CN101864480B - 一种癌症筛检的方法 - Google Patents
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Abstract
一种癌症筛检的方法,包含下列步骤:(1)提供一受测检体;(2)检测该受测检体的基因组DNA中至少一个目标基因的CpG序列甲基化状态,该目标基因是由PTPRR、ZNF582、PDE8B以及DBC1所组成;(3)根据至少一个目标基因甲基化状态的有无,判断该检体是否具有癌症或癌症前病变;甲基化状态检测方法为甲基化特异性聚合酶连锁反应(methylation-specific PCR,MSP)、定量甲基化特异性聚合酶连锁反应(quantitative methylation-specific PCR,QMSP)、亚硫酸盐定序(bisulfite sequencing,BS)、微阵列(microarrays)、质谱仪分析(massspectrometer)、变性高效液相色谱(denaturing high-performance liquidchromatography,DHPLC)等。
Description
技术领域
本发明涉及一种癌症筛检的方法,特别涉及一种以甲基化DNA作为生物标记的癌症筛检的方法。
背景技术
子宫颈癌是全球及台湾女性主要的死因之一,根据2002年世界卫生组织(WHO)的统计,子宫颈癌为全球女性癌症死因的第二位,仅次于乳癌;定期接受子宫颈癌筛检是预防子宫颈癌的最佳方法,习用子宫颈癌筛检的方式主要有两种,一是最常见的子宫颈抹片检查(Pap smear),另一则为人类乳突病毒检验(HPV testing);子宫颈抹片检查是取出子宫颈部的分泌物,以显微镜观察其中脱落的上皮细胞中,是否有癌病变产生,以早期侦测子宫颈癌;而HPV检验则是以聚合酶连锁反应(polymerase chain reaction,RT-PCR)或Hybrid Capture的方式检查样本中是否存在有人类乳突病毒(human papilloma virus,HPV)病毒的存在。
然而,由于子宫颈抹片检查(Pap smear)需要靠医师取样、检验师/病理医师判读抹片,除了容易产生高伪阴性率(High false negative rate)而延迟癌前病变的诊断与治疗之外,再者,所需的人力素质与成本太高,这对许多发展中的国家来说,有推广上的困难;另一方面,人类乳突病毒检验(HPV testing)虽具有高敏感度,但却容易造成高伪阳性率(High false positive rate),不仅让病患白白担心,也会浪费许多医疗资源在伪阳性患者的追踪检查上;因此,如何提高子宫颈癌检验方法的准确性及方便性,是推广子宫颈癌检验的重要课题之一。
基因的缺失(genomic deletions)被认为是肿瘤形成的重要因素,长久以来,我们都习惯了基因组中的编码是仰赖ATCG四个碱基排列的观念,Knudson早在1975年即提出双重受创理论(two-hit theory),指出一些同源肿瘤抑制基因伴随的突变或缺失可能造成或易造成癌症的发生;然而,其他影响表现型(phenotype)的讯息可能存于被修饰过的碱基5-甲基胞嘧啶(5-methylcytosine)中,5-甲基胞嘧啶被发现存在于哺乳类动物细胞内的回文序列5’-CpG-3’中,在哺乳类动物细胞内除了一些被称为“CpG岛”(CpG islands,CGIs)的区域之外,大多数的CpG双核苷酸对都被甲基化,CpG岛是指在大约1000个碱基对(1Kb)的区域内含有大量的GC-以及CpG-,通常位于基因的附近,且在广泛表现的基因的启动子附近被发现。胞嘧啶的甲基化发生在DNA合成后,自一甲基捐赠者s-腺核苷甲硫胺酸(S-adenosylmethionine,SAM)将一甲基经酵素转移到胞嘧啶第5个碳的位置上,该酵素反应是由DNA甲基转移酶(DNA methyltransferase,DNMTs)执行,DNMT1是哺乳类动物主要的甲基转移酶,是负责将半甲基化位置复制后修复(post-replicative restoration)为全甲基化,被称为维持甲基化(maintenancemethylation);反之,DNMT3A及DNMT3B则被认为主要负责甲基化新的位置,进行一种称为重新甲基化(de novo methylation)的步骤。
CpG双核苷酸对甲基化的遗失(loss of methylation),意即一般的低度甲基化,是癌细胞内的第一个超遗传异常(epigenetic abnormality);然而,在过去几年内的研究却显示,特定位置(例如:一些肿瘤抑制基因)的高度甲基化(site-specific hypermethylation)与其功能的丧失有关,这可能会在癌症生成时提供选择优势(selective advantages);在启动子区域上CpG岛的高度甲基化,可以借由组蛋白修饰(histone modification)伴随接续而来的基因默化现象(genesilencing),来引起染色质改造(chromatin remodeling);除了染色体缺失及基因突变之外,经由启动子的高度甲基化所造成肿瘤抑制基因的超遗传默化现象(epigenetic silencing)也常见于人类癌症中。
最近的流行病学研究显示,血清叶酸盐(serum folate)的浓度(一种甲基的主要来源)与HPV的感染和清除有关联;在甲基周期(methyl cycle)的代谢作用中,酵素的基因多型性(genetic polymorphisms)也曾被报导与子宫颈上皮内病变的发展有关;如同超基因演化的观念一般,DNA甲基化与子宫颈癌间关联的研究也同样盛行,子宫颈癌的DNA甲基化研究与日俱增,显示使用甲基化作为子宫颈癌筛检的可能性;由于遗传与环境交互作用的特性,肿瘤抑制基因甲基化程度因不同的基因及不同的族群而异,不同的疾病也会有不同的甲基化表现型(methylator phenotypes);然而,子宫颈癌的甲基化表现型以及其与HPV基因型的关联仍未知,而子宫颈癌中有何特定的基因会被甲基化,以及需要多少基因方可达到临床应用的需求,这些问题仍是未来需要被确认的议题。
本案发明人先前已于台湾(TW Pat.Pub.No.200831900)、中国(CN Appl.No.200810094659.2)、马来西亚(UI20085354)及美国(US Pat.Pub.No.20080311570)提出相关专利申请(下称前案),本案为前案的延伸,本案发明人发现新颖的癌症筛检生物指标及其筛检的方法。
由此可见,上述现有子宫颈癌筛检方法仍有诸多缺失,实非一良善的设计,而亟待加以改良。
本案发明人鉴于上述现有子宫颈癌筛检方法所衍生的各项缺点,乃亟思加以改良创新,并经多年苦心孤诣潜心研究后,终于成功研发完成本发明癌症筛检的方法。
发明内容
本发明的目的即在于提供一种子宫颈癌筛检的方法,以作为第一线子宫颈癌的筛检(cancer screen)。
本发明的次一目的是在于提供一种子宫颈癌筛检的方法,该方法除了可作为第一线子宫颈癌的筛检之外,也可作为第二线子宫颈癌的筛检,辅助人类乳突病毒检验(HPV testing)或不确定的抹片结果,以达到更准确的子宫颈癌筛检效果。
本发明的另一目的是在于提供一种癌症诊断的方法,该方法除可应用在子宫颈癌的检测上,亦可应用于其他癌症(如:卵巢癌,大肠癌)的检测,以辅助异常检体的诊断。
可达成上述发明目的的一种癌症筛检的方法,是检测受测检体细胞中目标基因甲基化的状态,以作为癌症有无的筛检指标,该方法包含下列步骤:
步骤1提供一受测检体;
步骤2检测该受测检体的基因组DNA中至少一个目标基因的CpG序列甲基化状态,该目标基因是由DBC1、PDE8B、PTPRR以及ZNF582所组成;以及
步骤3根据该目标基因甲基化状态的有无,判断该检体是否具有癌症或癌前病变病变,或作为治疗预后的指标。
其中该受测检体为子宫颈抹片、卵巢癌组织、腹水、血液、尿液、粪便、痰、口腔粘膜细胞、胃液、胆汁、子宫颈上皮细胞、或手术后的癌症组织等。
其中该目标基因的CpG序列甲基化状态检测方法包含但不限于甲基化特异性聚合酶连锁反应(methylation-specific PCR,MSP)、定量甲基化特异性聚合酶连锁反应(quantitative methylation-specific PCR,QMSP)、亚硫酸盐定序(bisulfitesequencing,BS)、微阵列(microarrays)、质谱仪分析(mass spectrometer)、变性高效液相色谱(denaturing high-performance liquid chromatography,DHPLC)。
其中该目标基因DBC1具有如SEQ ID No:1所示的核苷酸序列。
其中该目标基因PDE8B具有如SEQ ID No:2所示的核苷酸序列。
其中该目标基因PTPRR具有如SEQ ID No:3所示的核苷酸序列。
其中该目标基因ZNF582具有如SEQ ID No:4所示的核苷酸序列。
此外,前述筛检指标及筛检方法进一步可用于子宫颈癌、大肠癌的筛检。
本发明进一步提供一种卵巢癌筛检的方法,是检测受测检体细胞中目标基因甲基化的状态,以作为卵巢癌有无的筛检指标,该方法包含下列步骤:
步骤1提供一受测检体;
步骤2检测该受测检体的基因组DNA中至少一个目标基因的CpG序列甲基化状态,该目标基因是由DBC1、PTPRR以及ZNF582所组成;以及
