CN110714077B - 同时检测brca1/2外显子序列和化疗用药位点的引物及应用 - Google Patents
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Abstract
一种同时检测BRCA1/2外显子序列和化疗用药位点位点的引物,包括:为BRCA1设计的外显子扩增引物对、为BRCA2设计的外显子扩增引物对、针对SNP位点设计的特异性引物对;每一种外显子扩增引物对分别分为两组,且同一基因的每一组引物内部无扩增区域重叠;每一组引物对中,第一部分引物上游引物5’端添加SEQ ID.NO1,下游5’端添加SEQ ID.NO2;第二部分引物上游引物5’端添加SEQ ID.NO2,下游5’端添加SEQ ID.NO1;BRCA1基因的一组引物对与BRCA2基因的一组引物对随机组合,组成两个引物组对;所述特异性引物上游引物的5’端添加SEQ ID.NO1,在所述特异性引物下游引物5’端添加SEQ ID.NO2。本申请成本低,通量高,检测范围广,检测时间短;可同时检测BRCA1/2和癌症化疗用药检测位点。
Description
技术领域
本发明涉及生物技术领域,具体涉及一种BRCA1/BRCA2基因外显子区域和化疗用药位点检测引物设计方法/引物组/试剂盒及其应用。
背景技术
1990年,研究者发现了一种直接与遗传性乳腺癌有关的基因,命名为乳腺癌1号基因,英文简称BRCA1。1994年,又发现另外一种与乳腺癌有关的基因,称为BRCA2,很多情况下人们把两种基因统称BRCA1/2一起讨论。BRCA1定位于染色体17q221.31,基因大小为81.19kb,共有24个外显子,BRCA2定位于染色体13q13.1,基因大小为84.19kb,共有27个外显子。
BRCA1/2是两种具有抑制恶性肿瘤发生的基因,在调节人体细胞的复制、遗传物质DNA损伤修复、细胞的正常生长方面有重要作用。拥有这个基因突变的家族倾向于具有高乳腺癌发生率,通常发生在较年轻时,病人的两侧乳房都确癌,且同时患有卵巢癌。有人总结了BRCA1和BRCA2基因突变相关的癌症的终身风险,显示有BRCA1基因突变者,患乳腺癌和卵巢癌的风险分别是50%-85%和15%-45%,有BRCA2基因突变者,患乳腺癌和卵巢癌的风险分别是50%-85%和10%-20%。因此,对高风险人群进行BRCA1/2基因突变位点检测,对于癌症的预防和早期监查具有重要意义。
早期BRCA1/2基因突变的筛查,常规方法是直接进行Sanger测序,但是BRCA1/2基因尺寸大,直接测序成本高,变性高效液相色谱分析技术、高分辨率熔炼分析等方法也经常被采用。如今,二代测序(NGS)技术越来越高通量、低成本、多样化,已经成为分子诊断领域广泛应用的技术,目前有很多实验室采用多重PCR技术来检测BRCA1/BRCA2的突变,例如,晏蓉(基于多重PCR靶向测序的乳腺癌易感基因BRCA1/2全外显子区位点变异检测,东南大学硕士论文,2017年)研究了设计了58对引物,覆盖BRCA1/2外显子和剪接位点区域,其中包括BRCA1引物25对和BRCA2引物33对,分别覆盖基因长度17763bp和24383bp,扩增子长度在400-1500bp之间,共检出24个突变,其中有1例BRCA1致病突变rs80357303,该易感突变被报道会增加乳腺癌的发病风险。
二代测序技术由于其具有通量高的特性,且随着测序时间的逐步缩短,使用二代测序进行BRCA1和BRCA2进行价格低廉周期较短的普遍筛查可行性增强。
化疗是指使用化学药物抑制细胞生长繁殖、杀死肿瘤细胞或者促进肿瘤细胞分化的一种治疗方法,该方法被广泛应用于癌症治疗,但是越来越多的临床实验表明,化疗药物使用后的治疗效果因人而异。目前发现,某些SNP位点的基因型与特定化疗药物的治疗效果有明显的相关性,如果在确定治疗方案前对这些SNP位点进行检测,就能协助诊疗方案确定,减少无效治疗的时间,提高患者生存周期。
因此,将BRAC1/2和化疗相关SNP位点同时检测对乳腺癌高风险人群具有重要意义。
发明内容
本发明提供一种同时检测BRCA1/2外显子序列和所述化疗相关SNP位点的引物设计方法、引物组合和检测方法及试剂盒。本发明主要是使用多重PCR技术对目的区域进行富集后进行二代测序,通过生物信息学手段BRCA1/2外显子序列进行鉴定、并对化疗SNP位点进行分型,从而同时实现对乳腺癌易感程度和化疗药物敏感程度的预测。
本申请第一个方面是提供一种同时检测BRCA1/2外显子序列和化疗用药位点的引物,包括:为BRCA1设计的外显子扩增引物对,为BRCA2设计的外显子扩增引物对;其中,为BRCA1设计的外显子扩增引物对能够覆盖BRCA1全部外显子区域、为BRCA2设计的外显子扩增引物对能够覆盖BRCA2全部外显子区域;并且,为BRCA1设计的外显子扩增引物对,为BRCA2设计的外显子扩增引物对分别分为两组,且同一基因的每一组引物内部无扩增区域重叠;每一组引物对中,第一部分引物上游引物5’端添加SEQ ID.NO1,下游5’端添加SEQID.NO2;第二部分引物上游引物5’端添加SEQ ID.NO2,下游5’端添加SEQ ID.NO1;BRCA1基因的一组引物对与BRCA2基因的一组引物对随机组合,组成两个引物组对;
还包括,针对如下所述SNP位点设计的特异性引物对:rs1801133、rs2072671、rs60369023、rs1934951、rs1113129、rs183484、rs9937、rs1042858、rs2306283、rs11045879、rs116855232、rs4646、rs8060157、rs2232228、rs1042522、rs183205964、rs151264360、rs25487、rs1799782、rs13181、rs1052555、rs1056836、rs1872328、rs10497203、rs7582141、rs6432512、rs7270101、rs1056892、rs3892097、rs738409、rs2228001、rs1801019、rs924607、rs1801394、rs442767、rs1142345、rs1800460、rs2234693、rs9340799、rs4880、rs121434568、rs7779029、rs1045642、rs7853758、rs885004;并在所述特异性引物上游引物的5’端添加SEQ ID.NO1,在所述特异性引物下游引物5’端添加SEQ ID.NO2。
在一种优选实施例中,针对所述SNP位点设计的特异性引物作为一组,可以与上述两个引物组对中的其中一个进行组合。
在一种优选实施例中,所述同时检测BRCA1/2外显子序列和所述位点的引物,还包括通用建库引物。
本申请第二个方面是提供一种可以同时检测BRCA1/2和癌症化疗检测位点(化疗用药位点)的引物的设计方法,包括:
设计BRCA1外显子扩增引物对,能够覆盖BRCA1全部外显子区域,设计BRCA2外显子扩增引物对,能够覆盖BRCA2全部外显子区域;将BRCA1外显子扩增引物对、BRCA2外显子扩增引物对分别分为两组,且同一基因的每一组引物内部无扩增区域重叠;每一组引物对中,第一部分引物上游引物5’端添加SEQID.NO1,下游5’端添加SEQ ID.NO2;第二部分引物上游引物5’端添加SEQID.NO2,下游5’端添加SEQ ID.NO1;BRCA1基因的一组引物对与BRCA2基因的一组引物对随机组合,共组成两个引物组对;
针对如下所述SNP位点设计的特异性引物:rs1801133、rs2072671、rs60369023、rs1934951、rs1113129、rs183484、rs9937、rs1042858、rs2306283、rs11045879、rs116855232、rs4646、rs8060157、rs2232228、rs1042522、rs183205964、rs151264360、rs25487、rs1799782、rs13181、rs1052555、rs1056836、rs1872328、rs10497203、rs7582141、rs6432512、rs7270101、rs1056892、rs3892097、rs738409、rs2228001、rs1801019、rs924607、rs1801394、rs442767、rs1142345、rs1800460、rs2234693、rs9340799、rs4880、rs121434568、rs7779029、rs1045642、rs7853758、rs885004;并在所述特异性引物上游引物的5’端添加SEQ ID.NO1,在所述特异性引物下游引物5’端添加SEQ ID.NO2。
在一种优选实施例中,所述方法还包括,将针对所述SNP位点设计的特异性引物作为一组,与上述两个引物组对中的其中一个进行组合。
在一种优选实施例中,所述第一组引物对由选自SEQ ID.NO3至SEQID.NO40中的任意两个或更多个引物组成。
在一种优选实施例中,所述第二组引物对由选自SEQ ID.NO41至SEQID.NO72中的任意两个或更多个引物组成。
在一种优选实施例中,所述第三组引物对由选自SEQ ID.NO73至SEQID.NO130中的任意两个或更多个引物组成。
在一种优选实施例中,所述第四组引物对由选自SEQ ID.NO131至SEQID.NO180中的任意两个或更多个引物组成。
在一种优选实施例中,所述特异性引物由选自SEQ ID.NO181至SEQID.NO266中的任意两个或更多个引物组成。
在一种优选实施例中,所述通用建库引物选自SEQ ID.NO.267、SEQID.NO.268中的任意一种或更多种。
本发明第三个方面是提供一种可以同时检测BRCA1/2和癌症化疗检测位点(化疗用药位点)的试剂盒,包括所述可以同时检测BRCA1/2和癌症化疗检测位点的引物。
在一种优选实施例中,所述试剂盒还可以包括蒸馏水。
在一种优选实施例中,所述试剂盒还包括一轮PCR扩增酶,优选为多重PCR扩增酶,更优选为Vazyme多重PCR扩增酶。
在一种优选实施例中,所述试剂盒还包括二轮PCR扩增酶,优选为PCR扩增酶Q5。
在一种优选实施例中,所述试剂盒还可以包括gDNA提取试剂或gDNA提取试剂盒。
本发明第四个方面是提供一种用于连接到BRCA1外显子扩增引物对、BRCA1外显子扩增引物对、所述特异性引物上的核苷酸序列,包括SEQID.NO.1、SEQ ID.NO.2。
和现有发明相比,本申请具有如下优点:
1)成本低,检测范围广;
2)通量高;
3)检测时间短;
4)采用多种PCR策略组合的方式,增加检测范围;
5)检测灵活性高,可根据不同的要求设置不同的引物组。
