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人類特發(fā)性基底節(jié)鈣化致病基因及其檢測方法與流程

文檔序號:12644725閱讀:208來源:國知局
人類特發(fā)性基底節(jié)鈣化致病基因及其檢測方法與流程
本發(fā)明涉及基因及其檢測方法,特別涉及人類特發(fā)性基底節(jié)鈣化致病基因及其檢測方法。
背景技術(shù)
:人類特發(fā)性基底節(jié)鈣化(IdiopathicBasalGang1iaCalcification,IBGC)是一種以影像學(xué)上基底節(jié)及腦其它部位對稱性鈣化為特征的神經(jīng)系統(tǒng)變性遺傳病,也稱Fahr病。鈣化最常見的部位為基底節(jié)區(qū)豆狀核,尤其是蒼白球,也常見于殼核、丘腦、尾狀核及小腦齒狀核等部位,甚至出現(xiàn)在大腦皮層及皮層下白質(zhì)。IBGC屬罕見病,呈散發(fā)或家族性發(fā)病,家族性患者發(fā)病年齡逐代提前,進展緩慢、病程較長。該病無明顯性別差異,表現(xiàn)為復(fù)雜多樣的運動損害和行為癥狀的組合,同一家族中的不同成員之間臨床癥狀往往存在差異。臨床表現(xiàn)以神經(jīng)、精神功能紊亂為主,輕者僅僅為輕度注意力或記憶力的下降,重者則會出現(xiàn)人格及行為改變,終致精神病或癡呆。IBGC有家族性發(fā)病的現(xiàn)象,常染色體顯性遺傳和隱性遺傳的病例均有報道,以常染色體顯性遺傳居多。遺傳性IBGC相關(guān)致病基因和發(fā)病機制長期以來并未得到解析,直到2012年Liu等人在IBGC3基因位點中,通過連鎖分析、單倍體分析、Sanger測序等成功克隆特發(fā)性基底節(jié)鈣化的致病基因SLC20A2,并且在不同種族中得到驗證;此后,PDGFRB、PDGFB和XPR1這三個致病基因陸續(xù)被發(fā)現(xiàn)和得到驗證。其中,SLC20A2基因編碼的蛋白為鈉-磷共轉(zhuǎn)運體(PiT2),參與機體的鈣磷調(diào)節(jié);PDGFRB基因編碼血小板衍生生長因子受體PDGFRβ,屬于III型受體酪氨酸激酶家族;PDGFB基因則編碼血小板衍生生長因子PDGF-B,與PDGFBβ結(jié)合可激活下游信號通路,參與細胞的增殖、分化、生存以及遷移;XPR1基因編碼的蛋白含有SPX結(jié)構(gòu)域,主要參與磷穩(wěn)態(tài)的維持,具有Pi輸出功能。為了系統(tǒng)解析IBGC致病基因突變,本發(fā)明設(shè)計和合成了覆蓋4個已知致病基因的PCR捕獲引物組,并結(jié)合二代測序技術(shù)對10個IBGC患者樣本進行捕獲測序,共分析得出了1種SLC20A2基因突變、2種PDGFRB基因突變、2種PDGFB基因突變和2種XPR1基因突變,所發(fā)現(xiàn)的突變均經(jīng)Sanger測序確證;之后針對各自的致病基因突變,利用Sanger測序分析對應(yīng)的家系中其它成員的樣本,全部符合基因型-表型共分離規(guī)律從而致病基因突變得到了驗證。進一步的,采用同樣的方法在另外12個IBGC患者和200個正常人樣本中進行了驗證。本發(fā)明提供的IBGC致病基因突變尚未見報道,其對于該病的解析、篩查、檢測和治療具有潛在的重要價值。此外,本發(fā)明基于PCR捕獲結(jié)合二代測序(簡稱PCR捕獲測序)構(gòu)建的IBGC致病基因檢測方法,可以同時全面解析4種IBGC致病基因突變譜,具有通量高、靈敏度高、可解析未知突變的優(yōu)點。目前,國內(nèi)外均沒有該方法在IBGC致病基因SLC20A2,PDGFRB,PDGFB,XPR1突變同時檢測方面應(yīng)用的報道。由于單次反應(yīng)測序長度的限制(1000bp以內(nèi)),傳統(tǒng)的基于Sanger測序的方法進行的全面解析,費用高昂且費時費力。而基于實時熒光定量PCR的突變檢測方法,則只能針對少量已知的突變。技術(shù)實現(xiàn)要素:本發(fā)明提供了IBGC相關(guān)的4個致病基因的7種突變基因形式,同時提供了基于PCR捕獲測序的檢測方法;這些可以為IBGC致病機理的解析和藥物開發(fā)、致病基因篩查和檢測以及治療方案的制定等提供依據(jù)和奠定基礎(chǔ)。本發(fā)明解決其技術(shù)問題所采用的技術(shù)方案如下:本發(fā)明提供了IBGC的7種致病基因形式,包括1種SLC20A2突變基因,其序列如SEQIDNO.1所示;2種PDGFRB突變基因,其序列如SEQIDNO.2和SEQIDNO.3所示;2種PDGFB突變基因,其序列如SEQIDNO.4和SEQIDNO.5所示;和2種XPR1突變基因,其序列如SEQIDNO.6和SEQIDNO.7所示。所述涉及SLC20A2、PDGFRB、PDGFB和XPR1這4個基因共7種突變基因類型經(jīng)檢測和驗證,均為IBGC致病基因。其中,SLC20A2突變基因的突變位點在Exon7中,具體是野生型基因第799位的G突變?yōu)镃;PDGFRB突變基因的突變位點其一在Exon2中,具體是野生型基因第3位的G突變?yōu)锳,其二在Exon16中,具體是野生型基因第2209位的G突變?yōu)锳;PDGFB突變基因的突變位點其一在Exon3中,具體是野生型基因第220位的G突變?yōu)門,其二也在Exon3中,具體是野生型基因第232位的C突變?yōu)門;XPR1突變基因的突變位點其一在Exon5中,具體是野生型基因第490位的G突變?yōu)門,其二在Exon13中,具體是野生型基因第1708位的C突變?yōu)門。由于突變發(fā)生在外顯子區(qū),因此本發(fā)明提供的SEQIDNO.1-7為編碼區(qū)序列。另外,提供的SEQIDNO.8-11分別是SLC20A2,PDGFRB,PDGFB,XPR1的野生型基因的編碼區(qū)序列,供參比。本發(fā)明提供的基于PCR捕獲測序的IBGC致病基因檢測方法(參見圖1)包括如下主要步驟:(1)4個致病基因的PCR捕獲提取待測樣本基因組DNA,利用設(shè)計合成的PCR捕獲引物組進行擴增;(2)二代測序解析基因突變譜將(1)中的多重PCR擴增產(chǎn)物進行二代測序,并通過信息分析找出突變位點;(3)突變位點的驗證對于結(jié)果是已知突變的,則將原樣本突變片段擴增后直接進行Sanger測序驗證;對于結(jié)果是新發(fā)現(xiàn)突變的,則根據(jù)情況設(shè)計引物將原樣本突變片段擴增后直接進行Sanger測序驗證后,再通過Sanger測序分析患者家系相關(guān)成員樣本的基因型-表型共分離情況進行驗證。其中,(1)中所述PCR捕獲引物組是利用美國ThermoFisher公司的在線軟件IonAmpliSeqDesigner進行設(shè)計的,至少應(yīng)該捕獲所述所有7種突變形式其突變位點所在區(qū)域,結(jié)果顯示至少需要SEQIDNO.12-23這12條序列。優(yōu)選地,可以針對SLC20A2、PDGFRB、PDGFB和XPR1這4個基因所有外顯子及其側(cè)翼序列,使得目標基因覆蓋率達100%,結(jié)果共獲得了69對引物,其序列如表1所示。表1PCR捕獲擴增引物組其中,(1)中進行的多重PCR擴增體系(20ul)如表2所示。5XIonAmpliSeqTMHiFiMix、2XIonAmpliSeqTM引物組和20XIonAmpliSeqTMSampleIDPanel均購置于美國ThermoFisher公司。