步骤3根据目标基因甲基化状态的有无,判断该检体是否具有卵巢癌病变或作为治疗预后的指标。
其中该受测检体为卵巢癌组织、腹水、血液、尿液、或手术后的癌症组织等。
其中该目标基因的CpG序列甲基化状态检测方法包含但不限于甲基化特异性聚合酶连锁反应(methylation-specific PCR,MSP)、定量甲基化特异性聚合酶连锁反应(quantitative methylation-specific PCR,QMSP)、亚硫酸盐定序(bisulfitesequencing,BS)、微阵列(microarrays)、质谱仪分析(mass spectrometer)、变性高效液相色谱(denaturing high-performance liquid chromatography,DHPLC)、焦磷酸定序(pyrosequencing)。
其中该目标基因DBC1具有如SEQ ID No:1所示的核苷酸序列。
其中该目标基因PTPRR具有如SEQ ID No:3所示的核苷酸序列。
其中该目标基因ZNF582具有如SEQ ID No:4所示的核苷酸序列。
术语“受测检体”是指离体的受测样本,该样本包括前述的子宫颈抹片、腹水、血液、尿液、粪便、痰、口腔粘膜细胞、胃液、胆汁、子宫颈上皮细胞、或手术后的癌症组织等离体的检体样本。本发明的癌症筛检方法是用于检测该些离体样本中目标基因甲基化的状态,以作为各类癌症的筛检指标。本发明所提供的癌症筛检方法及其筛检指标,可供检测研究人员于实验室中进行检测。
附图说明
图1A为本发明癌症筛检方法所使用的目标基因PTPRR,在各类子宫颈样本中,进行亚硫酸盐定序(BS)分析的结果;
图1B为本发明癌症筛检方法所使用的目标基因ZNF582,在各类子宫颈样本中,进行亚硫酸盐定序(BS)分析的结果;
图1C为本发明癌症筛检方法所使用的目标基因PDE8B,在各类子宫颈样本中,进行亚硫酸盐定序(BS)分析的结果;以及
图1D为本发明癌症筛检方法所使用的目标基因DBC1,在各类子宫颈样本中,进行亚硫酸盐定序(BS)分析的结果。
具体实施方式
本发明是以下面的实施例予以示范阐明,但本发明不受下述实施例所限制。
实施例一材料与方法
一、材料
试验材料包含一系列完整的子宫颈病变样本,包括:鳞状细胞癌(squamouscell carcinoma,SCC,n=20)、腺癌(adenocarcinoma,AC,n=20),以及正常子宫颈样本(n=10)。所有的子宫颈样本、卵巢样本、大肠癌样本均取自台北三军总医院,各样本的基因组DNA(genomic DNA)以QIAamp DNA套组(QIAGEN)抽取,并用以比对分析全基因组中DNA甲基化的情形。另于分析前皆以Bioanalyzer(Agilent)检测基因组DNA的品质。于本实施例是以10μg片段化的DNA进行MeDIP(Methyl DNA IP)。
二、借由MeDIP及CpG island-Plus-Promoter阵列(CpGisland-Plus-Promoter arrays)进行DNA甲基化分析
首先,以BioruptorTM UCS-200(Diagenode)将基因组DNA片段化至300~1,000bp。再以30μl polyclonal Anti-5’-methyl cytosine antibody(Abcam)于最终体积100μl IP缓冲液(IP buffer)(0.15%SDS,1%Triton X-100,150mM NaCl,1mMEDTA,0.5mM EGTA,10mM Tris and 0.1%BSA)、4℃环境下,将该些片段化基因组DNA进行免疫沉淀。将免疫沉淀后的混合物与120μl Protein G Sepharose(Amersham)混匀后,于4℃反应2小时,再以1ml低盐度、高盐度溶液、氯化锂(lithium chloride)以及TE缓冲液(TE buffer)洗涤2次。再以elution buffer(1%SDS,0.1M NaHCO3)于室温下处理protein G 15分钟两次。再以phenol-chloroformextraction及酒精沉淀来回收甲基化的DNA。借由Whole Genome AmplificationKit(Sigma)将enriched methylated DNA及input DNA扩增(amplify)。EnrichedDNA以及total DNA分别于尾端标记Cy5及Cy3,再将标记后的Enriched DNA及total DNA与CpG Island-Plus-Promoter Arrays共同杂合(co-hybridization)。CpG Island-Plus-Promoter Arrays是由NimbleGen Systems,Inc所设计合成的。此阵列包含385,000个50-75bp的寡核苷酸(探针),该些寡核苷酸约每100bp含跨24,659个HG18 RefSeq promoters(启动子上游800bp至启动子下游200bp)以及28,226个CpG岛。
该阵列中的每个特征值具有一对应的二进位对数(log2)比率,该二进位对数比率代表的是将比率资料往零集中的正规化(normalization)结果。根据一个二进位对数比率资料,可将固定长度的视窗(500bp)布于每个连续的探测点周围,并且使用单面柯尔莫诺夫-斯米尔诺夫检验(one-sided Kolmogorov-Smirnov test),以判断相较于其它二进位对数比率资料的对应探测点,这些探测点是否从更具意义的正向分布中所取得,最后得到的每个探测点的比数是根据在每个探测点周围所执行的固定视窗的柯尔莫诺夫-斯米尔诺夫检验所得到的p值(p-value)的负常用对数(-log10),详请参阅Scacheri等人于Methods Enzymol 2006的方法(Statistics for ChIP-chip and DNase hypersensitivity experiments on NimbleGenarrays),用以挑选分析阵列结果。于本发明中搜寻至少两个高于一最小二进位截断p值2(p-value minimum cutoff of 2)的探测点,以侦测该二进位对数比率资料的尖峰,且将位于300bp的多个尖峰合并在一起。比较鳞状细胞癌(SCC)、腺癌(AC),以及正常子宫颈样本间的结果,若位于转录起始区域上下游2500bp具有差异性的甲基化区域,即被挑选以进一步评估。最终以SignalMap(NimbleGen)检视p-value的资料。
三、亚硫酸盐修饰作用(Bisulfite modification)、甲基化特异性聚合酶连锁反应(methylation-specific PCR,MSP)以及亚硫酸盐定序(bisulfite sequencing,BS)
使用Chemicon公司出产的DNA修饰套组(DNA modification kit,Chemicon,Ternecula,CA)进行亚硫酸盐修饰作用:取1μg样本的基因组DNA(genomicDNA),以亚硫酸钠对基因组DNA进行化学修饰,在单链DNA中,所有非甲基化的胞嘧啶都会发生脱氨基作用而转变成尿嘧啶,而甲基化的胞嘧啶则不被修饰,仍保持5-甲基胞嘧啶的状态;最后,将反应后的样本DNA溶于70μl 55℃的TE缓冲液(TE buffer)中,以进行甲基化特异性PCR(MSP)。
另取人类周围血(peripheral blood)的正常DNA进行亚硫酸盐修饰作用,以作为具有非甲基化启动子序列的对照组。
取1μg经过亚硫酸盐修饰作用后的样本基因组DNA,以及对照组DNA,以MSP引子进行甲基化特异性PCR扩增,该MSP引子为可专一辨认甲基化基因序列的MSP引子(M),各目标基因的MSP引子序列如表一所示;甲基化特异性PCR反应物的总体积为25μl,包含1μl已修饰过的模版DNA、每一引子各1.5pmol、0.2mmol/L dNTPs以及1unit Gold Taq DNA polymerase(AppliedBiosystems,Foster City,CA);将混合好的反应物置于95℃下5分钟,接着以95℃解离(denature)30秒、适当引子粘合(annealing)温度粘合30秒、72℃合成30秒为循环,解离、粘合、合成步骤共重复35个循环,之后再置于72℃反应5分钟。扩增后的产物以含有溴化乙锭(ethidium bromide,EtBr)的2.5%琼脂胶体进行电泳分析,并置于紫外光下照射观察。
表一甲基化特异性PCR(MSP)所使用的MSP引子的序列
引子种类M代表可专一性辨认甲基化基因序列的MSP引子
所有的样本均进行至少两次独立的亚硫酸盐修饰作用及甲基化特异性PCR,在使用可专一辨认甲基化基因序列的MSP引子(M)所进行的PCR反应中,若同一样本无法合成出PCR产物两次以上,则视为该样本不具甲基化;将使用可专一辨认甲基化基因序列的MSP引子(M)所扩增的PCR产物选殖到pCR4-TOPO载体(Invitrogen,Carlsbad,CA)中,选取至少5个独立的选殖株(clones)进行亚硫酸盐定序(BS),亚硫酸盐定序(BS)所使用的引子如表二所示,使用377自动定序仪(Applied Biosystems,Foster City,CA)进行亚硫酸盐定序,其定序的结果如序列表所示,亚硫酸盐定序的序列编号分别为:DBC1_BS(SEQID No:21)、PDE8B_BS(SEQ ID No:22)、PTPRR_BS(SEQ ID No:23)以及ZNF582_BS(SEQ ID No:24)。
