專利名稱：Pah基因13種新突變的檢測(cè)方法
技術(shù)領(lǐng)域：
本發(fā)明涉及一種生物技術(shù)領(lǐng)域的基因突變檢測(cè)方法，具體是一種/^//(苯丙氨酸羥化酶)基因13種新突變的檢測(cè)方法。
背景技術(shù)：
苯酮尿癥(phenylketonuria, PKU)是一種可以造成兒童不同程度智力損害的常見的先代謝性疾病，屬于常染色體隱性遺傳，0MIM編號(hào)為261600。 PKU的主要致病基因是/^〃基因，1983年該基因被定位在2q22-24。隨著尸力#基因突變分子的不斷深入研究，至2007年7月為止，http:〃麗.pahdb. mcgill. ca/(國(guó)際PAH數(shù)據(jù)庫(kù))共記載513種突變。/^#基因定位于染色體12q24. 1，大約由1. 5Mb堿基組成。/^#基因的編碼區(qū)包含13個(gè)外顯子，被不編碼的12個(gè)內(nèi)含子分隔。轉(zhuǎn)錄剪接后形成長(zhǎng)1353bp的mRNA，后者經(jīng)翻譯，形成含451個(gè)氨基酸的酶單體，單體聚會(huì)成有功能的PAH酶。我國(guó)自80年代初以來(lái)對(duì)PKU開展了系統(tǒng)研究，隨著分子生物學(xué)技術(shù)的發(fā)展，至今已發(fā)現(xiàn)90余種突變，并不斷有新突變發(fā)現(xiàn)。
經(jīng)對(duì)現(xiàn)有文獻(xiàn)的檢索，未發(fā)現(xiàn)與/^#基因13種新突變的檢測(cè)方法主題有關(guān)的報(bào)道。

發(fā)明內(nèi)容
本發(fā)明的目的在于克服現(xiàn)有技術(shù)中的不足，提供一種/^//基因13種新突變的檢測(cè)方法。本發(fā)明為進(jìn)一步開發(fā)/%1/基因突變種類及其頻率的分子遺傳技術(shù)提供了依據(jù)，具有實(shí)用價(jià)值。
本發(fā)明是通過(guò)以下技術(shù)方案實(shí)現(xiàn)的
本發(fā)明包括步驟如下
步驟一，以GenBank數(shù)據(jù)庫(kù)中/ 4〃基因第4或5或6或7或10或11或12號(hào)外顯子以及外顯子與內(nèi)含子接合部位序列為模板設(shè)計(jì)一對(duì)等位基因特異性的核酸引物，利用引物對(duì)來(lái)擴(kuò)增/^^基因相應(yīng)外顯子的DNA序列，對(duì)擴(kuò)增產(chǎn)物進(jìn)行分離純化，得到相應(yīng)分離核酸；
步驟二，對(duì)分離核酸的/^#基因外顯子的核苷酸進(jìn)行是否存在突變結(jié)果的 檢測(cè)。
步驟一中，所述的特異性的核酸引物具體為長(zhǎng)度為15bp-25bp，且能特異 性地雜交并擴(kuò)增出含州v7基因外顯子以及相應(yīng)外顯子與內(nèi)含子接合部位的序列。
步驟一中，所述分離純化具體為在基因組DNA水平上，利用引物來(lái)擴(kuò)增 每個(gè)突變位點(diǎn)的基因序列和利用純化試劑盒對(duì)擴(kuò)增產(chǎn)物進(jìn)行提純。
步驟一中，所述分離核酸具體為
當(dāng)外顯子為第4號(hào)時(shí)，該核酸具有序列表SEQ ID N0.3所示的序列； 當(dāng)外顯子為第5號(hào)時(shí)，該核酸具有序列表SEQ ID NO. 4或SEQ ID NO. 5所 示的序列；
當(dāng)外顯子為第6號(hào)時(shí)，該核酸具有序列表SEQ ID NO. 6或SEQ ID NO. 7或 SEQ ID NO. 8所示的序列;
當(dāng)外顯子為第7號(hào)時(shí)，該核酸具有序列表SEQ ID NO. 9或SEQ ID NO. 10所 示的序列；
當(dāng)外顯子為第10號(hào)時(shí)，該核酸具有序列表SEQ ID NO.ll所示的序列； 當(dāng)外顯子為第11號(hào)時(shí)，該核酸具有序列表SEQ ID NO. 12或SEQ ID NO. 13
或SEQ ID NO. 14所示的序列；
當(dāng)外顯子為第12號(hào)時(shí)，該核酸具有序列表SEQ ID N0.15所示的序列。 步驟二中，所述檢測(cè)，其方法包括DNA測(cè)序法、雜交測(cè)序法、酶促錯(cuò)配切
割法、異源雙鏈分析法、點(diǎn)雜交法、寡核苷酸陣列法、微測(cè)序法、Taqman技術(shù)、
分子信標(biāo)法、變性高效液相色譜法的一種或者幾種。 步驟二中，所述突變結(jié)果的檢測(cè)是指
當(dāng)為第4號(hào)外顯子時(shí)，檢測(cè)SEQ ID NO. 1所示序列的第361位核苷酸是否 存在突變，即T—C;
當(dāng)為第5號(hào)外顯子時(shí)，檢測(cè)SEQ ID NO. 1所示序列的第461位核苷酸是否 存在突變，即A—G;或檢測(cè)SEQ ID NO. 1所示序列的第466位核苷酸是否存在 突變，即G—C;
當(dāng)為第6號(hào)外顯子時(shí)，檢測(cè)SEQ ID NO. 1所示序列的第548位核苷酸是否存在突變，即A—G;或檢測(cè)SEQ ID NO. 1所示序列的第680位核苷酸是否存在 突變，即T—A;或檢測(cè)SEQ ID NO. 1所示序列的第682位核苷酸是否存在突變， 即G—T;
當(dāng)為第7號(hào)外顯子時(shí)，檢測(cè)SEQ ID NO. 1所示序列的第808位核苷酸是否 存在突變，即A—G;或檢測(cè)SEQ ID NO. 1所示序列的第823位核苷酸是否存在 突變，即C—G;
當(dāng)為第10號(hào)外顯子時(shí)，檢測(cè)SEQ ID NO. 1所示序列的第971位核苷酸是否 存在突變，即T—A;
當(dāng)為第11號(hào)外顯子時(shí)，檢測(cè)SEQ ID NO. 1所示序列的第1070位核苷酸是 否存在突變，即G—A;或檢測(cè)SEQ ID NO. 1所示序列的第1071位核苷酸是否存 在突變，即C—A;或檢測(cè)SEQ ID NO. 1所示序列的第1085位核苷酸是否存在突 變，即C—T;
當(dāng)為第12號(hào)外顯子時(shí)，檢測(cè)SEQ ID NO. 1所示序列的第1242位核苷酸是 否存在突變，即C—A。
本發(fā)明以GenBank數(shù)據(jù)庫(kù)中/^〃基因第4或5或6或7或10或11或12號(hào) 外顯子以及外顯子與內(nèi)含子接合部位序列為模板設(shè)計(jì)一對(duì)等位基因特異性的核 酸引物，利用引物對(duì)來(lái)擴(kuò)增M^基因相應(yīng)外顯子的DNA序列，對(duì)擴(kuò)增產(chǎn)物進(jìn)行分 離純化，得到相應(yīng)分離核酸；對(duì)分離核酸的/^^基因外顯子的核苷酸進(jìn)行檢測(cè)， 確定其是否存在突變。用于本發(fā)明的測(cè)試樣品對(duì)于檢測(cè)突變和單核苷酸多態(tài)性而 言，可以從測(cè)試樣品中抽提的DNA或mRNA。對(duì)于PAH酶活性而言，可以是任何 含有/^#基因的樣品。
本發(fā)明通過(guò)對(duì)/^^基因測(cè)序結(jié)果分析，發(fā)現(xiàn)13種不同的突變，經(jīng)功能分析， 此13種突變都是改變了原有的氨基酸序列，導(dǎo)致了氨基酸的置換，最終導(dǎo)致了 /^#基因編碼的苯丙氨酸羥化酶活性的改變，影響氨基酸的正常代謝。本發(fā)明為 進(jìn)一步開發(fā)/^7/基因突變種類及其頻率的分子遺傳技術(shù)提供了依據(jù)，具有重要的 實(shí)用價(jià)值。
具體實(shí)施例方式
下面結(jié)合具體實(shí)施例，進(jìn)一步闡述本發(fā)明。應(yīng)理解，這些實(shí)施例僅用于說(shuō) 明本發(fā)明而不用于限制本發(fā)明的范圍。下列實(shí)施例中未注明具體條件的實(shí)驗(yàn)方法，通常照常規(guī)條件，例如分子克隆，實(shí)驗(yàn)室手冊(cè)(New York: Cold Spring Harbor Laboratory Press, 1989)中所述的條件，或按照制造廠商所建議的條件。 實(shí)施例
步驟一，以GenBank數(shù)據(jù)庫(kù)中尸力^基因第4或5或6或7或10或11或12 號(hào)外顯子以及外顯子與內(nèi)含子接合部位序列為模板設(shè)計(jì)一對(duì)等位基因特異性的 核酸引物，利用引物對(duì)來(lái)擴(kuò)增M〃基因相應(yīng)外顯子的DNA序列，對(duì)擴(kuò)增產(chǎn)物進(jìn)行 分離純化，得到相應(yīng)分離核酸
引物設(shè)計(jì)
通過(guò)Primer5. 0軟件，以Genebank數(shù)據(jù)庫(kù)Z^〃基因(醒—000277)第4、 5、 6、 7、 10、 11、 12號(hào)外顯子以及相應(yīng)外顯子與內(nèi)含子接合部位序列為模板設(shè)計(jì)7 對(duì)等位基因特異性的核酸引物，由上海生工生物技術(shù)有限公司合成。/^〃基因序 列可由GeneBank數(shù)據(jù)庫(kù)中取得，州^基因序列如SEQ NO. 1所示，M^基因的外 顯子的基因組序列可從Genebank中獲得。 引物信息
擴(kuò)增W〃基因的第4號(hào)外顯子突變位點(diǎn)的DNA序列引物
正向引物 5' -AGAGAACATCTCCAGGAAAGA-3'
反向引物 5'-CCCAGCCCTCGTGTAAATAG-3'; 擴(kuò)增/^#基因的第5號(hào)外顯子突變位點(diǎn)的DNA序列引物
正向引物 5' -AGGGTATAACCAAGGGAAGG-3'
反向引物 5'-ATGAGGGCAAGGGAGAAGC-3'; 擴(kuò)增W〃基因的第6號(hào)外顯子突變位點(diǎn)的DNA序列引物
正向引物 5' -CCCCGACTCCCTCTGCTAA-3'
反向引物 5'-CCTCTGCCTCAATCCTCCC-3'; 擴(kuò)增W〃基因的第7號(hào)外顯子突變位點(diǎn)的DNA序列引物
正向引物5'-CTGCCTAGCGTCAAAGCC-3'
反向引物5'-ECAGCCAGCAATGAACCCAAA-3'; 擴(kuò)增/^#基因的第10號(hào)外顯子突變位點(diǎn)的DNA序列引物
正向引物5' -AGGTATCCCTTCATCCAGTC-3'
反向引物5'-CCCACAGCCATCATCAAATC-3';擴(kuò)增"^基因的第11號(hào)外顯子突變位點(diǎn)的DNA序列引物
正向引物5' -ATTTGGGCTGTGATGTAGAAG-3'
反向引物5' -TTCAGTGTCTTGACTTGGTGG-3' 擴(kuò)增M〃基因的第12號(hào)外顯子突變位點(diǎn)的DNA序列引物
正向引物5' -CTAGGGAGGTGTCCGTGTT—3，
反向引物5，-GGCGATGGTAGGGAAAGAC-3'; 第4、 7、 10、 11、 12號(hào)外顯子PCR擴(kuò)增條件為 體系各項(xiàng)試劑除DNA外均由Bio-Asia公司提供 反應(yīng)體系總體積為15W，其中 ddH20 8 .
