CN111440856B - 一种用于检测甲基丙二酸血症相关致病基因的探针组及试剂盒 - Google Patents
一种用于检测甲基丙二酸血症相关致病基因的探针组及试剂盒 Download PDFInfo
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Abstract
本发明公开了一种用于检测甲基丙二酸血症相关致病基因的探针组及试剂盒。本发明提供了一种甲基丙二酸血症相关基因捕获的捕获探针。本发明提供的捕获探针,由序列表中序列1至序列99所示的探针组成。本发明的试剂盒能同时检测MUT,MMAA,MMAB,MMADHC和MMACHC等甲基丙二酸血症的致病基因;2、对于多种点突变类型的甲基丙二酸血症均可检测;检测MUT,MMAA,MMAB,MMADHC和MMACHC等多个基因的序列,有利于新的致病突变的发现,为甲基丙二酸血症的遗传学基础提供新的发现手段。
Description
技术领域
本发明属于基因工程、分子遗传学及基因检测领域,具体涉及一种用于检测甲基丙二酸血症相关致病基因的探针组及试剂盒。
背景技术
甲基丙二酸血症(methylmalonic acidemia,MMA),也称为甲基丙二酸尿症(methylmalonicaciduria),是先天有机酸代谢异常中最常见的病种,是多种原因所致体内甲基丙二酸积蓄的总称,是一种常染色体隐性遗传病。
甲基丙二酸是甲基丙二酰辅酶A的代谢产物,正常情况下在甲基丙二酰甲基钴胺辅酶A变位酶及维生素B12的作用下转化生成琥珀酸,参与三羧酸循环。甲基丙二酰辅酶A变位酶缺陷或维生素B12代谢障碍导致甲基丙二酸、丙酸、甲基枸橼酸等代谢物异常蓄积,琥珀酸脱氢酶活性下降,线粒体能量合成障碍,引起神经、肝脏、肾脏、骨髓等多脏器损伤。患者脑组织病理分析可见脑萎缩、弥漫性神经胶质细胞增生、星形细胞变性、脑出血、苍白球坏死、丘脑及内囊细胞水肿,均与线粒体功能不良有关。
研究显示MUT,MMAA,MMAB,MMADHC和MMACHC等基因的突变可导致甲基丙二酸血症。根据甲基丙二酸辅酶A变位酶缺陷分为完全缺失Mut0和部分却失Mut1型,其中最严重的是Mut0型。
目前,甲基丙二酸血症伴同型半胱氨酸血症CblC型的诊断主要通过尿气相色谱/质谱技术检测尿液中甲基丙二酸、血串联质谱技术检测血中丙酰肉碱与乙酰肉碱的比值,两者同时增高并排除维生素B12缺乏等继发因素伴血清同型半胱氨酸升高,可合并甲硫氨酸降低时方可诊断。同时应完善患者血、尿常规、血气分析、血氨、血糖、血乳酸、肝肾功能和心肌酶谱测定,脑CT或MRT扫描、脑电图、智力测定等辅助检查。但是该方法的操作复杂,明确诊断费时且价格昂贵,并对于甲基丙二酸血症伴同型半胱氨酸血症CblC型的筛查及预防不具备普遍的意义。
通过对患者进行基因检测,可明确基因突变类型,对甲基丙二酸血症伴同型半胱氨酸血症CblC型患者明确诊断及个体化防治提供有力的证据。传统的基因检测方法基于Sanger测序需要先明确想要检测的基因,通过聚合酶链扩增技术将目标区段扩增出来,再使用双脱氧终止法进行测序确定致病突变。该方法通量低,效率差,成本高,且检测率不理想。
发明内容
本发明的一个目的是提供一种甲基丙二酸血症相关基因捕获的捕获探针。
本发明提供的捕获探针,由序列表中序列1至序列99所示的探针组成。
捕获检测MUT的探针组包含29个寡核苷酸探针,碱基序列如序列1-序列29;
捕获检测MMACHC的探针组包含15个寡核苷酸探针,碱基序列如序列30-序列40;
捕获检测MMAB的探针组包含42个寡核苷酸探针,碱基序列如序列41-序列82;
捕获检测MMADHC的探针组包含2个寡核苷酸探针,碱基序列如序列83-序列84;
捕获检测MMAA的探针组包含11个寡核苷酸探针,碱基序列如序列85-序列99。
上述捕获探针中,所述每条探针均标记生物素。
本发明另一个目的是提供一种甲基丙二酸血症相关基因捕获的试剂盒。
本发明提供的试剂盒,包括上述的捕获探针所示的组分1)。
上述试剂盒还包括如下独立包装的各个组分:
2)富集缓冲液,其包括人cot-1DNA、鲑鱼精DNA、引物1和引物2;具体由30-45%(体积百分比)人cot-1DNA(可为30%、35%、40%、45%)(Invitrogen货号:15279011)、5-25%(体积百分比)鲑鱼精DNA(可为5%、10%、15%、20%、25%)(Invitrogen货号:15634017)、0.5nmol/μl引物1、0.5nmol/μl引物2和水组成;
所述引物1的核苷酸序列为序列100;
所述引物2的核苷酸序列为序列101;
3)杂交缓冲液,包括Tris-HCl缓冲液、NaCl、柠檬酸钠、BSA和Tween20;具体由溶剂和溶质组成,溶剂为100mM Tris-HCl(pH 7.6),溶质及各溶质的浓度分别为1.0-1.5M NaCl(可为1M、1.25M、1.5M)、0.1-0.3M柠檬酸钠(可为0.1M、0.2M、0.3M)、0.08-0.12g/100mL BSA(可为0.08、0.1、0.12)和5-9%(体积百分比)Tween20(可为5%、6%、7%、8%、9%)。
4)结合缓冲液,包括Tris-HCl缓冲液、NaCl和EDTA;具体由溶剂和溶质组成,溶剂为10mM Tris-HCl(pH 7.5),溶质及其浓度分别为1.0-1.5M NaCl(可为1M、1.25M、1.5M),1mM EDTA;
5)漂洗液1,包括SDS和柠檬酸三钠缓冲液;具体由溶剂和溶质组成,溶剂为1×SSC溶液,溶质为SDS,SDS在漂洗液1中的质量百分比浓度为0.1%(g/L);
6)漂洗液2,包括SDS和10倍稀释的柠檬酸三钠缓冲液;具体由溶剂和溶质组成,溶剂为0.1×SSC溶液,溶质为SDS,SDS在漂洗液2中的质量百分比浓度为0.1%;
7)NaOH水溶液,具体为0.1-0.5M NaOH溶液(可为0.1M、0.3M、0.5M);
8)Tris-HCl缓冲液,具体为浓度为1M、pH值为7.5的Tris-HCl缓冲液;
9)PCR反应液;所述PCR反应液包括引物3和引物4;具体PCR反应液由溶剂和溶质组成,溶剂为1倍浓度的Phusion缓冲液(New England Biolabs),溶质及其浓度分别为0.2mMdATP、0.2mM dTTP、0.2mM dCTP、0.2mM dGTP、2.5pmol引物3、2.5pmol引物4、0.05U/μlHotstart Phusion酶(New England Biolabs)、5%(体积比)DMSO;
所述引物3的核苷酸序列为序列102;
所述引物4的核苷酸序列为序列103;
所述引物3和所述引物4的最后一位核苷酸为硫代修饰;
10)TE缓冲液,具体由溶剂和溶质组成,溶剂为水,溶质为Tris、EDTA和用于调节pH的HCl,Tris的浓度为10mM,EDTA的浓度为1mM,pH为8.0。
上述捕获探针或上述试剂盒在制备捕获待测样本中甲基丙二酸血症相关基因的产品中的应用也是本发明保护的范围。
上述捕获探针或上述试剂盒在制备测序待测样本中甲基丙二酸血症相关基因产品中的应用也是本发明保护的范围。
上述中,所述甲基丙二酸血症相关基因为MUT基因、MMAA基因、MMAB基因、MMADHC基因和/或MMACHC基因。
上述捕获探针或上述试剂盒在制备检测或预测待测者是否患有甲基丙二酸血症产品中的应用也是本发明保护的范围。
本发明第3个目的是提供一种捕获待测样本中甲基丙二酸血症相关基因的方法。
本发明提供的方法,包括如下步骤:
利用上述捕获探针对待测样本的基因组DNA进行捕获,得到捕获片段;实现捕获待测样本中甲基丙二酸血症相关基因。
本发明第4个目的是提供一种测序待测样本中甲基丙二酸血症相关基因或基因突变位点的方法。
本发明提供的方法,包括如下步骤:
1)利用上述捕获探针对待测样本的基因组DNA进行捕获,得到捕获片段;
2)用上述试剂盒中的PCR反应液进行扩增,得到测序文库;
3)对所述测序文库进行测序,得到待测样本中甲基丙二酸血症相关基因或基因突变位点的测序结果。
本发明的有益效果如下:
1、本发明的试剂盒能同时检测MUT,MMAA,MMAB,MMADHC和MMACHC等甲基丙二酸血症的致病基因;
2、对于多种点突变类型的甲基丙二酸血症均可检测;
3、检测MUT,MMAA,MMAB,MMADHC和MMACHC等多个基因的序列,有利于新的致病突变的发现,为甲基丙二酸血症的遗传学基础提供新的发现手段。
附图说明
图1为5例的已经确定的MMA携带者/患者样本的gDNA用Sanger测序验证。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
