CN109554443B - 一种基于高通量测序技术检测dmd基因变异的引物及其应用 - Google Patents
一种基于高通量测序技术检测dmd基因变异的引物及其应用 Download PDFInfo
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Abstract
本发明公开了一种基于高通量测序技术检测DMD基因变异的试剂盒和方法。该检测试剂盒基于Illumina高通量测序平台采用多重PCR捕获建库技术,检测DMD基因的79个外显子和3个内含子区域的外显子缺失/重复、点突变的基因变异信息。该检测的多重PCR引物组为SEQ ID NO:1~SEQ ID NO:260所示的核酸序列,该试剂盒中含有SEQ ID NO:1~SEQ ID NO:260所示的核酸序列。本发明的检测试剂盒灵敏度高、准确性好、扩增子均一性好,能一次性对DMD基因变异信息包括外显子缺失/重复、点突变的变异进行检测,可以用于产前诊断、阻止患儿出生,降低假肥大型肌营养不良症的发病率。
Description
技术领域
本发明属于基因测序领域,具体涉及一种基于Illumina高通量测序平台的多重PCR 捕获技术快速检测假肥大型肌营养不良症致病基因变异的引物及其应用。
背景技术
假肥大型肌营养不良症(pseudohypertrophy muscular dystrophy)包括杜兴型肌营养不良症(Duchenne muscular dystrophy,DMD)和贝克型肌营养不良症(Beackermuscular dystrophy,BMD),二者均是由于抗肌萎缩蛋白(dystrophin,dys)基因突变所致的X 连锁的隐性遗传病。DMD/BMD患者dys缺乏主要导致了骨骼肌细胞膜缺陷,细胞内的肌酸激酶(creatine kinase)等外漏,肌细胞坏死、脂肪组织和纤维结缔组织增生。DMD 是较严重的类型,其主要临床特征表现为进行性四肢近端骨骼肌萎缩无力、小腿腓肠肌假性肥大,同时累及心肌和呼吸肌,部分患者伴有智力障碍,发病率在活产男婴中高达 1/3500,通常从3-5岁起病,12岁左右失去行走能力,20多岁死亡。BMD与DMD互为等位基因异质性疾病,发病年龄比DMD晚,进展速度慢,其发病率有报道为1/30000。目前假肥大型肌营养不良症的发病机制仍不明了,且国内外尚无有效治疗手段,通过对先证者的确诊、携带者的产前诊断和提供正确的产前基因诊断、婚育指导和遗传咨询是防止患儿出生及降低本病发病率的关键措施。
DMD基因定位于Xp21.1~21.3,长度为2.4Mb,是人类最大的基因之一。cDNA全长1kb,含79个外显子,这样的基因易出现高频率的突变和异位断裂点,大约65-75%的DMD患儿是由于DMD基因外显子缺失/重复所致,约30%是由于DMD基因点突变所致。
目前DMD基因诊断的最有效方式是多重连接依赖式探针扩增技术(multiplexligation-dependent probe amplification,MLPA),MLPA是一种定量和半定量技术,可以检测DMD基因79个外显子拷贝数改变(缺失、重复),但是MLPA技术不能检测点突变,而且有可能出现假阳性结果。其他DMD基因检测的方法还有:多重PCR、Southern blot、变性高效液相色谱(DHPLC)、抗肌萎缩蛋白的免疫组化、Sanger测序等方法,但这些方法均只能检出部分突变类型和已知突变,且检测通量低、检测周期长、准确率低。
近年来随着高通量测序技术的发展,一次检测可涵盖40万到400万的序列,并且可以同时检测基因的缺失、重复和点突变多种突变类型,具有无可替代的高通量、检测变异类型全面和准确率高等优势,已广泛应用于各类生物学研究和医学检测。目前针对高通量测序技术的靶向捕获技术主要包括多重PCR捕获技术、探针捕获技术。相对于探针捕获技术,多重PCR捕获技术具有操作简单、灵活、开发成本低等显著优势。因此,基于高通量测序平台采用多重PCR捕获技术开发的检测DMD基因变异的试剂盒是一种快速、准确、全面检测的DMD基因各类型突变(缺失、重复、点突变)的产品,具有良好的市场应用价值。
发明内容
本发明要解决的技术问题是提供基于高通量测序技术检测DMD基因变异的PCR引物。
本发明还要解决的技术问题是提供DMD基因变异检测方法。
为解决上述技术问题,本发明采用如下技术方案:
一种基于高通量测序技术检测DMD基因变异的PCR引物,包括SEQ ID NO:1~ SEQID NO:260所示的核苷酸序列。
作为优选,SEQ ID NO:1~SEQ ID NO:260所示核苷酸序列的5’端连接有Illumina测序平台建库所需的序列,正向引物5’端连接引物序列为:acacgacgctcttccgatct;反向引物5’端连接引物序列为:gacgtgtgctcttccgatct。
一种检测DMD基因变异的试剂盒,该试剂盒中包括,SEQ ID NO:1~SEQ ID NO:260所示核苷酸序列。
检测DMD基因变异的的方法,该方法包括以下步骤:
(1)将SEQ ID NO.1~SEQ ID NO:260所示核苷酸序列分为两组,SEQ ID NO: 1~SEQ ID NO:130所示核苷酸序列为A组引物,SEQ ID NO:131~SEQ ID NO:260 所示核苷酸序列为B组引物,以样本DNA为模板,进行第一轮多重PCR反应,得到第一轮PCR产物;
(2)使用P5通用引物、P7index引物,以与第一轮PCR产物为模板进行第二轮 PCR反应,对第二轮扩增产物进行纯化,获得DMD基因变异测序文库;
(3)文库质控、高通量测序,对下机数据进行生物信息学分析。
文库质控:用Qsep100全自动核酸蛋白分析系统(BiOptic)对文库进行片段分布检测,正常文库片段分布在200-400bp之间,100-500bp片段的平均大小为330-4000bp。
生物信息分析:对下机的原始数据进行质量控制和数据筛选,以获得后续分析的fastq文件,利用序列比对软件将fastq文件中的序列比对到参考基因组上,再利用call 变异软件进行变异检测。经10个样本的下机数据质量详见表3;经比对后,进行SNV、 Indel的识别分析;CNV分析根据以往阴性(非CNV)样本获得的扩增子测序深度,与本次测序结果进行归一化处理后,按测序深度分析CNV(缺失/重复)。
其中,所述P5通用引物的核苷酸序列如SEQ ID NO:263所示,所述P7index引物的核苷酸序列为SEQ ID NO:264、六个随机碱基、SEQ ID NO:265依次连接。。
步骤(1)中,第一轮多重PCR的反应体系为:
PCR反应程序:98℃3min;98℃15s,60℃30s,72℃2min(15cycles);72℃5min, 4℃hold;
步骤(2)中,第二轮多重PCR的反应体系为:
PCR反应程序:98℃3min;98℃15s,60℃30s,72℃2min(8cycles);72℃5min,4℃hold;
作为上述方法的优选,所述P5通用引物5’-AATGATACGGCGACCACCGAGATCT ACACACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’
P7index引物:5’-CAAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3’;序列中N表示ATCG任意碱基,NNNNNN用于识别不同样本构建的文库。
有益效果:
本发明针对DMD基因的79个外显子和3个内含子区域的外显子缺失/重复、点突变在内的基因变异信息,提供了一种基于Illumina测序平台使用的多重PCR引物组,并基于引物组提供了一种DMD基因变异检测试剂盒及方法。本发明的检测试剂盒灵敏度高、准确性好、扩增子均一性好,能一次性对DMD基因变异信息包括外显子缺失/重复、点突变进行检测。
附图说明
图1实施例2中样本2归一化之后测序深度图,其中2号样本测序深度图52-54 号外显子缺失。
图2实施例2中样本3归一化之后测序深度图,其中3号样本测序深度图3-25号外显子缺失。
图3实施例2中样本5归一化之后测序深度图,5号样本检测结果5-7号外显子扩增。
图4实施例2中样本8归一化之后测序深度图,8号样本检测结果18-44号外显子缺失。
图5实施例2中样本10归一化之后测序深度图,10号样本检测结果阴性。
具体实施方式
一种检测DMD基因变异的多重PCR引物组,所述引物含有核苷酸序列如SEQ ID: 1~SEQ ID NO:260所示的引物序列。
一种检测DMD基因变异的多重PCR引物,其特征在于,所述引物5’端连接有Illumina测序平台建库所需的序列。
作为上述引物的优选,所述引物组分为A、B管,A管引物组含有的核苷酸序列为SEQ ID:1~SEQ ID NO:130;B管引物组含有的核苷酸序列为SEQ ID:131~SEQ ID NO:260。
一种检测DMD基因变异的试剂盒,含有核苷酸序列为SEQ ID NO.1~SEQ ID NO:260所示的核苷酸序列组。
一种检测检测DMD基因变异的方法,包括:根据Illumina高通量测序平台建库需求,利用权利要求1~2任一项所述的引物组构建文库,经文库质控、高通量测序和生物信息分析,获得DMD基因的变异信息,所述变异信息包括外显子的缺失/重复、点突变。
作为上述方法的优选,所述方法进一步包括步骤:
(1)使用多重PCR引物组,分两管捕获样本基因组DNA的靶区域,获得第一轮多重PCR扩增产物,对A、B管多重PCR进行合并后纯化;
(2)使用P5通用引物、P7index引物与多重PCR纯化产物进行第二轮PCR扩增,对第二轮扩增产物进行纯化,获得文库;
(3)文库质控、高通量测序,对下机数据进行生物信息学分析。
作为上述方法的优选,A、B管多重PCR引物混合比例为1:1;
作为上述方法的优选,任选地,步骤(1)中,20uL多重PCR的反应体系为:
PCR反应程序:98℃3min;98℃15s,60℃30s,72℃2min(15cycles);72℃5min, 4℃hold;
任选地,步骤(2)中,20uL多重PCR的反应体系为:
PCR反应程序:98℃3min;98℃15s,60℃30s,72℃2min(8cycles);72℃5min,4℃hold;