10 X Buffer 1.5^1，其中聚合反應(yīng)緩沖液包含KCl Tris-HCl溶液
DMSO 0.6^1
2mM dNTP 1. 5rt
10pM正反向引物各
5U Taq 0.
DNA 10ng/0.5ul
第5、 6號(hào)外顯子PCR擴(kuò)增條件為
體系各項(xiàng)試劑除DNA外均由Bio-Asia公司提供
反應(yīng)體系總體積為15W，其中
ddH20 8.
10 X Buffer H，其中聚合反應(yīng)緩沖液包含KCl Tris-HCl溶液
DMSO 0.
2mM藩P 1. 514
lOpM正反向引物各0.914
5U Taq 0.
DNA 10ng/0.5ul
上述外顯子PCR反應(yīng)條件為
94°C 3mins; 94°C 30 seconds; 64°C 30 seconds, -0. 5°C/CYCLE; 72 °C
lmin; 14timesto2 ; 30X (94°C 30 seconds; 57°C 30 seconds; 72 °C lmin;)
72°C lOmins。測(cè)序反應(yīng)均作正反向，條件如下
擴(kuò)增產(chǎn)物酶純化法PCR產(chǎn)物2)4 ，加入2^1純化酶系，酶系內(nèi)含SAP (Shrimp alkaline phosphatase,堿性磷酸酶)0. 15u和Exo-nuclease I(核酸外切酶I) 0.75u/ul， 37°C 30mins， 85°C 20mins， 4°C保存?zhèn)溆谩?br> 測(cè)序采用ABI公司的Prisra BigDye terminator卿T) cycle sequencing reaction kit試劑盒進(jìn)行測(cè)序，測(cè)序總體積為5W，其中純化后的PCR產(chǎn)物#1， BDT 0. 5W，正向或反向引物O. 5W。反應(yīng)條件是94。C 2 mins， 35 X (94°C 30 seconds, 55°C 40 seconds, 60°C, 1.5 mins) ， 4°C保存?zhèn)溆谩?br> 上樣前純化測(cè)序產(chǎn)物中加入25W無(wú)水乙醇和3. 0 mol / L醋酸鈉混 合液(V:V二25:1)，室溫、避光、靜置15min， 4000rpm/min 45mins， 700rpm/min 5 seconds, 棄上清；2X (75%Alcohol 25l4， 4000 rpm/min 10 tnins， 700rpm/min 5 seconds,棄上清)；室溫、避光、靜置、風(fēng)干20mins;上樣于3100測(cè)序 儀進(jìn)行測(cè)序。
步驟二，對(duì)分離核酸的/^#基因外顯子的核苷酸進(jìn)行是否存在突變結(jié)果的
檢測(cè)
對(duì)/^^基因進(jìn)行直接測(cè)序，將測(cè)定的各樣本序列與GeneBank數(shù)據(jù)庫(kù)/%￥基 因序列(NM_000277)進(jìn)行比較，州〃基因序列如SEQ NO. 1所示，對(duì)應(yīng)編碼的 氨基酸序列如SEQ ID NO. 2所示。
對(duì)樣本的測(cè)序結(jié)果標(biāo)明，在/^^基因第4、 5、 6、 7、 10、 11、 12號(hào)外顯子 中發(fā)現(xiàn)13種新突變，頻率均為0.05%, /^//基因的上述外顯子的基因組序列可從 Genebaiik中獲得。經(jīng)功能分析，13種突變都改變了原有的氨基酸序列，導(dǎo)致了 氨基酸的置換，具體為
(1) SEQ ID NO. 1所示序列的第361位核苷酸發(fā)生突變，即T—C;此時(shí)， SEQ ID NO. 1所示序列編碼的氨基酸序列SEQ ID NO. 2第121位由Phe (F) —Leu
(U，具體序列如SEQ ID NO. 16所示；
(2) SEQ ID NO. 1所示序列的第461位核苷酸發(fā)生突變，即A—G;此時(shí)， SEQ ID NO. 1所示序列編碼的氨基酸序列SEQ ID NO. 2第154位由Tyr (Y) —Cys
(C);具體序列如SEQ ID NO. 17所示;
(3) SEQ ID NO. l所示序列的第466位核苷酸發(fā)生突變，即G—C;此時(shí)，
9SEQ ID NO. 1所示序列編碼的氨基酸序列SEQ ID NO. 2第156位由Ala (A) —Pro (P);具體序列如SEQ ID NO. 18所示；
(4) SEQ ID NO. l所示序列的第548位核苷酸發(fā)生突變，即A—G;此時(shí)， SEQ ID NO. 1所示序列編碼的氨基酸序列SEQ ID NO. 2第183位由Glu (E) —Gly
(G);具體序列如SEQ ID N0.19所示；
(5) SEQ ID NO. l所示序列的第680位核苷酸發(fā)生突變，即T—A;此時(shí)， SEQ ID NO. 1所示序列編碼的氨基酸序列SEQ ID NO. 2第227位由Leu (L) —Gin
(Q);具體序列如SEQ ID NO. 20所示；
(6) SEQ ID NO. l所示序列的第682位核苷酸發(fā)生突變，即G—T;此時(shí)， SEQ ID NO. 1所示序列編碼的氨基酸序列SEQ ID NO. 2第270位由Arg (R) —Gly
(G);具體序列如SEQ ID NO. 21所示；
(7) SEQ ID NO. l所示序列的第808位核苷酸發(fā)生突變，即A—G;此時(shí)， SEQ ID NO. 1所示序列編碼的氨基酸序列SEQ ID NO. 2第275位由Pro (P) —Ala
(A);具體序列如SEQ ID N0.22所示;
(8) SEQ ID NO. l所示序列的第823位核苷酸發(fā)生突變，即C—G;此時(shí)， SEQ ID NO. 1所示序列編碼的氨基酸序列SEQ ID NO. 2第324位由lie (I) —Asn
(N);具體序列如SEQ ID NO. 23所示；
(9) SEQ ID NO. 1所示序列的第971位核苷酸發(fā)生突變，即T—A;此時(shí)， SEQ ID NO. 1所示序列編碼的氨基酸序列SEQ ID NO. 2第357位由Cys (C) —Tyr
(Y);具體序列如SEQ ID N0.24所示;
(10) SEQ ID NO. 1所示序列的第1070位核苷酸發(fā)生突變，即G—A;此時(shí)， SEQ ID NO. 1所示序列編碼的氨基酸序列SEQ ID NO. 2第362位由Pro (P) —Thr
(T);具體序列如SEQ ID NO. 25所示；
(11) SEQIDNO. 1所示序列的第1071位核苷酸發(fā)生突變，即C—A;此時(shí)， SEQ ID NO. 1所示序列編碼的氨基酸序列SEQ ID NO. 2第228位氨基酸由Glu (E) —終止(X);具體序列如SEQ ID N0.26所示；
(12) SEQ ID NO. 1所示序列的第1085位核苷酸發(fā)生突變，即C—T;此時(shí)， SEQ ID NO. 1所示序列編碼的氨基酸序列SEQ ID NO. 2第357位氨基酸由Cys (C) —終止(X);具體序列如SEQ ID NO. 27所示；(13) SEQ ID NO. 1所示序列的第1242位核苷酸發(fā)生突變，即C—A;此時(shí)， SEQ ID NO. 1所示序列編碼的氨基酸序列SEQ ID NO. 2第414位氨基酸由Tyr (Y) —終止(X);具體序列如SEQ ID N0.28所示。
最終這些突變都導(dǎo)致了 /^v7基因編碼的苯丙氨酸羥化酶活性的改變，影響 體內(nèi)氨基酸的正常代謝。序列表
<110〉上海交通大學(xué)
<120> PAH基因13種新突變的檢測(cè)方法
〈160> 28
<210〉 1
<211> 1359
<212>腿
〈213>人 (Homo sapiens)<400〉 1
atgtccactgcggtcctggaaaacccaggcttgggc鄉(xiāng)aaactctctga50
ctttggacaggaaacaagctcaactgcaatcaaaatggtg100
ccatatcactgatcttctcactcaaag肌gaagttggtgcattggccaaa150
gt3ttgcgcttatttgaggaaacctgacccacattgaatc200
tagaccttctcgtttaaagatgaatttttcacccatttgg250
at犯acgtagcctgcctgctctgacaaacatcatcaagatCttg3ggC3t300
g3C3ttggtgccactgtccatgagctttcacgagataagaaga犯g3cac350
agtgccctggttcccaagaagctggac卿tttgccaatc400
agattctcagctatggagcgg肌Ctgg3tgctgaccaccctggtttta犯450
gatcctgtgtaccgtgcaagacggaagcagtttgctgacattgcctacaa500
ctaccgccatgggC鄉(xiāng)CC3tccctcgagtgg犯toca^g550
gggcacagtgttcaagactctgaagtccttgtataaaacc600
catgcttgcttcacatttttccacttcttga朋agtsctg650
tggcttccatttccccagctgg卿3Cgtttctcagttcc700
tgcagacttgcactggtttccgcctccgacctgtggctggcctgctttcc750
tctcgggatttcttgggtggcctggccttccgagtcttccactgcacaca800
gtacatcagacatggatccaagcccatgtatacccccgaacctgacatct850
gccatgagctgttgggacatgtgcccttgttttcagatcgcagctttgcc900
cagttt"tcccccttgcctctctgggtgcacctgatgaata950
cattg犯aagctcgccacaatttactggttt8Ctgtgg3gtttgggctct1000
gc犯ac犯ggagactccataaaggcatatggtgctgggctcctgtcatcc1050
tttggtgaattacagtactgcttatcagagaagccaaagcttctccccct1100
ggsgctggag犯gacagcc3t c c aaaat t acactgtcacgg3gttCC3gC1150
ccctctattacgtggc卿gagttttaatgatgccaagga1200
aactttgctgtcggcccttctcagttcgctacgacccata1250
cacccaaaggattgaggtcttggacaatacccagcagcttaagattttgg1300
ctgattccattaacagtg犯3ttgg犯tcctttgcagtgccctccagaaa1350
1359
<210〉 2<211> 452<212> PRT
<213〉人 (Homo sapiens)<400> 2Met Ser ThrAlaValLeuGluAsnProGlyLeuGlyArgLysLeu
1 5 10 15
Ser Asp PheGlyGinGluThrSerTyrlieGluAspAsnCysAsn
20 25 30
Gin Asn GlyAlalieSerLeuliePheSerLeuLysGluGluVal
35 40 45
Gly Ala LeuAlaLysValLeuArgLeuPheGluGluAsnAspVal
50 55 60
Asn Leu ThrHislieGluSerArgProSerArgLeuLysLysAsp
65 70 75
Glu Tyr GluPhePheThrHisLeuAspLysArgSerLeuProAla
80 85 90
Leu Thr AsnlielieLyslieLeuArgHisAsplieGlyAlaThr
95 100 105
Val His GluLeuSerArgAspLysLysLysAspThrValProTrp
110 115 120
Phe Pro ArgThrlieGinGluLeuAspArgPheAlaAsnGinlie
125 130 135
Leu Ser TyrGlyAlaGluLeuAspAlaAspHisProGlyPheLys
140 145 150
Asp Pro ValTyrArgAlaArgArgLysGinPheAlaAsplieAla
155 160 165
Tyr Asn TyrArgHisGlyGinProlieProArgValGluTyrMet
170 175 180
Glu Glu GluLysLysThrTrpGlyThrValPheLysThrLeuLys
185 190 195
Ser Leu TyrLysThrHisAlaCysTyrGluTyrAsnHisliePhe
200 205 210
Pro Leu LeuGluLysTyrCysGlyPheHisGluAspAsnliePro
215 220 225
Gin Leu GluAspValSerGinPheLeuGinThrCysThrGlyPhe
230 235 240
Arg Leu ArgProValAlaGlyLeuLeuSerSerArgAspPheLeu
245 250 255
Gly Gly LeuAlaPheArgValPheHisCysThrGinTyrlieArg
260 265 270
His Gly SerLysProMetTyrThrProGluProAsplieCysHis
275 280 285
Glu Leu LeuGlyHisValProLeuPheSerAspArgSerPheAla
290 295 300
Gin Phe SerGinGlulieGlyLeuAlaSerLeuGlyAlaProAsp
305 310 315
Glu Tyr lieGluLysLeuAlaThrlieTyrTrpPheThrValGlu
320 325 330<formula>formula see original document page 14</formula>cgagtcttcc
CCtg￡LC￡ltct
cagttttcccctcgccacaaaaggca^tgaagccaaagcgagttccagcaactttgctgattgaggtctattgg犯tcc
actgcacaca gtacatcaga catggatcca agcccatgte tacccccgaa 840gccatgagct gttgggacat gtgcccttgt tttcagatcg cagctttgcc 900
aggaaattgg ccttgcctct ctgggtgcac ctgatg犯tatttactggtt tactgtggag tttgggctct gca犯c犯gggtgctgggct cctgtcatcc tttggtgaat tacagtactgttctccccct ggagctggag aagacagcca tccaaaattaccctctatta cgtggcagag兆tttt犯tg atgccaaggaccacaatacc tcggcccttc tcagttcgct acgacccatatggacaatac ccagcagctt aagattttgg ctgattccattttgcagtgc cctccaga犯ata犯gtaa
cattga^犯g 960agactccata 1020cttatcagag 1080cactgtcacg 1140gaaagtaagg 1200cacccaaagg 1260taacagtgaa 1320
〈210〉 4〈211〉 1359<212>腿
〈213〉人 (Homo sapiens)<220〉
〈221〉 mutation<222> (461)