表二亚硫酸盐定序(BS)所使用的引子的序列
实施例二子宫颈癌甲基化目标基因的筛选
借由CpG island-Plus-Promoter阵列(CpG island-Plus-Promoter arrays)进行筛选后,筛选出四个目标基因于子宫颈癌细胞可能具有高度甲基化现象,分别为DBC1(SEQ ID No:1)、PDE8B(SEQ ID No:2)、PTPRR(SEQ ID No:3)以及ZNF582(SEQ ID No:4),其详细资料如表三所示;由表三可知,这四个基因除了DBC1已知与膀胱癌有关外,目前很少有研究显示该些基因与子宫颈癌之间的关连。
表三以CpG island-Plus-Promoter阵列筛选出子宫颈癌细胞中具甲基化的基因的详细资料
实施例三亚硫酸盐定序(BS)分析子宫颈病变样本中目标基因甲基化状态
目标基因:PTPRR
试验样本组别:
1.HeLa子宫颈癌细胞株(HeLa_0);
2.处理10μM的DNA甲基转移酶抑制剂5’-aza-2’-deoxycytidine(AZC)的HeLa子宫颈癌细胞株(HeLa_10D);
3.同时处理10μM的DNA甲基转移酶抑制剂5’-aza-2’-deoxycytidine(AZC)及0.33μM的TSA(Sigma Chemical Co.,St.Louis,MO)的HeLa子宫颈癌细胞株(HeLa_DT);
4.子宫颈腺癌样本(AC);
5.子宫颈鳞状细胞癌样本(SCC);
6.对照组(normal):以正常子宫颈血液DNA作为无甲基化的对照组。
前述各试验样本经亚硫酸盐修饰后,续以亚硫酸盐定序(BS)分析目标基因(PTPRR)在各试验样本中是否存在高度甲基化(hypermethylation)现象,结果如图1所示,黑色表示甲基化区域、白色表示为未甲基化区域。目标基因PTPRR于对照组及腺癌中无甲基化现象,于HeLa子宫颈癌细胞株(HeLa_0)及子宫颈鳞状细胞癌样本(SCC)中,目标基因PTPRR呈现高度甲基化现象。因此,可借由PTPRR的甲基化程度用以筛检是否罹患子宫颈癌。
此外,为了确认子宫颈癌样本中目标基因的甲基化程度是否透过DNA甲基化作用来调节,以10μM的DNA甲基转移酶抑制剂5’-aza-2’-deoxycytidine(AZC)(Sigma Chemical Co.)处理HeLa子宫颈癌细胞株,再将萃取自该细胞的DNA样本经亚硫酸盐修饰后,续以亚硫酸盐定序(BS)。结果如图1A所示,相较于子宫颈鳞状细胞癌样本(SCC)及HeLa子宫颈癌细胞株(HeLa_0),经过AZC处理后的HeLa子宫颈癌细胞株(HeLa_10D),其目标基因PTPRR显示已有部分区域去除甲基化。
trichostatin A(TSA)为去乙酰基酵素抑制剂(histone deacetylase(HDAC)inhibitors),亦可用以降低、弱化甲基化程度。将HeLa子宫颈癌细胞株同时处理AZC及TSA(HeLa_DT),其结果如图1A所示,相较于子宫颈鳞状细胞癌样本(SCC)及HeLa子宫颈癌细胞株(HeLa_0),经过AZC及TSA处理后的HeLa子宫颈癌细胞株(HeLa_DT),其目标基因PTPRR显示高幅度地被去甲基化。
综合上述结果,在子宫颈癌样本中,目标基因PTPRR确实会经由DNA甲基化作用被甲基化。
目标基因:ZNF582
试验样本组别:
1.HeLa子宫颈癌细胞株(HeLa_0);
2.处理10μM的DNA甲基转移酶抑制剂5’-aza-2’-deoxycytidine(AZC)的HeLa子宫颈癌细胞株(HeLa_10D);
3.同时处理10μM的DNA甲基转移酶抑制剂5’-aza-2’-deoxycytidine(AZC)及0.33μM的TSA的HeLa子宫颈癌细胞株(HeLa_DT);
4.子宫颈鳞状细胞癌样本1(SCC 1);
5.子宫颈鳞状细胞癌样本2(SCC 2);
6.对照组(normal):以正常子宫颈血液DNA作为无甲基化的对照组。
其中子宫颈鳞状细胞癌样本1及样本2分别为不同病患的样本。
上述试验样本借由亚硫酸盐定序(BS)分析目标基因(ZNF582)在各试验样本中是否存在高度甲基化(hypermethylation)现象,结果如图1B所示,相较于对照组,目标基因ZNF582于子宫颈鳞状细胞癌样本1(SCC)、子宫颈鳞状细胞癌样本2(SCC)以及HeLa子宫颈癌细胞株(HeLa_0)呈现高度甲基化现象。故,目标基因ZNF582于子宫颈癌样本中会被高度地甲基化。
目标基因:PDE8B
试验样本组别:
1.SiHa子宫颈癌细胞株(SiHa_0);
2.处理10μM的DNA甲基转移酶抑制剂5’-aza-2’-deoxycytidine(AZC)的SiHa子宫颈癌细胞株(SiHa_10D);
3.同时处理10μM的DNA甲基转移酶抑制剂5’-aza-2’-deoxycytidine(AZC)及0.33μM的TSA的SiHa子宫颈癌细胞株(SiHa_DT);
4.子宫颈鳞状细胞癌样本1(SCC 1);
5.子宫颈鳞状细胞癌样本2(SCC 2);
6.对照组(normal):以正常子宫颈血液DNA作为无甲基化的对照组。
其中子宫颈鳞状细胞癌样本1及样本2分别为不同病患的样本。
上述试验样本借由亚硫酸盐定序(BS)分析目标基因(PDE8B)在各试验样本中是否存在高度甲基化(hypermethylation)现象,结果如图1C所示,相较于对照组,目标基因PDE8B于子宫颈鳞状细胞癌样本1(SCC)、子宫颈鳞状细胞癌样本2(SCC)以及SiHa子宫颈癌细胞株(SiHa_0)呈现高度甲基化现象。故,目标基因PDE8B于子宫颈癌样本中会被高度地甲基化。
目标基因:DBC1
试验样本组别:
1.SiHa子宫颈癌细胞株(SiHa_0);
2.处理10μM的DNA甲基转移酶抑制剂5’-aza-2’-deoxycytidine(AZC)的SiHa子宫颈癌细胞株(SiHa_10D);
3.同时处理10μM的DNA甲基转移酶抑制剂5’-aza-2’-deoxycytidine(AZC)及0.33μM的TSA的SiHa子宫颈癌细胞株(SiHa_DT);
4.子宫颈鳞状细胞癌样本1(SCC 1);
5.子宫颈鳞状细胞癌样本2(SCC 2);
6.对照组(normal):以正常子宫颈血液DNA作为无甲基化的对照组。
其中子宫颈鳞状细胞癌样本1及样本2分别为不同病患的样本。
上述试验样本借由亚硫酸盐定序(BS)分析目标基因(DBC1)在各试验样本中是否存在高度甲基化(hypermethylation)现象,结果如图1D所示,相较于对照组,目标基因DBC1于子宫颈鳞状细胞癌样本1(SCC)、子宫颈鳞状细胞癌样本2(SCC)以及SiHa子宫颈癌细胞株(SiHa_0)呈现高度甲基化现象。故,目标基因DBC1于子宫颈癌样本中会被高度地甲基化。
实施例四子宫颈癌样本内目标基因的甲基化分析
以甲基化特异性PCR(MSP)分析该四个目标基因在子宫颈鳞状细胞癌(SCC)样本中的甲基化状态,其甲基化状态分析结果如表四所示,结果显示,于正常子宫颈样本中,DBC1、PDE8B、PTPRR以及ZNF582的甲基化的频率分别为11%、0%、9%以及6%;于子宫颈鳞状细胞癌样本中,DBC1、PDE8B、PTPRR以及ZNF582的甲基化的频率分别为100%、47%、100%以及97%。由此可知,于子宫颈鳞状细胞癌样本中,该4个基因皆大幅地被甲基化。因此,DBC1、PDE8B、PTPRR以及ZNF582的甲基化程度确实可作为筛检子宫颈癌的筛检指标。
表四子宫颈鳞状细胞癌样本中目标基因的甲基化状态分析
实施例五卵巢肿瘤样本内目标基因的甲基化分析
以甲基化特异性PCR(MSP)分析目标基因在卵巢肿瘤样本中的甲基化状态,其甲基化状态分析结果如表五所示,分析在卵巢恶性肿瘤样本及卵巢良性肿瘤样本中DBC1、PTPRR以及ZNF582这3个基因的甲基化状态,结果显示,在卵巢恶性肿瘤样本中DBC1、PTPRR以及ZNF582的甲基化频率分别为50.3%、50.0%以及56.3%;在卵巢良性肿瘤样本中DBC1、PTPRR以及ZNF582的甲基化频率分别为2.5%、0.0%以及12.5%。其甲基化差异程度分别为53.8%、50.0%以及43.8%。因此,与卵巢良性肿瘤样本比较,该3个基因于卵巢恶性肿瘤样本中被甲基化的情况明显高出许多。故,DBC1、PTPRR及ZNF582的甲基化程度确实可作为筛检卵巢癌的筛检指标。
表五卵巢肿瘤样本中目标基因的甲基化状态分析
实施例六大肠癌样本内目标基因的甲基化分析
以甲基化特异性PCR(MSP)分析目标基因在大肠癌样本中的甲基化状态,其甲基化状态分析结果如表六所示,分析在大肠癌样本中DBC1、PDE8B、PTPRR以及ZNF582这4个基因的甲基化状态,结果显示,在大肠癌样本中DBC1、PDE8B、PTPRR以及ZNF582的甲基化频率分别为100.0%、100.0、100.0%以及100.0%;在正常大肠组织样本中DBC1、PDE8B、PTPRR以及ZNF582的甲基化频率分别为25.0%、25.0%、25.0%以及25.0%。因此,与正常大肠组织样本比较,该4个基因于大肠癌样本中被甲基化的情况明显高出许多。故,DBC1、PDE8B、PTPRR及ZNF582的甲基化程度确实可作为筛检大肠癌的筛检指标。