6)可同时检测BRCA1/2和癌症化疗检测位点。
附图说明
图1为采用本申请引物和试剂盒所得待测序产物的Q-Sep鉴定结果。
具体实施方式
为了更清楚的理解本发明的目的、特征和优点,下面结合附图和具体实施方式对本发明进行进一步的详细描述。
后续的描述中阐述了很多具体细节以便充分了解本发明,但是本发明还可以采用其他不同于在此描述的其他方式来实施,因此本发明并不限于下面公开的具体实施例的限制。
实施例1
使用primer3设计目标扩增子大小为220-300bp的引物,保证所有引物能覆盖BRCA1/2的全部外显子区域,将BRCA1和BRCA2的引物对分别分为两组,保证同一基因的每组引物内部无扩增区域重叠。其中,第一组和第二组为BRCA1外显子扩增引物:第一组引物为SEQ ID NO.3-SEQ ID NO.40,第二组引物为SEQ ID NO.41-SEQ ID NO.72。第三组和第四组为BRCA2外显子扩增引物:第三组引物为SEQ ID NO.73-SEQ ID NO.130,第四组引物为SEQID NO.131-SEQ ID NO.180。
将4个引物组引物对分别进行编号,按照基因组坐标顺序编号1,2,3……n,排序为奇数号引物对上游引物5’端添加SEQ ID.NO1,下游5’端添加SEQID.NO2,排序为偶数号引物对上游引物5’端添加SEQ ID.NO2,下游5’端添加SEQ ID.NO1。
本发明针对目前国内常用的化疗药物筛选出45个SNP位点。具体如表1所示:
表1,SNP位点:
根据上述SNP位点,本申请使用primer3设计了86条特异性引物,作为第五组引物,第五组包括86条特异性引物(SEQ ID NO.181-SEQ ID NO.266)。其中,第一组为第一轮PCR引物,每条第一轮PCR上游引物的5’端添加SEQID.NO.1,下游引物5’端添加SEQ ID.NO.2。
将BRCA1一组引物与BRCA2一组引物随机组合,剩下的BRCA1和BRCA2引物进行组合,得到两个引物组的组合,将第五组引物与其中任意一个组合进行再次组合,共计得到如下四个组合。
组合I:引物管1(SEQ ID NO.3-SEQ ID NO.40,SEQ ID NO.73-SEQ IDNO.130,SEQID NO.181-SEQ ID NO.266),引物管2(SEQ ID NO.41-SEQID NO.72,SEQ ID NO.131SEQ IDNO.180);
组合II:引物管1(SEQ ID NO.3-SEQ ID NO.40,SEQ ID NO.73-SEQ IDNO.130),引物管2(SEQ ID NO.41-SEQ ID NO.72,SEQ ID NO.131SEQ IDNO.180,SEQ ID NO.181-SEQID NO.266);
组合II:引物管1(SEQ ID NO.3-SEQ ID NO.40,SEQ ID NO.131SEQ IDNO.180,SEQID NO.181-SEQ ID NO.266),引物管2(SEQ ID NO.41-SEQID NO.72,SEQ ID NO.73-SEQ IDNO.130);
组合IV:引物管1(SEQ ID NO.3-SEQ ID NO.40,SEQ ID NO.131SEQID NO.180),引物管2(SEQ ID NO.41-SEQ ID NO.72,SEQ ID NO.73-SEQID NO.130,SEQ ID NO.181-SEQID NO.266)。
设计一对通用引物SEQ ID NO.267-SEQ ID NO.268,作为第六组引物。
实施例2
本发明所述引物进行多重PCR扩增方法如下:
1)一轮PCR:使用组合I至IV中的任意一组引物进行PCR反应,优选的其PCR程序为95℃5min;95℃30s,60℃30s,58℃30s,56℃30s,70℃1min(3cycles);95℃30s,68℃1min(15cycles);72℃5min;4℃∞。如表2所示:
表2,第一轮PCR扩增反应体系和扩增反应条件
2)一轮纯化,将PCR产物使用Ampure beads进行第一次纯化,然后使用Beadsbuffer和水进行第二次纯化;具体地:将两管PCR产物混合至一管;加入30μL(Ampurebeads)磁珠混合均匀静置5min然后置于磁力架上,待澄清后吸弃上清;使用80%乙醇清洗两边后晾干;使用15μL蒸馏水回溶3min后加入18μL Beads buffer混匀,静置5min;使用80%乙醇清洗两边后晾干;使用22μL蒸馏水回溶后吸取20μL上清准备进行第二轮PCR反应。
3)二轮PCR,使用引物组6进行二轮PCR,优选的PCR反应程序为95℃5min;95℃30s,65℃1min(8cycles);72℃5min;4℃∞。如表3所示:
表3,第一轮PCR扩增反应体系和扩增反应条件
4)二轮纯化,将PCR产物使用Ampure beads进行第一次纯化,然后使用Beadsbuffer和水进行第二次纯化;具体地:加入45μL(Ampure beads)磁珠混合均匀静置5min,然后置于磁力架上,待澄清后吸弃上清;使用80%乙醇清洗两边后晾干;使用20μL蒸馏水回溶3min后加入24μLBeads buffer混匀,静置5min;使用80%乙醇清洗两边后晾干;使用22μL蒸馏水回溶后吸取20μL上清准备测序使用。
5)测序,对步骤4)获得的产物进行二代测序,测序方式为PE150。
6)分析,对步骤5获得的数据进行化疗用药SNP位点分型分析。
实施例3
使用5mL EDTA采血管收集某健康自愿者血液,使用QIAGEN试剂盒提取血细胞gDNA,作为多重PCR扩增的DNA模板。采用实施例2多重PCR技术进行检测。
测定样品浓度为15ng/μL。Q-Sep结果如图1所示。
表4,下机数据质量评估
测序原始reads(条) | 质控后剩余reads数(条) | 质量控制后剩余数据比例 |
2254879 | 2097802 | 93.03% |
样品使用illumina测序仪进行测序,共测得reads数目未2254879条,经过质控(去除质量不合格的reads),剩余2097802条reads,质控合格reads占总reads数的93.03%。
表5,参考序列比对和depth数据统计
建库完成后完整文库大小为322-700bp,质控合格的reads有94.08%都能对应比对到人类基因组序列上,实验中扩增子PCR区域共为49342bp,其中有49240bp能被二代测序仪检测到,共占实际捕获区域的99.79%,每个扩增子平均被检测到8601次。
表6,分型结果
以上为该样本中检测到突变的SNP位点,第一列为SNP位点名称,第二列和第三列为染色体编号和对应的位置坐标,第四列为野生型的碱基类型,第五列为突变型碱基类型,最后一列为该样本对应SNP位点的基因型。(列表中未出现的SNP位点为纯合野生型)。
以上对本发明的具体实施例进行了详细描述,但其只是作为范例,本发明并不限制于以上描述的具体实施例。对于本领域技术人员而言,任何对本发明进行的等同修改和替代也都在本发明的范畴之中。因此,在不脱离本发明的精神和范围下所作的均等变换和修改,都应涵盖在本发明的范围内。
序列表
<110> 上海艾汭得基因科技有限公司
<120> 同时检测BRCA1/2外显子序列和化疗用药位点的引物及应用
<130> IPI20190813
<160> 268
<170> SIPOSequenceListing 1.0
<210> 1
<211> 38
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
gatctacact ctttccctac acgacgctct tccgatct 38
<210> 2
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
gtgactggag ttcagacgtg tgctcttccg atct 34
<210> 4
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
ggacgttgtc attagttctt tggtttg 27
<210> 5
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
gcataggaga taatcatagg aatccca 27
<210> 6
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
tgactcagtc ataacagctc aaagttg 27
<210> 7
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
gcttctataa agttaggtgt ttcctggg 28
<210> 8
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
cattactttt taaatggctc ttaaggg 27
<210> 9
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
gaatggtttt ataggaacgc tatgtta 27
<210> 10
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
gggtttctct tggtttcttt gattata 27
<210> 11
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
gaagaagaag aagaagaaaa caaatgg 27
<210> 12
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
ctgatgatgg tcaatttatt ttgtcca 27
<210> 13
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
gaccaagatt tttggcaaaa ctataag 27
<210> 14
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
ccacagtaga tgctcagtaa atatttc 27
<210> 15
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
cacatacatc cctgaaccta aaataaa 27
<210> 16