表2PCR捕獲擴增體系成分添加量(ul)5XIonAmpliSeqTMHiFiMix42XIonAmpliSeqTM引物組1020XIonAmpliSeqTMSampleIDPanel1濃度>20ng/ul的樣本基因組DNA0.5滅菌的Nuclease-freeWater4.5其中,(1)中所述的多重PCR擴增階段和條件如表3所示:表3PCR捕獲擴增階段和條件其中,(2)中所述的PCR擴增捕獲產(chǎn)物進行的二代測序和信息分析,有二代測序儀和配套分析軟件則按照規(guī)范操作和分析進行,或可由商業(yè)化的高通量測序公司完成,最終需得到所檢測樣本的致病基因突變信息。其中,(3)中所述的已知突變指的是前述的7種突變形式(可詳見表4),所述新發(fā)現(xiàn)突變指的是這7種已發(fā)現(xiàn)突變外的其它突變形式。所述的原樣本是指(1)中提取的待測樣本基因組,除用于PCR捕獲測序模板外剩余的部分;或者原檢測樣本再次提取的基因組DNA。所述突變包括點突變、插入或缺失,但不限于此。其中,(3)中所述對于已知突變則將原樣本突變片段擴增后直接進行Sanger測序驗證,是指利用特定的引物對擴增突變片段并進行Sanger測序,驗證是否同二代測序檢出的突變結(jié)果一致;有Sanger測序儀則按規(guī)范操作進行測序或可送商業(yè)化測序公司完成,最終需得到對應(yīng)的擴增子序列。所述原樣本突變片段擴增是指,對于SLC20A2基因的c.799G>C突變,擴增引物對為SEQIDNO.24-25;對于PDGFRB基因的c.3G>A和c.2209G>A突變,擴增引物對分別為SEQIDNO.26-27和SEQIDNO.28-29;對于PDGFB基因的c.220G>T和c.232C>T突變,擴增引物對分別為SEQIDNO.28-29和SEQIDNO.30-31;對于XPR1基因的c.490G>T和c.1708C>T突變,擴增引物對分別為SEQIDNO.32-33和SEQIDNO.34-35。其中,(3)中所述的患者家系相關(guān)成員必須包括IBGC患者和非IBGC患者。所述基因型-表型共分離是指基因型決定表型,由此兩者符合共同對應(yīng)的遺傳學(xué)傳遞現(xiàn)象和規(guī)律。本發(fā)明提供的IBGC相關(guān)4個致病基因的7種突變基因類型,可以為該病致病機理的解析和藥物開發(fā)、致病基因篩查和檢測以及治療方案的制定等提供依據(jù)和奠定基礎(chǔ);此外本發(fā)明提供的基于PCR捕獲測序的致病基因檢測方法,覆蓋了4個致病基因的全部外顯子,可以高效全面、快速準確地獲取突變信息。附圖說明圖1是本發(fā)明IBGC致病基因檢測方法流程圖。圖2-8是各種基因突變形式的Sanger測序驗證圖譜突變位點局部。其中圖2對應(yīng)于實施例1中的1號患者,為SLC20A2基因的c.799G>C突變,2號、3號和4號患者的圖譜與其類似;圖3、圖4、圖5、圖6、圖7、和圖8分別對應(yīng)于實施例1中的5、6、7、8、9和10號患者,分別為PDGFRB的c.3G>A、PDGFRB的c.2209G>A、PDGFB的c.220G>T、PDGFB的c.232C>T、XPR1的c.490G>T和XPR1的c.1708C>T;各圖中的序列位點數(shù)字只代表對應(yīng)擴增子,并不代表突變位點在實際基因中的位置。圖9是實施例1中1號IBGC患者顱腦CT平掃影像。圖10是實施例1中1號IBGC患者所在家系的遺傳圖譜。此圖中正方形表示男性,圓形表示女性;箭頭表示先證者,帶斜線表示死亡,帶問號表示未行顱腦CT檢查個體;實心表示IBGC患病個體,空心表示正常個體;“+”表示SLC20A2基因檢出c.799G>C突變,“-”表示SLC20A2基因未檢出c.799G>C突變;I、II和III分別代表第1、2和3代,數(shù)字為成員編號。具體實施方式以下結(jié)合具體實施例,進一步闡述本發(fā)明。實施例1根據(jù)詳細的病史和家族史調(diào)查情況以及規(guī)范的神經(jīng)系統(tǒng)體格檢查和相關(guān)輔助檢查信息,選擇所在醫(yī)院10名IBGC患者在其簽署了知情同意書的情況下,進行致病基因檢測和分析。(1)4個致病基因的PCR捕獲取患者肘靜脈血10ml(EDTA抗凝),用QIAampDNABloodMiniKit(德國Qiagen公司)提取基因組DNA,以其為模板采用表1的PCR捕獲引物組按照表2配制擴增體系,并在常規(guī)PCR儀上按照表3條件進行反應(yīng)。(2)二代測序解析基因突變譜將(1)中的多重PCR擴增產(chǎn)物送上海晶能生物技術(shù)有限公司進行二代測序,采用的是IonPGMTemplateOT2200試劑盒進行乳液PCR,DynabeadsMyOneStreptavidinC1磁珠進行陽性模板富集;以上兩步反應(yīng)均在IonOneTouch系統(tǒng)上進行。建庫后采用IonPGMSequencingkit及IonTorrent316v2芯片,在PGM測序儀上進行測序反應(yīng)。采用PGM服務(wù)器自帶分析軟件對測序結(jié)果初步分析,再用千人基因組數(shù)據(jù)庫、SNP數(shù)據(jù)庫和COSMIC(version64)數(shù)據(jù)庫等進行突變位點解析,結(jié)果如表4所示,共計有7種突變。表410例IBGC患者致病基因檢測及驗證結(jié)果(3)突變位點的驗證根據(jù)所檢出的突變,利用前述特定引物對將原樣本突變片段擴增后直接進行Sanger測序驗證。采用Takara的rTaq聚合酶擴增,其擴增體系詳見表5,擴增引物對詳見表6,PCR產(chǎn)物送上海生工生物工程股份有限公司進行Sanger測序。Sanger測序結(jié)果發(fā)現(xiàn)均與二代測序結(jié)果一致(表4)。表5擴增體系編號組份添加量110XPCRbuffer(Mg+)2.5ul2dNTPMix0.5ul3Primer(50uM)F0.25ul4Primer(50uM)R0.25ul5rTaq0.25ul6TemplateDNA(30ng/ul)1ul7滅菌ddH2O20ul表6擴增引物對擴增條件為:94℃預(yù)變性3min;30個循環(huán):94℃變性30s,60℃/65℃退火30s,72℃延伸30s;72℃終延伸5min。由于這7種突變均未被報道,因此收集這10例IBGC患者家系相關(guān)成員樣本,再通過Sanger測序突變片段。以S1號患者為例,如圖9所示其為家系先證者III4,無明顯癥狀,顱腦CT平掃示雙側(cè)蒼白球及小腦齒狀核對稱性大面積鈣化。根據(jù)PGM數(shù)據(jù)可知,其SLC20A2基因第799位的G突變?yōu)镃;利用引物對SEQIDNO.24-25擴增SLC20A2基因c.799G>C突變片段,其直接Sanger測序結(jié)果(如圖2所示),與二代測序結(jié)果一致。收集該家系中的其他成員資料,能獲得該家系6個陽性成員,4個陰性成員的影像資料(如圖10中所示):即先證者III4、II1、II3、II8、III2及III7的顱腦CT平掃均顯示雙側(cè)基底節(jié)區(qū)及小腦存在大片鈣化灶,II2、III1、III5、III6顱內(nèi)均無鈣化灶。采集這10個家系成員的血液樣本提取基因組DNA,利用引物對SEQIDNO.24-25擴增SLC20A2基因c.799G>C突變片段,Sanger測序顯示6個陽性患者均存在該突變位點,而4個陰性成員均無該位點突變,即出現(xiàn)了基因型-表型的家系共分離現(xiàn)象,由此證明了SLC20A2基因的c.799G>C突變是該家系的致病性突變。表4中其余9例患者,經(jīng)過同樣的分析均出現(xiàn)家系共分離現(xiàn)象(表4)。因此,通過本實施例獲得了7種IBGC的致病基因,其序列對應(yīng)信息詳見表4。