下述实施例中,如无特殊说明,序列表中各核苷酸序列的第1位均为相应DNA的5′末端核苷酸,末位均为相应DNA的3′末端核苷酸。
实施例1、甲基丙二酸相关基因捕获试剂盒及其专用探针的制备
一、甲基丙二酸相关基因捕获探针的设计和制备
根据Hg19参考基因组数据库中MUT,MMAA,MMAB,MMADHC和MMACHC基因的全长序列为目标序列,参考利WO2013/003585中的方法:沿着各个序列依次堆叠过去设计捕获检测该基因的探针,该探针能结合该基因。本发明采用的每条探针序列长度为78bp。
捕获检测MUT的探针组包含29个寡核苷酸探针,碱基序列如序列1-序列29;
捕获检测MMACHC的探针组包含15个寡核苷酸探针,碱基序列如序列30-序列40;
捕获检测MMAB的探针组包含42个寡核苷酸探针,碱基序列如序列41-序列82;
捕获检测MMADHC的探针组包含2个寡核苷酸探针,碱基序列如序列83-序列84;
捕获检测MMAA的探针组包含11个寡核苷酸探针,碱基序列如序列85-序列99。
将上述合成的探针进行生物素标记,具体将生物素标记在探针核苷酸序列的5'末端,形成本实施例的捕获探针。
利用PCR的方法扩增出大量的带有生物素标记的探针,具体方法如下:
将上述合成的探针等摩尔比混合在总体积1.2ml的ddH2O中,取其中5μl利用通用的PCR引物(5’端序列为GACTACATGGGACAT、3’端的序列为GGAACCTACGACGTA),分三管进行PCR扩增,其中引物GACTACATGGGACAT为带有生物素标记的引物。
上述PCR扩增体系:DNA模板,5μl;正向引物(25μM),2μl;反向引物(25μM),2μl;MgCl2(50mM),4μl;10x Platinum Taq缓冲液(购自Life Technologies),5μl;dNTPs(每种10mM),4μl;Platinum Taq(5U/μl,购自Life Technologies),1μl;H2O,27μl;总体积50μl。
PCR扩增条件:98℃,30s;(98℃,30s,60℃,25s,72℃,45s)35个循环;72℃,5min。
将PCR产物用MinElute PCR纯化试剂盒(购自Life Technologies)纯化后,取5500ng,用MyOne亲和素磁珠(购自Invitrogen货号:35602)将PCR产物结合下来。然后加入碱性NaOH将没有生物素的互补链变性、洗脱下来;然后将整个磁珠用100℃的甲酰胺液体洗涤,使探针从磁珠上分离下来。用乙醇沉淀后即得到生物素标记的探针组,作为捕获探针。
上述生物素标记的探针组由核苷酸序列为1-39所示的探针组成,且每条探针的5'末端标记生物素基团,该探针组中每条探针等摩尔比混合。
二、甲基丙二酸血症相关基因捕获试剂盒的制备及使用方法
A、甲基丙二酸血症相关基因捕获试剂盒的制备
甲基丙二酸血症相关基因捕获检测试剂盒,是通过检测甲基丙二酸血症相关基因MUT,MMAA,MMAB,MMADHC和MMACHC基因来进行分子遗传学检测的试剂盒,该试剂盒包括:上述一获得生物素标记的探针组、富集缓冲液、杂交缓冲液、结合缓冲液、漂洗液1、漂洗液2、NaOH溶液(0.1M)、Tris-HCl缓冲液(1M,pH 7.5)、PCR反应液、TE缓冲液。
1)生物素标记的探针组
生物素标记的探针组由序列1至序列99所示的探针组成,且每个单链DNA分子的5’端均标记生物素。
2)富集缓冲液
富集缓冲液由35%(体积百分比)人cot-1DNA(Invitrogen货号:15279011)、15%(体积百分比)鲑鱼精DNA(Invitrogen货号:15634017)、0.5nmol/μl引物1、0.5nmol/μl引物2和水组成;
其中引物1和引物2的序列如下:
引物1:AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCT(序列表中序列100);
引物2:CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGC(序列表中序列101);
3)杂交缓冲液
杂交缓冲液由溶剂和溶质组成,溶剂为100mM Tris-HCl(pH 7.6),溶质及各溶质的浓度分别为1.25M NaCl、0.125M柠檬酸钠、0.1g/100mL BSA和7%(体积百分比)Tween20;
100mMTris-HCl(pH 7.6)由溶剂和溶质组成,溶剂为水,溶质为三羟甲基氨基甲烷(Tris),Tris的浓度为100mM,HCl用于调节pH为7.6;
4)结合缓冲液
结合缓冲液由溶剂和溶质组成,溶剂为10mM Tris-HCl(pH 7.5),溶质及其浓度分别为1M NaCl,1mM EDTA;
10mM Tris-HCl(pH 7.5)由溶剂和溶质组成,溶剂为水,溶质为三羟甲基氨基甲烷(Tris),Tris的浓度为10mM,HCl用于调节pH为7.5;
5)漂洗液1
漂洗液1由溶剂和溶质组成,溶剂为1×SSC溶液,溶质为SDS,SDS在漂洗液1中的质量百分比浓度为0.1%(g/L);
其中,1×SSC溶液由溶剂和溶质组成,溶剂为水,溶质及其浓度分别为1.0M NaCl和柠檬酸三钠88g/L,pH 7.4;
6)漂洗液2:
漂洗液2由溶剂和溶质组成,溶剂为0.1×SSC溶液,溶质为SDS,SDS在漂洗液2中的质量百分比浓度为0.1%;
其中,0.1×SSC溶液为将1×SSC溶液稀释10倍得到的溶液;
7)0.1M NaOH溶液
0.1M NaOH溶液由溶剂和溶质组成,溶剂为水,溶质及其浓度为0.1M NaOH;
8)浓度为1M、pH值为7.5的Tris-HCl缓冲液
Tris-HCl缓冲液(1M,pH 7.5)由溶剂和溶质组成,溶剂为水,溶质为三羟甲基氨基甲烷(Tris),Tris的浓度为1M,HCl调节pH为7.5;
9)PCR反应液:
PCR反应液由溶剂和溶质组成,溶剂为1倍浓度的Phusion缓冲液(New EnglandBiolabs),溶质及其浓度分别为0.2mM dATP、0.2mM dTTP、0.2mM dCTP、0.2mM dGTP、2.5pmol引物3、2.5pmol引物4、0.05U/μl Hotstart Phusion酶(New England Biolabs)、5%(体积比)DMSO;
其中1倍浓度的Phusion缓冲液由溶剂和溶质组成,溶剂为50mM Tris-HCl(pH8.8),溶质及其浓度分别为4mM MgCl2,500mM KCl,0.8%(v/v)Nonidet P40;
50mM Tris-HCl(pH8.8)由溶剂和溶质组成,溶剂为水,溶质为三羟甲基氨基甲烷(Tris),Tris的浓度为50mM,HCl调节pH为8.8。
上述引物序列如下:
引物3:AATGATACGGCGACCACCGA*G(序列表中序列102);
引物4:CAAGCAGAAGACGGCATACG*A(序列表中序列103);*表示硫代修饰。
10)TE缓冲液
TE缓冲液由溶剂和溶质组成,溶剂为水,溶质为Tris、EDTA,Tris的浓度为10mM,EDTA的浓度为1mM,HCl调节pH为8.0。
上述试剂盒可以按照如下表1所列组分制备:
表1为试剂盒组成
B、检测方法的建立
1、全基因组文库制备:
提取待测者全血的gDNA作为样本;将gDNA片段化至150-250bp的片段后,使用KAPA建库试剂盒(货号KK8504)按照说明书进行全基因组文库的构建,得到gDNA文库。
2、甲基丙二酸血症相关致病基因探针组的捕获文库制备
使用实施例1中的生物素标记的探针组及捕获试剂盒进行捕获,具体步骤如下:
1)取1μg步骤1的gDNA文库,向其中加入13μl富集缓冲液和5μl生物素标记的探针组溶液(该溶液为利用水溶解生物素标记的探针组得到的液体,该溶液中探针的总浓度为100ng/μl),将得到的反应体系置于PCR仪上,反应条件为95℃7min,之后65℃2min,反应结束得到反应产物;
2)将65℃预热的杂交缓冲液23μl加入到上述步骤1)得到的反应产物中,然后于PCR仪上65℃杂交22小时,得到富集体系混合物;
3)将MyOne C1链霉亲和素磁珠(Invitrogen 35602)漩涡震荡使磁珠充分悬浮,取50μl MyOne C1链霉亲和素磁珠到新的1.5ml的离心管;
4)将步骤3)的装有50μl MyOne C1链霉亲和素磁珠的1.5ml离心管漩涡震荡至少5s,使磁珠充分悬浮,短暂离心后放入磁力架上保持静止一分钟(不要旋转离心管),小心吸弃上清;