<223〉n=a或g〈400〉 4
atgtccactg cggtcctgga aaacccaggc ttgggcagga aactctctga ctttggacag 60gaaacaagct atattgaaga c犯ctgcaat caaaatggtg ccatatcact gatcttctca 120ctc肌agaag aagttggtgc attggcca犯gtattgcgct tatttgagga g肌tgatgta 180犯cctgaccc acattgaatc tagaccttct cgtttaaaga aagatgagta tgaatttttc 240acccatttgg ata犯cgtag cctgcctgct ctgacaaaca tcatc犯gat cttgaggcat 300gacattggtg ccactgtcca tgagctttca cgagataaga agaaagacac agtgccctgg 360ttcccaagaa ccftttc犯ga gctggacaga tttgcc犯tc agattctcag ctatggagcg 420g犯ctggatg ctgaccaccc tggtttta犯 gatcctgtgt nccgtgcaag acgg犯gcag 480tttgctgaca ttgcctacaa ctaccgccat gggcagccca tccctcgagt ggaatacatg 540gaggaagsaa ag^iaaca^tg gggcacagtg ttcaagsctc tg犯gtcctt gtata^acc 600catgcttgct atgagtacaa tcacattttt ccacttcttg aaaagtactg tggcttccat 660g犯gat犯ca ttccccagct gg犯gacgtt tctcagttcc tgcagacttg cactggtttc 720cgcctccgac ctgtggctgg cctgctttcc tctcgggatt tcttgggtgg cctggccttc 780cgagtcttcc actgcacEica gtacatcaga catggatcca agcccatgta tacccccgaa 840cctgacatct gccatgagct gttgggacat gtgcccttgt tttcagatcg cagctttgcc 900cagttttccc agg犯attgg ccttgcctct ctgggtgcac ctgatgaata cattg犯aag 960ctcgccacaa cttactggtt tactgtggag tttgggctct gcaaacaagg agactccata 1020犯ggcatetg gtgctgggct cctgtcatcc tttggtgaat tacagtactt gttatcagag 1080朋gcca朋gc ttctccccct ggagctggag aagacagcca tccaaaatta cactgtcacg 1140gagttccagc ccctctatte cgtggcagag agtttt犯tg atgccaagga gaaagt犯gg 1200aactttgctg ccacaatacc tcggcccttc tcagttcgct acgacccata cacccaaagg 1260attgaggtct tggacaatac ccagcagctt aagattttgg ctgattccat taacagtgaa 1320attggeiatcc tttgcagtgc cctccagaaa ataaagtaa〈210〉 5 〈211〉 1359 <212〉 DNA
〈213>人 (Homo sapiens) 〈220〉
<221〉 mutation 〈222〉 (466)
〈223〉n二 g或c 〈400〉 5
atgtccactg cggtcctgga aaacccaggc ttgggcagga aactctctga ctttggacag 60 gaaacaagct atattgaaga caactgcaat caaaatggtg ccatatcact gatcttctca 120 ctcaaagaag aa^gttggtgc attggccaaa gtattgcgct tatttgagga g犯tgatgte 180 aacctgaccc acattgaatc tagaccttct cgtttaaaga aagatgagta tgaatttttc 240 acccatttgg ataaacgtag cctgcctgct ctgacaaaca tcatcaagat cttgaggcat 300 gacattggtg ccactgtccEi tgagctttca cga^t犯ga ag犯agacax; 3gtgccctgg 360 ttcccaag犯ccattcaaga gctggacaga tttgcc犯tc agattctcag ctatggagcg 420 gaactggatg ctgaccaccc tggttttaaa gatcctgtgt accgtncaag acggaagcag 480 tttgctgaca ttgcctacaa ctaccgccat gggcagccca tccctcgagt ggaatacatg 540 gaggaagaaa agaa肌catg gggcacagtg ttcaagacix tgaagtcctt gtataaetacc 600 catgcttgct atgagtacaa tcacattttt ccacttcttg aaaagtactg tggcttccat 660 g犯gataaca ttccccagct ggaagacgtt tctcagttcc tgcagacttg cactggtttc 720 cgcctccgac ctgtggctgg cctgctttcc tctcgggatt tcttgggtgg cctggccttc 780 cgagtcttcc actgcacaca gtacatcaga catggatcca agcccatgta tacccccgaa 840 cctgacatct gccatgagct gttgggacat gtgcccttgt tttcagatcg cagctttgcc 900 cagttttccc aggaaattgg ccttgcctct ctgggtgcac ctgatgaata cattgaaaag恥O ctcgccacaa tttactggtt tactgtggag tttgggctct gcaaacaagg agactccata 1020 aaggca/tatg gtgctgggct cctgtcatcc tttggtgaat tacagtactg cttatcagag 1080 aagccaaagc ttctccccct ggagctggag犯gacagcca tccaaaatta cactgtcacg 1140 gagttccagc ccctctatta cgtggcagag agttttaatg atgccaagga g犯agt犯gg 1200 aactttgctg ccac犯tacc tcggcccttc tcagttcgct acgacccata cacccaaagg 1260 attgaggtct tggac犯tac ccagcagctt aagattttgg ctgattccat taacagtgaa 1320 &ttggaa/tcc tttgcagtgc cctccagaaa ataaagt犯
〈210〉 6 <211> 1359 〈212〉腿
<213〉人 (Homo sapiens) <220〉
〈221〉 mutation <222〉 (548)
<223〉n= a或g <400〉 6
atgtccactg cggtcctgga aaacccaggc ttgggcagga aactctctga ctttggacag 60 gaaacaagct atattgaaga caactgcaat caaaatggtg ccatatcact gatcttctca 120aacctgaccc acccatttgg gacat;tggtg ttcccaagaa gaactggatg tttgctgaca
catgcttgct
cgcctccgac cgagtcttcc cctgacatct cagttttccc ctcgccacaa 犯ggcatstg 犯gcc朋agc gagttccagc 犯ctttgctg attgaggtct attggaatcc
犯gttggtgc 3ttggccaaa gtattgcgct城ttgagga gaatgatgta 180 acattg犯tc tagaccttct cgtttaaaga朋gatgagtei tgaatttttc 240
ataaacgtag cctgcctgct ctgacaaaca tcatcaagat cttgaggcat 300 ccactgtcca tgagctttca cgagataaga agaaagacac agtgccctgg 360 ccattcaaga gctggacaga tttgccaatc agattctcag ctatggagcg 420 ctgaccaccc tggttttaaa gatcctgtgt accgtgcaag acggaagcag 480 ttgcctacaa ctaccgccat gggcagccca tccctcgagt ggaatacatg 540
agaaaacatg gggcacagtg ttcaagactc tgaagtcctt gtataaaacc 600 atgagtacaa tcacatttt"t ccacttcttg aaaagtactg tggcttccat 660 ttccccagct ggaagacgtt tctcagttcc tgcagacttg cactggtttc 720 ctgtggctgg cctgctttcc tctcgggatt tcttgggtgg cctggccttc TOO actgcacaca gtacatcaga catggatcca agcccatgta tacccccgaa 840
gccatgagct gttgggacat gtgcccttgt tttcagatcg cagctttgcc 900 aggaaattgg ccttgcctct ctgggtgcac ctgatgaata cattgaaaag 960
tttactggtt tactgtggag gtgctgggct cctgtcatcc ttctccccct ggagctggag ccctctatta cgtggcagag ccacaatacc tcggcccttc tggacaatac ccagcagctt tttgcagtgc cctccagaaa
tttgggctct tttggtg犯t 犯g3C3gcca 3gttttaatg tcagttcgct aagattttgg
gca^casgg tacagtactg tccaaaatta
acgacccata ctg3ttccat
agactccata 1020 cttatcagag 1080 cactgtcacg 1140 gaaagtaagg 1200 cacccaaagg 1260 taacagtgaa 1320
〈210〉 7 〈211〉 1359 <212>醒
<213>人 (Homo sapiens) <220〉
<221〉 mutation <222> (680)
〈223>n: t或a 〈400〉 7
atgtccactg cggtcctgga aaacccaggc ttgggcagga aactctctga ctttggacag 60 gaaacaagct atattgaaga caactgcaat caaaatggtg ccatatcact gatcttctca 120 ctc犯agaag aagttggtgc attggcc服a gtattgcgct tatttgagga gaatgatgto 180 犯cctgaccc acattgaatc tagaccttct cgtttaaaga犯gatgagta tgaatttttc 240 acccatttgg ataaacgtag cctgcctgct ctgacaaaca tcatc犯gat cttgaggcat 300 gacattggtg ccactgtcca tgagctttca cgagataaga agaaagacac agtgccctgg 360 ttccc肌gaa ccattc肌ga gctggacaga tttgcc肌tc agattctcag ctatggagcg 420 g肌ctggatg ctgaccaccc tggtttt犯a gatcctgtgt accgtgc肌g acggaagcag 480 tttgctgaca ttgcctacaa ctaccgccat gggcagccca tccctcgagt ggaatacatg 540 gagga^ga犯 agaaaax;atg gggcscagtg ttc犯gactc tgaagtcctt gtota犯acc 600 catgcttgct atgagtacaa tcacattttt ccacttcttg aaaagtactg tggcttccat 660 gaagataaca ttccccagcn ggaagacgtt tctcagttcc tgcagacttg cactggtttc 720 cgcctccgac ctgtggctgg cctgctttcc tctcgggatt tcttgggtgg cctggccttc 780cgagtcttcc cctgacatct cagttttccc ctcgccacaa 犯ggcatatg 犯gcca犯gc gagttccagc aactttgctg attgaggtct attggaatcc
actgcacaca gtacatcaga catggatcca agcccatgta tacccccg犯840 gccatgagct gttgggacat gtgcccttgt tttcagatcg cagctttgcc 900
aggaaattgg ccttgcctct ctgggtgcac ctgatgaata tttactggtt tectgtggag tttgggctct gcaaacaagg gtgctgggct cctgtcatcc tttggtg犯t tacagtactg ttctccccct ggagctggag aagacagcca tcca^aatte ccctctatta cgtggcagag agttttaatg atgccaagga ccacaatacc tcggcccttc tcagttcgct acgacccata tggacaatac ccagcagctt aagattttgg ctgattccat tttgca_gtgc cctccagaaa ata肌gtaa
cattgaa犯g 960 agactccata 1020 cttatcagag 1080 cactgtcacg 1140 gaaagtaagg 1200 cacccaaagg 1260 taacaLgtgaa 1320
〈210〉 8 〈211〉 1359 〈212〉腿
〈213〉人 (Homo sapiens) 〈220〉
〈221〉 mutation 〈222〉 (682) <223〉n= g或t <400〉 8
cggtcctgg3 a^cccaggc ata/ttgaaga c犯ctgcaat 犯gttggtgc attggccaaa acattgaatc tagaccttct
aactctctga ctttggacag 60
ccatatcact gatcttctca 120 tatttgagga gaatgatgta 180 aagatgagta tgaatttttc 240
