CN110396540B - 一种血友病基因突变位点的检测方法 - Google Patents

一种血友病基因突变位点的检测方法 Download PDF

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CN110396540B
CN110396540B CN201910364285.XA CN201910364285A CN110396540B CN 110396540 B CN110396540 B CN 110396540B CN 201910364285 A CN201910364285 A CN 201910364285A CN 110396540 B CN110396540 B CN 110396540B
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胡洋
陈翀
谢婵芳
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Abstract

本发明公开了一种血友病基因突变位点的检测方法。并基于该方法构建了基于CRISPR/Cas12a的血友病基因突变位点检测试剂盒。该技术对血友病基因突变位点检测特异性好、灵敏度高，可在室温实现高灵敏、高精度的分子检测，检测限值可达到阿摩尔级，实现靶标单分子检测。同时还能够实现多位点同时检测，对血友病的检测及筛查具有重要的意义。同时该方案检测成本低廉、无需大型设备支持、操作方便快捷、实现床旁检测，便于广泛应用和快速推广，是一种具有巨大商业应用价值的检测方法及检测试剂盒，具有非常好的广阔应用前景。

Description

一种血友病基因突变位点的检测方法
技术领域
本发明属于分子检测诊断技术领域。更具体地，涉及一种血友病基因突变位点的检测方法。
背景技术
血友病(Hemophilia)，是一组由于血液中某些凝血因子的缺乏而导致患者产生严重凝血障碍的遗传性出血性疾病，男女均可发病，但绝大部分患者为男性。血友病在先天性出血性疾病中最为常见，出血是该病的主要临床表现。血友病有两种分型。血友病A(hemophilia A，HA)又称甲型血友病，是最为常见的X连锁隐性遗传性出血性疾病，其主要病因是由于凝血因子VIII(FVIII)基因突变而引起的FVIII含量不足或功能缺陷。FVIII基因全长186kb，位于X染色体长臂远端(Xq28)，含有26个外显子和25个内含子，编码2351个氨基酸。
目前血友病规范化诊断已建立了包括临床诊断、家系调查、实验检测等系列技术平台。实验检测分为基因诊断和非基因间接诊断。目前市场上常见的血友病诊断产品均采用非基因间接诊断，包括活化部分凝血活酶时间(APTT)、血浆凝血酶原时间(PT)、血浆FVIII：C以及FVIII抗原检测等。这些产品可以对HA进行确诊，但无法确定HA患者的基因突变类型更无法检出携带者。因此，开发可以方便、准确诊断血友病的基因突变位点产品，有效应用于产前诊断，对避免重型血友病胎儿出生，减低血友病发病率具有重要意义。
目前传统的遗传病相关突变基因检测方法有Sanger测序法、荧光定量PCR法、Fish法、micro array等。传统的基因突变检测方法因技术单次只能检测较少的突变位点、灵敏度低。如Sanger测序法灵敏度低，仅能检测突变率大于20％的高频突变，假阴性率较高，且该技术手段难以胜任多基因多位点的检测，操作繁琐。而聚合酶链反应(PCR)：包括普通PCR、等位基因特异性PCR、实时荧光定量PCR、PCR-基因芯片技术等，主要缺点在于需要依赖PCR仪或昂贵的实时定量PCR仪，及其它多种配套设备，以及专门的PCR实验室和专业操作人员。PCR检测无法实现即时检验、床旁诊断和无特定实验室检测条件的场景应用，因此无法满足基层、用户终端、现场的检验需求。同时，PCR检测可能存在假阳性和灵敏度不足等问题。
另外，CRISPR/Cas是进行基因编辑的强大工具，可以对基因进行定点的精确检测与编辑。在向导RNA(guide RNA，gRNA)和Cas9蛋白的参与下，待编辑的细胞基因组DNA将被看作病毒或外源DNA，被精确剪切。但是，CRISPR/Cas9的应用也有一些限制条件，首先，待编辑的区域附近需要存在相对保守的PAM序列(NGG)，其次向导RNA要与PAM上游的序列碱基互补配对，设计、制备出精确、特异性靶向目标基因的gRNA是CRISPR/Cas12a基因敲除的关键技术，等等。目前尚未有基于CRISPR/Cas12a系统的血友病相关突变基因检测方法的相关报道。
发明内容
本发明要解决的技术问题是克服现有血友病基因突变位点检测技术的缺陷和不足，设计、制备出精确、高效、特异性靶向血友病相关突变基因RNA的gRNA，这是CRISPR/Cas12a识别靶基因的关键；并基于此构建了一种基于CRISPR/Cas12a系统的血友病相关突变基因检测方法及检测试剂盒。
本发明的目的是提供一种基于CRISPR/Cas12a检测血友病基因突变位点的gRNA组合。
本发明的另一目的是提供一种基于CRISPR/Cas12a的血友病基因突变位点检测方法。
本发明再一目的是一种基于CRISPR/Cas12a的血友病基因突变位点检测试剂盒。
本发明上述目的通过以下技术方案实现：
本发明提供一种血友病基因突变位点的检测方法，该方法基于CRISPR/Cas12a技术进行检测，具体是利用Cas12a蛋白和gRNA进行CRISPR检测，检测靶标位点的核苷酸序列如SEQ ID NO.1-43任一个或任几个所示。
所述gRNA以所述靶标位点为靶序列进行设计，设计原则为：在选取gRNA靶向序列时，靶向序列5’端应具有5’-TTTN-3’序列，且靶向序列本身、靶向序列和其余序列间不形成稳定二级结构。
作为优选的可选择方案，所述gRNA的序列如SEQ ID NO.44-97任一个或任几个所示。该gRNA组合也应在本发明的保护范围之内。
另外，优选地，检测体系包括：2μl RPA产物，45nM纯化的LbCas12a，22.5nM gRNA，100nM在LbCas12a切割时可发出荧光的报告DNA链，即非特异单链DNA荧光探针(DNAseAlertQC System,Thermo Scientific)，0.5μl RNase抑制剂(Promega)，及核酸酶检测缓冲液(20mM Tris,60mM NaCl,10mM MgCl 2,pH 7.3)。
优选地，检测程序为：在37℃下反应1.5小时，荧光动力检测5分钟一次。
同时本发明还提供一种基于CRISPR/Cas12a的血友病基因突变位点检测试剂盒，包括Cas12a蛋白和上述gRNA。
另外，上述Cas12a蛋白为具有核酸内切酶活性且具有附属切割活性的Cas12a蛋白。比如LbCas12a、SsCas12a、ScCas12a、FnCas12a、AsCas12a等。
所述ScCas12a的序列如SEQ ID NO.98所示，所述SsCas12a的序列如SEQ ID NO.99所示，所述LbCas12a的序列参照Addgene号pMAL-his-LbCpf1-EC(Plasmid#79008)，FnCas12a的序列参照Addgene号6-His-MBP-TEV-FnCpf1(Plasmid#90094)、AsCas12a的序列参照Addgene号AsCpf1-2NLS(Plasmid#102565)。
为了实现对多个血友病基因突变位点的同时检测，便于实现快速多位点初筛的应用场景，我们开发了针对上述血友病基因突变靶标的多重检测体系，对选择的多种血友病基因突变序列进行分析，以及对应gRNA序列设计分析，根据这些序列的相似性、GC含量、碱基均一性、有无形成二级发夹结构、同一反应有无交叉反应等参数，对反应体系和gRNA组合方式进行优化。本发明的方案可实现对SEQ ID NO.1-43所示43个靶标位点实现多位点同时检测，对于血友病的检测筛查具有重要的意义。
本发明具有以下有益效果：
本发明提供了一种用于靶向血友病相关突变基因RNA的gRNA组合，同时还提供了一种基于CRISPR/Cas12a系统的血友病相关突变基因检测方法、检测试剂盒。本发明的检测方法综合了gRNA靶向识别血友病相关突变基因转录产物RNA(靶标RNA序列)的优势以及当CRISPR/Cas12a复合物检测到靶标RNA序列时，复合物会切割带有检测标记的报告RNA，释放可检测信号的特点，将CRISPR/Cas12a系统应用在血友病相关突变基因检测中，灵敏度高、准确度高、特异性好，可在25-37℃的室温下实现高灵敏、高精度的分子检测，检测限值可达到阿摩尔级(10-18摩尔/L)，实现靶标单分子检测。同时还能实现多位点同时检测，临床检测效果优异，对于血友病的检测筛查具有重要的意义。
而且，该方案检测成本低廉、无需大型设备支持、操作方便、快捷、实现床旁检测，便于广泛应用和快速推广，是一种具有巨大商业应用价值的检测方法及检测试剂盒。
附图说明
图1为实施例1中对血友病突变位点的不同gRNA检测效果。
图2为实施例2中对血友病突变位点多gRNA同时检测效果。
具体实施方式
以下结合说明书附图和具体实施例来进一步说明本发明，但实施例并不对本发明做任何形式的限定。本领域技术人员可以理解的是，可以采用本领域常规的替代方法替换本发明实施例中常规的Cas12a基因的克隆、重组表达载体的构建、Cas12a蛋白的表达及纯化、靶核苷酸/目标基因片段的扩增等步骤中的一种或多种，以期获得类似或等同的效果。
除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。除非特别说明，以下实施例所用试剂和材料均为市购。
实施例1基于CRISPR/Cas12a系统的血友病基因突变位点检测
1、CRISPR/Cas12a基因克隆及蛋白表达
采用源自Lachnospiraceae bacterium的Cas12a蛋白基因，经过密码子优化，使基因更适合在哺乳动物细胞中表达。优化后的Cas12a蛋白基因克隆入带6-His组氨酸标签的pET28a质粒，方便蛋白纯化表达。Cas12a蛋白重组表达载体转化，表达菌采用BL21star(DE3)。
将Cas12a蛋白重组表达载体转化后，进行蛋白表达、SDS-PAGE检测以及凝胶柱纯化，获得的纯化后的Cas12a蛋白放-80℃保存。
具体蛋白表达条件为：在培养菌液OD600＝0.6时加入0.5mMIPTG培养4小时。收集菌体进行蛋白纯化。
纯化条件为：将菌体重悬于裂解液(50mM Tris,pH8.0，300mM NaCl，5％甘油，20mM咪唑)，进行超声破碎(70％振幅，2s On/4s Off，3分钟，Sonics 750w超声仪)，离心分离上清液，以镍柱纯化，以含250mM咪唑的裂解液洗脱，浓缩洗脱组分，以Superdex 200，Tricorn10/300凝胶色谱柱进行纯化。SDS-PAGE检测以及凝胶柱纯化，获得的纯化后的Cas12a蛋白，放-80℃保存。
2、靶标序列扩增