5)取下步骤4)的离心管,向其中加入50μl结合缓冲液,旋窝震荡至少5s,短暂离心后放入磁力架上静止一分钟,小心吸弃上清,重复三次;
6)取下步骤5)的离心管,向其中加入100μl的2倍浓度的结合缓冲液(即将实施例1中结合缓冲液的各溶质浓度加倍得到的溶液,即2倍浓度的结合缓冲液中各溶质浓度为实施例结合缓冲液相应溶质浓度的2倍),旋窝震荡至少5s,短暂离心后放入磁力架上静止一分钟;
7)将步骤2)得到的富集体系混合物加入到骤6)的离心管中,旋窝震荡至少5s,然后置于旋转仪上室温旋转1小时(60转/分钟);
8)步骤7)完成后,利用漂洗液1室温清洗步骤7)的磁珠一次,15分钟,然后再用漂洗液2于65℃下清洗3次,每次15分钟;
9)步骤8)完成后,将磁珠用0.1M NaOH溶液室温下洗脱10分钟,然后将得到的洗脱液漩涡震荡至少5s,短暂离心后放入磁力架上静止一分钟,然后将上清液转移到含有70μlTris-HCl缓冲液(1M,pH 7.5)的干净离心管中,即得到DNA溶液;
10)采用Qiagen MinElute Column(Qiagen产品)对步骤9)得到的DNA溶液进行纯化,将得到DNA利用TE缓冲液溶解,得到纯化DNA溶液;
11)向30μl步骤10)得到的纯化DNA溶液中加入70ul PCR反应液进行PCR扩增,得到PCR产物;PCR反应条件为:98℃30s,1个循环;98℃25s,65℃30s,72℃30s,15个循环;72℃5min,1个循环;
12)利用Agencourt AMPure XP核酸纯化试剂盒(Beckman Coulter)纯化步骤11)的PCR产物,得到纯化产物。
3、上机测序
将步骤2得到的纯化产物在Illumina NextSeq 500测序仪上进行测序,所用测序引物为测序引物1和测序引物2,测序引物1:AATGATACGGCGACCACCGA;测序引物2:CAAGCAGAAG ACGGC ATACG AGAT。
得到5个待测样本(编号为A-E)全血全基因样本的测序结果。
4、生物信息学分析
高通量测序得到的原始图像数据文件经碱基识别分析转化为原始测序序列,称之为Raw Data,结果以FASTQ文件格式存储,其中包含测序序列的序列信息以及其对应的测序质量信息。具体分析流程如下:
测序结果用GATK软件对SNP和InDel进行变异检测,用ANNOVAR软件同时关联多个数据库(如dbSNP,1000g,ESP6500,HGMD,OMIM等)对变异结果进行注释。变异过滤:在进行后续分析之前需对变异数据集进行必要的过滤。过滤标准如下:
第一步:留下致病性分析(pathogenic_analysis)中的致病性突变(pathogenic)位点。
第二步:筛选突变碱基测序次数大于5,突变频率大于等于30%以及1000g2015apr、ESP6500si、Inhouse、ExAC_ALL、ExAC_EAS五个正常人突变数据库中保留没有出现或小于5%的位点;去除数据集中的同义突变位点,另外有文献报道的突变位点留下。
统计分析结果如下表2所示,其中覆盖率均大于99%,平均深度大于400*,平均覆盖20*比例也均在95%以上,表明,探针组能够很好的覆盖到目的基因区域,可为后续的分析提供一个准确度的保证。
表2测序数据基本统计结果
实施例2、试剂盒的应用
下面通过具体实施方式对本发明作进一步详细说明
一、本发明的试剂盒检测
利用实施例1的试剂盒检测5例的已经确定的MMA携带者/患者样本的MUT,MMAA,MMAB,MMADHC和MMACHC基因改变情况,按照实施例1的方法步骤进行。
测序结果如表2所示。
从测序结果中选取已知MMA携带者/患者对应的MUT,MMAA,MMAB,MMADHC和MMACHC基因的特定突变位点,如表3所示。
表3.利用本发明试剂盒对甲基丙二酸血症样本的检测结果
二、Sanger测序验证
将上述5例的已经确定的MMA携带者/患者样本的gDNA用Sanger测序验证,结果如图1所示。
比较图1和表3,可以看出,本发明试剂盒的结果与图1一致,表明本发明的方法正确。注:由于Sanger验证采用正向测序或反向测序,图1的峰图显示的碱基有可能为被检测碱基的反向互补序列,如:c.163G>A,峰图可显示为G>A或其反向互补序列C>T。
以上内容是结合具体的实施方式对本发明所作的进一步详细说明,不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干简单推演或替换,都应当视为属于本发明的保护范围。
序列表
<110>北京迈基诺基因科技股份有限公司
<120>一种用于检测甲基丙二酸血症相关致病基因的探针组及试剂盒
<160> 103
<170> PatentIn version 3.5
<210> 1
<211> 120
<212> DNA
<213> 人工序列
<400> 1
taatatttta gacaaaagct aaaacaaaaa gaggatatta tacagattgc tgcttctttt 60
ccaaacactt ctcaatatca tcaagcacct gaacggcagc ctttggaatt cgagtcccag 120
<210> 2
<211> 120
<212> DNA
<213> 人工序列
<400> 2
ttggaattcg agtcccagga ccaaatacat tggaaacacc aacttcaaac agaaattcat 60
aatcctgttg aaagaatgtg tttaattaat aagagccact atttccattt tcacggaaat 120
<210> 3
<211> 120
<212> DNA
<213> 人工序列
<400> 3
tagaaaaata gagataaaaa atacctgagg tggtatcacc cctccacaca tgacaagaat 60
atctggccgt ccaagggagt taagttcttt gatgagttca ggaactaggg ttttatgacc 120
<210> 4
<211> 120
<212> DNA
<213> 人工序列
<400> 4
aactagggtt ttatgaccag cagcgagggt gcttatgccc acagcatgca catccgcatc 60
cacagcctgc tgggccactt cacgaggagt ctaaacagtc agaaagtaaa gataaatcat 120
<210> 5
<211> 120
<212> DNA
<213> 人工序列
<400> 5
catcatttta ctacattttc taaatgtaag tgactagtaa atacttttga aattcccccc 60
aacagttttt agtacctgga aaagagggcc tatgtccaca tcaaaaccaa gatcagcaaa 120
<210> 6
<211> 120
<212> DNA
<213> 人工序列
<400> 6
aaaaccaaga tcagcaaatc ctgtagcaat aacttttgct cctctgtcat ggccatcttg 60
tcccattttt gctacaagaa gacgaggtct gcgaccttca cgttccatga atttatgaac 120
<210> 7
<211> 120
<212> DNA
<213> 人工序列
<400> 7
agctcccagt agattcaagg ggattgtgct aacagaaaag atgaaattct ggcctaagaa 60
accttacata ttacctcttg atagcagatg ttatctcttt actttctcca aattcctggc 120
<210> 8
<211> 120
<212> DNA
<213> 人工序列
<400> 8
tttctccaaa ttcctggcga tatgctccac tcaccattcg atcattcgct ttatgttcac 60
caaatacctt tttcagggca tctgtgattt ctcccactgt acatctgaaa catgaaatgg 120
<210> 9
<211> 120
<212> DNA
<213> 人工序列
<400> 9
aaaatgataa gaattaaatt tacgatggat gccattattt tcttttgggc tcacatggtt 60