atgtccactg cggtcctgga a^cccaggc ttgggcagga gaasc肌gct a_ta/ttg^ga c犯ctgcaat C3犯atggtg ctcaaagaag aagttggtgc attggcca^a gtattgcgct aacctgaccc acattgaatc tagaccttct cgtttaaaga acccatttgg ataaacgtag cctgcctgct ctgacaaaca tcatcaagat cttgaggcat 300 gacattggtg ccactgtcca tgagctttca cgagataaga agaaagacac agtgccctgg 360 ntcccaagaa ccattcaaga gctggacaga tttgccaatc agattctcag ctatggagcg 420 gaactggatg ctgaccaccc tggttttaaa gatcctgtgt accgtgcaag acggaagcag 480 tttgctgaca ttgcctacaa ctaccgccat gggcagccca tccctcgagt ggaatacatg 540 g3gg犯gaaa agaaagLcatg gggceica^gtg ttc犯gax:tc tgaagtcctt gtata^aacc 600 catgcttgct eitgagtacaa tcacattttt ccacttcttg aaaagtactg tggcttccat 660 gaagataaca ttccccagct gnaagacgtt tctcagttcc tgcagacttg cactggtttc 720 cgcctccgac ctgtggctgg cctgctttcc tctcgggatt tcttgggtgg cctggccttc 780 cgagtcttcc actgcacaca gtacatcaga catggatcca agcccatgta tacccccgaa 840 cctgacatct gccatgagct gttgggacat gtgcccttgt tttcagatcg cagctttgcc 900 cagttttccc aggaaattgg ccttgcctct ctgggtgcac ctgatgaMa cattgaa犯g 960 ctcgccacaa tttactggtt tactgtggag tttgggctct gcaaacaagg agactccata 1020 aaggcatatg gtgctgggct cctgtcatcc tttggtgaat tacagtactg cttatcagag 1080 犯gccaaagc ttctccccct ggagctggag aagacagcca tccaaaatta cactgtcacg 1140 gagttccagc ccctctatta cgtggcagag agttttaatg atgccaagga gaaagtaagg 1200 aactttgctg ccacaatacc tcggcccttc tcagttcgct acgacccata cacccaaagg 1260 attgaggtct tggac犯tac ccagcagctt朋gattttgg ctgattccat taacagtg肌1320 attggaatcc tttgcagtgc cctccagaaa ataaagtaa
18〈210〉 9 <211〉 1359 <212〉 DNA
〈213〉人 (Homo sapiens) 〈220〉
<221〉 mutation 〈222〉 (808)
〈223〉n= a或g 〈400〉 9
atgtccactg cggtcctgga aaacccaggc ttgggcagga aactctctga ctttggacag 60 gaaacaagct atattgaaga caactgcaat caaaatggtg ccatatcact gatcttctca 120 ctc8aagaag aagttggtgc attggccaaa gtattgcgct tatttgagga gaatgatgta 180 aacctgaccc acattgaatc tagaccttct cgtttaaaga aagatgagta tgaatttttc 240 acccatttgg ata肌cgtag cctgcctgct ctgacaaaca tcatcaagat cttgaggcat 300 gacattggtg ccactgtcca tgagctttca cgagataaga aga^agacac agtgccctgg 360 ttccc犯gaa ccattcaaga gctggacaga tttgccaatc agattctcag ctatggagcg 420 gaactggatg ctgaccaccc tggttttaaa gatcctgtgt accgtgcaag acggaagcag 480 tttgctgaca ttgcctacaa ctaccgccat gggcagccca tccctcgagt ggaatacatg 540 gaggaag3肌 3ga^aacatg gggcacagtg ttc犯g3ctc tga^gtcctt gt3taaaacc 600 catgcttgct atgagtacaa tcacattttt ccacttcttg aaaagtactg tggcttccat 660 gaagataaca ttccccagct gg犯gacgtt tctcagttcc tgcagacttg cactggtttc 720 cgcctccgac ctgtggctgg cctgctttcc tctcgggatt tcttgggtgg cctggccttc 780 cgagtcttcc actgcacaca gtacatcnga catggatcca agcccatgta tacccccgaa 840 cctgacatct gccsitgagct gttgggacat gtgcccttgt tttcagatcg cagctttgcc 900 cagttttccc aggaaattgg ccttgcctct ctgggtgcac ctgatgaata cattgaaaag %0 ctcgccac犯tttactggtt tactgtggag tttgggctct gcaaacaagg agactccata 1020 aaggcatatg gtgctgggct cctgtcatcc tttggtgaat tacagtactg cttatcagag 1080 aagccaaagc ttctccccct ggagctggag aagacagcca tccaaaatta cactgtcacg 1140 gagttccagc ccctctatta cgtggcagag agttttaatg atgccaagga gaaagtaagg 1200 aactttgctg ccacaatacc tcggcccttc tcagttcgct acgacccata cacccaaagg 1260 attgaggtct tggacaatac ccagcagctt aagattttgg ctgattccat taacagtgaa 1320 attggaatcc tttgcagtgc cctccagaaa eitaaagtaa
〈210〉 10 〈211〉 1359 〈212> DNA
〈213〉人 (Homo sapiens) <220〉
〈221〉 mutation 〈222〉 (823)
〈223〉n= c或g 〈400〉 10
atgtccactg cggtcctgga aaacccaggc ttgggcagga肌ctctctga ctttggacag GO gaaacaagct atattgaaga caactgcaat caaaatggtg ccatatcact gatcttctca 120ctcaB3ga^g aacctgaccc acccatttgg g織ttggtg ntcccaagaa g犯ctggatg tttgctgaca gaggaagaaa catgcttgct
cgcctccgac cgagtcttcc cctgacatct cagttttccc ctcgccacaa aaggcatatg aagcc犯agc gagttccagc aactttgctg attgaggtct attggaatcc
aagttggtgc attggcc犯a gtattgcgct tatttgagga gaatgatgta 180 acattgaatc tagaccttct cgtttaaaga aagatgagta tgaatttttc 240
ataaacgtag cctgcctgct ctgacaaaca tcatcaagat cttgaggcat 300 ccactgtcca tgagctttca cgsgataaga agaeiagacac agtgccctgg 360 ccattcaaga gctggacaga tttgccaatc agattctcag ctatggagcg 420 ctgaccaccc tggttttaaa gatcctgtgt accgtgcaag acggaagcag 480 ttgcctacaa ctaccgccat gggcagccca tccctcgagt ggaatacatg 540
agaaaacatg gggcacagtg ttcaagactc tgaagtcctt gtataaaacc 600 atgagtacaa tcacattttt ccacttcttg aa犯gtactg tggcttccat 660 ttccccagct ggaagacgtt tctcagttcc tgcagacttg cactggtttc 720 ctgtggctgg cctgctttcc tctcgggatt tcttgggtgg cctggccttc 780 actgcacaca gtacatcaga catggatcca agnccatgta tacccccgaa 840
gccatgagct gttgggacat gtgcccttgt tttcagatcg cagctttgcc 900 aggaaattgg ccttgcctct ctgggtgcac ctgatgaata cattgaaaag 960
tttactggtt tactgtggag gtgctgggct cctgtcatcc ttctccccct ggagctggag ccctctatta cgtggcagag ccacaatacc tcggcccttc tggacaatac ccagcagctt tttgcagtgc cctccagaaa
tttgggctct tttggtg肌t aagacagcca agttttaatg tcagttcgct aagattttgg
gC犯8C犯gg
tacagtactg tccaaaatta a_tgcca^gga<
ctgattccat
agactccata 1020 cttatcagag 1080 cactgtcacg 1140 gaa3gt^1gg 1200 cacccaaagg 1260 taacagtg犯1320
〈210〉 11 〈211〉 1359 〈212〉 DNA
〈213>人 (Homo sapiens) <220〉
〈221〉 mutation 〈222〉 (971)
〈223>n= t或a 〈400〉 11
atgtccactg cggtcctgga犯acccaggc ttgggcagga aactctctga ctttggacag 60 gaaac肌gct ata/ttgaaga caactgcaat caaaatggtg ccatatcact gatcttctca 120 ctcaa3ga3g aagttggtgc attggccaaa gtattgcgct tatttgagga gaatgatgta 180 aacctgaccc acattgaatc tagaccttct cgtttaaaga aagatgagta tgaatttttc 240 acccatttgg ataaacgtag cctgcctgct ctgacaaaca tcatcaagat cttgaggcat 300 gacattggtg ccactgtcca tgagctttca cgagataaga ftga^eigacac agtgccctgg 360 ntccc犯gaa ccattcaaga gctggacaga tttgccaatc agattctcag ctatggagcg 420 gaactggatg ctgaccaccc tggtttta犯 gatcctgtgt accgtgcaag acggaagcag 480 tttgctgaca ttgcctacaa ctaccgccat gggcagccca tccctcgagt ggaa/taca/tg 540 gagg肌ga犯 Eigaaaacatg gggcacagtg ttc肌gactc tgaagtcctt gtataa肌cc 600 catgcttgct atgagtacaa tcacattttt ccacttcttg aaaagtactg tggcttccat 660 gaagataaca ttccccagct ggaagacgtt tctcagttcc tgcagacttg cactggtttc 720 cgcctccgac ctgtggctgg cctgctttcc tctcgggatt tcttgggtgg cctggccttc 780
20cgagtcttcc actgcacaca gtacatcaga catggatcca agcccatgta tacccccgaa 840cctgacatct gccatgagct gttgggacat gtgcccttgt tttcagatcg cagctttgcc 900cagttttccc aggaaattgg ccttgcctct ctgggtgcac ctgatgaata cattgaaaag 960ctcgccsLcaa nttactggtt tactgtgg恥tttgggctct gceia^ca^gg agactccata 1020aaggcatatg gtgctgggct cctgtcatcc tttggtgaat tacagtactg cttatcagag 1080aagccaaagc ttctccccct ggagctggag aagacagcca tccaaaatta cactgtcacg 1140gagttccagc ccctct3tta cgtggcagag agttttaatg atgccaagga gaaagt犯gg 1200aactttgctg ccacaatacc "tcggcccttc tcagttcgct acgacccata caccc犯agg 1260attgaggtct tggacaatac ccagcagctt aagattttgg ctgattccat taacagtgaa 1320attgg犯tcc tttgcagtgc cctccagaaa ataaagtaa
〈210〉 12〈211〉 1359<212〉 DNA
〈213〉人 (Hotno sapiens)〈220〉
〈221〉 mutation〈222〉 (1070)
<223〉n= g或a〈400〉 12
cggtcctgga a犯cccaggc3ta<ttgaaga caactgcaat犯gttggtgc attggccaaaacattgaatc tagaccttct
aactctctga ctttggacag 60ccatatcact gatcttctca 120tatttgagga gaatgatgta 180aagatgagta tgaatttttc 240