表六大肠癌样本中目标基因的甲基化状态分析
本发明所提供的癌症诊断的方法,与前述习用技术相互比较时,更具有下列的优点:
1.本发明所提供的癌症筛检的方法是以离体的检体中特定基因的甲基化程度作为癌症有无的诊断指标,与习用子宫颈抹片及人类乳突病毒检验(HPVtesting)方法比较,本发明的癌症诊断方法的敏感性及专一性均较前述两者高。
2.本发明所提供的癌症筛检的方法除了可作为第一线子宫颈癌的筛检之外,亦可合并或辅助人类乳突病毒检验(HPV testing)检验,作为第二线子宫颈癌的筛检,以达到更准确的子宫颈癌筛检效果。
3.本发明所提供的癌症诊断的方法除可应用在子宫颈癌的检测上,亦可应用于其他癌症(如:卵巢癌、大肠癌)的检测,以辅助异常检体的诊断。
以上所述仅为本发明的较佳实施例,对本发明而言仅仅是说明性的,而非限制性的。本专业技术人员理解,在本发明权利要求所限定的精神和范围内可对其进行许多改变,修改,甚至等效,例如:受测者检体中各目标基因甲基化程度的判断方式等变化的等效性实施例,但都将落入本发明的保护范围内。
序列表
<110>赖鸿政
<120>一种癌症筛检的方法
<160>24
<210>1
<211>5000
<212>DNA
<213>人属智慧种(Homo sapiens)
<400>1
ggtcttcacc tgaagttgcc tgactacgtt gtctcagatt gatttcagaa ctgcattatc 60
agcagggcag ctgttgccta ctaacagtca tggaagtaga tctcagctgg gggatgatgg 120
ggaaacaaag agaaagatag ggacgaataa aggaaggaat ggggcacaga cagaaagaga 180
gagagagaaa agaaagaaag gaggagaggc aagagatagt gacctggggt ttcataccgc 240
ggtgggtggg agaaggagtg tgtgtggtgg gcgtatttct tcgtggtcag aatcaaagtc 300
agaactgacc tggggaacct gtgttcaaga cctgaacctg gaccagaagc caagggaaag 360
agaaagttgc ccaagaaaaa agggagaaat ttccaccagg cgaaaataga aatcgctgac 420
tgggtttgtg gctggagagc tgtccgtggt gctgactcct cctcattggg attccagtga 480
aggctgagaa gctctgcact ggctgctcct cctctccctt tcctctcccc tcagtcctga 540
ccttctgaga cccaggatca ttctggctag ctcgatgtct tctcctctct ttctcccttt 600
ctctttgtcg attaaataat ttttcccgag ttcccagttc atcactcaaa catttctctt 660
ccattttgtt tcaagactcc tcttcctcca attagttcac tggctcctag attgttctag 720
gcctccctgg tggtcacatc tctctcttat catcctttga attaaaaaac aaacaaaaaa 780
catacaatgg caatcataaa agcagacaac atgcactgag cactcactag gtgtcaggga 840
gcagtctaag tgcctttgca tacattaact catgagcagt ctaagtgcct ttgcatatat 900
taactcatta caattccaca tcaaccctat tagatgggta gatatattat tattctatta 960
aacagacaag gaattgaggc ttaaggggta agtgatttca ccaagaccag acagctacag 1020
gactccaacc tctctggagc ctgcacttct actgaatctg gctcttttac atattccaac 1080
accttttcct atgacttcct gcccctccat ctggtagcct taatgctttc tattcttgcc 1140
taatcttcag gggactaggg atctgcattg ctgatttggt ttaaatcaga agggaagcaa 1200
agtaaacaaa cataccaaat gtgcgctgtg tgggaattat catacgaggg ctttattttc 1260
tgcttcagga agaggcccta tgttagcagc cccagcctgc attcaggctg attgcagagt 1320
attttgcttt ttattttcat gtcttagtcc ctgtaccctc gccccttccc cgcctctggt 1380
ggtctccaga gaacttcgtg tcccctcagc ttctccctcc tacatcctgc ctacgtagag 1440
aagctcttgc ttcattctgg gaggttacgt gggctctcgc ctacacaccg agagaaacaa 1500
acagtgtcaa acactcacag agagacgcgc agacacaaac ggacccacac gggcaactcc 1560
cgagacaaaa cccacactcg atggatccac gcggccgtgg aaacacctgc cgccccagaa 1620
acactcaggt actcgcgaca cacacagtac agtcacgctt aagggcacca ggattccggg 1680
tttgcgcgta tgcgcggtcc ctttggatgc tcgtgcgcat agacacaaca ccctacacgc 1740
cccagaccca cgaaactccc tacggctcag ccccagccca cccgggccgc ccttccctcg 1800
aggcggcctc ccgtctctcc tcctctcgct tctcctcctc ctccgcctaa agatgtacaa 1860
aacactcctc ggaagcaacc ccggcgttca gctcctccct ccccgccccc cggccgccgc 1920
tcccccattc attttcggcc gtcgccggct aagtccctcc cccggcgtag cccggcctcc 1980
gccgctcccc gcccggagac cgcggcgcac ttggacttcc ctctccattc gccagccgcc 2040
tcgctcccgg accccacggc tgcaaactga tctggcgcgc ggggaggagg agagcgcagg 2100
cgagcgaacc cgcgagagag ggagagagcg agcgagcaac agcgagagcg agagcgagag 2160
agccgggagg cagagggagt agtgaccgcc ttccggagcc gggattcatg cctgtcctcg 2220
ggaccagcga aggggacttt acggctgagt atgagccagg ctgctaggag ccaggtaccc 2280
ccacgcctgc agtccccgcg ccgtgcccgg aatgcgagct gcacgcaggg ctctcccaag 2340
ttcccaccga gccgaataaa aagcgtcctc ccgcagctct ccgccaaaga cggacattga 2400
ctccaggtaa ggcggcgccg ggtgcagcgc cccgcagccc cgctgccctt ggacccggcc 2460
ccgggccgca ttcggggcgt ccccgcgctc ctctgcccct ccccctaccg gcacccttgt 2520
ccgctcttca cctggccgcc ccgccgcctc caagtcttct ccagttctag ggagggggtt 2580
cctgtgcctg gggctcaaag ggctaattgc gggtttgagt gagtggcgtg tgtgtccccg 2640
cgcgctcccg acgtgtgcac catggtggga acttgatgtg gtgctagtgt gtttgcgtgt 2700
gcggcgtcgt ttgcgtttga tgcgtgtgtt cgtggtgtgt gtgtgtgtcc gtgtgtgtaa 2760
gggaggggtg aagagagaga ggtcctataa cctacttacg gcgcgatgtg tgtgtgcatg 2820