tacaggatca accttttatt taagtatact ctgaaaagct aaactggtca acttttagtc 120
<210> 10
<211> 120
<212> DNA
<213> 人工序列
<400> 10
cacatggttt acaggatcaa ccttttattt aagtatactc tgaaaagcta aactggtcaa 60
cttttagtct ttggaaacct ccaaacttat atatcttcac cttgcccgag atgcatccac 120
<210> 11
<211> 120
<212> DNA
<213> 人工序列
<400> 11
tgcccgagat gcatccactg caagagccag gatatttcca tctccgctag cagcacattc 60
ggttagtgca gcaagacaac gttcagccaa agcttgatcc ctgctggatt tgatctatgg 120
<210> 12
<211> 120
<212> DNA
<213> 人工序列
<400> 12
ttcgcactga agtattatca attgccagaa cttctacagc gtcttctttt tccaactggt 60
acttatttac tccaacaatt acttcagaac ctggtaattt cccaaagaaa aattttattc 120
<210> 13
<211> 120
<212> DNA
<213> 人工序列
<400> 13
aaatgaattt ctttttaact actggatttc atatatgaac tttctcacta tcttaccaga 60
atctattcta gcttgtcttc gggcagcaca ttcttcaatt cgaagtttag gtattccctc 120
<210> 14
<211> 120
<212> DNA
<213> 人工序列
<400> 14
aagtttaggt attccctcag ctacagcttt ggccattcca cccatttctt caatttcatt 60
aatgagctaa aaagaaaaac attaacaaaa ctaaaagaga atattaaaaa tgtgtgtaaa 120
<210> 15
<211> 120
<212> DNA
<213> 人工序列
<400> 15
aagttgcaaa gtggaaaaac ttacctttaa agcagcatca taaacatcat ttgtgagaca 60
ttccatcatg taagaacctc cccaaggatc agccactttg ggaatcccag attcttcttg 120
<210> 16
<211> 120
<212> DNA
<213> 人工序列
<400> 16
aatcccagat tcttcttgaa tgatgatttg tgtgttcctg gcaattcgag cacttttcac 60
agttggcaaa cccaaagctt catcaaaaga atttgtgtgc aaagactgag tccctccaaa 120
<210> 17
<211> 120
<212> DNA
<213> 人工序列
<400> 17
agactgagtc cctccaaata ctgctgccat tgcttctatt gcagtacgga caatattatt 60
gtagggatcc taaaatattt gataaaaaac aaaaactcaa agaaacaggt gatagatatt 120
<210> 18
<211> 120
<212> DNA
<213> 人工序列
<400> 18
gcttgtgcca cattgctcag aaaaaatata tatatataac tttattaaaa ttctacattt 60
taaattatat acatacctgc tcagtaagtg accatccaga tgtctgacag tgtgctctta 120
<210> 19
<211> 120
<212> DNA
<213> 人工序列
<400> 19
tctgacagtg tgctcttaga agaagagatt ttgagttttt aggctgaaac attttctcta 60
ttaagtgagc ccagagtctt ctaccagctc tcatctttgc tatttccata tagaaattca 120
<210> 20
<211> 120
<212> DNA
<213> 人工序列
<400> 20
tttcttaatg ttgaaacaat atctaggttt aatttactca ccttggtgca aattcatcaa 60
ttgtcaggcc agcctggagt ccagttctag agtactccaa tccatctgct aaagtatagg 120
<210> 21
<211> 120
<212> DNA
<213> 人工序列
<400> 21
catctgctaa agtataggcc agctccagaa tggcatcagc ccctgcttcc tgcatatggt 60
atccactaat tgaaattgaa ttaaattttg gcatgtgcta cataaaaaaa aaaattgtaa 120
<210> 22
<211> 120
<212> DNA
<213> 人工序列
<400> 22
aaacctataa taaccacaaa gtataccttt gctgtatatt caaatatgtc agcaataatt 60
ttcatggatg gttctggagg aaaaatgtat gtatttcgaa ccataaattc ctttagtata 120
<210> 23
<211> 120
<212> DNA
<213> 人工序列
<400> 23
ataaattcct ttagtatatc attttggatg gtaccagtaa gcttctcttt aggtacacct 60
tgttcttctc cagttactat aaaatttgca agaactggaa taactgctcc attcatagtc 120
<210> 24
<211> 120
<212> DNA
<213> 人工序列
<400> 24
actgctccat tcatagtcat ggaaactgac attttttcta aaggaattcc atcaaaaaga 60
attttggtat cttccacagt gtcaatagca actccagcca ttccaacatc accacgaact 120
<210> 25
<211> 120
<212> DNA
<213> 人工序列
<400> 25
ccaacatcac cacgaactcg agggttgtct gaatcatagc cacgatgtgt cgccagatca 60
aaggcaactg ataatccctg ctgaccagct aaatatataa agaaaaataa tgtaagattc 120
<210> 26
<211> 120
<212> DNA
<213> 人工序列
<400> 26
aatattatgt cttacattaa aatctcaccc ttaatgttgt ccttatagaa cttattgctt 60
tcttccacag tactaaaacc agcatactgg cggatggtcc agggcctaaa ggtatacatg 120
<210> 27
<211> 120
<212> DNA
<213> 人工序列
<400> 27
ggcctaaagg tatacatggt aggatatggt ccacgtgtga atggcttcac tcctggaagt 60
tcttcaggta agtccatagt atctctcttg gaatacaagg gttttataga gatcccttcc 120
<210> 28
<211> 120
<212> DNA
<213> 人工序列
<400> 28
tttatagaga tcccttccgg ggtgtgccat attaggtctt ctgggttttt gcctttcagc 60
tgctttttag ccagggcagc ccattctggg tgaaggggct gttgctggtg tagaagtcgt 120
<210> 29
<211> 120
<212> DNA
<213> 人工序列
<400> 29
acctgcctca ggtaatgagg tgaaagtaaa aaaagctgat tcttagctct taacatggtg 60
gagcatggaa acacccaata gaaataagaa ctgacctaga aaaagaaaca tagagtaaaa 120
<210> 30
<211> 120
<212> DNA
<213> 人工序列
<400> 30
gctgactcaa aacgtaacgg cccaattgtc cttgagactt cattccccag caagctcagc 60
gtgtaacgtg cgctatggag ccgaaagtcg cagagctgaa gcagaagatc gaggacacgc 120
<210> 31
<211> 120
<212> DNA
<213> 人工序列
<400> 31
gcagaagatc gaggacacgc tatgtccttt tggcttcgag gtttacccct tccaggttag 60
tttatccctc ctgctgttct agggcgaaat atatgattgg cttcgttgca actggcgtga 120
<210> 32
<211> 120
<212> DNA
<213> 人工序列
<400> 32
gtgtgaggcc tgaaggttaa ggtgtgggcc aggctgaggc ctagactggc cctctccagc 60
ctggcctgaa ctttctgttt caggtggcat ggtacaatga actcttgcct ccagccttcc 120
<210> 33
<211> 120
<212> DNA
<213> 人工序列
<400> 33
actcttgcct ccagccttcc acctaccgct gccaggacct accctggcct tcctggtact 60
cagcacgcct gccatgtttg accgggccct caagcccttc ttgcagagct gccacctccg 120
<210> 34
<211> 120
<212> DNA
<213> 人工序列
<400> 34
ttgcagagct gccacctccg aatgctgact gacccagtgg accagtgtgt ggcctaccat 60
ctgggccgtg ttagagaggt gaggaaggct cagttttccc ccagctccca aacctacagc 120
<210> 35
<211> 120
<212> DNA
<213> 人工序列
<400> 35
tccctcggac aaggtcataa ctcccctcat gctgacagta ccctctattt tgtccactgt 60
tccagagcct cccagagctg cagatagaaa tcattgctga ctacgaggtg caccccaacc 120
<210> 36
<211> 120
<212> DNA
<213> 人工序列
<400> 36
ctacgaggtg caccccaacc gacgccccaa gatcctggcc cagacagcag cccatgtagc 60
tggggctgct tactactacc aacgacaaga tgtggaggct gacccatggg ggaaccaggt 120
<210> 37
<211> 120
<212> DNA
<213> 人工序列
<400> 37
ctagcttgca atgatggcag ttgacttggt gccaagggga cctccatgac cttgcttttc 60
ttcaccctct ccccagcgca tatcaggtgt gtgcatacac ccccgatttg ggggctggtt 120
<210> 38
<211> 120
<212> DNA
<213> 人工序列
<400> 38
ccccgatttg ggggctggtt tgccatccga ggggtagtgc tgctgccagg gatagaggtg 60
ccagatctgc cacccagaaa acctcatgac tgtgtaccta caagagctga ccgtatcgcc 120
<210> 39
<211> 120
<212> DNA
<213> 人工序列
<400> 39
gcagaaggcc tacttctcca ctccacctgc ccaacgattg gccctattgg gcttggctca 60
gccctcagag aagcctagtt ctccctcccc ggaccttccc tttaccacac ccgcccccaa 120
<210> 40
<211> 120
<212> DNA
<213> 人工序列
<400> 40
tttaccacac ccgcccccaa gaagcctggg aatcccagca gagcccggag ctggctcagc 60
cccagggtct caccacctgc atcccctggc ccttgatttt ctcccatgtg gaccctgatt 120
<210> 41
<211> 120
<212> DNA
<213> 人工序列
<400> 41
ttatgtagca ttcatgcgga atttattata caaagaattt tattcaatta aacttgaaat 60
gcatctggat tcttaaaggt tcagtagtga tcactgggac agggcgatca taaaactgaa 120
<210> 42
<211> 120
<212> DNA
<213> 人工序列
<400> 42
agggcgatca taaaactgaa tgggctgtcg gaaggtgtcg aggcagcagc aagggtgaca 60
ttgccactga cgggggcttc cgaactgggg acgtttgtca ttgggatgtg ttacaagttc 120
<210> 43
<211> 120
<212> DNA
<213> 人工序列
<400> 43
ttgggatgtg ttacaagttc gggctgtgga aattactcga tgaaaaacgc acattaacga 60
tagccatgaa atattagtta agggaaacta ggttgagaaa tgagacagca ggatctatca 120
<210> 44
<211> 120
<212> DNA
<213> 人工序列
<400> 44
tgagacagca ggatctatca gagcctggca ttgttcgcca cagcccaggt agtgattaaa 60
acgactgtca agcggcagtg ggtgggagct gaggagcacg gggctgtgag tgacgaggcc 120
<210> 45
<211> 120
<212> DNA
<213> 人工序列
<400> 45
gggctgtgag tgacgaggcc gcgtccgggg ctcacatctt ggcactgact ccccaggaca 60
acttggttcc ccaccctcca cagtgcgggc tggtgtcact gtgactgtgg gcttctctct 120
<210> 46
<211> 120
<212> DNA
<213> 人工序列
<400> 46
gtgactgtgg gcttctctct gacacaagag ccaactgcca gcttgtctct ggaaatgaca 60
ctaaacaccc cattccaggg agcctgacct gctgctgttt gtttcaaaac cccgtgttcc 120
<210> 47
<211> 120
<212> DNA
<213> 人工序列
<400> 47
gtttcaaaac cccgtgttcc cgtgcaatgg gactgattgt ttctgagagt tgccagtggt 60
gtgcaaacac tggggcagcg ggggcttcgc agtcacattt cctgactgta gggctttcat 120
<210> 48
<211> 120
<212> DNA
<213> 人工序列
<400> 48
cctgactgta gggctttcat taacttttcc cacctggttc aaaatatggg aggacatttg 60