atgtccactg cggtcctgga a犯cccaggc ttgggcaggag犯3ca3gct a^ttgaaga^ caactgcaat caaaatggtgctcaaagaag aagttggtgc attggccaaa gtattgcgctaacctgaccc acattgaatc tagaccttct cgtttaaagaax;ccatttgg at338cgtag cctgcctgct ctgacaaaca tcatcaagat cttgaggcst 300gacattggtg ccactgtcca tgagctttca cgagataaga agaaagacac agtgccctgg 360ttcccaagaa ccattcaaga gctggacaga tttgccaatc agattctcag ctatggagcg 420g肌ctggatg ctgaccaccc tggttttaaa gatcctgtgt accgtgc肌g acggaagcag 480tttgctgaca ttgcctacaa ctaccgccat gggcagccca tccctcgagt ggaatacatg 540gaggaagaaa agaaaacatg gggcacagtg ttcaagactc tgaagtcctt gtataaaacc 600catgcttgct atgagtacaa tcacattttt ccacttcttg aaaagtactg tggcttccat 660g犯gataaca ttccccagct ggaagacgtt tctcagttcc tgcagacttg cactggtttc 720cgcctccgac ctgtggctgg cctgctttcc tctcgggatt tcttgggtgg cctggccttc 780cgagtcttcc actgcacaca gtacatcaga catggatcca agcccatgta tacccccgaa 840cctgacatct gccatgagct gttgggacat gtgcccttgt tttcagatcg cagctttgcc 900cagttttccc aggaaattgg ccttgcctct ctgggtgcac ctgatgaata cattgaaaag %0ctcgccacaa tttactggtt tactgtggag tttgggctct gcaaacaagg agactccata 1020aaggcatatg gtgctgggct cctgtcatcc tttggtgaat tacagtactn cttatcagag 1080aagccaaagc ttctccccct ggagctggag aagacagcca tccaaaatta cactgtcacg 1140gagttccagc ccctctatta cgtggcagag agttttaatg atgccaagga gaaagtaagg 1200aactttgctg ccacaatacc tcggcccttc tcagttcgct acgacccata cacccaaagg 1260attgaggtct tggacaatac ccagcagctt aagattttgg ctgattccat taacagtgaa 1320attggaatcc tttgcagtgc cctccagaaa ataaagtaa〈210〉 13〈211〉 1359<212> DNA
<213〉人 (Homo sapiens)〈220〉
<221〉 mutation〈222〉 (1071)
<223〉n= c或g〈400〉 13
atgtccactg cggtxctgga犯acccaggc ttgggcagga aactctctga ctttggacag 60gaaacaagct atattgaaga caactgcaat caaaatggtg cca/tatcact gatcttctca 120ctcaaagaag aagttggtgc attggcca犯gtattgcgct tatttgagga gaatgatgta 180aacctgaccc acattgaatc tagaccttct cgttt犯aga aagatgagta tgaatttttc 240acccatttgg ataaacgtag cctgcctgct ctgacaaaca tcatcaagat cttgaggcat 300g6Lcattggtg ccactgtcca tgagctttca cgagat犯ga ag犯agacac agtgccctgg 360ttcccaagaa ccattcaaga gctggacaga tttgccaatc agattctcag ctatggagcg 420g33ctggatg ctgaccaccc tggtttt3aa gatcctgtgt accgtgcaag acggaagceig 480tttgctgaca ttgcctacaa ctaccgccat gggcagccca tccctcgagt ggaatacatg 540gaggaaga犯 agaaaacatg gggcacagtg ttcaagsctc tg犯gtcctt gtotaaaacc 600catgcttgct atgagtecaa tcacattttt ccacttcttg aaaagtactg tggcttccat 660gaagataaca ttccccagct ggaagacgtt tctcagttcc tgcagacttg cactggtttc 720cgcctccgac ctgtggctgg cctgctttcc tctcgggatt tcttgggtgg cctggccttc 780cgagtcttcc actgcacaca gtacatcaga catggatcca agcccatgta tacccccgaa 840cctgacatct gccatgagct gttgggacat gtgcccttgt tttcagatcg cagctttgcc 900cagttttccc aggaaattgg ccttgcctct ctgggtgcac ctgatgaata cattgaaaag %0ctcgccacaa tttactggtt tactgtggag tttgggctct gcaaacaagg agactccata 1020aaggcatatg gtgctgggct cctgtcatcc tttggtg犯t tacagtactg nttatcagag 1080aagccaaagc ttctccccct ggagctggag aagacagcca tccaaaatta cactgtcacg 1140gagttccagc ccctctatta cgtggcagag agttttaatg atgccaagga gaaagtaagg 1200aactttgctg ccacaatacc tcggcccttc tcagttcgct acgacccata cacccaaagg 1260aittgaggtct tggacaatac ccagcagctt aagattttgg ctgattccat taacagtgaa 1320attggaatcc tttgcagtgc cctccagaaa ataaagt犯
〈210〉 14〈211〉 1359〈212〉醒
<213〉人 (Homo sapiens)〈220〉
〈221〉 mutation〈222〉 (1085)
〈223〉n二 c或t<400> 14
atgtccactg cggtcctgga aaacccaggc ttgggcagga aactctctga ctttggacag 60g肌acaagct atattgaaga c￡tactgcaa/t caaaatggtg ccatatcact gatcttctca 120ctcaaaga^g肌cctgacccacccatttgg
g3C3ttggtg
ttcccaagaagaactggatgtttgctgaca
catgcttgct
cgcctccgaccgagtcttcccctgacatctcagttttcccctcgccacaaa3ggcatatgaagcrma^gcgagttccagcaactttgctgattgaggtctattggaatcc
犯gttggtgc attggcca犯gteittgcgct tatttgagg3 gaatgatgta 180acattgaa1:c tegaccttct cgtttaaaga aagatgagta tgaatttttc 240
ataaacgtag cctgcctgct ctgacaaaca tcatcaagat cttgaggcat 300ccactgtcca tgagctttca cgagataaga agaaagacac agtgccctgg 360ccattcaaga gctggacaga tttgccaatc agattctcag ctatggagcg 420ctgaccaccc tggtttta犯 gatcctgtgt accgtgcaag acggaagcag 480ttgcctacaa ctaccgccat gggcagccca tccctcgagt ggaatacatg 540
agaaaacatg gggcacagtg ttc犯gactc tgaagtcctt gtataaaacc 600atgagtacaa tcacattttt ccacttcttg aaaagtactg tggcttccat 660ttccccagct ggaagacgtt tctcagttcc tgcagacttg cactggtttc 720ctgtggctgg cctgctttcc tctcgggatt tcttgggtgg cctggccttc 780actgcacaca gtacatcaga catggatcca agcccatgta tacccccgaa 840
gccatgagct gttgggacat gtgcccttgt tttcagatcg cagctttgcc 900aggaaattgg ccttgcctct ctgggtgcac ctgatgaata cattg犯aag 960
tttactggtt tactgtggaggtgctgggct cctgtcatccttctccccct ggagctggagccctctatta cgtggcagagccacaatacc tcggcccttctggacaatac ccagcagctttttgcagtgc cctccaggi犯
tttgggctcttttggtgaataagac3gccaagttttaatgtcagttcgctaagattttggataaagtaa
tacagtactgtcc犯aattaatgcc朋gga3cgaccc3tactgattccat
agactccata 1020cttatcagag 1080cactgtcacg 1140gaaagtaagg 1200caccc333gg 1260taacagtgaa 1320
〈210〉 15〈211〉 1359〈212〉 DMA
<213〉人 (Homo sapiens)〈220〉
<221〉 mutation<222〉 (1242)
〈223〉n= c或a〈400〉 15
atgtccfictg cggtcctgg3 aaaccraggc ttgggcsgga ￡iactctctg￡i ctttggeicag 60gaa^caagct atattgaaga caactgcaat caaaatggtg ccatatcact gatcttctca 120ctc朋agaag犯gttggtgc attggcca犯gtattgcgct tatttgagga g犯tgatgta 180aacctgaccc acattgaatc tagaxxttct cgtttaaaga aagatgagta tgaatttttc 240acccatttgg ataaacgtag cctgcctgct ctgacaaaca tcatcaagat cttgaggcat 300gacattggtg ccactgtcca tgagctttca cgagataaga agaaagacac agtgccctgg 360ttcccaagaa ccattcaaga gctggacaga tttgccaatc agattctcag ctatggagcg 420gaactggatg ctgaccaccc tggtttta肌 gatcctgtgt accgtgc犯g acggaagcag 480tttgctgaca ttgcctacaa ctaccgccat gggcagccca tccctcgagt ggaatacatg 540gagg朋ga肌 agaaaacatg gggcacagtg ttcaagactc tg服gtcctt gtata^aacc 600catgcttgct atgagtacaa tcacattttt ccacttcttg aaaagtactg tggcttccat 660gaagataaca ttccccagct ggaagacgtt tctcagttcc tgcagacttg cactggtttc 720cgcctccgac ctgtggctgg cctgctttcc tctcgggatt tcttgggtgg cctggccttc TOOcgagtcttcc actgcacaca gtacatcaga catggatcca agccca_tgta_ tacccccg犯840 cctgacatct gccatgagct gttgggacat gtgcccttgt tttcagatcg cagctttgcc 900
cagttttcccagga犯ttggccttgcctctctgggtgcacctgatgaatacattgaa朋g 960ctcgccacaatttactggtttactgtgg觀tttgggctctgcaaacaaggagactccata1020
犯ggcatatggtgctgggctcctgtcatcctttggtg犯tt3C3gt3Ctgcttatcagag翻
犯gccaaagcttctccccctggagctggagaagacagccatccaaaattacactgtcacg1140
gagttccagcccctctattacgtggc,g3gtttt犯tgatgccaagga1200
aactttgctgccac3atacctcggcccttctcagttcgctangacccatacaccc肌agg1260
attgaggtcttggacaatacccagcagcttaagatt"ttggctgattccat1320
attggaatcctttgcagtgccctccagaaaa/taaagtaa
<210> 16 <211〉 452 <212> PRT
<213〉人 (Homo sapiens) 〈220〉
<221> VARIANT 〈222> 121
<223〉 Xaa二Phe或Leu <400> 16
Met Ser ThrAlaValLeuGluAsnProGlyLeuGlyArgCysLeu
1 5 10 15
Ser Asp PheGlyGinGluThrSerTyrlieGluAspAsnCysAsn
20 25 30
Gin Asn GlyAlalieSerLeuliePheSerLeuLysGluGluVal
35 40 45
Gly Ala LeuAlaLysValLeuArgLeuPheGluGluAsnAspVal
50 55 60
Asn Leu ThrHislieGluSerArgProSerArgLeuLysLysAsp
65 70 75
Glu Tyr GluPhePheThrHisLeuAspLysArgSerLeuProAla
80 85 90
Leu Thr AsnlielieLyslieLeuArgHisAsplieGlyAlaThr
95 100 105
Val His GluLeuSerArgAspLysLysLysAspThrValProTrp
110 115 120
Xaa Pro ArgThrlieGinGluLeuAspArgPheAlaAsnGinlie
125 130 135
Leu Ser TyrGlyAlaGluLeuAspAlaAspHisProGlyPheLys
140 145 150
Asp Pro ValTyrArgAlaArgArgLysGinPheAlaAsplieAla
155 160 165
Tyr Asn TyrArgHisGlyGinProlieProArgValGluTyrMet
170 175 180<formula>formula see original document page 25</formula><221> VARIANT
<222〉154
<223〉 Xaa^yr或Cys
<400> 17
Met Ser ThrAlaValLeuGluAsnProGlyLeuGlyArgLysLeu