作为上述方法的优选,所述P5通用引物5’-AATGATACGGCGACCACCGAGATCT ACACACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’
P7index引物:5’-CAAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3’;序列中N表示ATCG任意碱基,NNNNNN用于识别不同样本构建的文库。
上述方法中,模板DNA可源于外周血、干血斑、组织、羊水等,但不限于此。
下面结合具体实施例详细说明本发明,可以更好的理解本发明。然而,实施例所描述的内容仅用于说明本发明,而不是为了限制本发明的范围和应用。
实施例中所用的材料、试剂、一起等,如无特殊说明,均可从商业途径得到,无特别说明的实验操作均按厂商说明书或本领域常规技术实施。
实施例1:DMD基因多重PCR扩增引物组
发明人根据UCSC数据库DMD基因的参考序列,针对DMD基因的79个外显子和 3个内含子目标DNA序列设计多重PCR扩增引物,经过大量实验筛选、优化、验证,最终优选出130对260条引物(引物序列如SEQ ID NO:1~SEQ ID NO:260所示)。
实施例2:一种基于高通量测序技术的DMD基因变异检测试剂盒
1、本试剂盒包括多重PCR酶Mix:该反应液中含有酶缓冲液、Mg2+、dNTPs、高保真DNA聚合酶。
2、多重PCR引物组:该引物组由实施例1提供的SEQ ID NO:1~SEQ ID NO 260 所示引物进行分组,引物SEQ ID NO:1~SEQ ID NO:130分别稀释至10uM后等量混合为A管,引物SEQ ID NO:131~SEQ ID NO:260分别稀释至10uM后等量混合为B 管。
3、P5通用引物和P7index引物,使用浓度为10uM。
实施例3:DMD基因变异检测方法
本实施例给出一种基于Illumina测序平台使用多重PCR捕获技术检测DMD基因变异的方法,本实施例以杭州迪安医学检验中心有限公司NGS室提供的10例DMD患者 /携带者为例进行DMD基因突变检测,以上样本已经通过MLPA和sanger测序明确了致病突变信息。
1、基因组DNA提取
取外周血100uL-1mL,采用TIANamp Blood DNA Kit(天根生化科技有限公司)进行基因组DNA提取,对提取的基因组用Nanodrop 2000进行纯度检测,并对Qubit(LifeTechnologies公司)对基因组进行浓度测定,结果见表1。
表1、10例样本基因组DNA纯度与浓度检测结果
编号 | Nanodrop 2000浓度(ng/uL) | OD260/280 | Qubit浓度(ng/uL) |
1 | 68.7 | 1.82 | 63.4 |
2 | 80.2 | 1.88 | 69.2 |
3 | 45.8 | 1.90 | 41.0 |
4 | 200.3 | 1.67 | 170 |
5 | 89.7 | 1.90 | 73.8 |
6 | 130.2 | 1.80 | 112 |
7 | 65.3 | 1.74 | 57 |
8 | 48.2 | 1.62 | 37.8 |
9 | 110.5 | 1.73 | 92.6 |
10 | 66.5 | 1.78 | 57.6 |
2、多重PCR捕获文库构建
使用实施例2中的DMD基因变异检测试剂盒进行多重PCR捕获文库的构建。
(1)第一轮多重PCR扩增
(a)按照下表配制20uL多重PCR反应体系:
每个样本需同时进行A、B管多重PCR扩增,将以上PCR混合物充分混匀,短暂离心后,置于PCR仪上。
(b)运行以下PCR反应程序:
98℃3min;98℃15s,60℃30s,72℃2min(15cycles);72℃5min,4℃hold;
(2)第一轮多重PCR产物纯化
利用Beckman纯化磁珠(AMPure XP Beads)或其他功能等效的纯化试剂盒对PCR产物进行纯化。该纯化可有效的富集目标区域片段,除去多于的PCR引物及二聚体,降低非特异性产物的量。
(a)将以上A、B两管扩增产物等体积混合(共40μl),涡旋混匀,并瞬时离心,然后加入40μl AMPure XP Beads(需事先从冰箱中取出并平衡至室温后使用),涡旋混匀,室温放置5分钟;
(b)以上离心管瞬时离心,放置于磁力架上,静置5分钟,待溶液澄清后,小心吸去上清(避免吸起磁珠),弃上清;
(c)然后加入200μl 80%的乙醇,磁力架反复(不少于两次)在1.5ml离心管不同的两面来回吸附磁珠(即在磁力架上旋转1.5ml离心管,使得磁珠在80%乙醇中来回游移),以充分洗涤磁珠,然后在磁力架上,静置1分钟;
(d)小心吸去上清(避免吸起磁珠),再次加入200μl 80%的无水乙醇,重复步骤“(c)”;
(e)用移液枪小心吸除上清,尽可能吸取干净(避免吸起磁珠),然后将1.5ml离心管置于通风橱中,静置5分钟,使乙醇挥发完全;
注:乙醇必须去除干净,否则残留的乙醇会严重影响得率和后续实验;
(f)向离心管中加入10μl去离子水,瞬时涡旋,充分悬浮磁珠,室温放置5分钟;
(g)将以上离心管瞬时离心,置于磁力架上,静置5分钟,直至溶液澄清,小心吸取9μl上清,至新的PCR管中(避免吸起磁珠,若不小心吸取磁珠,则重复步骤“(6)”),该纯化产物,应立即进行文库富集的扩增。
(3)第二轮PCR扩增
(a)按照下表配制20uL文库富集的扩增反应体系:
其中P5通用引物(SEQ ID NO:263)序列为:5’-AATGATACGGCGACCACCG AGATCTACACACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’;
P7index引物(SEQ ID NO:264、SEQ ID NO:265)序为:5’-CAAGCAGAAG ACGGCATACGAGATNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3’;序列中N表示ATCG任意碱基,NNNNNN用于识别不同样本构建的文库。以上引物也由英潍捷基(上海)贸易有限公司合成。
将以上PCR混合物充分混匀,短暂离心后,置于PCR仪上。
(b)运行以下PCR反应程序:
PCR反应程序:
98℃3min;98℃15s,60℃30s,72℃2min(8cycles);72℃5min,4℃hold;
(3)第二轮PCR产物的纯化
将富集文库的扩增产物用Beckman纯化磁珠(AMPure XP Beads)进行纯化,扩增产物与磁珠添加比例为1:1,操作步骤与“第一轮多重PCR产物纯化”一致,获得文库。
3、文库质控
对文库进行Qubit定量,定量结果见表3。
用Qsep100全自动核酸蛋白分析系统(BiOptic)对文库进行片段分布检测,正常文库片段分布在200-400bp之间,100-500bp片段的平均大小为330-4000bp,本实施例中单个文库经Qsep100全自动核酸蛋白分析系统检测结果的平均大小见表2,以上10个文库质量合格。
表2、上机前文库质控
编号 | Qubit浓度(ng/uL) | 100-500bp平均大小(bp) | 是否合格 |
1 | 4.18 | 377.57 | 合格 |
2 | 3.30 | 382.71 | 合格 |
3 | 3.22 | 375.29 | 合格 |
4 | 7.64 | 385.04 | 合格 |
5 | 8.92 | 367.06 | 合格 |
6 | 5.54 | 343.22 | 合格 |
7 | 3.1 | 366.45 | 合格 |
8 | 2.64 | 390.48 | 合格 |
9 | 3.00 | 371.78 | 合格 |
10 | 3.04 | 345.88 | 合格 |
4、高通量测序
上机文库稀释和上机操作流程详见MiSeq system guide操作说明。
5、生物信息分析
上述10个样本经过测序后,对下机的原始数据进行质量控制和数据筛选,以获得后续分析的fastq文件,利用序列比对软件将fastq文件中的序列比对到参考基因组上,再利用call变异软件进行变异检测。经10个样本的下机数据质量详见表3;经比对后,进行SNV、Indel的识别分析;CNV分析根据以往阴性(非CNV)样本获得的扩增子测序深度,与本次测序结果进行归一化处理后,按测序深度分析CNV(缺失/重复)。
本实施例10例样本的检测结果与常规方法(点突变检测的常规方法为:Sanger测序法;外显子缺失/重复的常规方法为:多重连接依赖探针扩增法MLPA)对比结果见表4,本发明检测结果与常规方法检测结果一致,本发明能同时对点突变、外显子缺失/重复进行准确检测。
表3、10例样本下机数据质量统计
文库编号 | 平均覆盖度 | Map率 | 均一性 |
1 | 839.4 | 74.38% | 99.1% |
2 | 468.9 | 65.0% | 98.7% |
3 | 433.7 | 58.23% | 98.8% |
4 | 791.4 | 71.26% | 99.0% |
5 | 1040.5 | 75.88% | 100% |
6 | 861.8 | 73.68% | 99.4% |
7 | 639.3 | 65.10% | 98.8% |
8 | 482.8 | 58.83% | 100% |
9 | 811.3 | 67.90% | 100% |
10 | 846.2 | 68.99% | 98.8% |
表4、10例样本DMD变异检测结果与常规方法检测结果对比
编号 | 本发明检测结果 | 常规方法检测结果 | 判定结果 |
1 | ex4.c94.1G>A | ex4.c94.1G>A | 一致 |
2 | ex52~54缺失 | ex52~54缺失 | 一致 |
3 | ex3~25缺失 | ex3~25缺失 | 一致 |
4 | ex30.c4150G>T | ex30.c4150G>T | 一致 |
5 | ex5~7扩增 | ex5~7扩增 | 一致 |
6 | 阴性 | 阴性 | 一致 |
7 | 阴性 | 阴性 | 一致 |
8 | ex18~44缺失 | ex18~44缺失 | 一致 |