(4)致病基因制備和獲取基于本發(fā)明新鑒定的IBGC的7種致病基因,為了開展致病機理機制、篩查檢測方法和試劑盒開發(fā)、治療藥物研發(fā)等科研生產(chǎn)工作需要,申請人將突變所在的擴增子進行純化,將其克隆到商業(yè)化質(zhì)粒等載體中,之后將其導(dǎo)入大腸桿菌等宿主菌中,置于-80℃冰箱中保存。另一方面,由于本發(fā)明對于致病基因序列的公布,可以采用定點突變的方式由野生型基因制備得到,或通過序列合成的方式獲取。實施例2另取與實施例1中不同的IBGC患者12例以及200例正常人血樣進行分析,方法同實施例2,其結(jié)果如表7所示。表712例IBGC患者致病基因檢測及驗證結(jié)果對于表5中IBGC患者的突變均經(jīng)過Sanger測序驗證了,所有突變基因均屬于所述7種基因突變形式;而在200例正常人中,均不存在所述的突變,從而從正反兩方面再次驗證了本發(fā)明提供的IBGC致病基因,以及本發(fā)明提供的檢測方法具有良好的可靠性和準確性。雖然本發(fā)明是通過優(yōu)選的具體實施例進行說明,但是對于本領(lǐng)域的技術(shù)人員來說,本發(fā)明可以有各種更改和變化。凡在本發(fā)明的精神和原則之內(nèi),所做的任何修改、等同替換、改進等,均應(yīng)包含在本發(fā)明的保護范圍之內(nèi)。SEQUENCELISTING<110>申請人福建醫(yī)科大學(xué)附屬第一醫(yī)院<120>發(fā)明名稱人類特發(fā)性基底節(jié)鈣化致病基因及其檢測方法<160>35<170>PatentInversion3.3<210>1<211>3702<212>DNA<213>智人(Homosapiens)<400>1cgcttccggaagcgggcgactcgcagctccacgcgacgccgaggggctccgcgccgggac60cgggcgggtgctcggagtttcggggaccgcacgggaccgagggcaggaggagacatcaca120gctttcccagatcgggaggaaaaatatggaatgtgttttaccgctgactgaacacaacca180aatgaactgtcctgacagtagtttgcaaaccagcagctagcagtttgtccagcctctaac240attgtccagcactttccagagcaaactcactgtttacaagaactcttggccttacgaagt300ttataacctcaagctttgtttatttaaaatattcctgcaaaagaaaagtacccggcaccc360actttccaaaatggccatggatgagtatttgtggatggtcattttgggtttcatcatagc420tttcatcttggccttttctgttggtgcaaacgatgttgccaactcctttggtacagccgt480gggctctggtgtggtgaccttgaggcaggcatgcattttagcttcaatatttgaaaccac540cggctccgtgttactaggcgccaaagtaggagaaaccattcgcaaaggtatcattgacgt600gaacctgtacaacgagacggtggagactctcatggctggggaagttagtgccatggttgg660ttccgctgtgtggcagctgattgcttccttcctgaggcttccaatctcaggaacgcactg720cattgtgggttctactataggattctcactggtcgcaatcggtaccaaaggtgtgcagtg780gatggagcttgtcaagattgttgcttcttggtttatatctccactgttgtctggtttcat840gtctggcctgctgtttgtactcatcagaattttcatcttaaaaaaggaagaccctgttcc900caatggcctccgggcactcccagtattctatgctgctaccatagcaatcaatgtcttttc960catcatgtacacaggagcaccagtgctcggccttgttctccccatgtgggccatagccct1020catttcctttggtgtcgccctcctgttcgctttttttgtgtggctcttcgtgtgtccgtg1080gatgcggaggaaaataacaggcaaattacaaaaagaaggtgctttatcacgagtatctga1140cgaaagcctcagtaaggttcaggaagcacagtccccagtatttaaagagctaccaggtgc1200caaggctaatgatgacagcaccatcccgctcacgggagcagcaggggagacactggggac1260ctcggaaggcacttctgcgggcagccaccctcgggctgcatacggaagagcactgtccat1320gacccatggctctgtgaaatcgcccatctccaacggcaccttcggcttcgacggccacac1380caggagcgacggtcatgtgtaccacaccgtgcacaaagactcggggctctacaaagatct1440gctgcacaaaatccacatcgacaggggccccgaggagaagccagcccaggaaagcaacta1500ccggctgctgcgccgaaacaacagttacacctgctacaccgcagccatttgtgggctgcc1560agtgcacgccacctttcgagctgcggactcatcggccccagaggacagtgagaagctggt1620gggcgacaccgtgtcctactccaagaagaggctgcgctacgacagctactcgagctactg1680taacgcggtggcagaggcggagatcgaggcggaggagggcggcgtggagatgaagctggc1740gtcggagctggccgaccctgaccagccgcgagaggaccctgcagaggaggagaaggagga1800gaaggacgcacccgaggttcacctcctgttccatttcctgcaggtcctcaccgcctgttt1860cgggtcctttgctcacggcggcaatgacgtgagtaatgccatcggtcccctggtagcctt1920gtggctgatttacaaacaaggcggggtaacgcaagaagcagctacacccgtctggctgct1980gttttatggaggagttggaatctgcacaggcctctgggtctgggggagaagagtgatcca2040gaccatggggaaggacctcactcccatcacgccgtccagcggcttcacgatcgagctggc2100ctcagccttcacagtggtgatcgcctccaacatcgggcttccagtcagcaccacgcactg2160taaggtgggctcggtggtggccgtgggctggatccgctcccgcaaggctgtggactggcg2220cctctttcggaacatcttcgtggcctggttcgtgaccgtccctgtggctgggctgttcag2280cgctgctgtcatggctcttctcatgtatgggatccttccatatgtgtgagctgtcatggc2340tcttctcatgtatgggatccttccatatgtgtgatttgtcttcttccagctgcaaacagc2400taaagggatggtctggtgttggcgtgtgggagacatgtgtgctcgtgccgcacatacaca2460tcctggccgtgcacggctctctcatgaccagctctctgcctcccttccaggaggctccat2520cccacactgttcacccaggctgcggagactcaccttcccgagctaacttaactactgtac2580ataataatatgtattaaactggtatcgtggtgatataatgtggtgcagttacttatatat2640taaatatctattgtatccatagaataggcagcattatttcaaacatattcaagttgggag2700tggagatcattgcctagaagtcaatattcaataaatcttgtacataactatttcgatggc2760aaatgttaagccttctaaaaggaaagtgtagattggaaaatgattttttttccaaatgat2820gtttttgccttctaatatactgtaaggtaatgagcttcagaacaggcaacctgaccctgc2880agaggtcgcgtgctgtgggatgacagcgggacgggagctcacaagtgctttcactgaaga2940tttgttcatatactgtgtattgattgttgtgtaatatatcatcattgcttttgtaaatac3000gtaaaactgtaattttttaatggtgtgcttcccttatacttttttgatcagagaattttg3060gaaagtaccaaagaagcaggggaatcattggccagtgttacgttttcacattgtctgtct3120cccaccctcactgatcacgcctgccccagagcagtgtgtggcggtgacaccgtcacccag3180catgcgccacgccgtggctcccaccagcagtgccaccgccaccacaccccagatcccacc3240caccttgcagtggcctttccttgtcatcagagtagagaatgcacaggtgttggtgagggc3300gtgtggctgagcactacatgtcaagtccagagtcagtttctatcccaattctccctgcag3360cctgaagaacggatccttgtctccaatgtcagcacaaaggaggctttttctgtgctttga3420cattctagcacttcagggatgagagggagggagaatcctggatgctggatggagtatttc3480tctgaggcccacacaaagctggacacccccaggctctactccatcccattggagtctctt3540ctttttttgatagcgggagggaggaagtacgactaatgttggagcctgaaactatggaaa3600tgctgctaaaatttttatattgacaaacattttcttggtacttcattgtcatttttcatt3660aatcaaccatattaaatttataataaaaaatgcccctcagaa3702<210>2<211>3791<212>DNA<213>智人(Homosapiens)<400>2gctcctgaggctgccagcagccagcagtgactgcccgccctatctgggacccaggatcgc60tctgtgagcaacttggagccagagaggagatcaacaaggaggaggagagagccggcccct120cagccctgctgcccagcagcagcctgtgctcgccctgcccaacgcagacagccagaccca180gggcggcccctctggcggctctgctcctcccgaaggatgcttggggagtgaggcgaagct240gggccgctcctctcccctacagcagcccccttcctccatccctctgttctcctgagcctt300caggagcctgcaccagtcctgcctgtccttctactcagctgttacccactctgggaccag360cagtctttctgataactgggagagggcagtaaggaggacttcctggagggggtgactgtc420cagagcctggaactgtgcccacaccagaagccatcagcagcaaggacaccatacggcttc480cgggtgcgatgccagctctggccctcaaaggcgagctgctgttgctgtctctcctgttac540ttctggaaccacagatctctcagggcctggtcgtcacacccccggggccagagcttgtcc600tcaatgtctccagcaccttcgttctgacctgctcgggttcagctccggtggtgtgggaac660ggatgtcccaggagcccccacaggaaatggccaaggcccaggatggcaccttctccagcg720tgctcacactgaccaacctcactgggctagacacgggagaatacttttgcacccacaatg780actcccgtggactggagaccgatgagcggaaacggctctacatctttgtgccagatccca840ccgtgggcttcctccctaatgatgccgaggaactattcatctttctcacggaaataactg900agatcaccattccatgccgagtaacagacccacagctggtggtgacactgcacgagaaga960aaggggacgttgcactgcctgtcccctatgatcaccaacgtggcttttctggtatctttg1020aggacagaagctacatctgcaaaaccaccattggggacagggaggtggattctgatgcct1080actatgtctacagactccaggtgtcatccatcaacgtctctgtgaacgcagtgcagactg1140tggtccgccagggtgagaacatcaccctcatgtgcattgtgatcgggaatgaggtggtca1200acttcgagtggacatacccccgcaaagaaagtgggcggctggtggagccggtgactgact1260tcctcttggatatgccttaccacatccgctccatcctgcacatccccagtgccgagttag1320aagactcggggacctacacctgcaatgtgacggagagtgtgaatgaccatcaggatgaaa1380aggccatcaacatcaccgtggttgagagcggctacgtgcggctcctgggagaggtgggca1440cactacaatttgctgagctgcatcggagccggacactgcaggtagtgttcgaggcctacc1500caccgcccactgtcctgtggttcaaagacaaccgcaccctgggcgactccagcgctggcg1560aaatcgccctgtccacgcgcaacgtgtcggagacccggtatgtgtcagagctgacactgg1620ttcgcgtgaaggtggcagaggctggccactacaccatgcgggccttccatgaggatgctg1680aggtccagctctccttccagctacagatcaatgtccctgtccgagtgctggagctaagtg1740agagccaccctgacagtggggaacagacagtccgctgtcgtggccggggcatgccccagc1800cgaacatcatctggtctgcctgcagagacctcaaaaggtgtccacgtgagctgccgccca1860cgctgctggggaacagttccgaagaggagagccagctggagactaacgtgacgtactggg1920