1 5 10 15
Ser Asp PheGlyGinGluThrSerTyrlieGluAspAsnCysAsn
20 25 30
Gin Asn GlyAlalieSerLeuliePheSerLeuLysGluGluVal
35 40 45
Gly Ala LeuAlaLysValLeuArgLeuPheGluGluAsnAspVal
50 55 60
Asn LeuThrHislieGluSerArgProSerArgLeuLysLysAsp
65 70 75
Glu Tyr GluPhePheThrHisLeuAspLysArgSerLeuProAla
80 85 90
Leu Thr AsnlielieLyslieLeuArgHisAsplieGlyAlaThr
95 100 105
Val HisGluLeuSerArgAspLysLysLysAspThrValProTrp
110 115120
Phe ProArgThrlieGinGluLeuAspArgPheAlaAsnGinlie
125 130135
Leu SerTyrGlyAlaGluLeuAspAlaAspHisProGlyPheLys
140 145150
Asp ProValXaaArgAlaArgArgLysGinPheAlaAsplieAla
155 160165
Tyr AsnTyrArgHisGlyGinProlieProArgValGluTyrMet
170 175180
Glu GluGluLysLysThrTrpGlyThrValPheLysThrLeuLys
185 190195
Ser LeuTyrLysThrHisAlaCysTyrGluTyrAsnHisliePhe
200 205210
Pro LeuLeuGluLysTyrCysGlyPheHisGluAspAsnliePro
215 220225
Gin LeuGluAspValSerGinPheLeuGinThrCysThrGlyPhe
230 235240
Arg LeuArgProValAlaGlyLeuLeuSerSerArgAspPheLeu
245 250255
Gly GlyLeuAlaPheArgValPheHisCysThrGinTyrlieArg
260 265270
His GlySerLysProMetTyrThrProGluProAsplieCysHis
275 280285
Glu LeuLeuGlyHisValProLeuPheSerAspArgSerPheAla
290 295300Gin Phe Ser Gin 305 310 315 Glu Tyr lie Glu 320 325 330 Phe Gly Leu Cys 335 340 345 Gly Leu Leu Ser 350 355 360 Lys Pro Lys Leu 365 370 375 Asn Tyr Thr Val 380 385 390 Ser Phe Asn Asp 395 400 405 lie Pro Arg Pro 410 415 420 lie Glu Val Leu 425 430 435 Ser lie Asn Ser 440 445 450 lie Lys 452
<210> 18 <211〉 452 <212〉 PRT <213>人(Homo sapiens) 〈220>
<221> VARIANT <222〉156
<223> Xaa=Ala或Pro <400> 18
Met Ser Thr Ala Val Leu Glu Asn Pro Gly Leu Gly Arg Lys Leu 1 5 10 15
Ser Asp Phe Gly Gin Glu Thr Ser Tyr lie Glu Asp Asn Cys Asn 20 25 30
Gin Asn Gly Ala lie Ser Leu lie Phe Ser Leu Lys Glu Glu Val 35 40 45
Gly Ala Leu Ala Lys Val Leu Arg Leu Phe Glu Glu Asn Asp Val 50 55 60
Asn Leu Thr His lie Glu Ser Arg Pro Ser Arg Leu Lys Lys Asp 65 70 75
Glu Tyr Glu Phe Phe Thr His Leu Asp Lys Arg Ser Leu Pro Ala 80 85 90
Glu lie Gly Leu Ala Ser Leu Gly Ala Pro Asp Lys Leu Ala Thr lie Tyr Trp Phe Thr Val Glu Lys Gin Gly A印Ser lie Lys Ala Tyr Gly Ala Ser Phe Gly Glu Leu Gin Tyr Cys Leu Ser Glu Leu Pro Leu Glu Leu Glu Lys Thr Ala lie Gin Thr Glu Phe Gin Pro Leu Tyr Tyr Val Ala Glu Ala Lys Glu Lys Val Arg Asn Phe Ala Ala Thr Phe Ser Val Arg Tyr Asp Pro Tyr Thr Gin Arg Asp Asn Thr Gin Gin Leu Lys lie Leu Ala Asp Glu lie Gly lie Leu Cys Ser Ala Leu Gin LysLeuThr AsnlielieLyslieLeuArgHisAsplieGlyAlaThr
95 100 105
ValHisGluLeuSerArgAspLysLysLysAspThrValProTrp
110115120
PheProArgThrlieGinGluLeuAspArgPheAlaAsnGinlie
125130135
LeuSerTyrGlyAlaGluLeuAspAlaAspHisProGlyPheLys
140145150
AspProValTyrArgXaaArgArgCysGinPheAlaAsplieAla
155160165
TyrAsnTyrArgHisGlyGinProlieProArgValGluTyrMet
170175180
GluGluGluLysLysThrTrpGlyThrValPheLysThrLeuLys
185190195
SerLeuTyrLysThrHisAlaCysTyrGluTyrAsnHisliePhe
200205210
ProLeuLeuGluLysTyrCysGlyPheHisGluAspAsnliePro
215220225
GinLeuGluAspValSerGinPheLeuGinThrCysThrGlyPhe
230235240
ArgLeuArgProValAlaGlyLeuLeuSerSerArgAspPheLeu
245250255
GlyGlyLeuAlaPheArgValPheHisCysThrGinTyrlieArg
260265270
HisGlySerLysProMetTyrThrProGluProAsplieCysHis
275280285
GluLeuLeuGlyHisValProLeuPheSerAspArgSerPheAla
290295300
GinPheSerGinGlulieGlyLeuAlaSerLeuGlyAlaProAsp
305310315
GluTyrlieGluLysLeuAlaThrlieTyrTrpPheThrValGlu
320325330
PheGlyLeuCysLysGinGlyAspSerlieLysAlaTyrGlyAla
335340345
GlyLeuLeuSerSerPheGlyGluLeuGinTyrCysLeuSerGlu
350355360
LysProLysLeuLeuProLeuGluLeuGluLysThrAlalieGin
365370375
AsnTyrThrValThrGluPheGinProLeuTyrTyrValAlaGlu
380385390
SerPheAsnAspAlaLysGluLysValArgAsnPheAlaAlaThr
395400405
lieProArgProPheSerValArgTyrAspProTyrThrGinArg
410415420<formula>formula see original document page 29</formula>ProLeuLeuGluLysTyrCys Gly Phe His GluAspAsnlie
215220225
GinLeuGluAspValSerGin Phe Leu Gin ThrCysThrGly
230235240
ArgI>euArgProValAlaGly Leu Leu Ser SerArgAspPhe
245250255
GlyGlyLeuAlaPheArgVal Phe His Cys ThrGinTyrlie
260265270
HisGlySerLysProMetTyr Thr Pro Glu ProAsplieCys
275280285
GluLeuLeuGlyHisValPro Leu Phe Ser AspArgSerPhe
290295300
GinPheSerGinGlulieGly Leu Ala Ser LeuGlyAlaPro
305310315
GluTyrlieGluLysLeuAla Thr lie Tyr TrpPheThrVal
320325330
PheGlyLeuCysLysGinGly Asp Ser lie LysAlaTyrGly
335340345
GlyLeuLeuSerSerPheGly Glu Leu Gin TyrCysLeuSer
350355360
LysProLysLeuLeuProLeu Glu Leu Glu LysThrAlalie
365370375
AsnTyrThrValThrGluPhe Gin Pro Leu TyrTyrValAla
380385390
SerPheAsnAspAlaLysGlu Lys Val Arg AsnPheAlaAla
395400405
lieProArgProPheSerVal Arg Tyr Asp ProTyrThrGin
410415420
lieGluValLeuAspAsnThr Gin Gin Leu LyslieLeuAla
425430435
SerlieAsnSerGlulieGly lie Leu Cys SerAlaLeuGin
440445450
lieLys
452
〈210〉 20 <211> 452 〈212> PRT
<213>人 (Homo sapiens) <220>
<221> VARIANT <222〉 227
<223> Xaa:Leu或Gln 〈400〉 20Met Ser ThrAlaValLeuGluAsnProGlyLeuGlyArgLysLeu
1 5 10 15
Ser Asp PheGlyGinGluThrSerTyrlieGluAspAsnCysAsn
20 25 30
Gin Asn GlyAlalieSerLeuliePheSerI>euLysGluGluVal
35 40 45
Gly Ala LeuAlaLysValI>euArgLeuPheGluGluAsnAspVal
50 55 60
Asn Leu ThrHislieGluSerArgProSerArgCeuLysLysAsp
65 70 75
Glu Tyr GluPhePheThrHisLeuAspLysArgSerLeuProAla
80 85 90
L.eu Thr AsnlielieLyslieLeuArgHisAsplieGlyAlaThr
95 100 105
Val His GluLeuSerArgAspLysLysLysAspThrValProTrp
110 115 120
Phe Pro ArgThrlieGinGluLeuAspArgPheAlaAsnGinlie
125 130 135
Leu Ser TyrGlyAlaGluLeuAspAlaAspHisProGlyPheLys
140 145 150
Asp Pro ValTyrArgAlaArgArgLysGinPheAlaAsplieAla
155 160 165
Tyr Asn TyrArgHisGlyGinProlieProArgValGluTyrMet
170 175 180
Glu Glu GluLysLysThrTrpGlyThrValPheLysThrLeuLys
185 190 195
Ser Leu TyrLysThrHisAlaCysTyrGluTyrAsnHisliePhe
200 205 210
Pro Leu LeuGluLysTyrCysGlyPheHisGluAspAsnliePro
215 220 225
Gin Xaa GluAspValSerGinPheLeuGinThrCysThrGlyPhe
230 235 240
Arg Leu ArgProValAlaGlyLeuLeuSerSerArgAspPheLeu
245 250 255
Gly Gly LeuAlaPheArgValPheHisCysThrGinTyrlieArg
260 265 270
His Gly SerLysProMetTyrThrProGluProAsplieCysHis
275 280 285
Glu Leu LeuGlyHisValProLeuPheSerAspArgSerPheAla
290 295 300
Gin Phe SerGinGlulieGlyLeuAlaSerLeuGlyAlaProAsp
305 310 315
Glu Tyr lieGluLysLeuAlaThrlieTyrTrpPheThrValGlu
320 325 330Phe Gly Leu Cys 335 340 345 Gly Leu Leu Ser 350 355 360 Lys Pro Lys Leu 365 370 375 Asn Tyr Thr Val 380 385 390 Ser Phe Asn Asp 395 400 405 lie Pro Arg Pro 410 415 420 lie Glu Val Leu 425 430 435 Ser lie Asn Ser 440 445 450 lie Lys 452
〈210〉 21 <211> 452 <212> PRT <213〉人 (Homo sapiens) <220>
〈221〉 VARIANT <222> 228 <223> Xaa二Glu或X <400> 21
Met Ser Thr Ala Val Leu Glu Asn Pro Gly Leu Gly Arg Lys Leu 1 5 10 15
Ser Asp Phe Gly Gin Glu Thr Ser Tyr lie Glu Asp Asn Cys Asn 20 25 30
Gin Asn Gly Ala lie Ser Leu lie Phe Ser Leu Lys Glu Glu Val 35 40 45
Gly Ala Leu Ala Lys Val Leu Arg Leu Phe Glu Glu Asn Asp Val 50 55 60
Asn Leu Thr His lie Glu Ser Arg Pro Ser Arg Leu Lys Lys Asp 65 70 75
Glu Tyr Glu Phe Phe Thr His Leu Asp Lys Arg Ser Leu Pro Ala 80 85 90
Leu Thr Asn lie lie Lys lie Leu Arg His Asp lie Gly Ala Thr 95 100 105
Val His Glu Leu Ser Arg Asp Lys Lys Lys Asp Thr Val Pro Trp 110 115 120