9 | ex57.c8498del | ex57.c8498del | 一致 |
10 | 阴性 | 阴性 | 一致 |
基于以上结果,本发明的一种基于高通量测序技术检测DMD基因变异是试剂盒,包括本发明的DMD基因捕获多重PCR引物,可实现DMD基因变异信息的检测。
以上内容是结合具体的实施方式对本发明作进一步的详细说明,本发明也不限于上述举例。本领域技术人员在本发明的实质范围内,作出的变化、改型、添加或替换,也应属于本发明的保护范围。最后所应当说明的是,以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制,尽管参照较佳实施例对本发明做了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的实质和范围。
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<213> 人工序列(Artificial Sequence)
<400> 57
acacgacgct cttccgatct accactcact ttcagaatgt acat 44
<210> 58
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 58
gacgtgtgct cttccgatct ctcaatagga aattgatcgg gaat 44
<210> 59
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 59
acacgacgct cttccgatct gccaaccaca ctatcaagta ttttt 45
<210> 60
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 60
gacgtgtgct cttccgatct gatgatggtg atgactgaag ac 42
<210> 61
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 61
acacgacgct cttccgatct cagttcagct catttgtctg aatt 44
<210> 62
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 62
gacgtgtgct cttccgatct aggtcggatt gacattattc atag 44
<210> 63
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 63
acacgacgct cttccgatct aggcaaactc tctcatcctg a 41
<210> 64
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 64
gacgtgtgct cttccgatct gcggcgtttt cattatgata taaag 45
<210> 65
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 65
acacgacgct cttccgatct tttcaccctg cttataatct ctc 43
<210> 66
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 66
gacgtgtgct cttccgatct aaagacatgg ggcttcattt ttgt 44
<210> 67
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 67
acacgacgct cttccgatct gaactatgaa taacctaatg ggc 43
<210> 68
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 68
gacgtgtgct cttccgatct gtcccagttt gcattaacaa atagt 45
<210> 69
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 69
acacgacgct cttccgatct aatccacata ccagcctcct 40
<210> 70
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 70
gacgtgtgct cttccgatct aagacaaggt agttggaatt gtg 43
<210> 71
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 71
acacgacgct cttccgatct ctctctatcc aacctcctgt aa 42
<210> 72
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 72
gacgtgtgct cttccgatct gaccttgggc agcttgaaaa aa 42
<210> 73
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 73
acacgacgct cttccgatct ttgcctaaga actggtggga 40
<210> 74
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 74
gacgtgtgct cttccgatct actctggtga cacaacctgt 40
<210> 75
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 75
acacgacgct cttccgatct ctgttcattt cagctttaac gtgat 45
<210> 76
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 76
gacgtgtgct cttccgatct cagaatcagt gggatgaagt acaa 44
<210> 77
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 77
acacgacgct cttccgatct cagtttagct ggattggaaa aacaa 45
<210> 78
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 78
gacgtgtgct cttccgatct taagacactc caactagaga tttc 44
<210> 79
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 79
acacgacgct cttccgatct caactatttt gttttgtccc tgg 43
<210> 80
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 80
gacgtgtgct cttccgatct taattgcatc tgaacatttg gtcc 44
<210> 81
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 81
acacgacgct cttccgatct acatgtgaga taccagttac ttg 43
<210> 82
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 82
gacgtgtgct cttccgatct catcgcttgt ttcttttgtt tggta 45
<210> 83
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 83
acacgacgct cttccgatct cccttgggtg agaagagtaa a 41
<210> 84
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 84
gacgtgtgct cttccgatct tgacatggta cgctgctgtt 40
<210> 85
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 85
acacgacgct cttccgatct tctagcttta actttgtggg aaga 44
<210> 86
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 86
gacgtgtgct cttccgatct aggaggtcaa tactgagtgg 40
<210> 87
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 87
acacgacgct cttccgatct atatactaag tcttagttgc ttcca 45
<210> 88
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 88
gacgtgtgct cttccgatct ctctgaaaga gaacgtgagc 40
<210> 89
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 89
acacgacgct cttccgatct tgcttctact gcatctgata aaaga 45
<210> 90
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 90
gacgtgtgct cttccgatct aatgttgctt tagtgttctc agtct 45
<210> 91