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
gagcagcttt cactaactaa ataagat 27
<210> 17
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
taattacagt actgtatcta cccactc 27
<210> 18
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
ttagtgagga aacaaaatgt tctgcta 27
<210> 19
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
ttagctcact tctataaata gactggg 27
<210> 20
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
gacctgttag atgatggtga aataaag 27
<210> 21
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
tatgcctagt agactgagaa ggtatat 27
<210> 21
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
cacaattagc cgtaataaca ttagaga 27
<210> 22
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
ctgaactact tcttcatatt cttgctt 27
<210> 23
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
aaaagtcact tttgaatgtg aacaaaa 27
<210> 24
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
ttaacaaatg acttgatggg aaaaagt 27
<210> 25
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
tatttcattg gtacctggta ctgatta 27
<210> 26
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
gaaaccttga atgtattctg caaatac 27
<210> 27
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
aactagtagt cagtagaaat ctaagcc 27
<210> 28
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
aggattgaca aattctttaa gttcact 27
<210> 29
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
taattatagg agcatttgtt actgagc 27
<210> 30
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
agattctttt tcgagtgatt ctattgg 27
<210> 31
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
gagagaaaag aatggaataa gcagaaa 27
<210> 32
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
tcacttttac atattaaagc ctcatga 27
<210> 33
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
acaattcagt ttttgagtac cttgtta 27
<210> 34
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 34
gcttttatta cagaattcag ccttttc 27
<210> 35
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 35
cattttgtgt gacatgaaag taaatcc 27
<210> 36
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 36
ttaattcaaa gagatgatgt cagcaaa 27
<210> 37
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 37
gcttctcaaa gtatttcatt ttcttgg 27
<210> 38
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 38
gagataaagg ggaaggaaag aattttg 27
<210> 39
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 39
gaatcttgcc cttaacttgt ttacag 26
<210> 40
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 40
caaacacagc tattaaaaag tcattcc 27
<210> 41
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 41
acggaaaagc gcgggaatta cagataa 27
<210> 42
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 42
cttacgcctc tcaggttccg cccctac 27
<210> 43
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 43
gtgtgtgtat aaatttggtt tgttctt 27
<210> 44
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 44
ctggtttcca ctataccaaa gtaaaaa 27
<210> 45
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 45
tttttgaaga tctagaacca cattgtt 27
<210> 46
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 46
cgtctctaca gaaaacacaa aatttag 27
<210> 47
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 47
ttctactgtt gctgcatctt attttta 27
<210> 48
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 48
taacctagac taaaaggtct tatcacc 27
<210> 49
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 49
aagagaactt atctagtgag gatgaag 27
<210> 50
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 50
tatttgcagt caagtcttcc aattc 25
<210> 51
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 51
aacctgaggt ctataaacaa agtcttc 27
<210> 52
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 52
ctaaaaacag cagaactttc cttaatg 27
<210> 53
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 53
gaaactggac tcattactcc aaataaa 27
<210> 54
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 54
tggaacctac ttcattaata ttgcttg 27
<210> 55
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 55
gaaacaagca tagaaatgga agaaagt 27
<210> 56
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 56
cctgcagtga tattaactgt ctgta 25
<210> 57
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 57
cctggttctt ttactaagtg ttcaaat 27
<210> 58
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 58
catttatttg gttctgtttt tgccttc 27
<210> 59
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 59
tagtggtcat gagaataaaa caaaagg 27
<210> 60
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 60
ttatctcttc actgctagaa caactat 27
<210> 61
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 61
ggacgttcta aatgaggtag atgaata 27