tttgtacgta tgtgtttgta tgtgtgccct cgtgtgtctt tttaattagg tctctccagc 2880
ttacacggaa tgggaccctt actataggat cacgtagtca ccgggaaacc cgctgtggac 2940
ttcctcttgg ggctctgggc ttggggtttg gggaggatta tggggctgta gatggcacct 3000
tatttagccc aatgttggta cgcttgaagg aaaattcctc caaacggtgg aatcctgcta 3060
cactgggacc cacagcttaa tatgaaagag acatggccaa cccccgaggc aaatgagacg 3120
ctgtcacttt aaattctaca ctgggcagac tccaagattc tgatgggaat ttggagacac 3180
tggatagtgg gtgacagaga aagggggaag tcagcggtgg gctccttatc tggggcttgg 3240
aaagtctggg atagggattt accctgcatc cccgtttgca atcaacagag cccctccggc 3300
ttctgttggt tttggggagg atctggcaag tttgcatgga tccccctcag gggaaaggag 3360
aaagcgtcct cggggacttg ctcatccatc acagttgcaa agggtctcag aggaaatttc 3420
atctggggcg gctgtggatg atatggaagg agatggatgg ggcactttcc taagacagat 3480
tcgtctttct ttccccattt caggcgggga agcccccaga gagtctctcc ctaaatatgc 3540
ctctccatgg ctcccctgga gttaggggga tattgagaga aggcagagag gtggagaggg 3600
agagagagag agcaagagcg agagagaggg agacagagag agagagtgtt tcagtactga 3660
gggagatcta caatttgaaa aggggctgtg agtgtggaac ccatgacaga atgtggcagt 3720
aattgactta aactgctgtc ggtttgcacc tcgcgtctct cacttggctc caaaatactg 3780
ccaagggcag gggggcggtg gaggaatctc agccaggaag gtagtttggg tcaaggccct 3840
ctttcttctt tcctccatct cctgggaggc tctttaggta ggtcccttgg ccaccctggc 3900
ttggtactgt tgggcttggg ctctggggcc agccattgat ttgattccca gctgcctctt 3960
aaaggcttgc cctactcagc aaaaatgctg agttttactg ctgttagcat ggcttccaga 4020
ctcggcagct gttactttct caaggtaagc ggcagccgtt ctgctctcag gcagggaggc 4080
taggagaaga caagggctgt gacgctggga cacagcctct tgtctagctc agcacggagg 4140
ggccgctgaa aagccctctt aggggaaaaa agtagaaata tatttggcct aaaaaatgta 4200
cacatatatt ctaggaagat atatatatat atatatatat caatcacaca cacacacaca 4260
cacacacatg tgttcatatt ctggggagag ataaaagcaa acatgtattt gtaggaacct 4320
gccctagtgg gtgggttcta cctgcaggca cttgcagaca taccattctc tgtccagagg 4380
ctgatacctc aggctgaagc cttcacatag ccacagagaa tctccttcag gaaactcatg 4440
tcagggcaga gctccctgga ttatcttggg tagggggtac tagggccaag gaggagaccc 4500
atttttggga actgcctcat tttctctctt tcctaattca cagacgatcg aaactggaag 4560
gaatattaaa gagcagaaag gcaggactct gtggcctaaa gagggaatga gaattcccca 4620
acattcagta gtgggttagt ggcatgccca ggactagaat ctaggcttat caatcagcta 4680
gatcaatacc ctttccactc ctcccagcaa tatacactct aggactgtat attgctccca 4740
cccctatctt gccaacctgc caccatgacc cctaccaaga caatcgcttg tcttccctca 4800
tcaaacttca cattttcaga gatgcatttc agggtttttc tatctggaaa catggccctg 4860
aagagaaaag atccaatgcc tcaatcacta ttcccgaaag acacacacac acacacacac 4920
acacacacac acacacacac acacacacac acacacacgg tggtgggggg aggactgaga 4980
gagagacaga gagagaacat 5000
<210>2
<211>5000
<212>DNA
<213>人属智慧种(Homo sapiens)
<400>2
agaacaactc tgaagcatca tcccaccttc agaatcctcc ctgcagggat gcctgaggct 60
tggtcaattt ctccctctgc tcaagcttgc ctccctccat tccctccctt tatttcctcc 120
cacaaatgtt gactacaaga gcacttccta aaaagcctcc ctctgcctgc taaacaccat 180
ctcagaatct gcttctagga ggacccaacc tgtgcagaaa ttttatgagc aatttgcctg 240
tccattaaaa aatacagcat agactttttc caggttagct ggagatggag tttttttgta 300
tcaaaaaaag agagctaaac tgagaagaga agcaaaggta gaagaataag gttagactct 360
ccctgtataa ctgaatttgg gattgggaat gatatcttgc ttctctatga aatttagaaa 420
ctgtcttttg aagtcttcat ccagtcacat ccaagagtgg tccaggaaga aaaaggaaaa 480
taagaatgaa aaaaaaacaa aaaacaaacc ttaaaattgg atacttcttt gtgccttgca 540
ttaagcctct atgttactgc ccttgtccgt ataagcaact ctagataaaa tcctttctta 600
gaccatctaa tttatctccc cgtgccattg tgaaaatcgg gcaggaatta ctgcgggaac 660
cagtgactga atgggcagtg gtgtctcgca gctgtaactc agagtttaca agggaaggat 720
gaacagcagc catccttcta aaggcattct tcctctcctc cagcacagct cagtctgtgc 780
atgcctcacc aaggagaatg agggaaggcc ttgttgactg cccagcccag agtgtgagga 840
acagcagttg acatgggcgg ctggacggga tagaaaccag catgaacatt tagtgacacc 900
taaggaagag gtgaaaatgg gggagaaagc caagaaccat cagacagctt ctttctgttc 960
ttgccttgga tcttagggga acgtatgtgt tcaagggtgc cttcctccgc agggcctccc 1020
atcccttggc acaaacttcc aaacgatgag tagacatgga atgggcttct tctgaagact 1080
gggtcaccat ccaactcagc cttcggagta ggcggtgggg ctgggggaag aggacggaac 1140
catgaggtct ctccatcact tcccgggtgt gttgagaggc aagcaaatgc ggtgggtggg 1200