靶核苷酸可经过PCR扩增、重组酶聚合酶扩增(RPA)，NASBA等温扩增、或环介导等温扩增(LAMP)、链置换扩增(SDA)、解旋酶依赖性扩增(HDA)和切口酶扩增反应(NEAR)方式扩增靶DNA。
重组酶聚合酶扩增RPA(Recombinase Polymerase Amplification)：
采用NCBI Primer blast设计RPA引物，扩增片段大小为80-120nt，引物的变性温度可为54-67℃、Opt＝60，长度为30-35nt、Opt＝32，引物中GC含量为40-60％，根据设计序列合成DNA引物。
分别参考
Figure BDA0002047729200000051
Basic和/>
Figure BDA0002047729200000052
BasicRT(TwistDx)试剂盒进行RPA反应，不同的是，在样本核酸加入之前，先加入280mM的MgAc，即乙酸镁。在37℃下反应30分钟。
胶分离以及纯化(采用MinElute gel extraction kit(Qiagen)试剂盒)，纯化后的dsDNA与T7聚合酶37℃孵育过夜(采用T7RNA polymerase(Thermo)试剂盒)，然后用RNeasy mini kit(Qiagen)试剂盒纯化RNA，从而获得靶核RNA。
3、制备gRNA
gRNA引物序列设计原则：选取靶向序列时，靶向序列5’端应具有5’-TTTN-3’序列。且靶向序列本身、靶向序列和其余序列间不形成稳定二级结构，可通过http://www.rgenome.net/cas-designer/在线软件辅助设计。
T7引物序列：TGTAATACGACTCACTATAGGG
gRNA引物结构：
5’-靶向序列-“ATCTACACTTAGTAGAAATTA”-CCCTATAGTGAGTCGTATTACA-3’
其中“ATCTACACTTAGTAGAAATTA”序列可替换为“ATCTACAACAGTAGAAATTA”或“ATCTACAACAGTAGAAATTA”或“ATCTACAACAGTAGAAATTA”或“GCATGAGAACCATGCATTTC”或“ACCTAATTACTAGGTAATTT”或“ATCTACAAAAGTAGAAATCC”或“ATCTACAATAGTAGAAATTA”或“ATCTACAAAGTAGAAATTAT”或“ATCTACAAACAGTAGAAATT”。
参照T7RNApolymerase kit(Thermo)试剂盒说明书，将带T7启动子的DNA片段、T7引物、T7聚合酶混合，37℃孵育过夜；再采用RNeasy mini kit(Qiagen)，获得纯化的gRNAs。
T7引物序列：TGTAATACGACTCACTATAGGG
T7gRNA引物序列：
“靶向序列”-5’-ATCTACACTTAGTAGAAATTACCCTATAGTGAGTCGTATTACA-3’
经过大量探索研究，得出了一组基于CRISPR/Cas12a的检测靶标基因及对应的靶向gRNA序列(如表1所示)，还能够实现多位点同时检测。
表1检测靶标基因及对应的靶向gRNA序列
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4、核酸检测
检测体系包括：2μl RPA产物，45nM纯化的LbCas12a，22.5nM gRNA，100nM在LbCas12a切割时可发出荧光的报告DNA链，即非特异单链DNA荧光探针(DNAseAlert QCSystem,Thermo Scientific)，0.5μl RNase抑制剂(Promega)，及核酸酶检测缓冲液(20mMTris,60mM NaCl,10mM MgCl 2,pH 7.3)。
反应体系置于荧光分析仪(BioTek)，在37℃(除非另有说明)下反应1.5小时，荧光动力检测5分钟一次。
分析反应荧光数据：为了计算去除背景的荧光数据，方便不同条件之间的比较，样品的初始荧光被去除。背景荧光(无靶核苷酸或无gRNA的条件下)会从样品中去除，从而获得扣除背景荧光的数据。
检测结果如图1所示，结果表明：所述Cas12a蛋白和所设计的gRNA可识别切割靶标位点并产生荧光信号，并且拥有良好的特异性。
实施例2基于CRISPR/Cas12a系统的血友病多基因突变位点同时检测
为了实现对多个血友病基因突变位点的同时检测，便于实现快速多位点初筛的应用场景，我们开发了针对上述血友病基因突变靶标的多重检测体系，对选择的多种血友病基因突变序列进行分析，以及对应gRNA序列设计分析，根据这些序列的相似性、GC含量、碱基均一性、有无形成二级发夹结构、同一反应有无交叉反应等参数，对反应体系和gRNA组合方式进行优化。实施例1中的gRNA对于所对应的靶标基因能够实现多位点同时检测。本实施例呈现了血友病多基因突变位点检测的一种gRNA组合方案的实验及结果。
1、模板准备待检测模板选用制备按照实施例1中步骤2，准备SEQ ID NO.4,SEQ IDNO.6，SEQ ID NO.9，SEQ ID NO.24作为模板。
2、gRNA制备及突变检测
按照实施例1中步骤3的方法制备好gRNA后，取对应模板的3种gRNA按等比例混合(SEQ ID NO.47、SEQ ID NO.49、SEQ ID NO.54)，然后按照实施例1中步骤4的方法配制CRISPR/Cas12a检测体系，检测多重gRNA方法检测效果。检测反应中的模板分别为SEQ IDNO.4,SEQ ID NO.6，SEQ ID NO.9。实验中无靶核酸的样本为阴性对照，阳性标准品SEQ IDNO.4单独加对应gRNA(SEQ ID NO.47)为阳性对照，SEQ ID NO.24为非特异性对照，其它条件不变。配置好体系后置于荧光分析仪(BioTek)，在37℃(除非另有说明)下反应1.5小时，荧光动力检测5分钟一次。
3、检测结果如图2所示。实验结果表明，特异性的靶标基因加入3重gRNA反应体系后，均有荧光产生，结果为阳性；非特异性的靶标(SEQ ID NO.24)加入多重gRNA反应体系后，结果为阴性。说明实验建立的基于CRISPR/Cas12a系统的血友病多基因突变位点同时检测有良好的检测效果与特异性。
本领域技术人员可以理解的是，可以采用本领域常规的替代方法替换本发明实施例中常规的Cas12a基因的克隆、重组表达载体的构建、Cas12a蛋白的表达及纯化、靶核苷酸/目标基因片段的扩增等步骤中的一种或多种，以期获得类似或等同的效果。
另外，上文中SEQ ID NO.98和SEQ ID NO.99所示序列如下：
SEQ ID NO.98：(ScCas12a的序列)
ATGCAGACCCTGTTTGAGAACTTCACAAATCAGTACCCAGTGTCCAAGACCCTGCGCTTTGAGCTGATCCCCCAGGGCAAGACAAAGGACTTCATCGAGCAGAAGGGCCTGCTGAAGAAGGATGAGGACCGGGCCGAGAAGTATAAGAAGGTGAAGAACATCATCGATGAGTACCACAAGGACTTCATCGAGAAGTCTCTGAATGGCCTGAAGCTGGACGGCCTGGAGGAATACAAGACCCTGTATCTGAAGCAGGAGAAGGACGATAAGGATAAGAAGGCCTTTGACAAGGAGAAGGAGAACCTGCGCAAGCAGATCGCCAATGCCTTCCGGAACAATGAGAAGTTTAAGACACTGTTCGCCAAGGAGCTGATCAAGAACGATCTGATGTCTTTCGCCTGCGAGGAGGACAAGAAGAATGTGAAGGAGTTTGAGGCCTTCACCACATACTTCACCGGCTTCCACCAGAACCGCGCCAATATGTACGTGGCCGATGAGAAGAGAACAGCCATCGCCAGCAGGCTGATCCACGAGAACCTGCCAAAGTTTATCGACAATATCAAGATCTTCGAGAAGATGAAGAAGGAGGCCCCCGAGCTGCTGTCTCCTTTCAACCAGACCCTGAAGGATATGAAGGACGTGATCAAGGGCACCACACTGGAGGAGATCTTTAGCCTGGATTATTTCAACAAGACCCTGACACAGAGCGGCATCGACATCTACAATTCCGTGATCGGCGGCAGAACCCCTGAGGAGGGCAAGACAAAGATCAAGGGCCTGAACGAGTACATCAATACCGACTTCAACCAGAAGCAGACAGACAAGAAGAAGCGGCAGCCAAAGTTCAAGCAGCTGTATAAGCAGATCCTGAGCGATAGGCAGAGCCTGTCCTTTATCGCCGAGGCCTTCAAGAACGACACCGAGATCCTGGAGGCCATCGAGAAGTTTTACGTGAATGAGCTGCTGCACTTCAGCAATGAGGGCAAGTCCACAAACGTGCTGGACGCCATCAAGAATGCCGTGTCTAACCTGGAGAGCTTTAACCTGACCAAGATCTATTTCCGCTCCGGCACCTCTCTGACAGACGTGAGCCGGAAGGTGTTTGGCGAGTGGAGCATCATCAATAGAGCCCTGGACAACTACTATGCCACCACATATCCAATCAAGCCCAGAGAGAAGTCTGAGAAGTACGAGGAGAGGAAGGAGAAGTGGCTGAAGCAGGACTTCAACGTGAGCCTGATCCAGACCGCCATCGATGAGTACGACAACGAGACAGTGAAGGGCAAGAACAGCGGCAAAGTGATCGTCGATTATTTTGCCAAGTTCTGCGACGATAAGGAGACAGACCTGATCCAGAAGGTGAACGAGGGCTACATCGCCGTGAAGGATCTGCTGAATACACCCTGTCCTGAGAACGAGAAGCTGGGCAGCAATAAGGACCAGGTGAAGCAGATCAAGGCCTTTATGGATTCTATCATGGACATCATGCACTTCGTGCGCCCCCTGAGCCTGAAGGATACCGACAAGGAGAAGGATGAGACATTCTACTCCCTGTTCACACCTCTGTACGACCACCTGACCCAGACAATCGCCCTGTATAACAAGGTGCGGAACTATCTGACCCAGAAGCCTTACAGCACAGAGAAGATCAAGCTGAACTTCGAGAACAGCACCCTGCTGGGCGGCTGGGATCTGAATAAGGAGACAGACAACACAGCCATCATCCTGAGGAAGGAAAACCTGTACTATCTGGGCATCATGGACAAGAGGCACAATCGCATCTTTCGGAACGTGCCCAAGGCCGATAAGAAGGACTCTTGCTACGAGAAGATGGTGTATAAGCTGCTGCCTGGCGCCAACAAGATGCTGCCAAAGGTGTTCTTTTCTCAGAGCAGAATCCAGGAGTTTACCCCTTCCGCCAAGCTGCTGGAGAACTACGAAAATGAGACACACAAGAAGGGCGATAATTTCAACCTGAATCACTGTCACCAGCTGATCGATTTCTTTAAGGACTCTATCAACAAGCACGAGGATTGGAAGAATTTCGACTTTAGGTTCAGCGCCACCTCCACCTACGCCGACCTGAGCGGCTTTTACCACGAGGTGGAGCACCAGGGCTACAAGATCTCTTTTCAGAGCATCGCCGATTCCTTCATCGACGATCTGGTGAACGAGGGCAAGCTGTACCTGTTCCAGATCTATAATAAGGACTTTTCCCCATTCTCTAAGGGCAAGCCCAACCTGCACACCCTGTACTGGAAGATGCTGTTTGATGAGAACAATCTGAAGGACGTGGTGTATAAGCTGAATGGCGAGGCCGAGGTGTTCTACCGCAAGAAGAGCATTGCCGAGAAGAACACCACAATCCACAAGGCCAATGAGTCCATCATCAACAAGAATCCTGATAACCCAAAGGCCACCAGCACCTTCAACTATGATATCGTGAAGGACAAGAGATACACCATCGACAAGTTTCAGTTCCACATCCCAATCACAATGAACTTTAAGGCCGAGGGCATCTTCAACATGAATCAGAGGGTGAATCAGTTCCTGAAGGCCAATCCCGATATCAACATCATCGGCATCGACAGAGGCGAGAGGCACCTGCTGTACTATGCCCTGATCAACCAGAAGGGCAAGATCCTGAAGCAGGATACCCTGAATGTGATCGCCAACGAGAAGCAGAAGGTGGACTACCACAATCTGCTGGATAAGAAGGAGGGCGACCGCGCAACCGCAAGGCAGGAGTGGGGCGTGATCGAGACAATCAAGGAGCTGAAGGAGGGCTATCTGTCCCAGGTCATCCACAAGCTGACCGATCTGATGATCGAGAACAATGCCATCATCGTGATGGAGGACCTGAACTTTGGCTTCAAGCGGGGCAGACAGAAGGTGGAGAAGCAGGTGTATCAGAAGTTTGAGAAGATGCTGATCGATAAGCTGAATTACCTGGTGGACAAGAATAAGAAGGCAAACGAGCTGGGAGGCCTGCTGAACGCATTCCAGCTGGCCAATAAGTTTGAGTCCTTCCAGAAGATGGGCAAGCAGAACGGCTTTATCTTCTACGTGCCCGCCTGGAATACCTCTAAGACAGATCCTGCCACCGGCTTTATCGACTTCCTGAAGCCCCGCTATGAGAACCTGAATCAGGCCAAGGATTTCTTTGAGAAGTTTGACTCTATCCGGCTGAACAGCAAGGCCGATTACTTTGAGTTCGCCTTTGACTTCAAGAATTTCACCGAGAAGGCCGATGGCGGCAGAACCAAGTGGACAGTGTGCACCACAAACGAGGACAGATATGCCTGGAATAGGGCCCTGAACAATAACAGGGGCAGCCAGGAGAAGTACGACATCACAGCCGAGCTGAAGTCCCTGTTCGATGGCAAGGTGGACTATAAGTCTGGCAAGGATCTGAAGCAGCAGATCGCCAGCCAGGAGTCCGCCGACTTCTTTAAGGCCCTGATGAAGAACCTGTCCATCACCCTGTCTCTGAGACACAATAACGGCGAGAAGGGCGATAATGAGCAGGACTACATCCTGTCCCCTGTGGCCGATTCTAAGGGCCGCTTCTTTGACTCCCGGAAGGCCGACGATGACATGCCAAAGAATGCCGACGCCAACGGCGCCTATCACATCGCCCTGAAGGGCCTGTGGTGTCTGGAGCAGATCAGCAAGACCGATGACCTGAAGAAGGTGAAGCTGGCCATCTCCAACAAGGAGTGGCTGGAGTTCGTGCAGACACTGAAGGGCAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGGGATCCTACCCATACGATGTTCCAGATTACGCTTATCCCTACGACGTGCCTGATTATGCATACCCATATGATGTCCCCGACTATGCC
SEQ ID NO.99：(SsCas12a的序列)
ATGCAGACCCTGTTTGAGAACTTCACAAATCAGTACCCAGTGTCCAAGACCCTGCGCTTTGAGCTGATCCCCCAGGGCAAGACAAAGGACTTCATCGAGCAGAAGGGCCTGCTGAAGAAGGATGAGGACCGGGCCGAGAAGTATAAGAAGGTGAAGAACATCATCGATGAGTACCACAAGGACTTCATCGAGAAGTCTCTGAATGGCCTGAAGCTGGACGGCCTGGAGAAGTACAAGACCCTGTATCTGAAGCAGGAGAAGGACGATAAGGATAAGAAGGCCTTTGACAAGGAGAAGGAGAACCTGCGCAAGCAGATCGCCAATGCCTTCCGGAACAATGAGAAGTTTAAGACACTGTTCGCCAAGGAGCTGATCAAGAACGATCTGATGTCTTTCGCCTGCGAGGAGGACAAGAAGAATGTGAAGGAGTTTGAGGCCTTCACCACATACTTCACCGGCTTCCACCAGAACCGCGCCAATATGTACGTGGCCGATGAGAAGAGAACAGCCATCGCCAGCAGGCTGATCCACGAGAACCTGCCAAAGTTTATCGACAATATCAAGATCTTCGAGAAGATGAAGAAGGAGGCCCCCGAGCTGCTGTCTCCTTTCAACCAGACCCTGAAGGATATGAAGGACGTGATCAAGGGCACCACACTGGAGGAGATCTTTAGCCTGGATTATTTCAACAAGACCCTGACACAGAGCGGCATCGACATCTACAATTCCGTGATCGGCGGCAGAACCCCTGAGGAGGGCAAGACAAAGATCAAGGGCCTGAACGAGTACATCAATACCGACTTCAACCAGAAGCAGACAGACAAGAAGAAGCGGCAGCCAAAGTTCAAGCAGCTGTATAAGCAGATCCTGAGCGATAGGCAGAGCCTGTCCTTTATCGCCGAGGCCTTCAAGAACGACACCGAGATCCTGGAGGCCATCGAGAAGTTTTACGTGAATGAGCTGCTGCACTTCAGCAATGAGGGCAAGTCCACAAACGTGCTGGACGCCATCAAGAATGCCGTGTCTAACCTGGAGAGCTTTAACCTGACCAAGATGTATTTCCGCTCCGGCGCCTCTCTGACAGACGTGAGCCGGAAGGTGTTTGGCGAGTGGAGCATCATCAATAGAGCCCTGGACAACTACTATGCCACCACATATCCAATCAAGCCCAGAGAGAAGTCTGAGAAGTACGAGGAGAGGAAGGAGAAGTGGCTGAAGCAGGACTTCAACGTGAGCCTGATCCAGACCGCCATCGATGAGTACGACAACGAGACAGTGAAGGGCAAGAACAGCGGCAAAGTGATCGCCGATTATTTTGCCAAGTTCTGCGACGATAAGGAGACAGACCTGATCCAGAAGGTGAACGAGGGCTACATCGCCGTGAAGGATCTGCTGAATACACCCTGTCCTGAGAACGAGAAGCTGGGCAGCAATAAGGACCAGGTGAAGCAGATCAAGGCCTTTATGGATTCTATCATGGACATCATGCACTTCGTGCGCCCCCTGAGCCTGAAGGATACCGACAAGGAGAAGGATGAGACATTCTACTCCCTGTTCACACCTCTGTACGACCACCTGACCCAGACAATCGCCCTGTATAACAAGGTGCGGAACTATCTGACCCAGAAGCCTTACAGCACAGAGAAGATCAAGCTGAACTTCGAGAACAGCACCCTGCTGGGCGGCTGGGATCTGAATAAGGAGACAGACAACACAGCCATCATCCTGAGGAAGGATAACCTGTACTATCTGGGCATCATGGACAAGAGGCACAATCGCATCTTTCGGAACGTGCCCAAGGCCGATAAGAAGGACTTCTGCTACGAGAAGATGGTGTATAAGCTGCTGCCTGGCGCCAACAAGATGCTGCCAAAGGTGTTCTTTTCTCAGAGCAGAATCCAGGAGTTTACCCCTTCCGCCAAGCTGCTGGAGAACTACGCCAATGAGACACACAAGAAGGGCGATAATTTCAACCTGAATCACTGTCACAAGCTGATCGATTTCTTTAAGGACTCTATCAACAAGCACGAGGATTGGAAGAATTTCGACTTTAGGTTCAGCGCCACCTCCACCTACGCCGACCTGAGCGGCTTTTACCACGAGGTGGAGCACCAGGGCTACAAGATCTCTTTTCAGAGCGTGGCCGATTCCTTCATCGACGATCTGGTGAACGAGGGCAAGCTGTACCTGTTCCAGATCTATAATAAGGACTTTTCCCCATTCTCTAAGGGCAAGCCCAACCTGCACACCCTGTACTGGAAGATGCTGTTTGATGAGAACAATCTGAAGGACGTGGTGTATAAGCTGAATGGCGAGGCCGAGGTGTTCTACCGCAAGAAGAGCATTGCCGAGAAGAACACCACAATCCACAAGGCCAATGAGTCCATCATCAACAAGAATCCTGATAACCCAAAGGCCACCAGCACCTTCAACTATGATATCGTGAAGGACAAGAGATACACCATCGACAAGTTTCAGTTCCACATCCCAATCACAATGAACTTTAAGGCCGAGGGCATCTTCAACATGAATCAGAGGGTGAATCAGTTCCTGAAGGCCAATCCCGATATCAACATCATCGGCATCGACAGAGGCGAGAGGCACCTGCTGTACTATGCCCTGATCAACCAGAAGGGCAAGATCCTGAAGCAGGATACCCTGAATGTGATCGCCAACGAGAAGCAGAAGGTGGACTACCACAATCTGCTGGATAAGAAGGAGGGCGACCGCGCAACCGCAAGGCAGGAGTGGGGCGTGATCGAGACAATCAAGGAGCTGAAGGAGGGCTATCTGTCCCAGGTCATCCACAAGCTGACCGATCTGATGATCGAGAACAATGCCATCATCGTGATGGAGGACCTGAACTTTGGCTTCAAGCGGGGCAGACAGAAGGTGGAGAAGCAGGTGTATCAGAAGTTTGAGAAGATGCTGATCGATAAGCTGAATTACCTGGTGGACAAGAATAAGAAGGCAAACGAGCTGGGAGGCCTGCTGAACGCATTCCAGCTGGCCAATAAGTTTGAGTCCTTCCAGAAGATGGGCAAGCAGAACGGCTTTATCTTCTACGTGCCCGCCTGGAATACCTCTAAGACAGATCCTGCCACCGGCTTTATCGACTTCCTGAAGCCCCGCTATGAGAACCTGAATCAGGCCAAGGATTTCTTTGAGAAGTTTGACTCTATCCGGCTGAACAGCAAGGCCGATTACTTTGAGTTCGCCTTTGACTTCAAGAATTTCACCGAGAAGGCCGATGGCGGCAGAACCAAGTGGACAGTGTGCACCACAAACGAGGACAGATATGCCTGGAATAGGGCCCTGAACAATAACAGGGGCAGCCAGGAGAAGTACGACATCACAGCCGAGCTGAAGTCCCTGTTCGATGGCAAGGTGGACTATAAGTCTGGCAAGGATCTGAAGCAGCAGATCGCCAGCCAGGAGTCCGCCGACTTCTTTAAGGCCCTGATGAAGAACCTGTCCATCACCCTGTCTCTGAGACACAATAACGGCGAGAAGGGCGATAATGAGCAGGACTACATCCTGTCCCCTGTGGCCGATTCTAAGGGCCGCTTCTTTGACTCCCGGAAGGCCGACGATGACATGCCAAAGAATGCCGACGCCAACGGCGCCTATCACATCGCCCTGAAGGGCCTGTGGTGTCTGGAGCAGATCAGCAAGACCGATGACCTGAAGAAGGTGAAGCTGGCCATCTCCAACAAGGAGTGGCTGGAGTTCGTGCAGACACTGAAGGGCAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGGGATCCTACCCATACGATGTTCCAGATTACGCTTATCCCTACGACGTGCCTGATTATGCATACCCATATGATGTCCCCGACTATGCC
SEQUENCE LISTING
<110> 广州普世利华科技有限公司
<120> 一种血友病基因突变位点的检测方法
<130>
<160> 99
<170> PatentIn version 3.3
<210> 1
<211> 61
<212> DNA
<213> 靶基因序列
<400> 1
tacatgctgg taagcagaga ttttactccc tgagtagtta ctcctgtgac tttcattgtc 60
t 61
<210> 2
<211> 61
<212> DNA
<213> 靶基因序列
<400> 2
tggcacttac tatttaaatc acagcccatc aactccatgc gaagagtgct gcgaatgcta 60
t 61
<210> 3
<211> 61
<212> DNA
<213> 靶基因序列
<400> 3
atgcgaagag tgctgcgaat gctataatga gttgggtgca aacggatgta tcgagcaata 60
a 61
<210> 4
<211> 61
<212> DNA
<213> 靶基因序列
<400> 4
cataccatta aggttccagt ggaatttcct cgataagtct gccacttctt cccatcaaga 60
c 61
<210> 5
<211> 61
<212> DNA
<213> 靶基因序列
<400> 5
taagtctgcc acttcttccc atcaagacta tacatgatga taaactgaga gatgtagagg 60
c 61
<210> 6
<211> 61
<212> DNA
<213> 靶基因序列
<400> 6
atgccgtgaa taatcattgg tgccaacaga tccacctacc aattaaaata acactttatt 60
t 61
<210> 7
<211> 61
<212> DNA
<213> 靶基因序列
<400> 7
cattgattac attttctaac cttgatccaa gaaaagggct ccttggtgct ccaggcattg 60
a 61
<210> 8
<211> 61
<212> DNA
<213> 靶基因序列
<400> 8
aataatgaag tctggccagc tttggggccc actgtccttt gcccaagtaa agaaacagag 60
a 61
<210> 9
<211> 61
<212> DNA
<213> 靶基因序列
<400> 9
gtagatgctc gccaataagg cattccaccc gccaaattcc agctttggat ggtaacattt 60
c 61
<210> 10
<211> 61
<212> DNA
<213> 靶基因序列
<400> 10
gccaaattcc agctttggat ggtaacattt ccactgtctc aaaaacacct tataaaaacc 60
a 61
<210> 11
<211> 61
<212> DNA
<213> 靶基因序列
<400> 11
acagtgccat tttatactcc tctttttttc gtacagtgaa cacatgtcca ctgaaatgaa 60
t 61
<210> 12
<211> 61
<212> DNA
<213> 靶基因序列
<400> 12
actcctcttt ttttcgtaca gtgaacacat gtccactgaa atgaatagaa tggatgtttt 60
c 61
<210> 13
<211> 61
<212> DNA
<213> 靶基因序列
<400> 13
aaatgaatag aatggatgtt ttcattgctg cccatgctga gcagatacca tcgaatcctt 60
t 61
<210> 14
<211> 61
<212> DNA
<213> 靶基因序列
<400> 14
tattttcagt gaagtaccag cttttggtct catcaaagat ggtgaaaaac agagcaaatt 60
c 61
<210> 15
<211> 61
<212> DNA
<213> 靶基因序列
<400> 15
accaggtcaa catcagagaa ataagcccag gctttgcagt caaactcatc tttagtgggt 60
g 61
<210> 16
<211> 61
<212> DNA
<213> 靶基因序列
<400> 16
cccaggcttt gcagtcaaac tcatctttag tgggtgccat atgatgttgc actttccaaa 60
a 61
<210> 17
<211> 61
<212> DNA
<213> 靶基因序列
<400> 17
aggctttgca gtcaaactca tctttagtgg gtgccatatg atgttgcact ttccaaaagt 60
a 61
<210> 18
<211> 61
<212> DNA
<213> 靶基因序列
<400> 18
tttgcagtca aactcatctt tagtgggtgc catatgatgt tgcactttcc aaaagtaagt 60
t 61
<210> 19
<211> 61
<212> DNA
<213> 靶基因序列
<400> 19
agttttttct aggttctgct ccttgcctct gatcttcctc ataagaaata aggctagaat 60
a 61
<210> 20
<211> 61
<212> DNA
<213> 靶基因序列
<400> 20
ttatcttcaa cttctgctct tatatatggc cccaggagtc ccaaatgttc atttagttct 60
c 61
<210> 21
<211> 61
<212> DNA
<213> 靶基因序列
<400> 21
ccaggagtcc caaatgttca tttagttctc cacggtataa gggctgagta aaggagccat 60
c 61
<210> 22
<211> 61
<212> DNA
<213> 靶基因序列
<400> 22
agctactcat cccataatcc cagagcctct ccactgcagc aataaaatag tgtcgtgttt 60
t 61
<210> 23
<211> 61
<212> DNA
<213> 靶基因序列
<400> 23
aaatagtgtc gtgttttctt ttgaaagctg cgggggctct gattttcatc ctcatcataa 60
a 61
<210> 24
<211> 61
<212> DNA
<213> 靶基因序列
<400> 24
gtgttttctt ttgaaagctg cgggggctct gattttcatc ctcatcataa atgtcaaaat 60
c 61
<210> 25
<211> 61
<212> DNA
<213> 靶基因序列
<400> 25
ctgcgggggc tctgattttc atcctcatca taaatgtcaa aatcttcctt cttcatttca 60
a 61
<210> 26
<211> 61
<212> DNA
<213> 靶基因序列
<400> 26
tcttgtcaca actagaaacc ttcagtaagg cggtcatgcc tctgttccga aagtctgagt 60
t 61
<210> 27
<211> 61
<212> DNA
<213> 靶基因序列
<400> 27
gtgtgtcttc atagaccatt ttgtgtttga aggtatatcc agagaagaag acagaaagga 60
a 61