<210> 62
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 62
gtaggtctcc ttttacgctt taattta 27
<210> 63
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 63
aacagcagtt tattactcac taaagac 27
<210> 64
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 64
ttctgaatgc tgctatttag tgttatc 27
<210> 65
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 65
attgaaagtt ccccaattga aagtt 25
<210> 66
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 66
cctacataaa actctttcca gaatgtt 27
<210> 67
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 67
ggggttttag aatcataaat ccagatt 27
<210> 68
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 68
ttaggtgtta aacgttaggt gtaaaaa 27
<210> 69
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 69
taaattaaac atcaactctg tctccag 27
<210> 70
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 70
actatatgac tgaatgaata tctctggt 28
<210> 71
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 71
gaatgcctta aatatgacgt gtctg 25
<210> 72
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 72
agcactgtgt atgtatgtaa taagtct 27
<210> 73
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 73
cacataagga acagtttatg gttctaa 27
<210> 74
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 74
gagttttacc tcagtcacat aataagg 27
<210> 75
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 75
tttgaatatt attggagttg aagccag 27
<210> 76
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 76
agtaatccat agtcaagatc ttaagca 27
<210> 77
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 77
gctcttagcc aaaatattag cataaaa 27
<210> 78
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 78
tttttaaaca cttccaaaga atgcaaa 27
<210> 79
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 79
caattactaa gtcataaaaa taaaccaggt 30
<210> 80
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 80
aataagataa actagttttt gccagtttt 29
<210> 81
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 81
tagagatgac aattatcaac ctcatct 27
<210> 82
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 82
gcaattcagt aaacgttaag tgaaata 27
<210> 83
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 83
aaatcacata taggaccagg tttagag 27
<210> 84
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 84
tcaaatagta gatgtgcttt ttgatgt 27
<210> 85
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 85
aaaaacctgt agttcaacta aacagag 27
<210> 86
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 86
ccattaaaaa tttttggacc taggttg 27
<210> 87
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 87
agtcttgcta gttcttactt tttgtag 27
<210> 88
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 88
taaactgttt ctatgagaaa ggttgtg 27
<210> 89
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 89
atttaatggc ttctctgatt ttggtag 27
<210> 90
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 90
tccattagat tcaaatgtag caaatca 27
<210> 91
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 91
acttatttgt tttctttttc aaagtgga 28
<210> 92
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 92
cttcatttca gggtatcaaa aagtcta 27
<210> 93
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 93
gaatcagctt ctggggtaat aaataac 27
<210> 94
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 94
ccaaacacta cctttttaac ttagtga 27
<210> 95
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 95
cttagatttg tgttttggtt gaattgt 27
<210> 96
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 96
cttttatatg atcatgaaaa tgccagc 27
<210> 97
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 97
gcccatttgt tcatgtaatc attattt 27
<210> 98
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 98
agctaatgaa aggaataatc ttgcttt 27
<210> 99
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 99
agcttggttt tctaaactga gtaaatt 27
<210> 100
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 100
atatcctact agtttagctt gtgttga 27
<210> 101
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 101
taaacattga aacaacagaa tcatgac 27
<210> 102
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 102
gcaatttgaa ggtacagttg aaattaa 27
<210> 103
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 103
tttatatttt gctccgtttt agtagca 27
<210> 104
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 104
ctgccagtag aaattctcat aacttag 27
<210> 105
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 105
tttcatcaaa aaggtttttc actttgt 27
<210> 106
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 106
gaaaccagaa gaattgcata acttttc 27
<210> 107
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 107
