本发明涉及基因工程
技术领域：
，具体涉及一种前列腺癌检测探针序列及其应用。
背景技术：
：pca3即新型前列腺癌抗原3，pca3在1995年由johnshopkinshospital(baltimore)和radbouduniversity(nijmegen,netherlands)合作研究发现，最初命名为dd3(differentialdisplayclone，3)因为使用差别显示分析(在那时是一种新的描述方法，用于识别不同组织中的基因表达)对比正常vs癌变前列腺组织的mrna表达模式，发现一种mrna在前列腺癌症中高特异性表达。pca3在95％的检测的全部前列腺癌中过度表达，可从癌变前列腺细胞中无害测定，准确度接近100％。此外，在前列腺外组织(良性和恶性)中尚未检测到pca3转录，证明pca3是目前已知前列腺癌最具特异性的pca标志物。不像血清psa，pca3不受年龄，前列腺体积或其他前列腺病(前列腺炎)的影响。bussemakers等通过northernblot法分析正常前列腺、良性前列腺增生和前列腺癌细胞及其转移灶发现，pca3基因特异性地高表达于人类前列腺癌细胞和转移坏死灶中，在正常前列腺、良性前列腺增生细胞中不表达或低表达。且pca3mrna的表达水平与前列腺癌gleason病理分级有关。相比之下，在良性和恶性前列腺疾病的患者之间，psamrna水平差异无显著性。bussemakers等还发现在正常人，除前列腺外的18种组织器官中均检测不到pca3基因表达。hes-sels等还对108例psa>3ng/ml的患者行前列腺穿刺活检，用时间分辨荧光定量rt-pcr检测前列腺穿刺活检组织中的pca3mrna，发现pca3mrna在非恶性前列腺组织和良性前列腺增生组织之间差异无显著性。在前列腺癌中，癌细胞数大于10％的前列腺癌标本的pca3mrna拷贝数是非恶性前列腺组织的66倍。癌细胞数少于10％的前列腺癌标本的pca3mrna拷贝数是非恶性前列腺组织的11倍。landers等用实时定量pcr检测前列腺癌组织中的pca3mrna表达，证实前列腺癌组织中dd3是良性前列腺增生组织的140倍。由此可见，pca3特异性高表达于前列腺癌细胞，在正常前列腺或良性前列腺增生组织中不表达或低表达。因此pca3适合作为前列腺癌特异性标志物。现有专利cn105861669a公开了一种快速捕获杂交靶标物分支链dna信号放大的检测方法(quantimat技术)，其灵敏度和耗时均优于bdna技术。在其实施例中提供了检测高危型人乳头瘤病毒(hpv)16型e6e7区域的相关试验。但是不同的quantimat探针序列对于前列腺癌、宫颈癌等各种癌症的特异性有着最为直接的影响，依照probedesignerv1.0工具设计出的针对pca3的quantimat探针序列，在检测前列腺癌时并不能保证具备较高的特异性，致使定量限(loq)、检测限(lod)以及灵敏度受到影响。技术实现要素：有鉴于此，本发明的目的在于提供一种前列腺癌检测探针序列及其应用，使得所述探针序列能够检测前列腺癌，并具备较高的特异性，可应用于前列腺癌检测相关应用中。为实现上述发明目的，本发明提供如下技术方案：一种前列腺癌检测探针序列，包括捕获延伸探针、k型标记延伸探针和封闭探针；其中，所述捕获延伸探针序列如seqidno.1-14和seqidno.78-85所示，所述k型标记延伸探针如seqidno.15-64和seqidno.86-131所示，所述封闭探针如seqidno.65-77和seqidno.132-148所示。针对类似于bdna与quantimat技术中相关探针在检测前列腺癌时特异性不佳的问题，本发明提供了一组经过人为优化后的高特异性探针，能够在定量限(loq)、检测限(lod)以及灵敏度方面具备较优异的效果，同时检测信号和阳性细胞之间的线性关系也较优。由此，本发明提供了所述探针序列在制备检测前列腺癌试剂盒中的应用。根据上述应用，本发明提供了一种检测前列腺癌的试剂盒，包括本发明所述探针序列。更为具体地，所述试剂盒还包括信号放大前体、信号放大体、碱性磷酸酶(ap)标记的报告探针、化学发光底物以及偶联有通用探针的载体中的一种或两种以上；其中，所述通用探针与本发明所述捕获延伸探针的部分序列互补配对；所述信号放大前体包括放大前体前置引导区序列和多次重复的放大体前置引导区互补序列；所述信号放大体包括放大体前置引导区序列和多次重复的报告探针互补序列；所述放大前体前置引导区序列与本发明所述k型标记延伸探针部分序列互补配对；所述放大体前置引导区序列与所述多次重复的放大体前置引导区互补序列互补配对；所述碱性磷酸酶标记的报告探针与所述多次重复的报告探针重复区互补序列互补配对。根据本领域经常使用的各种载体形式，本发明所述载体优选为磁珠或细胞培养板，通用探针可通过一些修饰，比如氨基修饰既可以与磁珠表面的基团相偶联，也可通过包被液包被在细胞培养板上。在本发明具体实施方式中，本发明提供了一种具体的通用探针，其序列如seqidno.149所示。在发光底物的选择中，本发明选择金刚烷衍生物作为ap报告探针的发光底物，用于化学发光分析仪检测。本发明所述信号放大前体、信号放大体、碱性磷酸酶(ap)标记的报告探针可对应参考专利cn105861669a中公开的初级信号放大链、次级信号放大链和ap偶联的报告探针中的描述或具体的序列设计。作为优选，本发明所述多次重复为20次重复。作为优选，所述通用探针与本发明所述捕获延伸探针的部分序列互补配对，其中所述部分序列为本发明所述捕获延伸探针的3’端至5’方向的16bp序列；在本发明具体实施方式中，所有末端16bp序列为ctcttggaaagaaagt的探针都属于捕获延伸探针，其能够和seqidno.149所示的通用探针互补配对结合。作为优选，所述放大前体前置引导区序列与本发明所述k型标记延伸探针部分序列互补配对，其中所述部分序列为本发明所述k型标记延伸探针的3’端至5’方向的14bp序列；在本发明具体实施方式中，所有末端14bp序列为ctgagtcaaagcat或gaagttaccgtttt的探针都属于k型标记延伸探针，其能够和所述放大前体前置引导区序列互补配对结合，由此也可以设计出所述放大前体前置引导区序列。通过probedesignerv1.0工具(http://www.probedesigner.cn/)设计出一套针对前列腺癌pca3的quantimat探针序列作为对照，为保证可对比性，对照探针中所有末端16bp序列为ctcttggaaagaaagt的探针都属于捕获延伸探针(ce)，所有末端14bp序列为ctgagtcaaagcat或gaagttaccgtttt的探针都属于k型标记延伸探针(le)，其余为封闭探针(bl)，具体如下：捕获延伸探针：p0-ce：agggcactcttgtgagccactttttctcttggaaagaaagt；p640-ce：aagaactctgagtgatatcaacattaggtttttctcttggaaagaaagt；p701-ce：ggtcaaaaggaaccaagatacaaagaactttttctcttggaaagaaagt；p870-ce：tctcctcttgacacatattagcttctagtttttctcttggaaagaaagt；p973-ce：ttccccacaagaatttcaacgactctttttctcttggaaagaaagt；p1141-ce：attatcttctctttctttcacctccctgtttttctcttggaaagaaagt；p1217-ce：tagccatgcaaatgagaaacccagtttttctcttggaaagaaagt；p1286-ce：atgactgattacgcctcatgggtgtttttctcttggaaagaaagt；p1758-ce：gagggagagcattaggacaaatactttttctcttggaaagaaagt；k型标记延伸探针：p20-le：tttagggttcactcctggcaataaagtttttgaagttaccgtttt；p46-le：aatttacaaagagctactcaggaccagtttttctgagtcaaagcat；p73-le：ttgttaagagctctgtgtgtgtgtgtttttgaagttaccgtttt；p98-le：tgtgtgtgtgagtgtacatgccaatttttctgagtcaaagcat；p145-le：cattatttcagacttaaaaacaagcatgttttctttttgaagttaccgtttt；p178-le：aaatggcactatgagctgccaatgtttttctgagtcaaagcat；p202-le：atgtatcaccaccatatctcattattctctttttgaagttaccgtttt；p231-le：cagtaaatgtgataataatgtcatctgttaacatttttctgagtcaaagcat；p264-le：taaaaaaagtttgacttcacaaaagcagctgtttttgaagttaccgtttt；p295-le：gaaatggacaaccacaatatgcataaatctttttctgagtcaaagcat；p324-le：taactcctaccatcagctacacactttttgaagttaccgtttt；p348-le：tgcttgacatatattgttagaagcaccttttttctgagtcaaagcat；p400-le：aaatacttgcattaggtctcagctggtttttgaagttaccgtttt；p426-le：ggctgtgcatcaggcggttttttttctgagtcaaagcat；p446-le：gagaaatattcaattctcagcagaagcctttttgaagttaccgtttt；p474-le：agaatttgaattccctcatcttttaggaatctttttctgagtcaaagcat；p579-le：tgttcatggatagtccaataaataatgttatcttttttgaagttaccgtttt；p612-le：ttgaactgatgctcataggagagaatattttttctgagtcaaagcat；p729-le：tctgagctgtcatcgtcccctttttgaagttaccgtttt；p749-le：atctctgtgagccacaaccaacagtttttctgagtcaaagcat；p821-le：atgagttgaattctcctattatggatgctttttgaagttaccgtttt；p849-le：tagcttctggccatctctggctttttctgagtcaaagcat；p898-le：cctttgcttccacgacttttatcttttctttttgaagttaccgtttt；p926-le：tccaacacatcgcttaccaatccttttttctgagtcaaagcat；p998-le：tcaagtcttttcttccatccccactttttgaagttaccgtttt；p1022-le：cactaacctgaatgcctagaccctttttctgagtcaaagcat；p1045-le：ttatttttattaatttccaatagatgctgcctatgtttttgaagttaccgtttt；p1080-le：ggctatattgctttagatgaacattagatattttttttctgagtcaaagcat；p1169-le：ctcctctccctatattactgattgctttttgaagttaccgtttt；p1194-le：actgaacagcatggtccccaatgtttttctgagtcaaagcat；p1241-le：tggctccttgtggtacatgcatgtttttgaagttaccgtttt；p1264-le：caagactgctgaagccagaaggtttttctgagtcaaagcat；p1328-le：ccttcgtgattgtcaggagcaagtttttgaagttaccgtttt；p1351-le：acctgagatgctccctgccttttttctgagtcaaagcat；p1371-le：tcagtgtcctctgcatctccctttttgaagttaccgtttt；p1392-le：ctttctaatgaagatccatagaatttgctactttttctgagtcaaagcat；p1423-le：atttgagaattccaattaggaactcacatgtttttgaagttaccgtttt；p1453-le：ttttatctgccctatcaattttttaaacttgcttttttctgagtcaaagcat；p1551-le：atatcaactttgattctttgttacaacttttcttactttttgaagttaccgtttt；p1587-le：tcttttatcaccaaagtggcttttattctctttttctgagtcaaagcat；p1617-le：tttattattattattttcttttactactatattacgttgttattattttttgaagttaccgtttt；p1663-le：tttgttctctatagtatcaatttatttgatttagtttctttttctgagtcaaagcat；p1782-le：ctaatgcatgtgggacttaaaacctagtttttgaagttaccgtttt；p1809-le：atgatgggttgataggtgcagcaatttttctgagtcaaagcat；p1857-le：tgtaacaaacctacacattctgcacatgtttttgaagttaccgtttt；p1885-le：tatcccagaacgtaaagtaaaatttaaaaaaaagtgtttttctgagtcaaagcat；封闭探针：p122-bl：agtgtgcctctctctctttgacc；p376-bl：cgcatttgtgggttctcttaagca；p505-bl：atttaccaggtttggagaggattcag；p531-bl：acagctcaggtgctttcactaatg；p555-bl：tctctgaacttctgtccctctttg；p668-bl：gattcaaagaaatattagatttaagctcacact；p773-bl：caggacccaacgcatgtctg；p793-bl：agatccttaaatcaaggaaaccagtgtc；p950-bl：ctctctgctctgttgctttggac；p1113-bl：aaagctcaagaggttcaaaatccaactc；p1141-bl：attatcttctctttctttcacctccctg；p1310-bl：gaggggaccactcctggg；p1486-bl：gaaaattaagttttttcaaaatctgtccttgtaaatt；p1523-bl：actttttcttacagtgtcttggcatact；p1701-bl：aatttatttttattgctgacttttaaaataagtgattcg；p1740-bl：gggggtgggagaacaggg；p1833-bl：accactatggcacacgtatacctg；在其他通用探针、发光底物、信号放大前体、信号放大体以及ap报告探针均一致的前提下，以不同浓度的前列腺癌阳性细胞为试验对象进行化学发光检测，结果显示，同一浓度的阳性细胞标本，采用本发明探针序列所检测到的信号值明显高于对照探针序列，这表明了本发明所述探针序列对于检测前列腺癌细胞有很高的特异性，能够改善检测的定量限(loq)、检测限(lod)以及灵敏度。由以上技术方案可知，本发明针对前列腺癌pca3进行捕获延伸探针、k型标记探针以及封闭探针的设计和人为优化，进一步提高了基于类似bdna和quantimat技术对前列腺癌细胞检测的特异性，表现出较佳的定量限、检测限以及灵敏度，并可应用于此类试剂盒的制备中。附图说明图1所示为本发明所述试剂盒工作原理示意图(载体为磁珠)；其中，1表示通用探针，2表示捕获延伸探针，3表示目标rna，4表示k型标记探针，5表示信号放大前体，6表示信号放大体，7表示ap报告探针，8表示封闭探针；图2所示为本发明所述试剂盒工作原理示意图(载体为细胞培养板)；其中，1表示通用探针，2表示捕获延伸探针，3表示目标rna，4表示k型标记探针，5表示信号放大前体，6表示信号放大体，7表示ap报告探针，8表示封闭探针，9表示细胞培养板；图3所示为本发明探针序列和对照组探针序列的冷光仪检测试验结果。具体实施方式本发明公开了一种前列腺癌检测探针序列及其应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明所述探针序列及其应用已经通过实施例进行了描述，相关人员明显能在不脱离本
发明内容、精神和范围内对本文所述探针序列及其应用进行改动或适当变更与组合，来实现和应用本发明技术。依照本发明所提供的序列探针及其他配套试剂盒组分，其在具体检测时的工作步骤和原理如下：裂解待测样本释放目标rna；然后加入本发明所述探针序列，捕获延伸探针的一部分与偶联载体(磁珠或类似96孔板的细胞培养板)的通用探针结合，另一部分与目标rna结合；k型标记延伸探针与目标rna结合；封闭探针用来封闭一些位点提高特异性；探针组杂交后，逐步加入信号放大前体、放大体、碱性磷酸酶标记的探针和化学发光底物。根据上述原理，一组k型标记延伸探针可将信号放大400倍，一般一个目标rna会结合20组k型标记延伸探针，所以一个rna目标会被放大8000倍。最后用化学发光分析仪检测，原理示意图见图1和图2。定量检测可以得到实际的倍数差异，而不是常规方法taqman中ct的变化结果，结果更直接准确，灵敏度比taqman高100～1000倍。如未特别说明，本发明对比试验中除应有的技术特征区别外，其余试验环境和条件保持一致。以下就本发明所提供的一种前列腺癌检测探针序列及其应用做进一步说明。实施例1：本发明所述检测前列腺癌的试剂盒1、探针序列捕获延伸探针序列如seqidno.1-14和seqidno.78-85所示，k型标记延伸探针如seqidno.15-64和seqidno.86-131所示，封闭探针如seqidno.65-77和seqidno.132-148所示；2、试剂盒配套组分与磁珠或细胞培养板等载体偶联的通用探针：seqidno.149所示；信号放大前体：atgctttgactcag/aaaacggtaacttc(放大前体前置引导区序列，两种)+aggatcgtaagtaactaaggactacc(放大体前置引导区互补序列)+19次放大体前置引导区互补序列(seqidno.150-151所示)；信号放大体：ggtagtccttagttacttacgatcct(放大体前置引导区序列)+gaatccattgaatcctgtgatt(报告探针重复区互补序列)+19次报告探针重复区互补序列(seqidno.152所示)；ap标记的探针：aatcacaggattcaatggattc-ap(seqidno.153所示)；金刚烷衍生物。实施例2：特异性对比试验1、试验方法采用前列腺癌pc3阳性细胞，用科蒂亚公司生产的细胞标本保存液配制成浓度为26个/ml、78个/ml、150个/ml和300个/ml的四种细胞标本液；标本裂解：细胞标本液离心后沉淀细胞团，加入如下混合液：200μl裂解液(裂解液需提前放置37℃恒温箱20分钟)和400μl试验用水(混合比例为1：2)。用移液枪吹打细胞团，使细胞分散悬浮，再加入5μl蛋白酶k。65℃中放置1小时，裂解过程中进行2～3次震荡。1小时后取出，取上清液用于检测。