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 91
acacgacgct cttccgatct ccaggctaat gtcgcatttt aaa 43
<210> 92
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 92
gacgtgtgct cttccgatct aggacagaaa tttcagaaca gca 43
<210> 93
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 93
acacgacgct cttccgatct aagctgactg tcaattttgg atag 44
<210> 94
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 94
gacgtgtgct cttccgatct gattccgaat ggttcaaaag caaaa 45
<210> 95
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 95
acacgacgct cttccgatct gcaacaggaa ttgtgacagc t 41
<210> 96
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 96
gacgtgtgct cttccgatct ggcgtataca cactatatgt tttct 45
<210> 97
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 97
acacgacgct cttccgatct attaggtcca cagcttccag 40
<210> 98
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 98
gacgtgtgct cttccgatct ttgttttttg cagtggatct cttga 45
<210> 99
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 99
acacgacgct cttccgatct ctgtcaaata agaacagtct gtca 44
<210> 100
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 100
gacgtgtgct cttccgatct aaacagtggt caatagagtt cac 43
<210> 101
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 101
acacgacgct cttccgatct caaatatccc aaatcccata ccta 44
<210> 102
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 102
gacgtgtgct cttccgatct ctgtgttttg actgttgcaa ttttc 45
<210> 103
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 103
acacgacgct cttccgatct tcgatgttcc agcccaatca 40
<210> 104
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 104
gacgtgtgct cttccgatct tcctgtcttt gcaggctaat aata 44
<210> 105
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 105
acacgacgct cttccgatct gtgacaaatc acagcattat acatg 45
<210> 106
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 106
gacgtgtgct cttccgatct ggactaatga acattgcttt ctgtt 45
<210> 107
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 107
acacgacgct cttccgatct gtttgcattt gggagtgaag g 41
<210> 108
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 108
gacgtgtgct cttccgatct aacaagtgtc atggggcaga 40
<210> 109
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 109
acacgacgct cttccgatct ctgaaatgaa ggaaggggaa ttaa 44
<210> 110
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 110
gacgtgtgct cttccgatct taattcactt tacaccttgg tttgg 45
<210> 111
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 111
acacgacgct cttccgatct aacatgcagt ggaacggcat 40
<210> 112
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 112
gacgtgtgct cttccgatct aaagcattct aggccatgtg tataa 45
<210> 113
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 113
acacgacgct cttccgatct atgctaattc ctatatcctg tgcta 45
<210> 114
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 114
gacgtgtgct cttccgatct gcctcaaatg gttaagccaa c 41
<210> 115
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 115
acacgacgct cttccgatct gttcttcatc atactattgc atcca 45
<210> 116
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 116
gacgtgtgct cttccgatct aagtttgaac caggactccc 40
<210> 117
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 117
acacgacgct cttccgatct gatgcttgtt gcacctataa aaagt 45
<210> 118
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 118
gacgtgtgct cttccgatct atgaccgtct aaagcagcag 40
<210> 119
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 119
acacgacgct cttccgatct gagtcaaaca cttacggcca aat 43
<210> 120
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 120
gacgtgtgct cttccgatct agggtcaaaa tttatgagtc ctg 43
<210> 121
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 121
acacgacgct cttccgatct tgcgtgttgg cttccatatg 40
<210> 122
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 122
gacgtgtgct cttccgatct cctcattatt aagcttgagg gtttt 45
<210> 123
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 123
acacgacgct cttccgatct tcatgagctg caagtggaga 40
<210> 124
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 124
gacgtgtgct cttccgatct ttccagcaga gaataaatgg tcat 44
<210> 125
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 125
acacgacgct cttccgatct gggaatcagg agttgtaaaa cattt 45
<210> 126
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 126
gacgtgtgct cttccgatct acgtgtaatg gcttcattaa taagg 45
<210> 127
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 127
acacgacgct cttccgatct aaagacttgg ggaaggaaaa aattc 45
<210> 128