Lys Gin Gly Asp Ser lie Lys Ala Tyr Gly Ala Ser Phe Gly Glu Leu Gin Tyr Cys Leu Ser Glu Leu Pro Leu Glu Leu Glu Lys Thr Ala lie Gin Thr Glu Phe Gin Pro Leu Tyr Tyr Val Ala Glu Ala Lys Glu Lys Val Arg Asn Phe Ala Ala Thr Phe Ser Val Arg Tyr Asp Pro Tyr Thr Gin Arg Asp Asn Thr Gin Gin Leu Lys lie Leu Ala Asp Glu lie Gly lie Leu Cys Ser Ala Leu Gin LysPheProArgThrlieGinGluLeuAspArgPheAlaAsnGinlie
125130135
LeuSerTyrGlyAlaGluLeuAspAlaAspHisProGlyPheLys
140145150
AspProValTyrArgAlaArgArgLysGinPheAlaAsplieAla
155160165
TyrAsnTyrArgHisGlyGinProlieProArgValGluTyrMet
170175180
GluGluGluLys!>ysThrTrpGlyThrValPheLysThrLeuLys
185190195
SerLeuTyrLysThrHisAlaCysTyrGluTyrAsnHisliePhe
200205210
ProLeuLeuGluLysTyrCysGlyPheHisGluAspAsnliePro
215220225
GinLeuXaaAspValSerGinPheLeuGinThrCysThrGlyPhe
230235240
ArgLeuArgProValAlaGlyLeuLeuSerSerArgAspPheLeu
245250255
GlyGlyLeuAlaPheArgValPheHisCysThrGinTyrlieArg
260265270
HisGlySerLysProMetTyrThrProGluProAsplieCysHis
275280285
GluLeuLeuGlyHisValProLeuPheSerAspArgSerPheAla
290295300
GinPheSerGinGlulieGlyLeuAlaSerLeuGlyAlaProAsp
305310315
GluTyrlieGluLysLeuAlaThrlieTyrTrpPheThrValGlu
320325330
PheGlyLeuCysLysGinGlyAspSerlieLysAlaTyrGlyAla
335340345
GlyLeuLeuSerSerPheGlyGluLeuGinTyrCysLeuSerGlu
350355360
LysProLysLeuLeuProLeuGluLeuGluLysThrAlalieGin
365370375
AsnTyrThrValThrGluPheGinProLeuTyrTyrValAlaGlu
380385390
SerPheAsnAspAlaLysGluLysValArgAsnPheAlaAlaThr
395400405
lieProArgProPheSerValArgTyrAspProTyrThrGinArg
410415420
lieGluValLeuAspAsnThrGinGinLeuLyslieLeuAlaAsp
425430435
SerlieAsnSerGlulieGlylieLeuCysSerAlaLeuGinLys
440445450<formula>formula see original document page 34</formula><formula>formula see original document page 35</formula>Gin Asn GlyAlalieSerLeuliePheSerLeuLysGluGluVal
35 40 45
Gly Ala LeuAlaLysValLeuArgLeuPheGluGluAsnAspVal
50 55 60
Asn LeuThrHislieGluSerZVrgProSerArgLeuLysLysAsp
65 70 75
Glu Tyr GluPhePheThrHisLeuAspLysArgSerLeuProAla
80 85 90
Leu Thr AsnlielieLyslieLeuArgHisAsplieGlyAlaThr
95 100 105
Val HisGluLeuSerArgAspLysLysLysAspThrValProTrp
110 115120
Phe ProArgThrlieGinGluLeuAspArgPheAlaAsnGinlie
125 130135
Leu SerTyrGlyAlaGluLeuAspAlaAspHisProGlyPheLys
140 145150
Asp ProValTyrArgAlaArgArgLysGinPheAlaAsplieAla
155 160165
Tyr AsnTyrArgHisGlyGinProlieProArgValGluTyrMet
170 175180
Glu GluGluLysLysThrTrpGlyThrValPheLysThrLeuLys
185 190195
Ser LeuTyrLysThrHisAlaCysTyrGluTyrAsnHisliePhe
200 205210
Pro LeuLeuGluLysTyrCysGlyPheHisGluAspAsnliePro
215 220225
Gin LeuGluAspValSerGinPheLeuGinThrCysThrGlyPhe
230 235240
Arg LeuArgProValAlaGlyLeuLeuSerSerArgAspPheLeu
245 250255
Gly GlyLeuAlaPheArgValPheHisCysThrGinTyrlieArg
260 265270
His GlySerLysXaaMetTyrThrProGluProAsplieCysHis
275 280285
Glu LeuLeuGlyHisValProLeuPheSerAspArgSerPheAla
290 295300
Gin PheSerGinGlulieGlyLeuAlaSerLeuGlyAlaProAsp
305 310315
Glu TyrlieGluLysLeuAlaThrlieTyrTrpPheThrValGlu
320 325330
Phe GlyLeuCysLysGinGlyAspSerlieLysAlaTyrGlyAla
335 340345
Gly LeuLeuSerSerPheGlyGluLeuGinTyrCysLeuSerGlu
350 355360<formula>formula see original document page 37</formula>AspProVal Tyr Arg
155160165
TyrAsnTyr Arg His
170175180
GluGluGlu Lys Lys
185190195
SerLeuTyr Lys Thr
200205210
ProLeuLeu Glu Lys
215220225
GinLeuGlu Asp Val
230235240
ArgLeuArg Pro Val
245250255
GlyGlyLeu Ala Phe
260265270
HisGlySer Lys Pro
275280285
GluLeuLeu Gly His
290295300
GinPheSer Gin Glu
305310315
GluTyrlie Glu Lys
320325330
PheGlyLeu Cys Lys
335340345
GlyLeuLeu Ser Ser
350355360
LysProLys Leu Leu
365370375
AsnTyrThr Val Thr
380385390
SerPheAsn Asp Ala
395400艦
lieProArg Pro Phe
410415420
lieGluVal Leu Asp
425430435
SerlieAsn Ser Glu
440445450
lieLys
452
<210〉 25
Ala Arg Arg Lys Gin Gly Gin Pro lie Pro Thr Trp Gly Thr Val His Ala Cys Tyr Glu Tyr Cys Gly Phe His Ser Gin Phe Leu Gin Ala Gly Leu Leu Ser Arg Val Phe His Cys Met Tyr Thr Pro Glu Val Pro Leu Phe Ser lie Gly Leu Ala Ser Leu Ala Thr Xaa Tyr Gin Gly Asp Ser lie Phe Gly Glu Leu Gin Pro Leu Glu Leu Glu Glu Phe Gin Pro Leu Lys Glu Lys Val Arg Ser Val Arg Tyr Asp Asn Thr Gin Gin Leu lie Gly lie Leu Cys
Phe Ala Asp lie Ala Arg Val Glu Tyr Met Phe Lys Thr Leu Lys Tyr Asn His lie Phe Glu Asp Asn lie Pro Thr Cys Thr Gly Phe Ser Arg Asp Phe Leu Thr Gin Tyr lie Arg Pro Asp lie Cys His Asp Arg Ser Phe Ala Leu Gly Ala Pro Asp Trp Phe Thr Val Glu Lys Ala Tyr Gly Ala Tyr Cys Leu Ser Glu Lys Thr Ala lie Gin Tyr Tyr Val Ala Glu Asn Phe Ala Ala Thr Pro Tyr Thr Gin Arg Lys lie Leu Ala Asp Ser Ala Leu Gin Lys<211> 452 <212〉 PRT
<213>人 (Homo sapiens) <220>
<221> VARIANT <222> 357
<223〉 Xaa:Cys或Tyr <400> 25
Met SerThrAlaValLeuGluAsnProGlyLeuGlyArgLysLeu
1 5 10 15
Ser Asp PheGlyGinGluThrSerTyrlieGluAspAsnCysAsn
20 25 30
Gin Asn GlyAlalieSerLeuliePheSerLeuLysGluGluVal
35 40 45
Gly Ala LeuAlaLysValLeuArgLeuPheGluGluAsnAspVal
50 55 60
Asn Leu ThrHislieGluSerArgProSerArgLeuLysLysAsp
65 70 75
Glu Tyr GluPhePheThrHisLeuAspLysArgSerLeuProAla
80 85 90
Leu ThrAsnlielieLyslieLeuArgHisAsplieGlyAlaThr
95 100 105
Val HisGluLeuSerArgAspLysLysLysAspThrValProTrp
110 115120
Phe ProArgThrlieGinGluLeuAspArgPheAlaAsnGinlie
125 130135
Leu SerTyrGlyAlaGluLeuAspAlaAspHisProGlyPheLys
140 145150
Asp ProValTyrArgAlaArgArgLysGinPheAlaAsplieAla
155 160165
Tyr AsnTyrArgHisGlyGinProlieProArgValGluTyrMet
170 175180
Glu GluGluLysLysThrTi-pGlyThrValPheLysThrLeuLys
185 190195
Ser LeuTyrLysThrHisAlaCysTyrGluTyrAsnHisliePhe
200 205210
Pro LeuLeuGluLysTyrCysGlyPheHisGluAspAsnliePro
215 220225
Gin LeuGluAspValSerGinPheLeuGinThrCysThrGlyPhe
230 235240
Arg LeuArgProValAlaGlyLeuLeuSerSerArgAspPheLeu
245 250255
Gly GlyLeuAlaPheArgValPheHisCysThrGinTyrlieArg
260 265270His Gly Ser Lys 275 280 285 Glu Leu Leu Gly 290 295 300 Gin Phe Ser Gin 305 310 315 Glu Tyr lie Glu 320 325 330 Phe Gly Leu Cys 335 340 345 Gly Leu Leu Ser 350 355 360 Lys Pro Lys Leu 365 370 375 Asn Tyr Thr Val 380 385 390 Ser Phe Asn Asp 395 400 405 lie Pro Arg Pro 410 415 420 lie Glu Val Leu 425 430 435 Ser lie Asn Ser 440 445 450 lie Lys 452
<210> 26 <211> 452 <212> PRT <213〉人(Homo sapiens) 〈220〉
<221> VARIANT <222> 357 <223> Xaa-Cys或X <400> 26
Met Ser Thr Ala Val Leu Glu Asn Pro Gly Leu Gly Arg Lys Leu 1 5 10 15
Ser Asp Phe Gly Gin Glu Thr Ser Tyr lie Glu Asp Asn Cys Asn 20 25 30
Gin Asn Gly Ala lie Ser Leu lie Phe Ser Leu Lys Glu Glu Val 35 40 45
Gly Ala Leu Ala Lys Val Leu Arg Leu Phe Glu Glu Asn Asp Val 50 55 60
40
Pro Met Tyr Thr Pro Glu Pro Asp lie Cys His His Val Pro Leu Phe Ser Asp Arg Ser Phe Ala Glu lie Gly Leu Ala Ser Leu Gly Ala Pro Asp Lys Leu Ala Thr lie Tyr Trp Phe Thr Val Glu Lys Gin Gly Asp Ser lie Lys Ala Tyr Gly Ala Ser Phe Gly Glu Leu Gin Tyr Xaa Leu Ser Glu Leu Pro Leu Glu Leu Glu Lys Thr Ala lie Gin Thr Glu Phe Gin Pro Leu Tyr Tyr Val Ala Glu Ala Lys Glu Lys Val Arg Asn Phe Ala Ala Thr Phe Ser Val Arg Tyr Asp Pro Tyr Thr Gin Arg Asp Asn Thr Gin Gin Leu Lys lie Leu Ala Asp Glu lie Gly lie Leu Cys Ser Ala Leu Gin LysAsn Leu Thr 65 70 75 Glu Tyr Glu 80 85 90 Leu Thr Asn 95 100 105
ValHisGlu
110115120
PheProArg
125130135
LeuSerTyr
140145150
AspProVal
155160165
TyrAsnTyr
170175180
GluGluGlu
185190195
SerLeuTyr
200205210
ProLeuLeu
215220225