ctcacagaag tttttctact acaactt 27
<210> 108
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 108
caaagtgtaa agaaatgcag aattctc 27
<210> 109
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 109
ggacaattta atggctgcat ttttatt 27
<210> 110
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 110
aaaatcatct ctccgaaaaa caagata 27
<210> 111
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 111
aagtatttgc agatgagact gacttat 27
<210> 112
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 112
agaataaatc aaaaatttgc caaacga 27
<210> 113
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 113
acacgaggaa gtatttttga tacattt 27
<210> 114
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 114
agcaagtctt ttccaaagta ttgttta 27
<210> 115
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 115
gtttagaatc tgtcagttca tcatctt 27
<210> 116
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 116
agtttctcca tatctctctc aatttca 27
<210> 117
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 117
aagagtataa agaggtcctt gattagg 27
<210> 118
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 118
ttgagaaata aaactgatat tatttgcctt 30
<210> 119
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 119
aagtgttaac ttcttaacgt tagtgtc 27
<210> 120
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 120
acattcactg aaaattgtaa agcctat 27
<210> 121
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 121
tagttttaaa aggtggaaca aagactt 27
<210> 122
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 122
tgcaacaaag gcatattcct aaatatt 27
<210> 123
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 123
aaaattacac tctgtcataa aagccat 27
<210> 124
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 124
gggttgtgct ttttaaattt caatttt 27
<210> 125
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 125
tgagggaata cataaaagtt aacacac 27
<210> 126
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 126
tgtgtgatac atgtttactt taaattgtt 29
<210> 127
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 127
tactgccgta tatgattacg taatgta 27
<210> 128
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 128
ttgtagttgt tgaattcagt atcatcc 27
<210> 129
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 129
taccacccat ctgtaagttc aataatg 27
<210> 130
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 130
tcagtgactt gtttaaacag tggaatt 27
<210> 131
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 131
gtacaacttc cttggagatt ttgtc 25
<210> 132
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 132
tatgaaacag ttgtagatac ctctgaa 27
<210> 133
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 133
gaaaaacttg cattgaaagt ctcttta 27
<210> 134
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 134
gaaaaagacc tattagacac agagaac 27
<210> 135
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 135
cacagaagga atcgtcatct ataaaac 27
<210> 136
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 136
tagctttgaa gaatgcaggt ttaatat 27
<210> 137
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 137
ggctgaaaag acatagttta taacact 27
<210> 138
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 138
tactttgaaa cagaagcagt agaaatt 27
<210> 139
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 139
tccttggaag taggagttaa aataaga 27
<210> 140
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 140
ctctcaggat cttgattata aagaagc 27
<210> 141
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 141
tagagttctt gaaaatgggt tcgttta 27
<210> 142
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 142
tgaaaattat aaaaacgttg agctgtt 27
<210> 143
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 143
gatacagtat taattgactg aggcttg 27
<210> 144
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 144
aaatcggaca tctccttgaa tatagat 27
<210> 145
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 145
cacttttgtt acagtcattt ttcaaca 27
<210> 146
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 146
aaaaggcaga aattacagaa ctttcta 27
<210> 147
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 147
tatcattttt acttgaatca ctgccat 27
<210> 148
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 148
ttgagaatat tagtgaggaa acttctg 27
<210> 149
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 149
cctcataact tagaatgtcc attttgt 27
<210> 150
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 150
agcatgtcat ggtaatactt caaataa 27
<210> 151
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 151
acagtctcaa tagaaacaag gttttta 27
<210> 152
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 152
aagaacctac tctattgggt tttcata 27
