专利名称：Pah基因13种新突变的检测方法
技术领域：
本发明涉及一种生物技术领域的基因突变检测方法，具体是一种/^//(苯丙氨酸羟化酶)基因13种新突变的检测方法。
背景技术：
苯酮尿症(phenylketonuria, PKU)是一种可以造成儿童不同程度智力损害的常见的先代谢性疾病，属于常染色体隐性遗传，0MIM编号为261600。 PKU的主要致病基因是/^〃基因，1983年该基因被定位在2q22-24。随着尸力#基因突变分子的不断深入研究，至2007年7月为止，http:〃丽.pahdb. mcgill. ca/(国际PAH数据库)共记载513种突变。/^#基因定位于染色体12q24. 1，大约由1. 5Mb碱基组成。/^#基因的编码区包含13个外显子，被不编码的12个内含子分隔。转录剪接后形成长1353bp的mRNA，后者经翻译，形成含451个氨基酸的酶单体，单体聚会成有功能的PAH酶。我国自80年代初以来对PKU开展了系统研究，随着分子生物学技术的发展，至今已发现90余种突变，并不断有新突变发现。
经对现有文献的检索，未发现与/^#基因13种新突变的检测方法主题有关的报道。
发明内容
本发明的目的在于克服现有技术中的不足，提供一种/^//基因13种新突变的检测方法。本发明为进一步开发/%1/基因突变种类及其频率的分子遗传技术提供了依据，具有实用价值。
本发明是通过以下技术方案实现的
本发明包括步骤如下
步骤一，以GenBank数据库中/ 4〃基因第4或5或6或7或10或11或12号外显子以及外显子与内含子接合部位序列为模板设计一对等位基因特异性的核酸引物，利用引物对来扩增/^^基因相应外显子的DNA序列，对扩增产物进行分离纯化，得到相应分离核酸；
步骤二，对分离核酸的/^#基因外显子的核苷酸进行是否存在突变结果的 检测。
步骤一中，所述的特异性的核酸引物具体为长度为15bp-25bp，且能特异 性地杂交并扩增出含州v7基因外显子以及相应外显子与内含子接合部位的序列。
步骤一中，所述分离纯化具体为在基因组DNA水平上，利用引物来扩增 每个突变位点的基因序列和利用纯化试剂盒对扩增产物进行提纯。
步骤一中，所述分离核酸具体为
当外显子为第4号时，该核酸具有序列表SEQ ID N0.3所示的序列； 当外显子为第5号时，该核酸具有序列表SEQ ID NO. 4或SEQ ID NO. 5所 示的序列；
当外显子为第6号时，该核酸具有序列表SEQ ID NO. 6或SEQ ID NO. 7或 SEQ ID NO. 8所示的序列;
当外显子为第7号时，该核酸具有序列表SEQ ID NO. 9或SEQ ID NO. 10所 示的序列；
当外显子为第10号时，该核酸具有序列表SEQ ID NO.ll所示的序列； 当外显子为第11号时，该核酸具有序列表SEQ ID NO. 12或SEQ ID NO. 13
或SEQ ID NO. 14所示的序列；
当外显子为第12号时，该核酸具有序列表SEQ ID N0.15所示的序列。 步骤二中，所述检测，其方法包括DNA测序法、杂交测序法、酶促错配切
割法、异源双链分析法、点杂交法、寡核苷酸阵列法、微测序法、Taqman技术、
分子信标法、变性高效液相色谱法的一种或者几种。 步骤二中，所述突变结果的检测是指
当为第4号外显子时，检测SEQ ID NO. 1所示序列的第361位核苷酸是否 存在突变，即T—C;
当为第5号外显子时，检测SEQ ID NO. 1所示序列的第461位核苷酸是否 存在突变，即A—G;或检测SEQ ID NO. 1所示序列的第466位核苷酸是否存在 突变，即G—C;
当为第6号外显子时，检测SEQ ID NO. 1所示序列的第548位核苷酸是否存在突变，即A—G;或检测SEQ ID NO. 1所示序列的第680位核苷酸是否存在 突变，即T—A;或检测SEQ ID NO. 1所示序列的第682位核苷酸是否存在突变， 即G—T;
当为第7号外显子时，检测SEQ ID NO. 1所示序列的第808位核苷酸是否 存在突变，即A—G;或检测SEQ ID NO. 1所示序列的第823位核苷酸是否存在 突变，即C—G;
当为第10号外显子时，检测SEQ ID NO. 1所示序列的第971位核苷酸是否 存在突变，即T—A;