cctgggctgt agggcccctg ccatttcggg aggggcctcc tggtgtttag ctctgggatg 1260
tgtgaaaatg tgttggtgaa aagcgagagg cttacacagc ccttcagggg aagaggggct 1320
ggggcgctgg gggcggcgtc gggatgagtg cagaagagac gaggcctctg gacagcggag 1380
gaggagggga gggcgccgag gcgcggtgcc agctgccgcg cacaggggcc ccgcggcgga 1440
gccgagccgc gggcacgctc tgccctgtcg ggagagctcc gggagcggcg ggaggggcgg 1500
aggggcgcag tggggcccgg gcggctgcgg ccgcggagcc ggggcacctg aggaggaagg 1560
agggtgggag cgagggaggg aggggacggg cgcagaccga aagtggggaa agaaggtgca 1620
ggcaggcggg caggcgggcg ggcgccctgg cccagggccg cgggtgcggg agcccggcga 1680
ggtcgagctg ggcggcggcg ggggccgcgc cgagggagga ggggaaggcg gaggcgcggg 1740
gagcgtgttt ggggcgccgc ggcggggagg gtggcggccg ctggtgcgcg cggggcgctg 1800
tgtatgcgcg ctcccccgct cggggaggaa gatggcccaa aagggaaagt tggggtgacg 1860
cgcgcggtcc ccggaggctc ggcggggggc accgcggcca gcccgacgga gcggcggaca 1920
cacaggccgg ggggcgcgca gtccgggcgc cgccgcggcc gccccctcac tgcaggtggc 1980
agcgggtgcg ctgggtcccg gcggccgcgg gcgcgggcgg gcgcgcgggg gagcccggcc 2040
gagggatggg ctgcgccccc agcatccatg tctcgcagag cggcgtgatc tactgccggg 2100
actcggacga gtccagctcg ccccgccaga ccaccagcgt gtcgcagggc ccggcggcac 2160
ccctgcccgg cctcttcgtc cagaccgacg ccgccgacgc catccccccg agccgcgcgt 2220
cgggaccccc cagcgtagcc cgcgtccgca gggcccgcac cgagctgggc agcggtagca 2280
gcgcgggttc cgcagccccc gccgcgacca ccagcagggg ccggaggcgc cactgctgca 2340
gcagcgccga ggccgagact cagacctgct acaccagcgt gaaggtaaat gccccgcgct 2400
ggcacacgcc gtgggggccg tccgccccgt cggcggggct cgcacgggta gggggctccg 2460
gcggagttgg gtgaccgtga ggcggttggt ttggagaggt tgtcactaag gaggagttta 2520
cttttcattt gtggagatga tgggagccca ggaaatgtgg tcagaaaaag gcccctggag 2580
gggtcctgga agcgtcctta gctggtcctg ggggactggg cggggaaggg agcgcagaag 2640
gaagcaggtg ggctggcctg ttcctccttg agggcaggaa ggctgtggct tggtttatgc 2700
aggaagaggg gtggggacca ttgagagcat tcggtggcca gtcctgttga atgaaatctg 2760
agcactgagc tggatttgcg tgccttgtag gtgactggtg cagttgcagc accaggatag 2820
atagtgcccc atattccgat ttttacctgg gattaccagc caggctggag tctcagcaca 2880
ggaaccgagc gtagggattt gtgaatgaat gagtgttcgt gttttaagag atgtgggaac 2940
ggagcagagt ggaacctgtt gtttgtcact gtaacgtttc tctgggttgg ctgcatccta 3000
gacagaattg agagaaccgg gccatgagtt cggagtgtca gcagagccac cgtgagggga 3060
cgtggtttcc agtgcagtac agctgtctga ggatgattct gcacatacaa ctgatcttcc 3120
cagagagtgg gattttgagg aagtggaaaa aattgtttag atggttaaag cagtcgctaa 3180
atatttattt cttaaagaca gaagaaaaat aattatttaa atagtgtcct cccgtatgtt 3240
ctcaaagtac cgttaaatcc aagaggcttt tcattgtgta aatctgggca ctgggtcttt 3300
tttcctttca gcaaacaaga taacaatggc atatcctatt gtacagagag aaaaaaatac 3360
tcctaatgtc agatagaatc acagccttta cctggtcaat aggttaccaa gaaccattcc 3420
atggattatt tgtcaaagac acatttgtat ttttaatagt taaaaacttg gtcttcattg 3480
gatgaaggca gcccacagtg gaggagtgag gagaaggata acatgttttg gcttcaatct 3540
ggaagtccaa aagtttttta atgaacctat ttttttttta acagcatctg attgtttaac 3600
atgaagcttg actgatgttt gcttgtggtt gcagtgtttc tctggcagta atcataatca 3660
ccattatagt aaaaacatcg tttattgagc acctactatg tgccaggagt aagctttctc 3720
tcctaactat tgaatgttaa cagtgtgatc ttagcatagc atgtctgaag actagagtct 3780
aggatttatg acagtacaaa cctaccagtt gcccattcgt tacagaggca tgtgctgaac 3840
gctatacttt gcttttgtca agctctctct ctctccccct ccctgttcac acaaagttag 3900
ctttaggagc aattttcagg gatgaaaatg ttaaatttgg tgaaaatatt aagttgggat 3960
attatcagta aagaccatcc ttttgcctga cggttgaaga cctgaagcag tagttcaaaa 4020
ttgtgacagc tcatgaactt tgtgatactt tttttggttg ctccagagaa attgtgaatt 4080
tttgttttct agaatatcta taatctcagt ttagtacttg gcaactctag ctactccatg 4140
tttgttcagt agaagaaaga atactgaatg aacccattct gtacattttt aatacttccc 4200
tcaagaggct gcttagagac caaatgaagt aaaaactgaa gggacaatga gccccaatta 4260
ttagcagctt gtataaactt gtattaatct cattgctact tggcttaagt attaaaaggc 4320
ttgacctggg taggagttta actcattttg ctttatcaag aagattccca aataagagct 4380
taattcactc taacccatta ctgggaatgt tggaaatgaa acccctggtt cattgactta 4440
acactacttt gcattgtgtg gatctcagaa agtatatatt tgaagtgaaa tttgagacca 4500
gagttattta tgcaaatctt acagaattat tatcacagaa ctgccctaat atatttaata 4560