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 128
gacgtgtgct cttccgatct attgctgaca acataatgaa gagg 44
<210> 129
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 129
acacgacgct cttccgatct atttaggaaa aatcatggcc tgct 44
<210> 130
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 130
gacgtgtgct cttccgatct gatcatattg ggataaagac gtgt 44
<210> 131
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 131
acacgacgct cttccgatct atgcttcttt gcaaactact gtg 43
<210> 132
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 132
gacgtgtgct cttccgatct agatctggga ggcaattacc t 41
<210> 133
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 133
acacgacgct cttccgatct gacactatga gagaaataaa acgga 45
<210> 134
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 134
gacgtgtgct cttccgatct accagcatca ctcatgttta attc 44
<210> 135
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 135
acacgacgct cttccgatct cctagtcatt ctactagatg tcata 45
<210> 136
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 136
gacgtgtgct cttccgatct cgtcatcttc ggcagattaa tta 43
<210> 137
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 137
acacgacgct cttccgatct gaactggtct gatttacaag agaat 45
<210> 138
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 138
gacgtgtgct cttccgatct tatattaatg cctcacaggc tctg 44
<210> 139
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 139
acacgacgct cttccgatct gagcagggtt tgttattgtt agaaa 45
<210> 140
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 140
gacgtgtgct cttccgatct caatgctaag tctctgaaaa tagct 45
<210> 141
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 141
acacgacgct cttccgatct tgagagcatt caaagccagg 40
<210> 142
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 142
gacgtgtgct cttccgatct cttctggtag aatctattat gtctc 45
<210> 143
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 143
acacgacgct cttccgatct tcaggatcga gtagtttctc tat 43
<210> 144
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 144
gacgtgtgct cttccgatct cacccaatga gatcttccat taaaa 45
<210> 145
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 145
acacgacgct cttccgatct cctttacaca ctttacctgt tga 43
<210> 146
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 146
gacgtgtgct cttccgatct actgttcatt aatctatcgt cttcc 45
<210> 147
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 147
gacgtgtgct cttccgatct gatgatggtg atgactgaag ac 42
<210> 148
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 148
gacgtgtgct cttccgatct gatgatggtg atgactgaag ac 42
<210> 149
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 149
acacgacgct cttccgatct ctcatgagta tgaaactggt cttt 44
<210> 150
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 150
gacgtgtgct cttccgatct gataatgcca gtggacagtc 40
<210> 151
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 151
acacgacgct cttccgatct cccatcttga atttaggaga ttcat 45
<210> 152
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 152
gacgtgtgct cttccgatct taaaagtggt tttgggattc tgc 43
<210> 153
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 153
acacgacgct cttccgatct tcaagatcag gtccaagagg 40
<210> 154
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 154
gacgtgtgct cttccgatct agtctgagaa ataatagttc cgg 43
<210> 155
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 155
acacgacgct cttccgatct ttccaaagca gcagttgcgt 40
<210> 156
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 156
gacgtgtgct cttccgatct gtcttctcta gatactttcc agtta 45
<210> 157
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 157
acacgacgct cttccgatct gccagttttt gaagacttga taaca 45
<210> 158
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 158
gacgtgtgct cttccgatct agatcgatgg gcaaacatct g 41
<210> 159
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 159
acacgacgct cttccgatct ctgtgctgta ctcttttcaa gttt 44
<210> 160
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 160
gacgtgtgct cttccgatct tgcgggcacg attgaaatat ttt 43
<210> 161
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 161
acacgacgct cttccgatct accttttcct aatttcagaa tccac 45
<210> 162
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 162
gacgtgtgct cttccgatct gtgctatttt gatctgaagg tcaa 44
<210> 163
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 163
acacgacgct cttccgatct cgcattaatc tatcaactag tagaa 45
<210> 164
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 164