当为第11号外显子时，检测SEQ ID NO. 1所示序列的第1070位核苷酸是 否存在突变，即G—A;或检测SEQ ID NO. 1所示序列的第1071位核苷酸是否存 在突变，即C—A;或检测SEQ ID NO. 1所示序列的第1085位核苷酸是否存在突 变，即C—T;
当为第12号外显子时，检测SEQ ID NO. 1所示序列的第1242位核苷酸是 否存在突变，即C—A。
本发明以GenBank数据库中/^〃基因第4或5或6或7或10或11或12号 外显子以及外显子与内含子接合部位序列为模板设计一对等位基因特异性的核 酸引物，利用引物对来扩增M^基因相应外显子的DNA序列，对扩增产物进行分 离纯化，得到相应分离核酸；对分离核酸的/^^基因外显子的核苷酸进行检测， 确定其是否存在突变。用于本发明的测试样品对于检测突变和单核苷酸多态性而 言，可以从测试样品中抽提的DNA或mRNA。对于PAH酶活性而言，可以是任何 含有/^#基因的样品。
本发明通过对/^^基因测序结果分析，发现13种不同的突变，经功能分析， 此13种突变都是改变了原有的氨基酸序列，导致了氨基酸的置换，最终导致了 /^#基因编码的苯丙氨酸羟化酶活性的改变，影响氨基酸的正常代谢。本发明为 进一步开发/^7/基因突变种类及其频率的分子遗传技术提供了依据，具有重要的 实用价值。
具体实施例方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说 明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常照常规条件，例如分子克隆，实验室手册(New York: Cold Spring Harbor Laboratory Press, 1989)中所述的条件，或按照制造厂商所建议的条件。 实施例
步骤一，以GenBank数据库中尸力^基因第4或5或6或7或10或11或12 号外显子以及外显子与内含子接合部位序列为模板设计一对等位基因特异性的 核酸引物，利用引物对来扩增M〃基因相应外显子的DNA序列，对扩增产物进行 分离纯化，得到相应分离核酸
引物设计
通过Primer5. 0软件，以Genebank数据库Z^〃基因(醒—000277)第4、 5、 6、 7、 10、 11、 12号外显子以及相应外显子与内含子接合部位序列为模板设计7 对等位基因特异性的核酸引物，由上海生工生物技术有限公司合成。/^〃基因序 列可由GeneBank数据库中取得，州^基因序列如SEQ NO. 1所示，M^基因的外 显子的基因组序列可从Genebank中获得。 引物信息
扩增W〃基因的第4号外显子突变位点的DNA序列引物
正向引物 5' -AGAGAACATCTCCAGGAAAGA-3'
反向引物 5'-CCCAGCCCTCGTGTAAATAG-3'; 扩增/^#基因的第5号外显子突变位点的DNA序列引物
正向引物 5' -AGGGTATAACCAAGGGAAGG-3'
反向引物 5'-ATGAGGGCAAGGGAGAAGC-3'; 扩增W〃基因的第6号外显子突变位点的DNA序列引物
正向引物 5' -CCCCGACTCCCTCTGCTAA-3'
反向引物 5'-CCTCTGCCTCAATCCTCCC-3'; 扩增W〃基因的第7号外显子突变位点的DNA序列引物
正向引物5'-CTGCCTAGCGTCAAAGCC-3'
反向引物5'-ECAGCCAGCAATGAACCCAAA-3'; 扩增/^#基因的第10号外显子突变位点的DNA序列引物
正向引物5' -AGGTATCCCTTCATCCAGTC-3'
反向引物5'-CCCACAGCCATCATCAAATC-3';扩增"^基因的第11号外显子突变位点的DNA序列引物
正向引物5' -ATTTGGGCTGTGATGTAGAAG-3'
反向引物5' -TTCAGTGTCTTGACTTGGTGG-3' 扩增M〃基因的第12号外显子突变位点的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号外显子PCR扩增条件为 体系各项试剂除DNA外均由Bio-Asia公司提供 反应体系总体积为15W，其中 ddH20 8 .