检测缓冲液配制：取出检测探针、封闭反应液放在冰上融解，融解后需混匀离心。标本检测缓冲液50ul/孔。配置方法如下：1ul封闭反应液+17ul裂解液+31ul试验用水+1ul检测探针。探针配制：各组分浓度要求，探针组中ce探针：le探针：bl探针的大致比例是1:4:2。各组分每条探针终浓度分别是ce要求1fmol/ul、le要求4fmol/ul、bl要求2fmol/ul。布板：将包被有通用探针的96孔捕获包被板从4度冰箱中取出，在常温下复温。将上述缓冲液及待测上清液分别加入到96孔捕获包被板中：空白对照孔：98ul空白对照缓冲液+2ul试验用水。试验孔：98ul空白对照缓冲液+2ul待测上清。保温条件和时间：用封板贴纸封紧整块板，放入55度温箱保温3.5小时。信号放大：将稀释液、10×洗脱液置37℃预热20分钟；信号放大前体，信号放大体取出放在冰上融解，震荡混匀，轻微离心。配置所需液体：1×洗脱液：取10×洗脱液，用ddh2o稀释为1×洗脱液,其体积比为10×洗脱液:ddh2o＝1:9。信号放大前体：溶液体积配比为信号放大前体：稀释液＝1:1000，取试剂盒所配信号放大前体加入到适量稀释液中；信号放大体：溶液体积配比为信号放大体：稀释液＝1:1000，取试剂盒所配信号放大体加入到适量稀释液中；ap标记探针：溶液体积配比为ap标记探针：稀释液＝1:1000，取试剂盒所配探针标记物加入到适量稀释液中；(1)将96孔捕获包被板从温箱中取出，掀开封板贴纸，倒净板中液体，用1×洗脱液200ul/孔洗板洗3次。将所配信号放大前体每孔加入100ul，放入55度温箱保温40分钟。(2)将96孔捕获包被板从温箱中取出，掀开封板贴纸，倒净板中液体，用1×洗脱液200ul/孔洗板洗3次。将所配信号放大体每孔加入100ul，放入55度温箱保温40分钟。(3)将96孔捕获包被板从温箱中取出，掀开封板贴纸，倒净板中液体，用1×洗脱液200ul/孔洗板洗3次。将所配ap标记探针每孔加入100ul，放入50度温箱保温40分钟。读板：从4度冰箱取出底物金刚烷衍生物复温。复温后加入底物催化剂，底物与底物催化剂配比为1000:3，震荡混匀，使用前再次移液器吹打混匀。将96孔捕获包被板从温箱中取出，掀开封板贴纸，倒净板中液体，用1×洗脱液200ul/孔洗板洗3次。将所配底物每孔加入100ul，放入46度温箱保温20分钟后取出，上冷光仪检测。2、试验对象本发明：实施例1序列探针及其试剂盒配套组分；对照组：本发明
发明内容部分提供的对照序列探针及实施例1中试剂盒配套组分；3、结果表1阳性细胞数目(个/ml)本发明检测平均值对照组检测平均值26.006.792.1278.0015.684.15150.0032.858.83300.0065.9516.36根据表1数据绘制对比柱形图，见图3。根据试验结果可以明显看出，采用本发明探针序列能够获得更高的检测信号，这表明本发明所述探针序列具备更高的检测特异性。以上所述仅是本发明的优选实施方式，应当指出，对于本
技术领域：
的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。序列表<110>郑州科蒂亚生物技术有限公司<120>一种前列腺癌检测探针序列及其应用<130>mp1730590<160>153<170>siposequencelisting1.0<210>1<211>45<212>dna<213>人工序列(artificialsequence)<400>1acagaagaaatagcaagtgccgagtttttctcttggaaagaaagt45<210>2<211>41<212>dna<213>人工序列(artificialsequence)<400>2cgagggagaccaggaagatctttttctcttggaaagaaagt41<210>3<211>54<212>dna<213>人工序列(artificialsequence)<400>3tgtttcaatgaacaccaagataaataagtgaagtttttctcttggaaagaaagt54<210>4<211>43<212>dna<213>人工序列(artificialsequence)<400>4tcaccatcgacggcactttctgtttttctcttggaaagaaagt43<210>5<211>49<212>dna<213>人工序列(artificialsequence)<400>5tgagaaataagaaaggctgctgactttatttttctcttggaaagaaagt49<210>6<211>49<212>dna<213>人工序列(artificialsequence)<400>6atgagaggaaaacagacgagaaaatctttttttctcttggaaagaaagt49<210>7<211>41<212>dna<213>人工序列(artificialsequence)<400>7agataaccacggggcagaggtttttctcttggaaagaaagt41<210>8<211>54<212>dna<213>人工序列(artificialsequence)<400>8aatcatttcatatttctaaccctcaaaacaaagtttttctcttggaaagaaagt54<210>9<211>54<212>dna<213>人工序列(artificialsequence)<400>9atatatccagccacactcatttttaatatttagtttttctcttggaaagaaagt54<210>10<211>40<212>dna<213>人工序列(artificialsequence)<400>10gagtgttctggcccaggggtttttctcttggaaagaaagt40<210>11<211>46<212>dna<213>人工序列(artificialsequence)<400>11actagcacacagcatgatcattacgtttttctcttggaaagaaagt46<210>12<211>48<212>dna<213>人工序列(artificialsequence)<400>12ttcatgcaaagaagggacacatatgagtttttctcttggaaagaaagt48<210>13<211>49<212>dna<213>人工序列(artificialsequence)<400>13cccaaaggtaacctttatccatttcatgtttttctcttggaaagaaagt49<210>14<211>45<212>dna<213>人工序列(artificialsequence)<400>14gtgagtgcgctttagaattttggctttttctcttggaaagaaagt45<210>15<211>43<212>dna<213>人工序列(artificialsequence)<400>15aagctggcatcagaaaaacagaggtttttgaagttaccgtttt43<210>16<211>40<212>dna<213>人工序列(artificialsequence)<400>16ggagatttgtgtggctgcagctttttctgagtcaaagcat40<210>17<211>39<212>dna<213>人工序列(artificialsequence)<400>17tgcatggtgggaaggacctgtttttgaagttaccgtttt39<210>18<211>52<212>dna<213>人工序列(artificialsequence)<400>18atgatacagaggaattacaacacatatacttagtttttctgagtcaaagcat52<210>19<211>40<212>dna<213>人工序列(artificialsequence)<400>19agctagtccgctgtgagtctctttttgaagttaccgtttt40<210>20<211>39<212>dna<213>人工序列(artificialsequence)<400>20ctcagtgacacagggctggatttttctgagtcaaagcat39<210>21<211>40<212>dna<213>人工序列(artificialsequence)<400>21ccatctgaggccacacatctgtttttgaagttaccgtttt40<210>22<211>51<212>dna<213>人工序列(artificialsequence)<400>22ctgaaatggagataattaacatcactagaaactttttctgagtcaaagcat51<210>23<211>50<212>dna<213>人工序列(artificialsequence)<400>23agcaagatgacaatataatgtctaagtagtgtttttgaagttaccgtttt50<210>24<211>44<212>dna<213>人工序列(artificialsequence)<400>24acatgtttttgcacatttccagccctttttctgagtcaaagcat44<210>25<211>46<212>dna<213>人工序列(artificialsequence)<400>25ctttaaatatccacacacacaggaagctttttgaagttaccgtttt46<210>26<211>43<212>dna<213>人工序列(artificialsequence)<400>26acaaaaggaagcacagagatcccttttttctgagtcaaagcat43<210>27<211>39<212>dna<213>人工序列(artificialsequence)<400>27ccgcttgtgagggaaggacatttttgaagttaccgtttt39<210>28<211>52<212>dna<213>人工序列(artificialsequence)<400>28ttagaaaatgaattgatgtgttccttaaaggattttttctgagtcaaagcat52<210>29<211>51<212>dna<213>人工序列(artificialsequence)<400>29tggatatttatttgaacgggattacagatttgtttttgaagttaccgtttt51<210>30<211>47<212>dna<213>人工序列(artificialsequence)<400>30aaatgaagtcacaaagtgagcattaccatttttctgagtcaaagcat47<210>31<211>39<212>dna<213>人工序列(artificialsequence)<400>31gtcaggattctggccctgcttttttgaagttaccgtttt39<210>32<211>43<212>dna<213>人工序列(artificialsequence)<400>32gcctaaactgtgcgttcataaccatttttctgagtcaaagcat43<210>33<211>47<212>dna<213>人工序列(artificialsequence)<400>33ctgttgtaatatctgatctctacggttctttttgaagttaccgtttt47<210>34<211>40<212>dna<213>人工序列(artificialsequence)<400>34cttctgggcccaacattctcctttttctgagtcaaagcat40<210>35<211>43<212>dna<213>人工序列(artificialsequence)<400>35ttcccagatctgtactgtgacctttttttgaagttaccgtttt43<210>36<211>52<212>dna<213>人工序列(artificialsequence)<400>36tctacactgtagaataacattactcattttgtttttttctgagtcaaagcat52<210>37<211>41<212>dna<213>人工序列(artificialsequence)<400>37caaagacccttcgtgttgctgctttttgaagttaccgtttt41<210>38<211>48<212>dna<213>人工序列(artificialsequence)<400>38ctaatatgtagctgactgtttttcctaagtttttctgagtcaaagcat48<210>39<211>41<212>dna<213>人工序列(artificialsequence)<400>39atctgtgaacaggctgggaagctttttgaagttaccgtttt41<210>40<211>47<212>dna<213>人工序列(artificialsequence)<400>40atctcaagatctttccagggttatactttttttctgagtcaaagcat47<210>41<211>48<212>dna<213>人工序列(artificialsequence)<400>41gagtgaattatctaatcaacatcatcctctttttgaagttaccgtttt48<210>42<211>47<212>dna<213>人工序列(artificialsequence)<400>42agtgtctttgcccatactgaaattcatttttttctgagtcaaagcat47<210>43<211>43<212>dna<213>人工序列(artificialsequence)<400>43tcccacttttgtgcccattctcaatttttgaagttaccgtttt43<210>44<211>49<212>dna<213>人工序列(artificialsequence)<400>44gacctcaaaatgtcattccattaatatcactttttctgagtcaaagcat49<210>45<211>51<212>dna<213>人工序列(artificialsequence)<400>45aatgttacatgcagctatgggaatttaattactttttgaagttaccgtttt51<210>46<211>51<212>dna<213>人工序列(artificialsequence)<400>46atattttgttttccagtgcaaagatgactaagtttttctgagtcaaagcat51<210>4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