatgg gaagtcctgg cacagttttt
39721 gtaaagccct tgcacagctg gagaaatggc atcattataa gctatgagtt gaaatgttct
39781 gtcaaatgtg tctcacatct acacgtggct tggaggcttt tatggggccc tgtccaggta
39841 gaaaagaaat ggtatgtaga gcttagattt ccctattgtg acagagccat ggtgtgtttg
39901 taataataaa accaaagaaa catacgtcct gtgtgcatgg tacagtgtgc tgacctgagg
39961 ccgtcatgct cctccacacc tcaattctgc tctggagaag ctcagaaagg agccccgagg
40021 gatggttttg gggagattcc agcagccagc cctcagacag ccagacagct catgggggtt
40081 tgagcctgtc tttgccaaac aggtttttat ttcaccctcc tccggtcctg gggtttcaag
40141 ttttcagtgt tgccttcacc ccgcacttta ttcctcttat tacttggaag taccttccct
40201 ccagcatggt gatcccctgc ctgtgtgctg gacttttgag tcctcagcac caacctgtga
40261 agtggttgcc agcataatcc cattatgcag atgaggagac caaggcccag ggaagggaga
40321 accaccagca gcacgtaaaa tagctgagct gggactggaa ctcacacctc ctgactctca
40381 gtgaccacca ctgacaacag cataagtcca ggttttccag gcccatcccc tctgtgccaa
40441 cccacattca gattccttcc ccggctcccg taatctctgg catctagaat atcctcagga
40501 ctctgagagg tgatatcatg tggttgtggt gccattgccc cctacctgtg tggcctgggg
40561 ccagtcatgt gacctcccag ggtctcctct tctgtaatag ggagatgacc gtcacatcta
40621 cttcatgggt ccatcgtgag gatgaaatga gatgatctat ataaaatgct tggtacaaca
40681 ttaggtggcc ttatttttat cctgccgtct gggactgctc aggatcaatg cgccagagag
40741 cctttatttg tgtctttccc acaggtgggc tggcccactt tcctagagaa tgggacagac
40801 ctccttccca cccacaccca tctctgccaa ggctgattca ctccagcagg cggagctcat
40861 ttcacttcat ggaaccaatg acccaaagat atatccccag cactactgct ggtcagtcca
40921 ctgctgctgg gaatacagca atggtagtgg cagacagagg ccctctctta aatagcttcc
40981 agtctgagga aagagagata tgacatcaat ccattaaaat cattcatcca ttggttccac
41041 aaatatttgt tgagggctac ctatgtgcac ccccatgtta gaccctgggg aatagacatg
41101 tcattctcat gaggcttctc tactgatggg ggggaagaga attgtcaacc agataatggc
41161 actacagcct gtgtgttctt agtgactctg aggatagcac tgtggttctg tgacagataa
41221 tgaaggattt ggaagcagga atgcccagga gctcccagaa gtgggaagag atgagaggaa
41281 tggaaggaac ttacctgaag gtgaaggcat caggctaggg gaccaaggga gaaggtgtcc
41341 tgagaggtaa ggcttaacct tgggtgtgaa ttcagttccc gtcactctcc catagctctg
41401 tcctgctgtt cccacctccc ctgcagccat gcgggcttgg gcggctagtg agggccttgc
41461 tcatgctggg tatcctatgc tatgcttcac tttgagcacc taaaatacac acactgcact
41521 ttaccaagat gacctcggaa accaaagagg tgatcagcat aagttttaaa gacccttaaa
41581 tttaaagtaa aaatcactac aggatccatt ataaatgcca aacactaaga tgtgtgtttc
41641 cagttctccc cttcatttgt ccctgccact ccctgccctg actttgcccc accccctagt
41701 aatgtgggct ccactctatg ctccaaactc tccctggaga gaaatcctcc ctgtggttga
41761 ggacaaggcg cagccttccc ctcccaccaa agaaggtcag attccctttt ttggttccta
41821 accatccata ccccttcttt tctcatgaag actcgggcta agcattcatt agggctgcca
41881 tctggaggat ggacccttag agctgagggg ccagcactgt gtgt