10 X Buffer 1.5^1，其中聚合反应缓冲液包含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号外显子PCR扩增条件为
体系各项试剂除DNA外均由Bio-Asia公司提供
反应体系总体积为15W，其中
ddH20 8.
10 X Buffer H，其中聚合反应缓冲液包含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
上述外显子PCR反应条件为
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。测序反应均作正反向，条件如下
扩增产物酶纯化法PCR产物2)4 ，加入2^1纯化酶系，酶系内含SAP (Shrimp alkaline phosphatase,碱性磷酸酶)0. 15u和Exo-nuclease I(核酸外切酶I) 0.75u/ul， 37°C 30mins， 85°C 20mins， 4°C保存备用。
测序采用ABI公司的Prisra BigDye terminator卿T) cycle sequencing reaction kit试剂盒进行测序，测序总体积为5W，其中纯化后的PCR产物#1， BDT 0. 5W，正向或反向引物O. 5W。反应条件是94。C 2 mins， 35 X (94°C 30 seconds, 55°C 40 seconds, 60°C, 1.5 mins) ， 4°C保存备用。
上样前纯化测序产物中加入25W无水乙醇和3. 0 mol / L醋酸钠混 合液(V:V二25:1)，室温、避光、静置15min， 4000rpm/min 45mins， 700rpm/min 5 seconds, 弃上清；2X (75%Alcohol 25l4， 4000 rpm/min 10 tnins， 700rpm/min 5 seconds,弃上清)；室温、避光、静置、风干20mins;上样于3100测序 仪进行测序。
步骤二，对分离核酸的/^#基因外显子的核苷酸进行是否存在突变结果的
检测
对/^^基因进行直接测序，将测定的各样本序列与GeneBank数据库/%￥基 因序列(NM_000277)进行比较，州〃基因序列如SEQ NO. 1所示，对应编码的 氨基酸序列如SEQ ID NO. 2所示。
对样本的测序结果标明，在/^^基因第4、 5、 6、 7、 10、 11、 12号外显子 中发现13种新突变，频率均为0.05%, /^//基因的上述外显子的基因组序列可从 Genebaiik中获得。经功能分析，13种突变都改变了原有的氨基酸序列，导致了 氨基酸的置换，具体为
(1) SEQ ID NO. 1所示序列的第361位核苷酸发生突变，即T—C;此时， SEQ ID NO. 1所示序列编码的氨基酸序列SEQ ID NO. 2第121位由Phe (F) —Leu
(U，具体序列如SEQ ID NO. 16所示；
(2) SEQ ID NO. 1所示序列的第461位核苷酸发生突变，即A—G;此时， SEQ ID NO. 1所示序列编码的氨基酸序列SEQ ID NO. 2第154位由Tyr (Y) —Cys
(C);具体序列如SEQ ID NO. 17所示;
(3) SEQ ID NO. l所示序列的第466位核苷酸发生突变，即G—C;此时，
9SEQ ID NO. 1所示序列编码的氨基酸序列SEQ ID NO. 2第156位由Ala (A) —Pro (P);具体序列如SEQ ID NO. 18所示；
(4) SEQ ID NO. l所示序列的第548位核苷酸发生突变，即A—G;此时， SEQ ID NO. 1所示序列编码的氨基酸序列SEQ ID NO. 2第183位由Glu (E) —Gly
(G);具体序列如SEQ ID N0.19所示；
(5) SEQ ID NO. l所示序列的第680位核苷酸发生突变，即T—A;此时， SEQ ID NO. 1所示序列编码的氨基酸序列SEQ ID NO. 2第227位由Leu (L) —Gin
(Q);具体序列如SEQ ID NO. 20所示；
(6) SEQ ID NO. l所示序列的第682位核苷酸发生突变，即G—T;此时， SEQ ID NO. 1所示序列编码的氨基酸序列SEQ ID NO. 2第270位由Arg (R) —Gly