<210> 28
<211> 61
<212> DNA
<213> 靶基因序列
<400> 28
agtctgtgct ccaatgctta gaatgtacca gtatgccacc tcatgcaaac aaactgacaa 60
c 61
<210> 29
<211> 61
<212> DNA
<213> 靶基因序列
<400> 29
gctccaatgc ttagaatgta ccagtatgcc acctcatgca aacaaactga caactgcaaa 60
c 61
<210> 30
<211> 61
<212> DNA
<213> 靶基因序列
<400> 30
taagtgctgc tttactcact gtgcatgatg ttggaggctt ggaactctgg atcctcaagc 60
t 61
<210> 31
<211> 61
<212> DNA
<213> 靶基因序列
<400> 31
gttgtatatt ctctgtgagg taccagcttc ggttctcatc aaatacagaa aacaggatga 60
c 61
<210> 32
<211> 61
<212> DNA
<213> 靶基因序列
<400> 32
aacaggatga cattcctctt gtctgacatt atctgttaat tacatatatt gaaaggggta 60
a 61
<210> 33
<211> 61
<212> DNA
<213> 靶基因序列
<400> 33
aactcacctg gtttcctctt tgatctacag attctttgta gcagatgagg agagggccaa 60
t 61
<210> 34
<211> 61
<212> DNA
<213> 靶基因序列
<400> 34
aggagagggc caatgagtcc tgaagctaga tctctctcca tattaacgaa actagagtaa 60
t 61
<210> 35
<211> 61
<212> DNA
<213> 靶基因序列
<400> 35
cttaccaaca gtgtgtctcc aacttcccca taaagtaaag gtcccaagat tcctgattca 60
t 61
<210> 36
<211> 61
<212> DNA
<213> 靶基因序列
<400> 36
cttcacgagt cttaaaggtt tcatctgtgt atgccataaa tcggactttt ttgtacttcc 60
t 61
<210> 37
<211> 61
<212> DNA
<213> 靶基因序列
<400> 37
cttcctacca atccgctgag ggccattgtt caaatattga cttttataac ttctgtataa 60
g 61
<210> 38
<211> 61
<212> DNA
<213> 靶基因序列
<400> 38
cccaagtttt aggatgcttc ttggcaactg agcgaatttg gataaaggaa ggagagttgt 60
c 61
<210> 39
<211> 61
<212> DNA
<213> 靶基因序列
<400> 39
aagttttagg atgcttcttg gcaactgagc gaatttggat aaaggaagga gagttgtcat 60
c 61
<210> 40
<211> 61
<212> DNA
<213> 靶基因序列
<400> 40
agtcttccgc ttcttcatta tttttcattc gtagttgggg ttcctctgga cagctgtcta 60
c 61
<210> 41
<211> 61
<212> DNA
<213> 靶基因序列
<400> 41
gggttcctct ggacagctgt ctactttgac ataagcttcc atgccatctg gagtcagaca 60
a 61
<210> 42
<211> 61
<212> DNA
<213> 靶基因序列
<400> 42
atattaccat gttggtggga agagatatga caaaacagta gaaactgtcc aaggtccatc 60
a 61
<210> 43
<211> 61
<212> DNA
<213> 靶基因序列
<400> 43
tattaccatg ttggtgggaa gagatatgac aaaacagtag aaactgtcca aggtccatca 60
a 61
<210> 44
<211> 21
<212> DNA
<213> gRNA的序列
<400> 44
ctccctgagt agttactcct g 21
<210> 45
<211> 21
<212> DNA
<213> gRNA的序列
<400> 45
aatcacagcc catcaactcc a 21
<210> 46
<211> 21
<212> DNA
<213> gRNA的序列
<400> 46
cacccaactc attatagcat t 21
<210> 47
<211> 21
<212> DNA
<213> gRNA的序列
<400> 47
ctcgataagt ctgccacttc t 21
<210> 48
<211> 21
<212> DNA
<213> gRNA的序列
<400> 48
tcatcatgta tagtcttgat g 21
<210> 49
<211> 21
<212> DNA
<213> gRNA的序列
<400> 49
attggtaggt ggatctgttg g 21
<210> 50
<211> 21
<212> DNA
<213> gRNA的序列
<400> 50
ttggatcaag gttagaaaat g 21
<210> 51
<211> 21
<212> DNA
<213> gRNA的序列
<400> 51
taaccttgat ccaagaaaag g 21
<210> 52
<211> 21
<212> DNA
<213> gRNA的序列
<400> 52
gggcccactg tcctttgccc a 21
<210> 53
<211> 21
<212> DNA
<213> gRNA的序列
<400> 53
cttgggcaaa ggacagtggg c 21
<210> 54
<211> 21
<212> DNA
<213> gRNA的序列
<400> 54
gcgggtggaa tgccttattg g 21
<210> 55
<211> 21
<212> DNA
<213> gRNA的序列
<400> 55
cactgtctca aaaacacctt a 21
<210> 56
<211> 21
<212> DNA
<213> gRNA的序列
<400> 56
agacagtgga aatgttacca t 21
<210> 57
<211> 21
<212> DNA
<213> gRNA的序列
<400> 57
gatggtaaca tttccactgt c 21
<210> 58
<211> 21
<212> DNA
<213> gRNA的序列
<400> 58
gtacagtgaa cacatgtcca c 21
<210> 59
<211> 21
<212> DNA
<213> gRNA的序列
<400> 59
agtggacatg tgttcactgt a 21
<210> 60
<211> 21
<212> DNA
<213> gRNA的序列
<400> 60
attgctgccc atgctgagca g 21
<210> 61
<211> 21
<212> DNA
<213> gRNA的序列
<400> 61
gtctcatcaa agatggtgaa a 21
<210> 62
<211> 21
<212> DNA
<213> gRNA的序列
<400> 62
atgagaccaa aagctggtac t 21
<210> 63
<211> 21
<212> DNA
<213> gRNA的序列
<400> 63
accatctttg atgagaccaa a 21
<210> 64
<211> 21
<212> DNA
<213> gRNA的序列
<400> 64
actgcaaagc ctgggcttat t 21
<210> 65
<211> 21
<212> DNA
<213> gRNA的序列
<400> 65
cagtcaaact catctttagt g 21
<210> 66
<211> 21
<212> DNA
<213> gRNA的序列
<400> 66
gtgggtgcca tatgatgttg c 21
<210> 67
<211> 21
<212> DNA
<213> gRNA的序列
<400> 67
gaaagtgcaa catcatatgg c 21
<210> 68
<211> 21
<212> DNA
<213> gRNA的序列
<400> 68
ttatgaggaa gatcagaggc a 21
<210> 69
<211> 21
<212> DNA
<213> gRNA的序列
<400> 69
ggactcctgg ggccatatat a 21
<210> 70
<211> 21
<212> DNA
<213> gRNA的序列
<400> 70
gttctccacg gtataagggc t 21
<210> 71
<211> 21
<212> DNA
<213> gRNA的序列
<400> 71
ctcagccctt ataccgtgga g 21
<210> 72
<211> 21
<212> DNA
<213> gRNA的序列
<400> 72
ttgctgcagt ggagaggctc t 21
<210> 73
<211> 21
<212> DNA
<213> gRNA的序列
<400> 73
aaagctgcgg gggctctgat t 21
<210> 74
<211> 21
<212> DNA
<213> gRNA的序列
<400> 74
tgatgaggat gaaaatcaga g 21
<210> 75
<211> 21
<212> DNA
<213> gRNA的序列
<400> 75
acatttatga tgaggatgaa a 21
<210> 76
<211> 21
<212> DNA
<213> gRNA的序列
<400> 76
atcctcatca taaatgtcaa a 21
<210> 77
<211> 21
<212> DNA
<213> gRNA的序列
<400> 77
ggaacagagg catgaccgcc t 21
<210> 78
<211> 21
<212> DNA
<213> gRNA的序列
<400> 78