GinLeuGlu
230235240
ArgLeuArg
245250255
GlyGlyLeu
260265270
HisGlySer
275280285
GluL>euLeu
290295300
GinPheSer
305310315
GluTyrlie
320325330
PheGlyLeu
335340345
GlyLeuLeu
350355360
LysProLys
365370375
AsnTyrThr
380385390
His lie Phe Phe lie lie Leu Ser Thr lie Gly Ala Tyr Arg Arg His Lys Lys Lys Thr Glu Lys Asp Val Pro Val Ala Phe Lys Pro Gly His Gin Glu Glu Lys Cys Lys Ser Ser Leu Leu Val Thr
Glu Ser Thr His Lys lie Arg Asp Gin Glu Glu Leu Ala Arg Gly Gin Thr Trp His Ala Tyr Cys Ser Gin Ala Gly Arg Val Met Tyr Val Pro lie Gly Leu Ala Gin Gly Phe Gly Pro Leu Glu Phe
Arg Pro Leu Asp Leu Arg Lys Lys Leu Asp Asp Ala Arg Lys Pro lie Gly Thr Cys Tyr Gly Phe Phe Leu Leu Leu Phe His Thr Pro Leu Phe Leu Ala Thr lie Asp Ser Glu Leu Glu Leu Gin Pro
Ser Arg Lys Arg His Asp Lys Asp Arg Phe Asp His Gin Phe Pro Arg Val Phe Glu Tyr His Glu Gin Thr Ser Ser Cys Thr Glu Pro Ser Asp Ser Leu Tyr Trp lie Lys Gin Tyr Glu Lys Leu Tyr
Leu Lys Ser Leu lie Gly Thr Val Ala Asn Pro Gly Ala Asp Val Glu Lys Thr Asn His Asp Asn Cys Thr Arg Asp Gin Tyr Asp lie Arg Ser Gly Ala Phe Thr Ala Tyr Xaa Leu Thr Ala Tyr Val
Lys Asp Pro Ala Ala Thr Pro Trp Gin lie Phe Lys lie Ala Tyr Met Leu Lys lie Phe lie Pro Gly Phe Phe Leu lie Arg Cys His Phe Ala Pro Asp Val Glu Gly Ala Ser Glu lie Gin Ala Glu<formula>formula see original document page 42</formula>GluGluGluLysLysThrTrpGlyThrValPheLysThrLeuLys
185190195
SerLeuTyrLysThrHisAlaCysTyrGluTyrAsnHisliePhe
200205210
ProLeuLeuGluLysTyrCysGlyPheHisGluAspAsnliePro
215220225
GinLeuGluAspValSerGinPheLeuGinThrCysThrGlyPhe
230235240
ArgLeuArgProValAlaGlyLeuLeuSerSerArgAspPheLeu
245250255
GlyGlyLeuAlaPheArgValPheHisCysThrGinTyrlieArg
260265270
HisGlySerLysProMetTyrThrProGluProAsplieCysHis
275280285
GluLeuLeuGlyHisValProLeuPheSerAspArgSerPheAla
290295300
GinPheSerGinGlulieGlyLeuAlaSerLeuGlyAlaProAsp
305310315
GluTyrlieGluLysLeuAlaThrlieTyrTrpPheThrValGlu
320325330
PheGlyLeuCysLysGinGlyAspSerlieLysAlaTyrGlyAla
335340345
GlyLeuLeuSerSerPheGlyGluLeuGinTyrCysLeuSerGlu
350355360
LysX朋LysLeuLeuProLeuGluLeuGluLysThrAlalieGin
365370375
AsnTyrThrValThrGluPheGinProLeuTyrTyrValAlaGlu
380385390
SerPheAsnAspAlaLysGluLysValArgAsnPheAlaAlaThr
395400405
lieProArgProPheSerValArgTyrAspProTyrThrGinArg
410415420
lieGluValLeuAspAsnThrGinGinLeuLyslieLeuAlaAsp
425430435
SerlieAsnSerGlulieGlylieLeuCysSerAlaLeuGinLys
440445450
lieLys
452
<210〉 28<211〉 452<212> PRT
<213〉人 (Homo sapiens)<220><221> VARIANT
<222> 414
<223> Xaa:Tyr或X
<400〉 28
Met Ser ThrAlaValLeuGluAsnProGlyLeuGlyArgLysLeu
1 5 10 15
Ser Asp PheGlyGinGluThrSerTyrlieGluAspAsnCysAsn
20 25 30
Gin Asn GlyAlalieSerLeuliePheSerLeuLysGluGluVal
35 40 45
Gly Ala LeuAlaLysValLeuArgLeuPheGluGluAsnAspVal
50 55 60
Asn Leu ThrHislieGluSerArgProSerArgLeuLysLysAsp
65 70 75
Glu Tyr GluPhePheThrHisLeuAspLysArgSerLeuProAla
80 85 90
Leu Thr AsnlielieLyslieLeuArgHisAsplieGlyAlaThr
95 100 105
Val His GluLeuSerArgAspLysLysLysAspThrValProTrp
110 115 120
Phe Pro ArgThrlieGinGluLeuAspArgPheAlaAsnGinlie
125 130 135
Leu Ser TyrGlyAlaGluLeuAspAlaAspHisProGlyPheLys
140 145 150
Asp Pro ValTyrArgAlaArgArgLysGinPheAlaAsplieAla
155 160 165
Tyr Asn TyrArgHisGlyGinProlieProArgValGluTyrMet
170 175 180
Glu Glu GluLysLysThrTrpGlyThrValPheLysThrLeuLys
185 190 195
Ser Leu TyrLysThrHisAlaCysTyrGluTyrAsnHisliePhe
200 205 210
Pro Leu LeuGluLysTyrCysGlyPheHisGluAspAsnliePro
215 220 225
Gin Leu GluAspValSerGinPheLeuGinThrCysThrGlyPhe
230 235 240
Arg Leu ArgProValAlaGlyLeuLeuSerSerArgAspPheLeu
245 250 255
Gly Gly LeuAlaPheArgValPheHisCysThrGinTyrlieArg
260 265 270
His Gly SerLysProMetTyrThrProGluProAsplieCysHis
275 280 285
Glu Leu LeuGlyHisValProLeuPheSerAspArgSerPheAla
290 295 300GinPheSerGinGlulieGlyLeuAlaSer Leu Gly AlaProAsp
305310315
GluTyrlieGluLysLeuAlaThrlieTyr Trp Phe ThrValGlu
320325330
PheGlyLeuCysLysGinGlyAspSerlie Lys Ala TyrGlyAla
335340345
GlyLeuLeuSerSerPheGlyGluLeuGin Tyr Cys LeuSerGlu
350355360
LysProLysLeuLeuProLeuGluLeuGlu Lys Thr AlalieGin
365370375
AsnTyrThrValThrGluPheGinProLeu Tyr Tyr ValAlaGlu
380385390
SerPheAsnAspAlaLysGluLysValArg Asn Phe AlaAlaThr
395400405
lieProArgProPheSerValArgX朋Asp Pro Tyr ThrGinArg
410415420
lieGluValLeuAspAsnThrGinGinLeu Lys lie LeuAlaAsp
425430435
SerlieAsnSerGlulieGlylieLeuCys Ser Ala LeuGinLys
440445450
lieCys
45權(quán)利要求
1、一種PAH基因13種新突變的檢測(cè)方法，其特征在于，包括步驟如下步驟一，以GenBank數(shù)據(jù)庫(kù)中PAH基因第4或5或6或7或10或11或12號(hào)外顯子以及外顯子與內(nèi)含子接合部位序列為模板設(shè)計(jì)一對(duì)等位基因特異性的核酸引物，利用引物對(duì)來(lái)擴(kuò)增PAH基因相應(yīng)外顯子的DNA序列，對(duì)擴(kuò)增產(chǎn)物進(jìn)行分離純化，得到相應(yīng)分離核酸；步驟二，對(duì)分離核酸的PAH基因外顯子的核苷酸進(jìn)行是否存在突變結(jié)果的檢測(cè)。
2、 根據(jù)權(quán)利要求1所述的尸/^基因13種新突變的檢測(cè)方法，其特征是，步 驟一中，所述的特異性的核酸引物具體為長(zhǎng)度為15bp-25bp，且能特異性地雜 交并擴(kuò)增出含/^//基因外顯子以及相應(yīng)外顯子與內(nèi)含子接合部位的序列。
3、 根據(jù)權(quán)利要求1所述的尸^^基因13種新突變的檢測(cè)方法，其特征是，步 驟一中，所述分離純化具體為在基因組DNA水平上，利用引物來(lái)擴(kuò)增每個(gè)突變 位點(diǎn)的基因序列和利用純化試劑盒對(duì)擴(kuò)增產(chǎn)物進(jìn)行提純。
4、 根據(jù)權(quán)利要求1所述的/^//基因13種新突變的檢測(cè)方法，其特征是，步 驟一中，所述分離核酸具體為-當(dāng)外顯子為第4號(hào)時(shí)，該核酸具有序列表SEQ ID N0.3所示的序列； 當(dāng)外顯子為第5號(hào)時(shí)，該核酸具有序列表SEQ ID NO. 4或SEQ ID NO. 5所示 的序列；當(dāng)外顯子為第6號(hào)時(shí)，該核酸具有序列表SEQ ID NO. 6或SEQ ID NO. 7或 SEQ ID NO. 8所示的序列；當(dāng)外顯子為第7號(hào)時(shí)，該核酸具有序列表SEQ ID NO. 9或SEQ ID NO. 10所 示的序列；當(dāng)外顯子為第10號(hào)時(shí)，該核酸具有序列表SEQ ID NO.ll所示的序列； 當(dāng)外顯子為第11號(hào)時(shí)，該核酸具有序列表SEQ ID圖.12或SEQ ID NO. 13 或SEQ ID NO. 14所示的序列；當(dāng)外顯子為第12號(hào)時(shí)，該核酸具有序列表SEQ ID NO. 15所示的序列。
5、 根據(jù)權(quán)利要求1所述的Z^^基因13種新突變的檢測(cè)方法，其特征是，步驟二中，所述檢測(cè)，其方法為DNA測(cè)序法、雜交測(cè)序法、酶促錯(cuò)配切割法、異 源雙鏈分析法、點(diǎn)雜交法、寡核苷酸陣列法、微測(cè)序法、Taqman技術(shù)、分子信 標(biāo)法、變性高效液相色譜法的一種或者幾種。
6、根據(jù)權(quán)利要求1所述的/^#基因13種新突變的檢測(cè)方法，其特征是， 步驟二中，所述突變結(jié)果的檢測(cè)是指當(dāng)為第4號(hào)外顯子時(shí)，檢測(cè)SEQ ID NO. 1所示序列的第361位核苷酸是否存 在突變，即T—C;當(dāng)為第5號(hào)外顯子時(shí)，檢測(cè)SEQ ID NO. 1所示序列的第461位核苷酸是否存 在突變，即A—G;或檢測(cè)SEQ ID NO. 1所示序列的第466位核苷酸是否存在突 變，即G—C;當(dāng)為第6號(hào)外顯子時(shí)，檢測(cè)SEQ ID NO. 1所示序列的第548位核苷酸是否存 在突變，即A—G;或檢測(cè)SEQ ID NO. 1所示序列的第680位核苷酸是否存在突 變，即T—A;或檢測(cè)SEQ ID NO. 1所示序列的第682位核苷酸是否存在突變， 即G—T;當(dāng)為第7號(hào)外顯子時(shí)，檢測(cè)SEQ ID NO. 1所示序列的第808位核苷酸是否存 在突變，即A—G;或檢測(cè)SEQ ID NO. 1所示序列的第823位核苷酸是否存在突 變，即C—G;當(dāng)為第10號(hào)外顯子時(shí)，檢測(cè)SEQ ID NO. 1所示序列的第971位核苷酸是否 存在突變，即T—A;當(dāng)為第11號(hào)外顯子時(shí)，檢測(cè)SEQ ID NO. 1所示序列的第1070位核苷酸是否 存在突變，即G—A;或檢測(cè)SEQ ID NO. 1所示序列的第1071位核苷酸是否存在 突變，即C—A;或檢測(cè)SEQIDNO. 1所示序列的第1085位核苷酸是否存在突變， 即C—T;當(dāng)為第12號(hào)外顯子時(shí)，檢測(cè)SEQ ID NO. 1所示序列的第1242位核苷酸是否 存在突變，即C—A。
全文摘要
一種生物技術(shù)領(lǐng)域的PAH基因13種新突變的檢測(cè)方法，步驟如下步驟一，以GenBank數(shù)據(jù)庫(kù)中PAH基因第4或5或6或7或10或11或12號(hào)外顯子以及外顯子與內(nèi)含子接合部位序列為模板設(shè)計(jì)一對(duì)等位基因特異性的核酸引物，利用引物對(duì)來(lái)擴(kuò)增PAH基因相應(yīng)外顯子的DNA序列，對(duì)擴(kuò)增產(chǎn)物進(jìn)行分離純化，得到相應(yīng)分離核酸；步驟二，對(duì)分離核酸的PAH基因外顯子的核苷酸進(jìn)行是否存在突變檢測(cè)。本發(fā)明為進(jìn)一步開發(fā)PKU基因突變種類及其頻率的分子遺傳技術(shù)提供了依據(jù)，具有重要的實(shí)用價(jià)值。
文檔編號(hào)C12Q1/68GK101481741SQ200910046100
公開日2009年7月15日 申請(qǐng)日期2009年2月12日 優(yōu)先權(quán)日2009年2月12日
發(fā)明者朱天聞, 秦勝營(yíng), 顧學(xué)范 申請(qǐng)人:上海交通大學(xué)醫(yī)學(xué)院附屬新華醫(yī)院;上海交通大學(xué)