gacgtgtgct cttccgatct gagtctcaga ttgagaaaag aatga 45
<210> 165
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 165
acacgacgct cttccgatct acaagtgctt gtctgatata attca 45
<210> 166
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 166
gacgtgtgct cttccgatct agattcacgt gataagctga cagag 45
<210> 167
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 167
acacgacgct cttccgatct gtggtgggtt ggattttcaa c 41
<210> 168
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 168
gacgtgtgct cttccgatct ggtttgaaat cattcatgtg gtgaa 45
<210> 169
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 169
acacgacgct cttccgatct tggcctgcac atcagaaaag 40
<210> 170
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 170
gacgtgtgct cttccgatct gtctatactg tcatggatgt ttctt 45
<210> 171
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 171
acacgacgct cttccgatct agtctctgct ccatgatttc atagt 45
<210> 172
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 172
gacgtgtgct cttccgatct ctggcatatt acacatgtag cag 43
<210> 173
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 173
acacgacgct cttccgatct catttgctcc tctagctttt gaca 44
<210> 174
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 174
gacgtgtgct cttccgatct gaagatcatc tactttgttt acatg 45
<210> 175
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 175
acacgacgct cttccgatct caaaatccac cccagctgta 40
<210> 176
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 176
gacgtgtgct cttccgatct gacatccttt aaacatggga attgt 45
<210> 177
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 177
acacgacgct cttccgatct gttctttgag ttctgtctca agt 43
<210> 178
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 178
gacgtgtgct cttccgatct gaacattttt gcaagactgt tagg 44
<210> 179
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 179
acacgacgct cttccgatct tgctttctgt aattcatctg gag 43
<210> 180
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 180
gacgtgtgct cttccgatct ccccatgagt tattttctta tgc 43
<210> 181
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 181
acacgacgct cttccgatct aaagaccaag aaaagcaact gact 44
<210> 182
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 182
gacgtgtgct cttccgatct taactgggat gttgtgagaa agaa 44
<210> 183
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 183
acacgacgct cttccgatct atttacaact tacatctagc acctc 45
<210> 184
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 184
gacgtgtgct cttccgatct atgatgaaac tgtctagctg catt 44
<210> 185
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 185
acacgacgct cttccgatct tcacttatct tcatacctct tcatg 45
<210> 186
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 186
gacgtgtgct cttccgatct ctccttggtg tttttaatcc caa 43
<210> 187
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 187
acacgacgct cttccgatct ggaagctgat tcccagatgt a 41
<210> 188
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 188
gacgtgtgct cttccgatct ctgtcttaca aaaaggtgat tgtg 44
<210> 189
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 189
acacgacgct cttccgatct aatgtcctca aatccaatct tgc 43
<210> 190
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 190
gacgtgtgct cttccgatct tacatggtag aggtggttga g 41
<210> 191
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 191
acacgacgct cttccgatct tcaaggggta cctgcgtatt 40
<210> 192
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 192
gacgtgtgct cttccgatct cttcagaaat aaaggcagat ctatc 45
<210> 193
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 193
acacgacgct cttccgatct taaggaaagt ggaaagaagt gtttg 45
<210> 194
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 194
gacgtgtgct cttccgatct tcaaacatgg aatagcaatt aaggg 45
<210> 195
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 195
acacgacgct cttccgatct attccttcaa ctgctgatct ctttg 45
<210> 196
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 196
gacgtgtgct cttccgatct tgtcatctgc cctagacagt ttt 43
<210> 197
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 197
acacgacgct cttccgatct ctatccagtt actattcaga agact 45
<210> 198
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 198
gacgtgtgct cttccgatct cccaacaatg aagcttcagt atata 45
<210> 199
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 199
acacgacgct cttccgatct ctggggtttc tttttctctg attc 44
<210> 200
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 200
gacgtgtgct cttccgatct aattctacgt atatgctggc tctta 45
<210> 201
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 201