tgtttgaagg tatatccaga g 21
<210> 79
<211> 21
<212> DNA
<213> gRNA的序列
<400> 79
aaggtatatc cagagaagaa g 21
<210> 80
<211> 21
<212> DNA
<213> gRNA的序列
<400> 80
catgaggtgg catactggta c 21
<210> 81
<211> 21
<212> DNA
<213> gRNA的序列
<400> 81
tttgcatgag gtggcatact g 21
<210> 82
<211> 21
<212> DNA
<213> gRNA的序列
<400> 82
ctcactgtgc atgatgttgg a 21
<210> 83
<211> 21
<212> DNA
<213> gRNA的序列
<400> 83
atgagaaccg aagctggtac c 21
<210> 84
<211> 21
<212> DNA
<213> gRNA的序列
<400> 84
tgtatttgat gagaaccgaa g 21
<210> 85
<211> 21
<212> DNA
<213> gRNA的序列
<400> 85
aatatatgta attaacagat a 21
<210> 86
<211> 21
<212> DNA
<213> gRNA的序列
<400> 86
atctacagat tctttgtagc a 21
<210> 87
<211> 21
<212> DNA
<213> gRNA的序列
<400> 87
gttaatatgg agagagatct a 21
<210> 88
<211> 21
<212> DNA
<213> gRNA的序列
<400> 88
ctttatgggg aagttggaga c 21
<210> 89
<211> 21
<212> DNA
<213> gRNA的序列
<400> 89
atctgtgtat gccataaatc g 21
<210> 90
<211> 21
<212> DNA
<213> gRNA的序列
<400> 90
tggcatacac agatgaaacc t 21
<210> 91
<211> 21
<212> DNA
<213> gRNA的序列
<400> 91
aacaatggcc ctcagcggat t 21
<210> 92
<211> 21
<212> DNA
<213> gRNA的序列
<400> 92
ggatgcttct tggcaactga g 21
<210> 93
<211> 21
<212> DNA
<213> gRNA的序列
<400> 93
tccaaattcg ctcagttgcc a 21
<210> 94
<211> 21
<212> DNA
<213> gRNA的序列
<400> 94
attcgtagtt ggggttcctc t 21
<210> 95
<211> 21
<212> DNA
<213> gRNA的序列
<400> 95
acataagctt ccatgccatc t 21
<210> 96
<211> 21
<212> DNA
<213> gRNA的序列
<400> 96
tcatatctct tcccaccaac a 21
<210> 97
<211> 21
<212> DNA
<213> gRNA的序列
<400> 97
tactgttttg tcatatctct t 21
<210> 98
<211> 3885
<212> DNA
<213> ScCas12a的序列
<400> 98
atgcagaccc tgtttgagaa cttcacaaat cagtacccag tgtccaagac cctgcgcttt 60
gagctgatcc cccagggcaa gacaaaggac ttcatcgagc agaagggcct gctgaagaag 120
gatgaggacc gggccgagaa gtataagaag gtgaagaaca tcatcgatga gtaccacaag 180
gacttcatcg agaagtctct gaatggcctg aagctggacg gcctggagga atacaagacc 240
ctgtatctga agcaggagaa ggacgataag gataagaagg cctttgacaa ggagaaggag 300
aacctgcgca agcagatcgc caatgccttc cggaacaatg agaagtttaa gacactgttc 360
gccaaggagc tgatcaagaa cgatctgatg tctttcgcct gcgaggagga caagaagaat 420
gtgaaggagt ttgaggcctt caccacatac ttcaccggct tccaccagaa ccgcgccaat 480
atgtacgtgg ccgatgagaa gagaacagcc atcgccagca ggctgatcca cgagaacctg 540
ccaaagttta tcgacaatat caagatcttc gagaagatga agaaggaggc ccccgagctg 600
ctgtctcctt tcaaccagac cctgaaggat atgaaggacg tgatcaaggg caccacactg 660
gaggagatct ttagcctgga ttatttcaac aagaccctga cacagagcgg catcgacatc 720
tacaattccg tgatcggcgg cagaacccct gaggagggca agacaaagat caagggcctg 780
aacgagtaca tcaataccga cttcaaccag aagcagacag acaagaagaa gcggcagcca 840
aagttcaagc agctgtataa gcagatcctg agcgataggc agagcctgtc ctttatcgcc 900
gaggccttca agaacgacac cgagatcctg gaggccatcg agaagtttta cgtgaatgag 960
ctgctgcact tcagcaatga gggcaagtcc acaaacgtgc tggacgccat caagaatgcc 1020
gtgtctaacc tggagagctt taacctgacc aagatctatt tccgctccgg cacctctctg 1080
acagacgtga gccggaaggt gtttggcgag tggagcatca tcaatagagc cctggacaac 1140
tactatgcca ccacatatcc aatcaagccc agagagaagt ctgagaagta cgaggagagg 1200
aaggagaagt ggctgaagca ggacttcaac gtgagcctga tccagaccgc catcgatgag 1260
tacgacaacg agacagtgaa gggcaagaac agcggcaaag tgatcgtcga ttattttgcc 1320
aagttctgcg acgataagga gacagacctg atccagaagg tgaacgaggg ctacatcgcc 1380
gtgaaggatc tgctgaatac accctgtcct gagaacgaga agctgggcag caataaggac 1440
caggtgaagc agatcaaggc ctttatggat tctatcatgg acatcatgca cttcgtgcgc 1500
cccctgagcc tgaaggatac cgacaaggag aaggatgaga cattctactc cctgttcaca 1560
cctctgtacg accacctgac ccagacaatc gccctgtata acaaggtgcg gaactatctg 1620
acccagaagc cttacagcac agagaagatc aagctgaact tcgagaacag caccctgctg 1680
ggcggctggg atctgaataa ggagacagac aacacagcca tcatcctgag gaaggaaaac 1740
ctgtactatc tgggcatcat ggacaagagg cacaatcgca tctttcggaa cgtgcccaag 1800
gccgataaga aggactcttg ctacgagaag atggtgtata agctgctgcc tggcgccaac 1860
aagatgctgc caaaggtgtt cttttctcag agcagaatcc aggagtttac cccttccgcc 1920
aagctgctgg agaactacga aaatgagaca cacaagaagg gcgataattt caacctgaat 1980
cactgtcacc agctgatcga tttctttaag gactctatca acaagcacga ggattggaag 2040
aatttcgact ttaggttcag cgccacctcc acctacgccg acctgagcgg cttttaccac 2100
gaggtggagc accagggcta caagatctct tttcagagca tcgccgattc cttcatcgac 2160
gatctggtga acgagggcaa gctgtacctg ttccagatct ataataagga cttttcccca 2220
ttctctaagg gcaagcccaa cctgcacacc ctgtactgga agatgctgtt tgatgagaac 2280
aatctgaagg acgtggtgta taagctgaat ggcgaggccg aggtgttcta ccgcaagaag 2340
agcattgccg agaagaacac cacaatccac aaggccaatg agtccatcat caacaagaat 2400
cctgataacc caaaggccac cagcaccttc aactatgata tcgtgaagga caagagatac 2460
accatcgaca agtttcagtt ccacatccca atcacaatga actttaaggc cgagggcatc 2520
ttcaacatga atcagagggt gaatcagttc ctgaaggcca atcccgatat caacatcatc 2580
ggcatcgaca gaggcgagag gcacctgctg tactatgccc tgatcaacca gaagggcaag 2640