gtatcctttt aggcctgtga ctcggtgttg tattgtcttt tctcaatcgt aaagttcaca 4620
tgttctgtgc ccttttgttg ctgagataat tttataaaaa ctttacattc atttttttgt 4680
agactaagca ttcttttttt gccgtaagag tttgagatag ttgctaggca cgtggtggtg 4740
ccccacccct ttccaaagca cacaggtgga aaaataaaat ctttaccatt tggtcctgtt 4800
tacagtggtg gaaggttgta atagtttcca ccccctcccc ctggccattt tacatcaatt 4860
atgaagtgta tcctcattat ctacttggcc tctggtattc cccagagtgc aaaaatgtgt 4920
gcttgatctc tctctctctc ttttctagca ataattcatc tcattaagct tttagaatga 4980
tgaggcatta tgatgtcaaa 5000
<210>3
<211>5000
<212>DNA
<213>人属智慧种(Homo sapiens)
<400>3
aagaaaaata gacctattgt aagtgggata caattatttt tccctaacct ccagccctgc 60
agcatttcta gtaacagatg ttagatgaga agagttattc ttctaagctt tacagaatgc 120
taaataaatt atctcaaagg ttgactcagt aagaatttca aagagttaat tgtcagtatt 180
gaactttgtc aactatcatt ttcatttatt catttttgag tatctatttt gcatgaggca 240
ttgctctagg gagttgggac atatcaataa agtcatcaaa gttcttcaac attggagctc 300
aaattctgtt ttgcgggggg gagtagggaa atagatacta aacaatagat tttataagta 360
attattttgg tttgttagaa agtgatacgg gaaaagcaaa ctggaggaca gtgaatcagg 420
aataggagaa gaggttgcag cttagagtgg tttgggatga aatgatagtg ccacagaaat 480
ggagtgtgcc acacgcatgg gggagtaggg ggtggagcac gttgctagag aggggctaga 540
catggagata tgtctctatt ttcagcagag aaggcctgtc aagatattaa aagtacatct 600
cattgtcccc ttctcccact tgttggagtg cttcctggcc ttgtctctca gagctgaaga 660
tgacataaat cataaaagaa ataaatggta ccgcctgggc tctctgatct tataaggtcc 720
cacctaattt aagctctggt ggatagccta tttcccatta gttgtctcaa ttcacagagg 780
atggagaaag aaagcactga atgagatgtg cagctcaact caatgggcac ttgttagccc 840
caattatttg gctaggacaa ctttattcaa gttaatggaa tacaaaactc agcagttctt 900
atggagactt caatgtgctg tagaacaaac cacagataaa tgaactgtct gaaagaataa 960
tatgaggcac ggctaaacaa gtgttgtgga agagaaagat cactgtaatc tagagttagt 1020
tagagttagg gctttctgga aaagtgagac caacattaca ccttcgaaaa ctagtgtggt 1080
ggtggtgcaa gtggaggaaa atgggaaaga ggcaacagtg tccataaaga cacagaggca 1140
gaaagggcag aggccctggg atagttaaaa gagaagtcta gctgagggac ttcttgactt 1200
gactagcagc aattagccat gactaataag gctttccaca ctccaaagac ttagatggag 1260
ggataaaaaa ccatctaatg gcagactgtg gtagcctccc tagagacaca gagctgggcc 1320
ggatgagtcc aggcactgac gtgatccatt atctttcacc ttaaagagta aaagggaaac 1380
taaagttaat tacctccacg aaacaaaaag gtgccttctt gtgcttcaat tacatggata 1440
tattctacta gtctaaaagt atcttctcac ttctttctgt cactgtgagg acttgagtca 1500
gaagaaagtt taaatacagt cattgagctg gaaagagtgg aaagagaagc aaagaggggg 1560
aagctgtagg aaggacgaag tcacccccaa gatacatggt tactgcttac accaagcaag 1620
ctgccttggg aacgcttccc ccgagcagcc agaatgctca gcagtggaag acacctctat 1680
tcctgtaggc gagtcctggg aagctggtca atctgcaaat gccaattccc agcagtgagc 1740
tcggtccacg tgtaaatcaa gatttgggga aagagtaggg tgggtggcat ggttgacaat 1800
gtcatcagct ccctcctctg actcctgtgg tcgtgccccc atctactctc actcagctac 1860
accccacctt cggatttgtg atggacgctg ggtccctagt aaccacagca agtgtctccc 1920
ccgcacttcc cccttcccca cccccacccc cacccccaac caccacccca gcgatggagc 1980
ctactctgct ccaagccgcc gctaagaccc ggagaagcgg aatttcactt tgaaattccc 2040
ttgcctcgtg agggccggcg ctgggcatgc tcagtagccg cggcgctgct gctgggctgc 2100
tgggctggcg cggagtccac cctgccgtct ccgccttggc ttctgggcgt ccagaaggcc 2160
aggcatttgc cgcctctgag cgcttctgtt ccccttaccc gcaacctcct actgctcttc 2220
ctctctccct ctcttaggga ggttgaagct ggtgctggtt tctgtcggcg ccacagactg 2280
actgctctgc aaaccccagc cgaggacctg aatcccggag actagaagac ccttggcggt 2340
ggctctttct aatagcactt tacctgaagt ggggtcgtgg tggagtttct cctccacctc 2400
tcaatgcaaa cactatgcgg agagcagtct gcttccctgc gctgtgcctg ctccttaatc 2460
ttcacgctgc aggtaagggg ttgccaggct tagagccgga gctgtgcatg agatgggaaa 2520
ctgcacatgc ttaaggactc taggaaaagc ttgccttgcg aagaaagcct ttctaaaaag 2580
gtaaaacagg acagtacctg acagggaagg ggtgagggag tcgtgcacct gttggaaaac 2640
tgaggccgaa aacttaacct aaaattagct cttgattttt ctttacttta tacgaaaata 2700
gtgaacattt tcaatatgaa tacaggtttg ctcccccctt cgcctcccct ccgtctagat 2760
tccctgctct tgtttctagg ctatgcactt aactgtcaat ttttcaggag agggataaga 2820
catcctgcta gatgtaacct tttctactgc agcggctact acattcataa ggtctcttgg 2880