MALFVRLLALALALALGPAATLAGPAKSPYQLVLQHSRLRGRQHGPNVCAVQKVIGTNRKYFTNCKQWYQRKICGKSTVISYECCPGYEKVPGEKGCPAALPLSNLYETLGVVGSTTTQLYTDRTEKLRPEMEGPGSFTIFAPSNEAWASLPAEVLDSLVSNVNIELLNALRYHMVGRRVLTDELKHGMTLTSMYQNSNIQIHHYPNGIVTVNCARLLKADHHATNGVVHLIDKVISTITNNIQQIIEIEDTFETLRAAVAASGLNTMLEGNGQYTLLAPTNEAFEKIPSETLNRILGDPEALRDLLNNHILKSAMCAEAIVAGLSVETLEGTTLEVGCSGDMLTINGKAIISNKDILATNGVIHYIDELLIPDSAKTLFELAAESDVSTAIDLFRQAGLGNHLSGSERLTLLAPLNSVFKDGTPPIDAHTRNLLRNHIIKDQLASKYLYHGQTLETLGGKKLRVFVYRNSLCIENSCIAAHDKRGRYGTLFTMDRVLTPPMGTVMDVLKGDNRFSMLVAAIQSAGLTETLNREGVYTVFAPTNEAFRALPPRERSRLLGDAKELANILKYHIGDEILVSGGIGALVRLKSLQGDKLEVSLKNNVVSVNKEPVAEPDIMATNGVVHVITNVLQPPANRPQERGDELADSALEIFKQASAFSRASQRSVRLAPVYQKLLERMKH
Claims (21)
- 対象において角膜ジストロフィーを検出するための反応混合物であって、配列番号25〜30、36および54からなる群から選択される変異ヌクレオチド配列を含む標識プローブを含む、反応混合物。
- 配列番号19〜24、33および50からなる群から選択される正常ヌクレオチド配列を含む、対応する標識プローブをさらに含む、請求項1に記載の反応混合物。
- 前記標識プローブが、配列番号25〜30、36および54からなる群から選択される変異ヌクレオチド配列からなり、および/または前記対応する標識プローブが、配列番号19〜24、33および50からなる群から選択される正常ヌクレオチド配列からなる、請求項2に記載の反応混合物。
- 配列番号36のヌクレオチド配列を含む標識TGFBI G623Dプローブおよび配列番号30のヌクレオチド配列を含む標識TGFBI M502Vプローブを含む、請求項1〜3のいずれか一項に記載の反応混合物。
- 前記標識プローブが、蛍光標識されている、請求項1〜4のいずれか一項に記載の反応混合物。
- 前記標識プローブのそれぞれが、異なるプローブを含み、そして独立してVIC、FAM、ABYまたはJUNで標識されている、請求項1〜5のいずれか一項に記載の反応混合物。
- 前記対象由来の生体試料からのTGFBI遺伝子配列を増幅するために、少なくとも1つの増幅プライマー対をさらに含む、請求項1〜6のいずれか一項に記載の反応混合物。
- (a)配列番号7〜12および41からなる群から選択されるヌクレオチド配列を含む対応するフォワードプライマー、および
(b)配列番号13〜18および47からなる群から選択されるヌクレオチド配列を含む対応するリバースプライマー
をさらに含む、請求項1〜7のいずれか一項に記載の反応混合物。 - (a)請求項4〜8のいずれか一項に記載の反応混合物と、(b)前記反応混合物とは別個に、R124S、A546D、H572RおよびH626Rからなる群から選択される1つ以上のTGFBI変異のための1つ以上の標識プローブとを含む、反応キット。
- 前記1つ以上の標識プローブが、配列番号19、25、20、26、21、27、23、29、50および54のヌクレオチド配列を含む標識プローブからなる群から選択される、請求項9に記載の反応キット。
- 配列番号19または25のヌクレオチド配列を含む標識TGFBI R124Sプローブを含む、請求項9に記載の反応キット。
- 配列番号20または26のヌクレオチド配列を含む標識TGFBI A546Dプローブを含む、請求項9に記載の反応キット。
- 配列番号21または27のヌクレオチド配列を含む標識TGFBI H572Rプローブを含む、請求項9に記載の反応キット。
- 配列番号23、29、50または54のヌクレオチド配列を含む標識TGFBI H626Rプローブを含む、請求項9に記載の反応キット。
- 第3の増幅プライマーセットをさらに含む、請求項8に記載の反応キット。
- 角膜ジストロフィーを検出するための方法であって、
(A−1)少なくとも第1の増幅プライマー対および少なくとも2つの検出プローブのセットを含む反応混合物を使用して、対象由来の生体試料からの第1のTGFBI遺伝子配列を増幅するステップと、
(B−1)前記少なくとも2つの検出プローブのセットの第1および第2の検出プローブを、G623D変異を有する第1のTGFBI遺伝子配列およびM502V変異を含む第2のTGFBI遺伝子配列とそれぞれハイブリダイズするステップと、
(C−1)前記第1および第2の検出プローブの、それぞれ前記第1および第2のTGFBI遺伝子配列とのハイブリダイゼーションに基づき、前記TGFBI遺伝子配列における1つ、2つまたはそれ以上の変異を検出するステップと、
を含む、方法。 - 前記反応混合物が、請求項1〜8のいずれか一項に記載の反応混合物を含む、請求項16に記載の方法。
- (A−2)前記生体試料からの第3のTGFBI遺伝子配列を増幅するステップであって、前記反応混合物が、R124S,A546D,H572RおよびH626Rからなる群から選択される第3のTGFBI変異のための第3の標識プローブをさらに含むステップと、
(B−2)前記第3の標識プローブを、前記第3のTGFBI遺伝子配列とハイブリダイズするステップと、
(C−2)前記第3の検出プローブの、前記第3のTGFBI遺伝子配列とのハイブリダイゼーションに基づき、前記第3のTGFBI遺伝子配列における変異を検出するステップと
をさらに含む、請求項16または17に記載の方法。 - 前記第1のTGFBI遺伝子配列を増幅するステップ(A−1)および前記第2のTGFBI遺伝子配列を増幅するステップ(A−2)が、別個に実施される、請求項18に記載の方法。
- 前記ハイブリダイズするステップ(B−1)および前記ハイブリダイズするステップ(B−2)が、別個に実施される、請求項18に記載の方法。
- 前記検出するステップ(C−1)および前記検出するステップ(C−2)が、別個に実施される、請求項18〜20のいずれか一項に記載の方法。
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