atcctgaagc aggataccct gaatgtgatc gccaacgaga agcagaaggt ggactaccac 2700
aatctgctgg ataagaagga gggcgaccgc gcaaccgcaa ggcaggagtg gggcgtgatc 2760
gagacaatca aggagctgaa ggagggctat ctgtcccagg tcatccacaa gctgaccgat 2820
ctgatgatcg agaacaatgc catcatcgtg atggaggacc tgaactttgg cttcaagcgg 2880
ggcagacaga aggtggagaa gcaggtgtat cagaagtttg agaagatgct gatcgataag 2940
ctgaattacc tggtggacaa gaataagaag gcaaacgagc tgggaggcct gctgaacgca 3000
ttccagctgg ccaataagtt tgagtccttc cagaagatgg gcaagcagaa cggctttatc 3060
ttctacgtgc ccgcctggaa tacctctaag acagatcctg ccaccggctt tatcgacttc 3120
ctgaagcccc gctatgagaa cctgaatcag gccaaggatt tctttgagaa gtttgactct 3180
atccggctga acagcaaggc cgattacttt gagttcgcct ttgacttcaa gaatttcacc 3240
gagaaggccg atggcggcag aaccaagtgg acagtgtgca ccacaaacga ggacagatat 3300
gcctggaata gggccctgaa caataacagg ggcagccagg agaagtacga catcacagcc 3360
gagctgaagt ccctgttcga tggcaaggtg gactataagt ctggcaagga tctgaagcag 3420
cagatcgcca gccaggagtc cgccgacttc tttaaggccc tgatgaagaa cctgtccatc 3480
accctgtctc tgagacacaa taacggcgag aagggcgata atgagcagga ctacatcctg 3540
tcccctgtgg ccgattctaa gggccgcttc tttgactccc ggaaggccga cgatgacatg 3600
ccaaagaatg ccgacgccaa cggcgcctat cacatcgccc tgaagggcct gtggtgtctg 3660
gagcagatca gcaagaccga tgacctgaag aaggtgaagc tggccatctc caacaaggag 3720
tggctggagt tcgtgcagac actgaagggc aaaaggccgg cggccacgaa aaaggccggc 3780
caggcaaaaa agaaaaaggg atcctaccca tacgatgttc cagattacgc ttatccctac 3840
gacgtgcctg attatgcata cccatatgat gtccccgact atgcc 3885
<210> 99
<211> 3885
<212> DNA
<213> SsCas12a的序列
<400> 99
atgcagaccc tgtttgagaa cttcacaaat cagtacccag tgtccaagac cctgcgcttt 60
gagctgatcc cccagggcaa gacaaaggac ttcatcgagc agaagggcct gctgaagaag 120
gatgaggacc gggccgagaa gtataagaag gtgaagaaca tcatcgatga gtaccacaag 180
gacttcatcg agaagtctct gaatggcctg aagctggacg gcctggagaa gtacaagacc 240
ctgtatctga agcaggagaa ggacgataag gataagaagg cctttgacaa ggagaaggag 300
aacctgcgca agcagatcgc caatgccttc cggaacaatg agaagtttaa gacactgttc 360
gccaaggagc tgatcaagaa cgatctgatg tctttcgcct gcgaggagga caagaagaat 420
gtgaaggagt ttgaggcctt caccacatac ttcaccggct tccaccagaa ccgcgccaat 480
atgtacgtgg ccgatgagaa gagaacagcc atcgccagca ggctgatcca cgagaacctg 540
ccaaagttta tcgacaatat caagatcttc gagaagatga agaaggaggc ccccgagctg 600
ctgtctcctt tcaaccagac cctgaaggat atgaaggacg tgatcaaggg caccacactg 660
gaggagatct ttagcctgga ttatttcaac aagaccctga cacagagcgg catcgacatc 720
tacaattccg tgatcggcgg cagaacccct gaggagggca agacaaagat caagggcctg 780
aacgagtaca tcaataccga cttcaaccag aagcagacag acaagaagaa gcggcagcca 840
aagttcaagc agctgtataa gcagatcctg agcgataggc agagcctgtc ctttatcgcc 900
gaggccttca agaacgacac cgagatcctg gaggccatcg agaagtttta cgtgaatgag 960
ctgctgcact tcagcaatga gggcaagtcc acaaacgtgc tggacgccat caagaatgcc 1020
gtgtctaacc tggagagctt taacctgacc aagatgtatt tccgctccgg cgcctctctg 1080
acagacgtga gccggaaggt gtttggcgag tggagcatca tcaatagagc cctggacaac 1140
tactatgcca ccacatatcc aatcaagccc agagagaagt ctgagaagta cgaggagagg 1200
aaggagaagt ggctgaagca ggacttcaac gtgagcctga tccagaccgc catcgatgag 1260
tacgacaacg agacagtgaa gggcaagaac agcggcaaag tgatcgccga ttattttgcc 1320
aagttctgcg acgataagga gacagacctg atccagaagg tgaacgaggg ctacatcgcc 1380
gtgaaggatc tgctgaatac accctgtcct gagaacgaga agctgggcag caataaggac 1440
caggtgaagc agatcaaggc ctttatggat tctatcatgg acatcatgca cttcgtgcgc 1500
cccctgagcc tgaaggatac cgacaaggag aaggatgaga cattctactc cctgttcaca 1560
cctctgtacg accacctgac ccagacaatc gccctgtata acaaggtgcg gaactatctg 1620
acccagaagc cttacagcac agagaagatc aagctgaact tcgagaacag caccctgctg 1680
ggcggctggg atctgaataa ggagacagac aacacagcca tcatcctgag gaaggataac 1740
ctgtactatc tgggcatcat ggacaagagg cacaatcgca tctttcggaa cgtgcccaag 1800
gccgataaga aggacttctg ctacgagaag atggtgtata agctgctgcc tggcgccaac 1860
aagatgctgc caaaggtgtt cttttctcag agcagaatcc aggagtttac cccttccgcc 1920
aagctgctgg agaactacgc caatgagaca cacaagaagg gcgataattt caacctgaat 1980
cactgtcaca agctgatcga tttctttaag gactctatca acaagcacga ggattggaag 2040
aatttcgact ttaggttcag cgccacctcc acctacgccg acctgagcgg cttttaccac 2100
gaggtggagc accagggcta caagatctct tttcagagcg tggccgattc cttcatcgac 2160
gatctggtga acgagggcaa gctgtacctg ttccagatct ataataagga cttttcccca 2220
ttctctaagg gcaagcccaa cctgcacacc ctgtactgga agatgctgtt tgatgagaac 2280
aatctgaagg acgtggtgta taagctgaat ggcgaggccg aggtgttcta ccgcaagaag 2340
agcattgccg agaagaacac cacaatccac aaggccaatg agtccatcat caacaagaat 2400
cctgataacc caaaggccac cagcaccttc aactatgata tcgtgaagga caagagatac 2460
accatcgaca agtttcagtt ccacatccca atcacaatga actttaaggc cgagggcatc 2520
ttcaacatga atcagagggt gaatcagttc ctgaaggcca atcccgatat caacatcatc 2580
ggcatcgaca gaggcgagag gcacctgctg tactatgccc tgatcaacca gaagggcaag 2640