ctcagtgtac agaggggtac acatcttttc ttttgcctcg ggctcttgat agagcttttg 120
<210> 49
<211> 120
<212> DNA
<213> 人工序列
<400> 49
ggctcttgat agagcttttg aaaggcactg cagaagagca aatgttttaa acaccccatc 60
cttccagccc ccaaagcaag ctttctccat ttttcccagg ttggtagcac aggagagaaa 120
<210> 50
<211> 120
<212> DNA
<213> 人工序列
<400> 50
ttggtagcac aggagagaaa cacctgctga gtggtggcat ctgccctgca cccaggtggt 60
ttctcagaga agtgtttgct ggtgaaccgg gagacagata cagaggcggg ggtctgagaa 120
<210> 51
<211> 120
<212> DNA
<213> 人工序列
<400> 51
cagaggcggg ggtctgagaa cgcgactgtt aacatccagg ctgtgacacc cggtcctgga 60
aggacaggac ttggcaacag gtgaacggcc ttgtttcaaa gattgtcact ttaggatgtt 120
<210> 52
<211> 120
<212> DNA
<213> 人工序列
<400> 52
gattgtcact ttaggatgtt gtatttactc aaaaagcttt ccttatcctt tggaggctgt 60
gctttcccat ggctaggcat gcagggctgc tgtgttattt tccaggaaga ttttgagatt 120
<210> 53
<211> 120
<212> DNA
<213> 人工序列
<400> 53
tccaggaaga ttttgagatt ttgaggtgta gacagaattg agtgattcgc tgcaagctct 60
ttgaacatac tccctgaccg agccttagtg attgcgtttt cagggccgtt ttcagctctg 120
<210> 54
<211> 120
<212> DNA
<213> 人工序列
<400> 54
cagggccgtt ttcagctctg ttttgcaaac actgagcacc gactccgtac cagacctggt 60
agtgacgatg ggggcaggga ggcacggaac aaagccacct cctggaaggc attcacacac 120
<210> 55
<211> 120
<212> DNA
<213> 人工序列
<400> 55
cctggaaggc attcacacac tcctggtcat ctgcacctca cccaccctgc ccaaggctca 60
agagggccat cacagcctgt cagcaatgaa aggacccaga agacacaagt gaatatacct 120
<210> 56
<211> 120
<212> DNA
<213> 人工序列
<400> 56
agacacaagt gaatatacct ggccctcctg caaactttgc aggccaggtg gtaagaatga 60
aggcaaaaat atgcacgtga ctaagaaggt aatgtttgct gacttgccag caagaaagat 120
<210> 57
<211> 120
<212> DNA
<213> 人工序列
<400> 57
gacttgccag caagaaagat ggccggaaag accagctgtc ccgagcctag cgcggtggcc 60
catgctaagc agccactcag tcaatatgtg atggctgaat ggagttcgcc aggcatttca 120
<210> 58
<211> 120
<212> DNA
<213> 人工序列
<400> 58
ggagttcgcc aggcatttca gccctgaaac tggacaagag cctgcccttc caaagtcatt 60
tcctgcaatt agcagcctgc agtctttagg ttcttggggc tgcctcttct gcccttcata 120
<210> 59
<211> 120
<212> DNA
<213> 人工序列
<400> 59
tgcctcttct gcccttcata aatattgcct ttcccaagga cactgctggc actggcagtg 60
tcgtttcata gcaggaggga gcagcagtga caactgaaat aaatacagcc gtggcaccga 120
<210> 60
<211> 120
<212> DNA
<213> 人工序列
<400> 60
aaatacagcc gtggcaccga tctcagaggc ataagccagc caagtgcaac atcggagaaa 60
tcagtctggt ggatgtcagc cttgctggaa actgacaacc tcattttctc aagctgaaga 120
<210> 61
<211> 120
<212> DNA
<213> 人工序列
<400> 61
tcattttctc aagctgaaga tgctgcaccg cagacccttg actcagtcca accagacagg 60
gaatgttcac cttcaagtgg tagtgttgtt ctcatcagca tctccatccc tttcagaggg 120
<210> 62
<211> 120
<212> DNA
<213> 人工序列
<400> 62
tctccatccc tttcagaggg ggtcctctaa gctgcacccc catcacacac acttcaatct 60
aagccaggag tttctgagcc tcgcctgtca atctgcagct atccttcccc cacactttgg 120
<210> 63
<211> 120
<212> DNA
<213> 人工序列
<400> 63
atccttcccc cacactttgg cggtgatggg tggctcagag gtgactaaac ttccaaatct 60
tgtccgcctt cccctttaca aatgtgacat ttaaagcacc tttggcacag gtaaatcaca 120
<210> 64
<211> 120
<212> DNA
<213> 人工序列
<400> 64
tttggcacag gtaaatcaca attacatttt caagggcctc tggtcccatt ccaaatctgt 60
gaacaacctg agctggcagt gggagggctc tctctggctc cagcctttcc aacttttatt 120
<210> 65
<211> 120
<212> DNA
<213> 人工序列
<400> 65
cagcctttcc aacttttatt ccttttccgc acagctcctt ctcattgcag caatcacttt 60
acctgtcttt cctgcaatgc caattcattc ccacatccct ccaagaccca ggagagcttg 120
<210> 66
<211> 120
<212> DNA
<213> 人工序列
<400> 66
ccaagaccca ggagagcttg gggaagcagg gtcagggaca gaatccccaa agggtcacag 60
tcccttgagg aaggtggcaa gaggtgtaga tcaaagctga gctgggacaa aaggctgctt 120
<210> 67
<211> 120
<212> DNA
<213> 人工序列
<400> 67
gctgggacaa aaggctgctt tgagcctctc tgggtgagct cttcaggaac caggacccca 60
gaagggcaag ctcctctctc cacggccatc gccatggagg gatcctccaa gctcccactt 120
<210> 68
<211> 120