acacgacgct cttccgatct atttagcaca agtttccacc ttg 43
<210> 202
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 202
gacgtgtgct cttccgatct ttctcgctca cttgctcact 40
<210> 203
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 203
acacgacgct cttccgatct tgctctgaaa attcagttgg agac 44
<210> 204
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 204
gacgtgtgct cttccgatct gcatgtgatt agtttagcaa cagg 44
<210> 205
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 205
acacgacgct cttccgatct ccacaggcaa ggtatattat aattt 45
<210> 206
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 206
gacgtgtgct cttccgatct gcacagtatc ttataatagc attcc 45
<210> 207
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 207
acacgacgct cttccgatct cgttaataga aacaagaaca tcaac 45
<210> 208
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 208
gacgtgtgct cttccgatct agatgaggga cgcaaattat tttga 45
<210> 209
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 209
acacgacgct cttccgatct acaatttgtg caaagttgag tcttc 45
<210> 210
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 210
gacgtgtgct cttccgatct acttcaaagg atgacaataa aggga 45
<210> 211
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 211
acacgacgct cttccgatct agactcctct tgcttaaaga gat 43
<210> 212
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 212
gacgtgtgct cttccgatct ttttcagccc agtttgtaga ttcaa 45
<210> 213
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 213
acacgacgct cttccgatct tttttcccat tggaaatcaa gctg 44
<210> 214
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 214
gacgtgtgct cttccgatct atgtcaaagt cactggactt cat 43
<210> 215
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 215
acacgacgct cttccgatct catgttccca attctcagga att 43
<210> 216
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 216
gacgtgtgct cttccgatct gtgtgtacat gctaggtgtg tatat 45
<210> 217
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 217
acacgacgct cttccgatct agattcaggc ttcccaattt ttc 43
<210> 218
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 218
gacgtgtgct cttccgatct tctttctgtc ttgtatcctt tgga 44
<210> 219
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 219
acacgacgct cttccgatct gcttcctcca accataaaac aaatt 45
<210> 220
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 220
gacgtgtgct cttccgatct gcctggccag gaatttttga a 41
<210> 221
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 221
acacgacgct cttccgatct tttatccact ggagatttgt ctg 43
<210> 222
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 222
gacgtgtgct cttccgatct cagctctgtg ctcagataga a 41
<210> 223
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 223
acacgacgct cttccgatct caaatggtcc ttcttggttt gg 42
<210> 224
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 224
gacgtgtgct cttccgatct gctgctaaaa taacacaaat cagta 45
<210> 225
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 225
acacgacgct cttccgatct cactgattat aaatagtcca cgtca 45
<210> 226
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 226
gacgtgtgct cttccgatct ttgtgccctt atgtaccagg 40
<210> 227
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 227
acacgacgct cttccgatct tgaaacaaat tttctctctc accca 45
<210> 228
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 228
gacgtgtgct cttccgatct ctacccacta aagttaattt agaag 45
<210> 229
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 229
acacgacgct cttccgatct caccctctgt gattttataa cttg 44
<210> 230
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 230
gacgtgtgct cttccgatct gtttggctca aattgttact cttca 45
<210> 231
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 231
acacgacgct cttccgatct gactgaaaat ctcagcacaa ttgtt 45
<210> 232
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 232
gacgtgtgct cttccgatct tggctggtct cacaattgta cttt 44
<210> 233
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 233
acacgacgct cttccgatct accctccttc catgactcaa 40
<210> 234
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 234
gacgtgtgct cttccgatct atcatcctag ccataacaca atgaa 45
<210> 235
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 235
acacgacgct cttccgatct ggcattgata ttctctgtta tcatg 45
<210> 236
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 236
gacgtgtgct cttccgatct gggttacctt atactgtcat gat 43
<210> 237
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 237
acacgacgct cttccgatct tttccttacg ggtagcatcc 40