<210> 153
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 153
tcctgaatca ttatatacct catcaga 27
<210> 154
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 154
ccttagcttt ttacacaagt tgtagta 27
<210> 155
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 155
gaagaatatc ctctgaatca tccaatg 27
<210> 156
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 156
aaactgtaaa tgaagatatt tgcgttg 27
<210> 157
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 157
ggatattaaa tgttctggag tacgtat 27
<210> 158
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 158
gtgatgttag tttggaaact tcagata 27
<210> 159
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 159
aacaagtgac actttggttc ctaata 26
<210> 160
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 160
gggagtgtta gaggaatttg atttaat 27
<210> 161
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 161
ttgtcataca atacctaaag gttcttc 27
<210> 162
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 162
caagtttctg ctacaagaaa tgaaaaa 27
<210> 163
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 163
atcaatgact gatttttacc aagagtg 27
<210> 164
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 164
cgcaaatata tctgaaactt ctagcaa 27
<210> 165
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 165
tgtatcaaaa gaaagaaata tatggtaagt 30
<210> 166
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 166
gcagttctag aagaatgaaa actctta 27
<210> 167
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 167
aaaatgttaa attcaaagtc tctaagac 28
<210> 168
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 168
ggcctgatac aattaacttg aatgtta 27
<210> 169
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 169
ctaaaacagc ttctcacctt gaataat 27
<210> 170
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 170
tcttatcttt aaatctccct tctttgg 27
<210> 171
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 171
taaaactgat aaaaacaaag catttaca 28
<210> 172
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 172
tttttgttct gattgctttt tattcca 27
<210> 173
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 173
ggtagctcca actaatcata agagatt 27
<210> 174
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 174
taagtgcttg ttagtttatg gaatctc 27
<210> 175
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 175
cattttgttg aatgtctctt gaaagtg 27
<210> 176
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 176
tcctttcttg catcttaaaa ttcatct 27
<210> 177
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 177
cagaatatac gatggcctcc atatata 27
<210> 178
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 178
tctgctttta aaggaaatac ttttgga 27
<210> 179
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 179
gaagcaaaag tataccaata cggaatc 27
<210> 180
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 180
gttgtactac atctctgatc aaagaac 27
<210> 181
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 181
gaaagatccc ggggacgatg 20
<210> 182
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 182
aagatcagag cccccaaagc 20
<210> 183
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 183
gaggagctgc aatcgtgtct 20
<210> 184
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 184
gggtgcccac ctttaccttt 20
<210> 185
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 185
gccctaattg ccctgtcctt 20
<210> 186
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 186
aacctggctt tcccactcac 20
<210> 187
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 187
gaaagttgtg cccttggagc 20
<210> 188
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 188
ctcctctggg gctggtagaa 20
<210> 189
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 189
agcagagatt gtggagctga 20
<210> 190
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 190
tcccactgtt cactcagaag c 21
<210> 191
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 191
agaagtagag atggagagca ggt 23
<210> 192
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 192
ccagtacttt caattcatgg agc 23
<210> 193
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 193
tgtagggttt gaagactggg 20
<210> 194
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 194
cagggtccag caaagcctta 20
<210> 195
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 195
ataacccact tagcctgggg t 21
<210> 196
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 196
aacaagtgga taaggtcgat gttg 24
<210> 197
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 197
cctggagacc actgtgcttt 20
<210> 198
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 198
aaaacgtccc ctctcaccac 20
<210> 199