(G);具体序列如SEQ ID NO. 21所示；
(7) SEQ ID NO. l所示序列的第808位核苷酸发生突变，即A—G;此时， SEQ ID NO. 1所示序列编码的氨基酸序列SEQ ID NO. 2第275位由Pro (P) —Ala
(A);具体序列如SEQ ID N0.22所示;
(8) SEQ ID NO. l所示序列的第823位核苷酸发生突变，即C—G;此时， SEQ ID NO. 1所示序列编码的氨基酸序列SEQ ID NO. 2第324位由lie (I) —Asn
(N);具体序列如SEQ ID NO. 23所示；
(9) SEQ ID NO. 1所示序列的第971位核苷酸发生突变，即T—A;此时， SEQ ID NO. 1所示序列编码的氨基酸序列SEQ ID NO. 2第357位由Cys (C) —Tyr
(Y);具体序列如SEQ ID N0.24所示;
(10) SEQ ID NO. 1所示序列的第1070位核苷酸发生突变，即G—A;此时， SEQ ID NO. 1所示序列编码的氨基酸序列SEQ ID NO. 2第362位由Pro (P) —Thr
(T);具体序列如SEQ ID NO. 25所示；
(11) SEQIDNO. 1所示序列的第1071位核苷酸发生突变，即C—A;此时， SEQ ID NO. 1所示序列编码的氨基酸序列SEQ ID NO. 2第228位氨基酸由Glu (E) —终止(X);具体序列如SEQ ID N0.26所示；
(12) SEQ ID NO. 1所示序列的第1085位核苷酸发生突变，即C—T;此时， SEQ ID NO. 1所示序列编码的氨基酸序列SEQ ID NO. 2第357位氨基酸由Cys (C) —终止(X);具体序列如SEQ ID NO. 27所示；(13) SEQ ID NO. 1所示序列的第1242位核苷酸发生突变，即C—A;此时， SEQ ID NO. 1所示序列编码的氨基酸序列SEQ ID NO. 2第414位氨基酸由Tyr (Y) —终止(X);具体序列如SEQ ID N0.28所示。
最终这些突变都导致了 /^v7基因编码的苯丙氨酸羟化酶活性的改变，影响 体内氨基酸的正常代谢。序列表
<110〉上海交通大学
<120> PAH基因13种新突变的检测方法
〈160> 28
<210〉 1
<211> 1359
<212>腿
〈213>人 (Homo sapiens)<400〉 1
atgtccactgcggtcctggaaaacccaggcttgggc鄉aaactctctga50
ctttggacaggaaacaagctcaactgcaatcaaaatggtg100
ccatatcactgatcttctcactcaaag肌gaagttggtgcattggccaaa150
gt3ttgcgcttatttgaggaaacctgacccacattgaatc200
tagaccttctcgtttaaagatgaatttttcacccatttgg250
at犯acgtagcctgcctgctctgacaaacatcatcaagatCttg3ggC3t300
g3C3ttggtgccactgtccatgagctttcacgagataagaaga犯g3cac350
agtgccctggttcccaagaagctggac卿tttgccaatc400
agattctcagctatggagcgg肌Ctgg3tgctgaccaccctggtttta犯450
gatcctgtgtaccgtgcaagacggaagcagtttgctgacattgcctacaa500
ctaccgccatgggC鄉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观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舰
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权利要求
1、一种PAH基因13种新突变的检测方法，其特征在于，包括步骤如下步骤一，以GenBank数据库中PAH基因第4或5或6或7或10或11或12号外显子以及外显子与内含子接合部位序列为模板设计一对等位基因特异性的核酸引物，利用引物对来扩增PAH基因相应外显子的DNA序列，对扩增产物进行分离纯化，得到相应分离核酸；步骤二，对分离核酸的PAH基因外显子的核苷酸进行是否存在突变结果的检测。