aggaggagcaggagtttgaggtggtgagcacactgcgtctgcagcacgtggatcggccac1980tgtcggtgcgctgcacgctgcgcaacgctgtgggccaggacacgcaggaggtcatcgtgg2040tgccacactccttgccctttaaggtggtggtgatctcagccatcctggccctggtggtgc2100tcaccatcatctcccttatcatcctcatcatgctttggcagaagaagccacgttacgaga2160tccgatggaaggtgattgagtctgtgagctctgacggccatgagtacatctacgtggacc2220ccatgcagctgccctatgactccacgtgggagctgccgcgggaccagcttgtgctgggac2280gcaccctcggctctggggcctttgggcaggtggtggaggccacggctcatggcctgagcc2340attctcaggccacgatgaaagtggccgtcaagatgcttaaatccacagcccgcagcagtg2400agaagcaagcccttatgtcggagctgaagatcatgagtcaccttgggccccacctgaacg2460tggtcaacctgttgggggcctgcaccaaaggaggacccatctatatcatcactgagtact2520gccgctacggagacctggtggactacctgcaccgcaacaaacacaccttcctgcagcacc2580actccgacaagcgccgcccgcccagcgcggagctctacagcaatgctctgcccgttgggc2640tccccctgcccagccatgtgtccttgaccggggagagcgacggtggctacatggacatga2700gcaaggacgagtcggtggactatgtgcccatgctggacatgaaaggagacgtcaaatatg2760cagacatcgagtcctccaactacatggccccttacgataactacgttccctctgcccctg2820agaggacctgccgagcaactttgatcaacgagtctccagtgctaagctacatggacctcg2880tgggcttcagctaccaggtggccaatggcatggagtttctggcctccaagaactgcgtcc2940acagagacctggcggctaggaacgtgctcatctgtgaaggcaagctggtcaagatctgtg3000actttggcctggctcgagacatcatgcgggactcgaattacatctccaaaggcagcacct3060ttttgcctttaaagtggatggctccggagagcatcttcaacagcctctacaccaccctga3120gcgacgtgtggtccttcgggatcctgctctgggagatcttcaccttgggtggcacccctt3180acccagagctgcccatgaacgagcagttctacaatgccatcaaacggggttaccgcatgg3240cccagcctgcccatgcctccgacgagatctatgagatcatgcagaagtgctgggaagaga3300agtttgagattcggccccccttctcccagctggtgctgcttctcgagagactgttgggcg3360aaggttacaaaaagaagtaccagcaggtggatgaggagtttctgaggagtgaccacccag3420ccatccttcggtcccaggcccgcttgcctgggttccatggcctccgatctcccctggaca3480ccagctccgtcctctatactgccgtgcagcccaatgagggtgacaacgactatatcatcc3540ccctgcctgaccccaaacccgaggttgctgacgagggcccactggagggttcccccagcc3600tagccagctccaccctgaatgaagtcaacacctcctcaaccatctcctgtgacagccccc3660tggagccccaggacgaaccagagccagagccccagcttgagctccaggtggagccggagc3720cagagctggaacagttgccggattcggggtgccctgcgcctcgggcggaagcagaggata3780gcttcctgtag3791<210>3<211>3791<212>DNA<213>智人(Homosapiens)<400>3gctcctgaggctgccagcagccagcagtgactgcccgccctatctgggacccaggatcgc60tctgtgagcaacttggagccagagaggagatcaacaaggaggaggagagagccggcccct120cagccctgctgcccagcagcagcctgtgctcgccctgcccaacgcagacagccagaccca180gggcggcccctctggcggctctgctcctcccgaaggatgcttggggagtgaggcgaagct240gggccgctcctctcccctacagcagcccccttcctccatccctctgttctcctgagcctt300caggagcctgcaccagtcctgcctgtccttctactcagctgttacccactctgggaccag360cagtctttctgataactgggagagggcagtaaggaggacttcctggagggggtgactgtc420cagagcctggaactgtgcccacaccagaagccatcagcagcaaggacaccatgcggcttc480cgggtgcgatgccagctctggccctcaaaggcgagctgctgttgctgtctctcctgttac540ttctggaaccacagatctctcagggcctggtcgtcacacccccggggccagagcttgtcc600tcaatgtctccagcaccttcgttctgacctgctcgggttcagctccggtggtgtgggaac660ggatgtcccaggagcccccacaggaaatggccaaggcccaggatggcaccttctccagcg720tgctcacactgaccaacctcactgggctagacacgggagaatacttttgcacccacaatg780actcccgtggactggagaccgatgagcggaaacggctctacatctttgtgccagatccca840ccgtgggcttcctccctaatgatgccgaggaactattcatctttctcacggaaataactg900agatcaccattccatgccgagtaacagacccacagctggtggtgacactgcacgagaaga960aaggggacgttgcactgcctgtcccctatgatcaccaacgtggcttttctggtatctttg1020aggacagaagctacatctgcaaaaccaccattggggacagggaggtggattctgatgcct1080actatgtctacagactccaggtgtcatccatcaacgtctctgtgaacgcagtgcagactg1140tggtccgccagggtgagaacatcaccctcatgtgcattgtgatcgggaatgaggtggtca1200acttcgagtggacatacccccgcaaagaaagtgggcggctggtggagccggtgactgact1260tcctcttggatatgccttaccacatccgctccatcctgcacatccccagtgccgagttag1320aagactcggggacctacacctgcaatgtgacggagagtgtgaatgaccatcaggatgaaa1380aggccatcaacatcaccgtggttgagagcggctacgtgcggctcctgggagaggtgggca1440cactacaatttgctgagctgcatcggagccggacactgcaggtagtgttcgaggcctacc1500caccgcccactgtcctgtggttcaaagacaaccgcaccctgggcgactccagcgctggcg1560aaatcgccctgtccacgcgcaacgtgtcggagacccggtatgtgtcagagctgacactgg1620ttcgcgtgaaggtggcagaggctggccactacaccatgcgggccttccatgaggatgctg1680aggtccagctctccttccagctacagatcaatgtccctgtccgagtgctggagctaagtg1740agagccaccctgacagtggggaacagacagtccgctgtcgtggccggggcatgccccagc1800cgaacatcatctggtctgcctgcagagacctcaaaaggtgtccacgtgagctgccgccca1860cgctgctggggaacagttccgaagaggagagccagctggagactaacgtgacgtactggg1920aggaggagcaggagtttgaggtggtgagcacactgcgtctgcagcacgtggatcggccac1980tgtcggtgcgctgcacgctgcgcaacgctgtgggccaggacacgcaggaggtcatcgtgg2040tgccacactccttgccctttaaggtggtggtgatctcagccatcctggccctggtggtgc2100tcaccatcatctcccttatcatcctcatcatgctttggcagaagaagccacgttacgaga2160tccgatggaaggtgattgagtctgtgagctctgacggccatgagtacatctacgtggacc2220ccatgcagctgccctatgactccacgtgggagctgccgcgggaccagcttgtgctgggac2280gcaccctcggctctggggcctttgggcaggtggtggaggccacggctcatggcctgagcc2340attctcaggccacgatgaaagtggccgtcaagatgcttaaatccacagcccgcagcagtg2400agaagcaagcccttatgtcggagctgaagatcatgagtcaccttgggccccacctgaacg2460tggtcaacctgttgggggcctgcaccaaaggaggacccatctatatcatcactgagtact2520gccgctacggagacctggtggactacctgcaccgcaacaaacacaccttcctgcagcacc2580actccgacaagcgccgcccgcccagcgcggagctctacagcaatgctctgcccgttgggc2640tccccctgcccagccatgtgtccttgaccggggagagcaacggtggctacatggacatga2700gcaaggacgagtcggtggactatgtgcccatgctggacatgaaaggagacgtcaaatatg2760cagacatcgagtcctccaactacatggccccttacgataactacgttccctctgcccctg2820agaggacctgccgagcaactttgatcaacgagtctccagtgctaagctacatggacctcg2880tgggcttcagctaccaggtggccaatggcatggagtttctggcctccaagaactgcgtcc2940acagagacctggcggctaggaacgtgctcatctgtgaaggcaagctggtcaagatctgtg3000actttggcctggctcgagacatcatgcgggactcgaattacatctccaaaggcagcacct3060ttttgcctttaaagtggatggctccggagagcatcttcaacagcctctacaccaccctga3120gcgacgtgtggtccttcgggatcctgctctgggagatcttcaccttgggtggcacccctt3180acccagagctgcccatgaacgagcagttctacaatgccatcaaacggggttaccgcatgg3240cccagcctgcccatgcctccgacgagatctatgagatcatgcagaagtgctgggaagaga3300agtttgagattcggccccccttctcccagctggtgctgcttctcgagagactgttgggcg3360aaggttacaaaaagaagtaccagcaggtggatgaggagtttctgaggagtgaccacccag3420ccatccttcggtcccaggcccgcttgcctgggttccatggcctccgatctcccctggaca3480ccagctccgtcctctatactgccgtgcagcccaatgagggtgacaacgactatatcatcc3540ccctgcctgaccccaaacccgaggttgctgacgagggcccactggagggttcccccagcc3600tagccagctccaccctgaatgaagtcaacacctcctcaaccatctcctgtgacagccccc3660tggagccccaggacgaaccagagccagagccccagcttgagctccaggtggagccggagc3720cagagctggaacagttgccggattcggggtgccctgcgcctcgggcggaagcagaggata3780gcttcctgtag3791<210>4<211>1542<212>DNA<213>智人(Homosapiens)<400>4gcagctcgcagctcgcagcccgcagcccgcagaggacgcccagagcggcgagcgggcggg60cagacggaccgacggactcgcgccgcgtccacctgtcggccgggcccagccgagcgcgca120gcgggcacgccgcgcgcgcggagcagccgtgcccgccgcccgggccccgcgccagggcgc180acacgctcccgcccccctacccggcccgggcgggagtttgcacctctccctgcccgggtg240ctcgagctgccgttgcaaagccaactttggaaaaagttttttgggggagacttgggcctt300gaggtgcccagctccgcgctttccgattttgggggcctttccagaaaatgttgcaaaaaa360gctaagccggcgggcagaggaaaacgcctgtagccggcgagtgaagacgaaccatcgact420gccgtgttccttttcctcttggaggttggagtcccctgggcgcccccacacggctagacg480cctcggctggttcgcgacgcagccccccggccgtggatgctcactcgggctcgggatccg540cccaggtagcggcctcggacccaggtcctgcgcccaggtcctcccctgccccccagcgac600ggagccggggccgggggcggcggcgcccgggggccatgcgggtgagccgcggctgcagag660gcctgagcgcctgatcgccgcggacccgagccgagcccacccccctccccagccccccac720cctggccgcgggggcggcgcgctcgatctacgcgtccggggccccgcggggccgggcccg780gagtcggcatgaatcgctgctgggcgctcttcctgtctctctgctgctacctgcgtctgg840tcagcgccgagggggaccccattcccgaggagctttatgagatgctgagtgaccactcga900tccgctcctttgatgatctccaacgcctgctgcacggagaccccggagaggaagatgggg960ccgagttggacctgaacatgacccgctcccactctggaggcgagctgtagagcttggctc1020gtggaagaaggagcctgggttccctgaccattgctgagccggccatgatcgccgagtgca1080agacgcgcaccgaggtgttcgagatctcccggcgcctcatagaccgcaccaacgccaact1140tcctggtgtggccgccctgtgtggaggtgcagcgctgctccggctgctgcaacaaccgca1200acgtgcagtgccgccccacccaggtgcagctgcgacctgtccaggtgagaaagatcgaga1260ttgtgcggaagaagccaatctttaagaaggccacggtgacgctggaagaccacctggcat1320gcaagtgtgagacagtggcagctgcacggcctgtgacccgaagcccggggggttcccagg1380agcagcgagccaaaacgccccaaactcgggtgaccattcggacggtgcgagtccgccggc1440cccccaagggcaagcaccggaaattcaagcacacgcatgacaagacggcactgaaggaga1500cccttggagcctaggggcatcggcaggagagtgtgtgggcag1542<210>5<211>1542<212>DNA<213>智人(Homosapiens)<400>5gcagctcgcagctcgcagcccgcagcccgcagaggacgcccagagcggcgagcgggcggg60cagacggaccgacggactcgcgccgcgtccacctgtcggccgggcccagccgagcgcgca120gcgggcacgccgcgcgcgcggagcagccgtgcccgccgcccgggccccgcgccagggcgc180acacgctcccgcccccctacccggcccgggcgggagtttgcacctctccctgcccgggtg240ctcgagctgccgttgcaaagccaactttggaaaaagttttttgggggagacttgggcctt300gaggtgcccagctccgcgctttccgattttgggggcctttccagaaaatgttgcaaaaaa360gctaagccggcgggcagaggaaaacgcctgtagccggcgagtgaagacgaaccatcgact420gccgtgttccttttcctcttggaggttggagtcccctgggcgcccccacacggctagacg480cctcggctggttcgcgacgcagccccccggccgtggatgctcactcgggctcgggatccg540cccaggtagcggcctcggacccaggtcctgcgcccaggtcctcccctgccccccagcgac600ggagccggggccgggggcggcggcgcccgggggccatgcgggtgagccgcggctgcagag660gcctgagcgcctgatcgccgcggacccgagccgagcccacccccctccccagccccccac720cctggccgcgggggcggcgcgctcgatctacgcgtccggggccccgcggggccgggcccg780gagtcggcatgaatcgctgctgggcgctcttcctgtctctctgctgctacctgcgtctgg840tcagcgccgagggggaccccattcccgaggagctttatgagatgctgagtgaccactcga900tccgctcctttgatgatctccaacgcctgctgcacggagaccccggagaggaagatgggg960ccgagttggacctgaacatgacccgctcccactctggaggcgagctggagagcttggctt1020gtggaagaaggagcctgggttccctgaccattgctgagccggccatgatcgccgagtgca1080agacgcgcaccgaggtgttcgagatctcccggcgcctcatagaccgcaccaacgccaact1140tcctggtgtggccgccctgtgtggaggtgcagcgctgctccggctgctgcaacaaccgca1200acgtgcagtgccgccccacccaggtgcagctgcgacctgtccaggtgagaaagatcgaga1260ttgtgcggaagaagccaatctttaagaaggccacggtgacgctggaagaccacctggcat1320gcaagtgtgagacagtggcagctgcacggcctgtgacccgaagcccggggggttcccagg1380agcagcgagccaaaacgccccaaactcgggtgaccattcggacggtgcgagtccgccggc1440cccccaagggcaagcaccggaaattcaagcacacgcatgacaagacggcactgaaggaga1500cccttggagcctaggggcatcggcaggagagtgtgtgggcag1542<210>6<211>8614<212>DNA<213>智人(Homosapiens)<400>6gcggggaggggcggggctatggagaggaggaggaagatggcgggcgggctgctctgaaga60gacctcggcggcggcggaggaggagagaagcgcagcgccgcgccgcgccggggcccatgt120ggggaggagtcggagtcgctgttgccgccgccgcctgtagctgctggacccgagtgggag180tgagggggaaacggcaggatgaagttcgccgagcacctctccgcgcacatcactcccgag240tggaggaagcaatacatccagtatgaggctttcaaggatatgctgtattcagctcaggac300caggcaccttctgtggaagttacagatgaggacacagtaaagaggtattttgccaagttt360gaagagaagtttttccaaacctgtgaaaaagaacttgccaaaatcaacacattttattca420gagaagctcgcagaggctcagcgcaggtttgctacacttcagaatgagcttcagtcatca480ctggatgcacagaaagaaagcactggtgttactacgctgcgacaacgcagaaagccagtc540ttccacttgtcccatgaggaacgtgtccaacatagaaatattaaagaccttaaactggcc600ttcagtgagttctacctcagtctaatcctgctgcagaactatcagaatctgaattttaca660gggtttcgaaaaatcctgaaaaagcattacaagatcctggaaacatctcgtggagcagat720tggcgagtggctcacgtagaggtggccccattttatacatgcaagaaaatcaaccagctt780atctctgaaactgaggctgtagtgaccaatgaacttgaagatggtgacagacaaaaggct840atgaagcgtttacgtgtcccccctttgggagctgctcagcctgcaccagcatggactact900tttagagttggcctattttgtggaatattcattgtactgaatattacccttgtgcttgcc960gctgtatttaaacttgaaacagatagaagtatatggcccttgataagaatctatcggggt1020ggctttcttctgattgaattcctttttctactgggcatcaacacgtatggttggagacag1080gctggagtaaaccatgtactcatctttgaacttaatccgagaagcaatttgtctcatcaa1140catctctttgagattgctggattcctcgggatattgtggtgcctgagccttctggcatgc1200ttctttgctccaattagtgtcatccccacatatgtgtatccacttgccctttatggattt1260atggttttcttccttatcaaccccaccaaaactttctactataaatcccggttttggctg1320cttaaactgctgtttcgagtatttacagcccccttccataaggtaggctttgctgatttc1380tggctggcggatcagctgaacagcctgtcagtgatactgatggacctggaatatatgatc1440tgcttctacagtttggagctcaaatgggatgaaagtaagggcctgttgccaaataattca1500gaagaatcaggaatttgccacaaatatacatatggtgtgcgggccattgttcagtgcatt1560cctgcttggcttcgcttcatccagtgcctgcgccgatatcgagacacaaaaagggccttt1620cctcatttagttaatgctggcaaatactccacaactttcttcatggtgacgtttgcagcc1680ctttacagcactcacaaagaacgaggtcactcggacactatggtgttcttttacctgtgg1740attgtcttttatatcatcagttcctgctataccctcatctgggatctcaagatggactgg1800ggtctcttcgataagaatgctggagagaacactttcctccgggaagagattgtatacccc1860caaaaagcctactactactgtgccataatagaggatgtgattctgcgctttgcttggact1920atccaaatctcgattacctctacaactttgttgcctcattctggggacatcattgctact1980gtctttgccccacttgaggttttccggcctactactactgtgccataatagaggatgtga2040ttctgcgctttgcttggactatccaaatctcgattacctctacaactttgttgcctcatt2100ctggggacatcattgctactgtctttgc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aaa8520gttaatgtggttatgtgtttttaaagaaaataagtgattggtcactaaactctgtccttt8580tttcattattacaatttctctctgttttccaaca8614<210>12<211>25<212>DNA<213>人工序列<400>12gggatccttttgtgcattaaaacca25<210>13<211>29<212>DNA<213>人工序列<400>13gaactctcttccagaatgttcagtatgac29<210>14<211>22<212>DNA<213>人工序列<400>14ctggagggccaatcaaaggatt22<210>15<211>22<212>DNA<213>人工序列<400>15ggccctgactcctgtttaactc22<210>16<211>23<212>DNA<213>人工序列<400>16gctcccttcttctcagggttttt23<210>17<211>28<212>DNA<213>人工序列<400>17gtgtgtgagtgagagaagaaattcatga28<210>18<211>22<212>DNA<213>人工序列<400>18agtcttacccaggctccttctt22<210>19<211>24<212>DNA<213>人工序列<400>19ccaaaaatgttggctaaagagcca24<210>20<211>26<212>DNA<213>人工序列<400>20tgtaggacatatgctcattggtatgc26<210>21<211>30<212>DNA<213>人工序列<400>21ctgaggagaaatttgagaaaagttggaaaa30<210>22<211>22<212>DNA<213>人工序列<400>22ccagctctgtcagtgcttagtg22<210>23<211>30<212>DNA<213>人工序列<400>23caggtaaggcatatatattcaaagctagca30<210>24<211>21<212>DNA<213>人工序列<400>24accacacctggcctcaacttc21<210>25<211>22<212>DNA<213>人工序列<400>25tccctagcttctgggttttctc22<210>26<211>19<212>DNA<213>人工序列<400>26tgggacgtggtggcttttg19<210>27<211>19<212>DNA<213>人工序列<400>27acagagcccactggaaatc19<210>28<211>20<212>DNA<213>人工序列<400>28ctctctgggttctaactgtc20<210>29<211>20<212>DNA<213>人工序列<400>29tctatgaaatgggcacgagg20<210>30<211>19<212>DNA<213>人工序列<400>30aggagggactgttctctgg19<210>31<211>20<212>DNA<213>人工序列<400>31ctgtgatgactgaacacagg20<210>32<211>20<212>DNA<213>人工序列<400>32aatctttgtccagccagctc20<210>33<211>19<212>DNA<213>人工序列<400>33gggccagctaaatactgtg19<210>34<211>20<212>DNA<213>人工序列<400>34tcgtaggacatgtgtgagtc20<210>35<211>19<212>DNA<213>人工序列<400>35cctggccaacatggttttc19當前第1頁1 2 3 
當前第1頁1 2 3 
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