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 199
tagccaagca aatgcaaagc atc 23
<210> 200
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 200
tgtatcccac cagatggttc aga 23
<210> 201
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 201
agagctctac tggggaacca 20
<210> 202
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 202
ctcagtggcc catggcattt 20
<210> 203
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 203
ggggtcagaa gttgtgggag 20
<210> 204
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 204
ctgggtggag ggcattactc 20
<210> 205
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 205
tatgtgacgg gctaccagtt c 21
<210> 206
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 206
cagggaagga gatgcgctg 19
<210> 207
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 207
aagtctcatg gaagccagcc 20
<210> 208
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 208
tcactgaaga cccaggtcca 20
<210> 209
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 209
ctcctgcgtt tccccctg 18
<210> 210
<211> 15
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 210
ctcggcgtcc cgctc 15
<210> 211
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 211
gggtatctga caatgctgag gtta 24
<210> 212
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 212
caaagcgtgg acgaatgcag a 21
<210> 213
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 213
tgctcctcca gccttttctg 20
<210> 214
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 214
taaggagtgg gtgctggact 20
<210> 215
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 215
cgttgtccga gctcacct 18
<210> 216
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 216
tgtacggttt catagccccc 20
<210> 217
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 217
aggagtcacc aggaaccgt 19
<210> 218
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 218
atgttgacag ggatggccc 19
<210> 219
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 219
catctgtggc ccctcgaatc 20
<210> 220
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 220
cacctcacag atgccaacct 20
<210> 221
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 221
ggtgagccag gatggagatg 20
<210> 222
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 222
acctctgtct tgggctacca 20
<210> 223
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 223
ttcatcctgt tcttgggccc 20
<210> 224
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 224
actcacggtt catgtggctt 20
<210> 225
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 225
tgagatcttc atgttgcaaa aggtt 25
<210> 226
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 226
tgtttcaaca aactcaaaac aggc 24
<210> 227
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 227
gagggggttt caggttgagg 20
<210> 228
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 228
gcatccggtt taccaccact 20
<210> 229
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 229
gggaggaact tacgggcatc 20
<210> 230
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 230
gtgcactaaa gcacgcatgt 20
<210> 231
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 231
agatgggcag cagagttatc g 21
<210> 232
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 232
ggtactccgg cacttatcag g 21
<210> 233
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 233
gggtcacggt cttatcgagg 20
<210> 234
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 234
tccgaagcag acgtttacca 20
<210> 235
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 235
ctgcagagac tcctcggtct 20
<210> 236
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 236
gatgggcaga agggcacaaa 20
<210> 237
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 237
cctgaagtcc gagggtgtat 20
<210> 238
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 238
agaaagccga cttaccacgc 20
<210> 239
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 239
tgggtgaatc tgacaagggc 20
<210> 240
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 240
agcagcttcc cacctgttc 19
<210> 241
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 241
ctgttggaca gagagcaggg 20
<210> 242
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 242
aagcttcgat gcaactgggt 20
<210> 243
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 243
tgtgcttctg aagagccagg 20
<210> 244
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 244
gtctctgcca cagcccttag 20
<210> 245
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 245
tgtgggtatt gttgcattgt ttct 24
<210> 246
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 246
cggctctaac cttatcggat tca 23
<210> 247
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 247
tcatattgtg cagttcccca gt 22
<210> 248
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 248
aaacacggta ggtggctaaa ca 22
<210> 249
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 249
tcatccatta cattttcagg cttt 24
<210> 250
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 250
tccctgatgt cattcttcat agta 24
<210> 251
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 251
acttaccatt tgcgatcacc tg 22
<210> 252
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 252
acaggctcct aaaaccatga gg 22
<210> 253
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 253
tgttctgtgt tgtccatcag ttc 23
<210> 254
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 254
ttgcagcaaa aggtgttgcc ta 22
<210> 255
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 255
ttgatgtgag gttccagggc 20
<210> 256
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 256
cacctgcagg cggttctc 18
<210> 257
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 257
cctggcatga acatgaccct 20
<210> 258
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 258
gccacctcct tactttgcct 20
<210> 259
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 259
tgttcccatt tccaggctcc 20
<210> 260
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 260
gtcagggctc tcagttccag 20
<210> 261
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 261
acaggaagtg tggccagatg 20
<210> 262
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 262
tgcctatgga gacaacagcc 20
<210> 263
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 263
caaaccagga cagggctgaa 20
<210> 264
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 264
gcaggggcct tagagtttgt 20
<210> 265
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 265
atcaggcagc ttccctatct ac 22
<210> 266
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 266
cagcacctac catgccttac c 21
<210> 267
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 267
aatgatacgg cgaccaccga gatctacact ctttccctac acgac 45
<210> 268
<211> 53
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 268
caagcagaag acggcatacg agatnnnnnn gtgactggag ttcagacgtg tgc 53
Claims (8)
1.一种同时检测BRCA1/2外显子序列和化疗用药位点的引物,包括:
为BRCA1设计的外显子扩增引物对,为BRCA2设计的外显子扩增引物对;其中,为BRCA1设计的外显子扩增引物对能够覆盖BRCA1全部外显子区域、为BRCA2设计的外显子扩增引物对能够覆盖BRCA2全部外显子区域;并且,为BRCA1设计的外显子扩增引物对,为BRCA2设计的外显子扩增引物对分别分为两组,且同一基因的每一组引物内部无扩增区域重叠;将4个引物组引物对分别进行编号,按照基因组坐标顺序编号1,2,3…… n,排序为奇数号引物对定义为第一部分引物,排序为偶数号引物对定义为第二部分引物,每一组引物对中,第一部分引物上游引物5’端添加SEQ ID.NO 1,下游5’端添加SEQ ID.NO 2;第二部分引物上游引物5’端添加SEQ ID.NO 2,下游5’端添加SEQ ID.NO 1;BRCA1基因的一组引物对与BRCA2基因的一组引物对随机组合,组成两个引物对组;
第一组引物对由SEQ ID.NO 3至SEQ ID.NO 40中的引物组成,第二组引物对由SEQID.NO 41至SEQ ID.NO 72中的引物组成,第三组引物对由SEQ ID.NO 73至SEQ ID.NO 130中的引物组成,第四组引物对由SEQ ID.NO 131至SEQ ID.NO 180中的引物组成,为化疗用药位点设计的特异性引物由SEQ ID.NO 181至SEQ ID.NO 266中的引物组成;
具体为针对如下SNP位点设计的特异性引物:rs1801133、rs2072671、rs60369023、rs1934951、rs1113129、rs183484、rs9937、rs1042858、rs2306283、rs11045879、rs116855232、rs4646、rs8060157、rs2232228、rs1042522、rs183205964、rs151264360、rs25487、rs1799782、rs13181、rs1052555、rs1056836、rs1872328、rs10497203、rs7582141、rs6432512、rs7270101、rs1056892、rs3892097、rs738409、rs2228001、rs1801019、rs924607、rs1801394、rs442767、rs1142345、rs1800460、rs2234693、rs9340799、rs4880、rs121434568、rs7779029、rs1045642、rs7853758、rs885004;并在所述特异性引物上游引物的5’端添加SEQ ID.NO 1,在所述特异性引物下游引物5’端添加SEQ ID.NO 2。
2.根据权利要求1所述同时检测BRCA1/2外显子序列和化疗用药位点的引物,其特征在于,还包括通用建库引物。
3.根据权利要求2所述同时检测BRCA1/2外显子序列和化疗用药位点的引物,其特征在于,所述通用建库引物选自SEQ ID.NO.267、SEQ ID.NO.268中的任意一种或更多种。
4.根据权利要求1所述同时检测BRCA1/2外显子序列和化疗用药位点的引物,其特征在于,将针对所述SNP位点设计的特异性引物作为一组,与上述两个引物对组中的其中一个进行组合。
5.根据权利要求1所述的一种同时检测BRCA1/2外显子序列和化疗用药位点的引物的设计方法,其特征在于,包括:
设计BRCA1外显子扩增引物对,能够覆盖BRCA1全部外显子区域,设计BRCA2外显子扩增引物对,能够覆盖BRCA2全部外显子区域;将BRCA1外显子扩增引物对、BRCA2外显子扩增引物对分别分为两组,且同一基因的每一组引物内部无扩增区域重叠;每一组引物对中,第一部分引物上游引物5’端添加SEQ ID.NO 1,下游5’端添加SEQ ID.NO 2;第二部分引物上游引物5’端添加SEQ ID.NO 2,下游5’端添加SEQ ID.NO 1;BRCA1基因的一组引物对与BRCA2基因的一组引物对随机组合,共组成两个引物组对;
针对如下SNP位点设计的特异性引物:rs1801133、rs2072671、rs60369023、rs1934951、rs1113129、rs183484、rs9937、rs1042858、rs2306283、rs11045879、rs116855232、rs4646、rs8060157、rs2232228、rs1042522、rs183205964、rs151264360、rs25487、rs1799782、rs13181、rs1052555、rs1056836、rs1872328、rs10497203、rs7582141、rs6432512、rs7270101、rs1056892、rs3892097、rs738409、rs2228001、rs1801019、rs924607、rs1801394、rs442767、rs1142345、rs1800460、rs2234693、rs9340799、rs4880、rs121434568、rs7779029、rs1045642、rs7853758、rs885004;并在所述特异性引物上游引物的5’端添加SEQ ID.NO 1,在所述特异性引物下游引物5’端添加SEQ ID.NO 2。
6.一种同时检测BRCA1/2外显子序列和化疗用药位点的试剂盒,其特征在于,包括权利要求1所述的同时检测BRCA1/2外显子序列和化疗用药位点的引物。
7.根据权利要求6所述的试剂盒,其特征在于,所述试剂盒还包括如下任意一种或更多种:蒸馏水、一轮PCR扩增酶、二轮PCR扩增酶。
8.根据权利要求6所述的试剂盒,其特征在于,所述试剂盒还包括gDNA提取试剂或gDNA提取试剂盒。
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