2、 根据权利要求1所述的尸/^基因13种新突变的检测方法，其特征是，步 骤一中，所述的特异性的核酸引物具体为长度为15bp-25bp，且能特异性地杂 交并扩增出含/^//基因外显子以及相应外显子与内含子接合部位的序列。
3、 根据权利要求1所述的尸^^基因13种新突变的检测方法，其特征是，步 骤一中，所述分离纯化具体为在基因组DNA水平上，利用引物来扩增每个突变 位点的基因序列和利用纯化试剂盒对扩增产物进行提纯。
4、 根据权利要求1所述的/^//基因13种新突变的检测方法，其特征是，步 骤一中，所述分离核酸具体为-当外显子为第4号时，该核酸具有序列表SEQ ID N0.3所示的序列； 当外显子为第5号时，该核酸具有序列表SEQ ID NO. 4或SEQ ID NO. 5所示 的序列；当外显子为第6号时，该核酸具有序列表SEQ ID NO. 6或SEQ ID NO. 7或 SEQ ID NO. 8所示的序列；当外显子为第7号时，该核酸具有序列表SEQ ID NO. 9或SEQ ID NO. 10所 示的序列；当外显子为第10号时，该核酸具有序列表SEQ ID NO.ll所示的序列； 当外显子为第11号时，该核酸具有序列表SEQ ID图.12或SEQ ID NO. 13 或SEQ ID NO. 14所示的序列；当外显子为第12号时，该核酸具有序列表SEQ ID NO. 15所示的序列。
5、 根据权利要求1所述的Z^^基因13种新突变的检测方法，其特征是，步骤二中，所述检测，其方法为DNA测序法、杂交测序法、酶促错配切割法、异 源双链分析法、点杂交法、寡核苷酸阵列法、微测序法、Taqman技术、分子信 标法、变性高效液相色谱法的一种或者几种。
6、根据权利要求1所述的/^#基因13种新突变的检测方法，其特征是， 步骤二中，所述突变结果的检测是指当为第4号外显子时，检测SEQ ID NO. 1所示序列的第361位核苷酸是否存 在突变，即T—C;当为第5号外显子时，检测SEQ ID NO. 1所示序列的第461位核苷酸是否存 在突变，即A—G;或检测SEQ ID NO. 1所示序列的第466位核苷酸是否存在突 变，即G—C;当为第6号外显子时，检测SEQ ID NO. 1所示序列的第548位核苷酸是否存 在突变，即A—G;或检测SEQ ID NO. 1所示序列的第680位核苷酸是否存在突 变，即T—A;或检测SEQ ID NO. 1所示序列的第682位核苷酸是否存在突变， 即G—T;当为第7号外显子时，检测SEQ ID NO. 1所示序列的第808位核苷酸是否存 在突变，即A—G;或检测SEQ ID NO. 1所示序列的第823位核苷酸是否存在突 变，即C—G;当为第10号外显子时，检测SEQ ID NO. 1所示序列的第971位核苷酸是否 存在突变，即T—A;当为第11号外显子时，检测SEQ ID NO. 1所示序列的第1070位核苷酸是否 存在突变，即G—A;或检测SEQ ID NO. 1所示序列的第1071位核苷酸是否存在 突变，即C—A;或检测SEQIDNO. 1所示序列的第1085位核苷酸是否存在突变， 即C—T;当为第12号外显子时，检测SEQ ID NO. 1所示序列的第1242位核苷酸是否 存在突变，即C—A。
全文摘要
一种生物技术领域的PAH基因13种新突变的检测方法，步骤如下步骤一，以GenBank数据库中PAH基因第4或5或6或7或10或11或12号外显子以及外显子与内含子接合部位序列为模板设计一对等位基因特异性的核酸引物，利用引物对来扩增PAH基因相应外显子的DNA序列，对扩增产物进行分离纯化，得到相应分离核酸；步骤二，对分离核酸的PAH基因外显子的核苷酸进行是否存在突变检测。本发明为进一步开发PKU基因突变种类及其频率的分子遗传技术提供了依据，具有重要的实用价值。
文档编号C12Q1/68GK101481741SQ200910046100
公开日2009年7月15日 申请日期2009年2月12日 优先权日2009年2月12日
发明者朱天闻, 秦胜营, 顾学范 申请人:上海交通大学医学院附属新华医院;上海交通大学