<210> 238
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 238
gacgtgtgct cttccgatct gactgcacaa tattatagtt gttgc 45
<210> 239
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 239
acacgacgct cttccgatct ttgagagact ttttccgaag ttc 43
<210> 240
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 240
gacgtgtgct cttccgatct gtatcttctt gttagtttag cctga 45
<210> 241
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 241
acacgacgct cttccgatct aaagtcgcct ccaataggtg 40
<210> 242
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 242
gacgtgtgct cttccgatct ctcaaaattg ccatcaaact atcct 45
<210> 243
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 243
acacgacgct cttccgatct atgagagcta tccagaccct 40
<210> 244
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 244
gacgtgtgct cttccgatct gaaattgacc tgggagtttc ataaa 45
<210> 245
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 245
acacgacgct cttccgatct agcttgaggt ccagctcatc 40
<210> 246
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 246
acacgacgct cttccgatct agcttgaggt ccagctcatc 40
<210> 247
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 247
acacgacgct cttccgatct aaggacaaca atgtttacaa tgtgc 45
<210> 248
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 248
gacgtgtgct cttccgatct ggacacaaac attcaaaccg tag 43
<210> 249
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 249
acacgacgct cttccgatct ccagttcaga cacatatcca c 41
<210> 250
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 250
gacgtgtgct cttccgatct ttcacgtttg gtgtttatta aaggg 45
<210> 251
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 251
acacgacgct cttccgatct gttacctaga aggtgaataa cttac 45
<210> 252
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 252
gacgtgtgct cttccgatct ccccactctg tggaaatact 40
<210> 253
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 253
acacgacgct cttccgatct ttggttccta atacctgaat ccaat 45
<210> 254
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 254
gacgtgtgct cttccgatct gctttgcaac cattgttctt tct 43
<210> 255
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 255
acacgacgct cttccgatct tctctctagc tcccctcttt 40
<210> 256
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 256
gacgtgtgct cttccgatct agtagaatgg tagatcacaa cct 43
<210> 257
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 257
acacgacgct cttccgatct tttattgtgg tcttccagga tttg 44
<210> 258
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 258
gacgtgtgct cttccgatct gatgcatcta tctaccatgg tatat 45
<210> 259
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 259
acacgacgct cttccgatct atcatctcac ttctcatttg ctttc 45
<210> 260
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 260
gacgtgtgct cttccgatct tgttttgttc tttttgtggc agg 43
<210> 261
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 261
acacgacgct cttccgatct 20
<210> 262
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 262
gacgtgtgct cttccgatct 20
<210> 263
<211> 62
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 263
aatgatacgg cgaccaccga gatctacaca cactctttcc ctacacgacg ctcttccgat 60
ct 62
<210> 264
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 264
caagcagaag acggcatacg agat 24
<210> 265
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 265
gtgactggag ttcagacgtg tgctcttccg atct 34
Claims (4)
1.一种基于高通量测序技术检测DMD基因变异的PCR引物组,其特征在于,由SEQ IDNO:1~SEQ ID NO:260所示的核苷酸序列及其5’端连接的Illumina测序平台建库所需的序列组成;
正向引物5’端连接的序列如SEQ ID NO:261所示;
反向引物5’端连接的序列如SEQ ID NO:262所示。
2.一种检测DMD基因变异的试剂盒,其特征在于,该试剂盒中包括权利要求1所述的基于高通量测序技术检测DMD基因变异的PCR引物组。
3.检测DMD基因变异的方法,其特征在于,所述方法用于非疾病诊断目的,该方法包括以下步骤:
(1)以权利要求1所述的基于高通量测序技术检测DMD基因变异的PCR引物组为引物,以样本DNA为模板,进行第一轮多重PCR,得到第一轮PCR产物;
(2)使用P5通用引物、P7index引物,以第一轮PCR产物为模板进行第二轮PCR扩增,对第二轮扩增产物进行纯化,获得DMD基因变异测序文库;
(3)文库质控、高通量测序,对下机数据进行生物信息学分析。
4.根据权利要求3所述检测DMD基因变异的方法,其特征在于,所述的P5通用引物的核苷酸序列如SEQ ID NO:263所示,所述的P7index引物的核苷酸序列为SEQ ID NO:264、六个随机碱基、SEQ ID NO:265依次连接后所得到的序列。
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CN110551804B (zh) * | 2019-07-19 | 2023-01-17 | 武汉拜肯生物科技有限公司 | 一种基于供体与受体的嵌合体的嵌合率的检测方法 |
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CN111139297B (zh) * | 2020-04-02 | 2020-07-03 | 北京大学第三医院(北京大学第三临床医学院) | 用于dmd的植入前胚胎遗传学诊断和产前诊断的试剂盒 |
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CN112458195B (zh) * | 2020-12-28 | 2023-03-24 | 广州迈景基因医学科技有限公司 | 基于高通量测序检测性传播病原体的多重pcr引物组、试剂盒及其方法 |
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