<212> DNA
<213> 人工序列
<400> 68
caggacccca gaagggcaag ctcctctctc cacggccatc gccatggagg gatcctccaa 60
gctcccactt tctgtgattt cagagtccct cagactcggc cgatgggtca tttttcatgt 120
<210> 69
<211> 120
<212> DNA
<213> 人工序列
<400> 69
cgatgggtca tttttcatgt atattttctc ttgattcccc tccttcatgg ctgcatatct 60
ggctagcgtg aagagatagt cactgagtct ggaggggcag agagagagaa gcaaacagaa 120
<210> 70
<211> 120
<212> DNA
<213> 人工序列
<400> 70
ttccacgcga gtacctgttt aagaacttgg ccacgttcgc atcggtctct cccatctgga 60
caagaggcac cacactagaa agggaggaga cactgagtca cgtgacatta tggggctcaa 120
<210> 71
<211> 120
<212> DNA
<213> 人工序列
<400> 71
taccgtctct cggcccggcg gcacacggcc cggcagaaat gcagcgccga gctgatcttg 60
cctcccgact gaaaggagaa agggacattg cctgagcagg gtgggaaagg tgtgcccact 120
<210> 72
<211> 120
<212> DNA
<213> 人工序列
<400> 72
gcctccagga gccctgccac ggcacaccca ccgggcacgc tgctccagag tgggcagggc 60
tgggagggac cggtgaggac ctggagggac ttggggaact ggaggacagc gtctgtcact 120
<210> 73
<211> 120
<212> DNA
<213> 人工序列
<400> 73
ccgggcacgc tgctccagag tgggcagggc tgggagggac cggtgaggac ctggagggac 60
ttggggaact ggaggacagc gtctgtcact gtgaaaagag ggatttattc agagcccatg 120
<210> 74
<211> 120
<212> DNA
<213> 人工序列
<400> 74
tttattcaga gcccatgtgt gtctgtcact gaacctgcct gcagccgccc ccggttaagc 60
ctgcccagta cctacaggca ggatgaaggc cgtgagtggt gggagctggc tggtgtactt 120
<210> 75
<211> 120
<212> DNA
<213> 人工序列
<400> 75
gggagctggc tggtgtactt gtcgatccac tgctccagct ccaggatggg ccccgccttg 60
aacgtggtat actctgagga gccaaggagc agagggaact gccatgaggc catcacccac 120
<210> 76
<211> 120
<212> DNA
<213> 人工序列
<400> 76
aggagagtcc cctgacccta gggccctctg aacacccaca ggagtttgag aattacttaa 60
gtgagcctcc cgggccgagg agcatggtgt cgccagggcc gagccgacgt cctgcaatgt 120
<210> 77
<211> 120
<212> DNA
<213> 人工序列
<400> 77
agtcccgtga tggccaccgg ggctgaagat tcccagcttg ggtgagatgg tgttactcac 60
tttctgaagc tcttcggcaa atgtatggcc cttttctgtg actaattcca gagcaaacct 120
<210> 78
<211> 120
<212> DNA
<213> 人工序列
<400> 78
gagagcttca cattcattac gtttactcat actcgactca aacgcaacct caaggccaat 60
cctgtccccc ttacccaata gctgaactta attcatctgt agttcccacg gcttcaaaca 120
<210> 79
<211> 120
<212> DNA
<213> 人工序列
<400> 79
agttcccacg gcttcaaaca cttggtcatc tttgggtctc ctttctcctg tgaaggtact 60
agaaaaccct gtggaaaaaa atgtttagcc acccaaatta agattctgtt ttcctgatat 120
<210> 80
<211> 120
<212> DNA
<213> 人工序列
<400> 80
agaactttaa aatggtgtat gccatgagta tttctttgca tttttcacct gtccccaccc 60
tacctttgtc tcccgttttg gtgtaaatct tggggatcct gggtgtcttc gaggaaggct 120
<210> 81
<211> 120
<212> DNA
<213> 人工序列
<400> 81
ctgtccccgt cttccacgcc ctgagggccg cggctctgga aacggggata caggagcctg 60
gcggcgccga agcacccgcg caggccaaga cggctcccca ggccaagacg gctccccagg 120
<210> 82
<211> 120
<212> DNA
<213> 人工序列
<400> 82
caggagcctg gcggcgccga agcacccgcg caggccaaga cggctcccca ggccaagacg 60
gctccccagg ccgcacacag ccatgagcca ggctgcttga cgggacctga ccccgccagg 120
<210> 83
<211> 120
<212> DNA
<213> 人工序列
<400> 83
atatatctgg aggtgcagca gccacatgag actcatccga acctgatgat cctgcagtcg 60
aaaaggcttt gggattgaca acccttttaa ctaaagagca aaatcctggg agataggaaa 120
<210> 84
<211> 120
<212> DNA
<213> 人工序列
<400> 84
acccgttctt tcattcctaa cattcccaaa cgagtttact atgctgattc ttaagagata 60
atttactcac attggccatc tccgctggag aagatagttc gcaaaatagc tttcctttgg 120
<210> 85
<211> 120
<212> DNA
<213> 人工序列
<400> 85
ataaaacgaa tatgcccatg ctgctaccac atcctcacca gcatttccta aaaggccttt 60
taagagcacc tttccgatgt taccacttca tctttcactc aagtactcat ctcggatcag 120
<210> 86
<211> 120
<212> DNA
<213> 人工序列
<400> 86
aagtactcat ctcggatcag gaatcccatg tgctcagccg tttaattctc ttggactcca 60
ttgtacaaag tggatgctgc tgtcagatgg cttaaagaga aaattatgtg tacaaacaac 120
<210> 87
<211> 120
<212> DNA
<213> 人工序列
<400> 87
aaattatgtg tacaaacaac cttaaaggac cacacagaag gactttctga taaagagcaa 60
agatttgtgg ataaacttta tactggttta atccaagggc aaagggcctg tttagcagag 120
<210> 88
<211> 120
<212> DNA
<213> 人工序列
<400> 88
aaagggcctg tttagcagag gccataactc ttgtagaatc aactcacagc aggaaaaagg 60
agttagccca ggtgcttctt cagaaagtat tactttacca cagagaacaa gaacaatcaa 120
<210> 89
<211> 120
<212> DNA
<213> 人工序列
<400> 89
cagagaacaa gaacaatcaa ataaaggaaa accactagca tttcgagtag gtcagtcttt 60
tttgtgtgtt ttctcagtaa atatttttac aaattctctg tattctgttt tttaaaaaaa 120
<210> 90
<211> 120
<212> DNA
<213> 人工序列
<400> 90
tattttactc agtaaaactg atcgtagttc tgatttcatt tgtttcattg aattagaaga 60
tctctttcca ccgtaggatt gtctgggccc cctggtgctg gaaaatcaac atttatagaa 120
<210> 91
<211> 120
<212> DNA
<213> 人工序列
<400> 91
gaaaatcaac atttatagaa tattttggaa aaatgcttac tgagagaggg cacaaattat 60
ctgtgctagc tgtggaccct tcttcttgta ctagtggtgg taagtatggc tgattctttt 120
<210> 92
<211> 120
<212> DNA
<213> 人工序列
<400> 92
actggctgat aattgacccg taaaactgtt ccatgattat aaaatgtaac tgtatgtttt 60
aggatcactc ttaggtgata aaacccgaat gactgagtta tcaagagata tgaatgcata 120
<210> 93
<211> 120
<212> DNA
<213> 人工序列
<400> 93
tcaagagata tgaatgcata catcaggcca tctcctacta gaggaacttt aggaggcgtg 60
acaaggacca caaatgaagc tattctgttg tgtgaaggag cgggatatga cataattctt 120
<210> 94
<211> 120
<212> DNA
<213> 人工序列
<400> 94
tattttagga tatagttgtg atttacaatt tcaggtgtgg gtcagtcgga gtttgctgtt 60
gctgacatgg ttgacatgtt tgttttacta ctgccaccag caggaggaga tgagctgcag 120
<210> 95
<211> 120
<212> DNA
<213> 人工序列
<400> 95
actttcaaaa tctgagcatt gactagtttt cccattttta tgttggttgt cattaaatgt 60
ttctgatctc tttagggtat caaaaggggt ataatcgaga tggcagatct ggtagctgta 120
<210> 96
<211> 120
<212> DNA
<213> 人工序列
<400> 96
tggcagatct ggtagctgta actaaatctg atggagactt gattgtgcca gctcgaagga 60
tacaagcgga atatgtgagt gcactgaaat tactccgcaa acgttcacaa gtctggaaac 120
<210> 97
<211> 120
<212> DNA
<213> 人工序列
<400> 97
ggcaggtatc agcgtccctg taaaaatttt ttctatcatt ttaagtaaaa tggtctggtt 60
cttccctttt cgataggtaa ttcgtatttc tgcccgaagt ggagagggga tctctgaaat 120
<210> 98
<211> 120
<212> DNA
<213> 人工序列
<400> 98
ggagagggga tctctgaaat gtgggataaa atgaaagatt tccaggacct aatgcttgcc 60
agtggggagc tgactgccaa acgacggaag caacagaaag tttggatgtg gaatctcatt 120
<210> 99
<211> 120
<212> DNA
<213> 人工序列
<400> 99
tttggatgtg gaatctcatt caggaaagtg tgttagagca tttcaggacc caccccacag 60
tccgggaaca gattccactt ctggaacaaa aggttctcat tggggccctg tccccaggac 120
<210> 100
<211> 50
<212> DNA
<213> 人工序列
<400> 100
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct 50
<210> 101
<211> 50
<212> DNA
<213> 人工序列
<400> 101
caagcagaag acggcatacg agatcggtct cggcattcct gctgaaccgc 50
<210> 102
<211> 21
<212> DNA
<213> 人工序列
<400> 102
aatgatacgg cgaccaccga g 21
<210> 103
<211> 21
<212> DNA
<213> 人工序列
<400> 103
caagcagaag acggcatacg a 21
Claims (6)
1.一种甲基丙二酸血症相关基因捕获的捕获探针,由序列表中序列1至序列99所示的探针组成;
所述甲基丙二酸血症相关基因为MUT基因、MMAA基因、MMAB基因、MMADHC基因和/或MMACHC基因。
2.根据权利要求1所述的捕获探针,其特征在于:
所述每条探针均标记生物素。
3.一种甲基丙二酸血症相关基因捕获的试剂盒,包括权利要求1或2所述的捕获探针所示的组分1);
所述甲基丙二酸血症相关基因为MUT基因、MMAA基因、MMAB基因、MMADHC基因和/或MMACHC基因;
所述试剂盒还包括如下独立包装的各个组分:
2)富集缓冲液,其包括人cot-1DNA、鲑鱼精DNA、引物1和引物2;
所述引物1的核苷酸序列为序列100;
所述引物2的核苷酸序列为序列101;
3)杂交缓冲液,包括Tris-HCl缓冲液、NaCl、柠檬酸钠、BSA和Tween20;
4)结合缓冲液,包括Tris-HCl缓冲液、NaCl和EDTA;
5)漂洗液1,包括SDS和柠檬酸三钠缓冲液;
6)漂洗液2,包括SDS和10倍稀释的柠檬酸三钠缓冲液;
7)NaOH水溶液;
8)Tris-HCl缓冲液;
9)PCR反应液;所述PCR反应液包括引物3和引物4;
所述引物3的核苷酸序列为序列102;
所述引物4的核苷酸序列为序列103;
所述引物3和所述引物4的最后一位核苷酸为硫代修饰;
10)TE缓冲液。
4.权利要求1或2所述的捕获探针或权利要求3所述的试剂盒在制备捕获待测样本中甲基丙二酸血症相关基因的产品中的应用;
所述甲基丙二酸血症相关基因为MUT基因、MMAA基因、MMAB基因、MMADHC基因和/或MMACHC基因。
5.权利要求1或2所述的捕获探针或权利要求3所述的试剂盒在制备测序待测样本中甲基丙二酸血症相关基因产品中的应用;
所述甲基丙二酸血症相关基因为MUT基因、MMAA基因、MMAB基因、MMADHC基因和/或MMACHC基因。
6.权利要求1或2所述的捕获探针或权利要求3所述的试剂盒在制备检测或预测待测者是否患有甲基丙二酸血症产品中的应用。
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