atcctgaagc aggataccct gaatgtgatc gccaacgaga agcagaaggt ggactaccac 2700
aatctgctgg ataagaagga gggcgaccgc gcaaccgcaa ggcaggagtg gggcgtgatc 2760
gagacaatca aggagctgaa ggagggctat ctgtcccagg tcatccacaa gctgaccgat 2820
ctgatgatcg agaacaatgc catcatcgtg atggaggacc tgaactttgg cttcaagcgg 2880
ggcagacaga aggtggagaa gcaggtgtat cagaagtttg agaagatgct gatcgataag 2940
ctgaattacc tggtggacaa gaataagaag gcaaacgagc tgggaggcct gctgaacgca 3000
ttccagctgg ccaataagtt tgagtccttc cagaagatgg gcaagcagaa cggctttatc 3060
ttctacgtgc ccgcctggaa tacctctaag acagatcctg ccaccggctt tatcgacttc 3120
ctgaagcccc gctatgagaa cctgaatcag gccaaggatt tctttgagaa gtttgactct 3180
atccggctga acagcaaggc cgattacttt gagttcgcct ttgacttcaa gaatttcacc 3240
gagaaggccg atggcggcag aaccaagtgg acagtgtgca ccacaaacga ggacagatat 3300
gcctggaata gggccctgaa caataacagg ggcagccagg agaagtacga catcacagcc 3360
gagctgaagt ccctgttcga tggcaaggtg gactataagt ctggcaagga tctgaagcag 3420
cagatcgcca gccaggagtc cgccgacttc tttaaggccc tgatgaagaa cctgtccatc 3480
accctgtctc tgagacacaa taacggcgag aagggcgata atgagcagga ctacatcctg 3540
tcccctgtgg ccgattctaa gggccgcttc tttgactccc ggaaggccga cgatgacatg 3600
ccaaagaatg ccgacgccaa cggcgcctat cacatcgccc tgaagggcct gtggtgtctg 3660
gagcagatca gcaagaccga tgacctgaag aaggtgaagc tggccatctc caacaaggag 3720
tggctggagt tcgtgcagac actgaagggc aaaaggccgg cggccacgaa aaaggccggc 3780
caggcaaaaa agaaaaaggg atcctaccca tacgatgttc cagattacgc ttatccctac 3840
gacgtgcctg attatgcata cccatatgat gtccccgact atgcc 3885

Claims (5)

1.一种基于CRISPR/Cas12a的血友病基因突变位点检测试剂盒，其特征在于，包括Cas12a蛋白和gRNA，所述gRNA以核苷酸序列如SEQ ID NO.1～43所示的靶标位点为靶序列进行设计，设计原则为：在选取gRNA靶向序列时，靶向序列5’端应具有5’-TTTN-3’序列，且靶向序列本身、靶向序列和其余序列间不形成稳定二级结构；
所述靶标位点的核苷酸序列如SEQ ID NO.1所示，则所述gRNA靶向序列的核苷酸序列如SEQ ID NO.44所示；
所述靶标位点的核苷酸序列如SEQ ID NO.2所示，则所述gRNA靶向序列的核苷酸序列如SEQ ID NO.45所示；
所述靶标位点的核苷酸序列如SEQ ID NO.3所示，则所述gRNA靶向序列的核苷酸序列如SEQ ID NO.46所示；
所述靶标位点的核苷酸序列如SEQ ID NO.4所示，则所述gRNA靶向序列的核苷酸序列如SEQ ID NO.47所示；
所述靶标位点的核苷酸序列如SEQ ID NO.5所示，则所述gRNA靶向序列的核苷酸序列如SEQ ID NO.48所示；
所述靶标位点的核苷酸序列如SEQ ID NO.6所示，则所述gRNA靶向序列的核苷酸序列如SEQ ID NO.49所示；
所述靶标位点的核苷酸序列如SEQ ID NO.7所示，则所述gRNA靶向序列的核苷酸序列如SEQ ID NO.51所示；
所述靶标位点的核苷酸序列如SEQ ID NO.8所示，则所述gRNA靶向序列的核苷酸序列如SEQ ID NO.52所示；
所述靶标位点的核苷酸序列如SEQ ID NO.9所示，则所述gRNA靶向序列的核苷酸序列如SEQ ID NO.54所示；
所述靶标位点的核苷酸序列如SEQ ID NO.10所示，则所述gRNA靶向序列的核苷酸序列如SEQ ID NO.55或SEQ ID NO.57所示；
所述靶标位点的核苷酸序列如SEQ ID NO.11所示，则所述gRNA靶向序列的核苷酸序列如SEQ ID NO.58所示；
所述靶标位点的核苷酸序列如SEQ ID NO.12所示，则所述gRNA靶向序列的核苷酸序列如SEQ ID NO.59所示；
所述靶标位点的核苷酸序列如SEQ ID NO.13所示，则所述gRNA靶向序列的核苷酸序列如SEQ ID NO.60所示；
所述靶标位点的核苷酸序列如SEQ ID NO.14所示，则所述gRNA靶向序列的核苷酸序列如SEQ ID NO.61或SEQ ID NO.62所示；
所述靶标位点的核苷酸序列如SEQ ID NO.15所示，则所述gRNA靶向序列的核苷酸序列如SEQ ID NO.64所示；
所述靶标位点的核苷酸序列如SEQ ID NO.16所示，则所述gRNA靶向序列的核苷酸序列如SEQ ID NO.65所示；
所述靶标位点的核苷酸序列如SEQ ID NO.17所示，则所述gRNA靶向序列的核苷酸序列如SEQ ID NO.66所示；
所述靶标位点的核苷酸序列如SEQ ID NO.18所示，则所述gRNA靶向序列的核苷酸序列如SEQ ID NO.67所示；
所述靶标位点的核苷酸序列如SEQ ID NO.19所示，则所述gRNA靶向序列的核苷酸序列如SEQ ID NO.68所示；
所述靶标位点的核苷酸序列如SEQ ID NO.20所示，则所述gRNA靶向序列的核苷酸序列如SEQ ID NO.69所示；
所述靶标位点的核苷酸序列如SEQ ID NO.21所示，则所述gRNA靶向序列的核苷酸序列如SEQ ID NO.70所示；
所述靶标位点的核苷酸序列如SEQ ID NO.22所示，则所述gRNA靶向序列的核苷酸序列如SEQ ID NO.72所示；
所述靶标位点的核苷酸序列如SEQ ID NO.23所示，则所述gRNA靶向序列的核苷酸序列如SEQ ID NO.73所示；
所述靶标位点的核苷酸序列如SEQ ID NO.24所示，则所述gRNA靶向序列的核苷酸序列如SEQ ID NO.74所示；
所述靶标位点的核苷酸序列如SEQ ID NO.25所示，则所述gRNA靶向序列的核苷酸序列如SEQ ID NO.75所示；
所述靶标位点的核苷酸序列如SEQ ID NO.26所示，则所述gRNA靶向序列的核苷酸序列如SEQ ID NO.77所示；
所述靶标位点的核苷酸序列如SEQ ID NO.27所示，则所述gRNA靶向序列的核苷酸序列如SEQ ID NO.78所示；
所述靶标位点的核苷酸序列如SEQ ID NO.28所示，则所述gRNA靶向序列的核苷酸序列如SEQ ID NO.80所示；
所述靶标位点的核苷酸序列如SEQ ID NO.29所示，则所述gRNA靶向序列的核苷酸序列如SEQ ID NO.81所示；
所述靶标位点的核苷酸序列如SEQ ID NO.30所示，则所述gRNA靶向序列的核苷酸序列如SEQ ID NO.82所示；
所述靶标位点的核苷酸序列如SEQ ID NO.31所示，则所述gRNA靶向序列的核苷酸序列如SEQ ID NO.84所示；
所述靶标位点的核苷酸序列如SEQ ID NO.32所示，则所述gRNA靶向序列的核苷酸序列如SEQ ID NO.所示85；
所述靶标位点的核苷酸序列如SEQ ID NO.33所示，则所述gRNA靶向序列的核苷酸序列如SEQ ID NO.所示86；
所述靶标位点的核苷酸序列如SEQ ID NO.34所示，则所述gRNA靶向序列的核苷酸序列如SEQ ID NO.87所示；
所述靶标位点的核苷酸序列如SEQ ID NO.35所示，则所述gRNA靶向序列的核苷酸序列如SEQ ID NO.88所示；
所述靶标位点的核苷酸序列如SEQ ID NO.36所示，则所述gRNA靶向序列的核苷酸序列如SEQ ID NO.89所示；
所述靶标位点的核苷酸序列如SEQ ID NO.37所示，则所述gRNA靶向序列的核苷酸序列如SEQ ID NO.91所示；
所述靶标位点的核苷酸序列如SEQ ID NO.38所示，则所述gRNA靶向序列的核苷酸序列如SEQ ID NO.92所示；
所述靶标位点的核苷酸序列如SEQ ID NO.39所示，则所述gRNA靶向序列的核苷酸序列如SEQ ID NO.93所示；
所述靶标位点的核苷酸序列如SEQ ID NO.40所示，则所述gRNA靶向序列的核苷酸序列如SEQ ID NO.94所示；
所述靶标位点的核苷酸序列如SEQ ID NO.41所示，则所述gRNA靶向序列的核苷酸序列如SEQ ID NO.95所示；
所述靶标位点的核苷酸序列如SEQ ID NO.42所示，则所述gRNA靶向序列的核苷酸序列如SEQ ID NO.96所示；
所述靶标位点的核苷酸序列如SEQ ID NO.43所示，则所述gRNA靶向序列的核苷酸序列如SEQ ID NO.97所示。
2.根据权利要求1所述检测试剂盒，其特征在于，所述Cas12a蛋白为LbCas12a、SsCas12a、ScCas12a、FnCas12a或AsCas12a。
3.根据权利要求1所述检测试剂盒，其特征在于，还包括非特异单链DNA荧光探针、RNase抑制剂和核酸酶检测缓冲液。
4. 根据权利要求1所述检测试剂盒，其特征在于，检测体系包括：2μl RPA产物，45nMCas12a，22.5nM gRNA，100nM在Cas12a切割时可发出荧光的报告DNA链，0.5μl RNase抑制剂，及核酸酶检测缓冲液。
5.根据权利要求1所述检测试剂盒，其特征在于，检测程序为：在37℃下反应1.5小时，荧光动力检测5分钟一次。
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