tctagcgagc gctcatagga aggcattggc tgtaacctga tggaccacat ctccgcccaa 2940
aagatcgaaa tacatggttc acattagtga tgctgcacca ggctcctttg gcccctgctc 3000
ttgtcactcg aattttctaa gccaataaga ggaagaaaag gtttcaaata gaatcctctt 3060
gtctctttca gcaccgtggc tagcgtccgc tggcatatta aaaaaaaaaa aaaaaaaaaa 3120
aaaaaaaaaa acaattacag ttccaaagct gacaaccctg ggttaggcac tgtccctcgg 3180
acagatttga taaggtgtaa ccaagagtaa cagtgagact gctcccaaat ataaatagat 3240
ttgaggcacc agaatcaata tcaagtctcg tgaggaagca aaccttggga agtacctgag 3300
agatattata tgtggtgtcc ctgctttttg catacatagc acctctattt aatgcagaca 3360
gacttgctag gctgaattcc ttatcattgt cacacacaca tgaaccaaaa taaaatatgg 3420
cagggagatt ttagaaaccc tactgtgaca gcagttgtcc atgcagtagt cctgtaacaa 3480
ccagagacga ggcattttcc ctgagtgtgc ttctcatcca atcacacata tccattcatt 3540
ccttcaagca acatttgctg agtgtatact atgttctagg ggtaatgagt aaaaataatt 3600
gttctgaaat tacaatcctt gcagtatctt ttttaaaaga gtgggtgaat tttattctct 3660
gtgtacaata gtaggagtga aatatggtac tttcttattc cagctaacgc tattatttaa 3720
atgtatcatg aatcttttga gcaaataatg cataggatta ttaaagctat tattaaatac 3780
acatgttaat tgttataatt atgatgttat attgtggagt tttcaatagt tctttggcac 3840
attttgagaa gtagaaaata gctatgactt gattattaaa ttatttgcaa acagctgtag 3900
tgaattattt cgttaccaca tacaattctt caatgctgaa atgtgggatg aaaacataca 3960
cattgggttg agttttattt atagtattgc ttaataatag actgacacaa acattcatta 4020
cagaaaaatc tcaattctta ctaaggtagc aatgatttag ctggtctgcc aacaatatca 4080
gtgtagtcat tttttcaaaa ctaacatata aaaaagttta aagatcacat ttgatacatc 4140
ttgagtaatt atatcttaaa agttataaat aaactgaagc atgtgaaaaa catgtcttga 4200
tttactgtag atgaatattc gggatgagaa aaatgaggac ttttgctcat cagatgagaa 4260
acctagaagc ctgtagccct agattttacc tggagttctg ccaaagggta tattaccaaa 4320
gccaatccaa tccatctcta cttcaacttt ttaatttgca aaacaaagat aaaaagtaag 4380
attaatcaac caattttaat ttaataagaa aatgcattta aggacatttt tatatttcaa 4440
ttgcaagata ctattcattg tattcatatt taatatagta aaatacattg gtgttttatc 4500
acagcagatt gtaagtttaa tattatagtt ctcttgccaa tttcatttta aatgcatttt 4560
aattttaaac agcaatttgt gggtcacaag ctgtcatcag attaaaattt atcacagtta 4620
tctaataaca ttgttagggc ttttcatcag aactaaaaca aacacagctc cttggtctaa 4680
gcttctatac atttggagat taagaaaata agtggatggg atgacaaaga tcaagaaatc 4740
aagatattaa tctctacttg ttgaaggtgt gagtttgtta cttttatgga agacttttct 4800
ctcagaacag aggctgactt cataaagaag ggaaatgaat tctatacatt caggcctctt 4860
taactgtaaa gctaattgat ccattttggt tacgcccagg gtatcagcaa ctgctttatg 4920
ctggtggaac tgatgacacc ttataatgct ttagagatgg gaggaatgtg ctaacgggat 4980
gtaagtgttc tttttagcta 5000
<210>4
<211>5000
<212>DNA
<213>人属智慧种(Homo sapiens)
<400>4
gacgaccctg aatgatccct gcctcctagt acttatactc ttgtgtagcc tccacctact 60
tagactcaca gctgattttg tgaccaagag tataatggtg tatgacttca gaggcttggt 120
tcagagtcag aagtggccat ttcatcatcc tctaaatttc tctttggcag attaatgaat 180
gtgagaaagt aaaaatggta tagaagaaag ttagcctgag agatggctgg ctttgtacct 240
gtttggtctt tttccttcct ttcctccata agcaaatact gacgcatgta catatagaca 300
cactcaacac agtgggatgt taaagggaaa ctagagccac tcgccccttc ccaagatcag 360
agaacatagg aaagatgatg actttttgca tctaccctct cctcttgaac tcgcaacact 420
gtcttgaacc acaaataagg tcaagtagga taaatctaca actgtctcct caactgtccg 480
ctcacaagtg tgaaattcaa ccatccttgc tgaagttgtc ttttaatatc agtaatcagc 540
aataaccaac ctctgagctt tgcttatatt attaaatcat tttatttgac cctcaggaca 600
aagctgcaag cccagcatta cattcttcct tttatggaca aaaaaaaaaa aacaaaacaa 660
aaccgagata ggcacgaaga aactaataaa tggctgaaat gctatttgaa tccacatctg 720
tctaattcta aaagtgtgct ttccaccaca tcaccgcacc tacaaggaaa atgcagcctc 780
taccctctcc tgaagaatgt gtaaagaggc aatatggtgt gttttggaaa aagcatcttt 840
gcagaaagaa agcattagct tcagtcatat tagcatcctt atccagcagc agtagcatta 900
ttttacatat cagttactcc tccccaacaa ctcaatgaat tagatgccac aatcatcctc 960
actttcaaat gtggagaccg aggcacatct ctaagactgg agttcaggca ttctggctcc 1020
agtcttagag atggttaagg gttcacactc ttaaccattt attacaccat agagctcacc 1080
aggtttgagg gaaacaggat caaatcaaaa gagtcactca ggactccagt cctcactcaa 1140
ggacaaactg ttccacctcg gacagggaga gtttccgcat tctgagaccc agcataacag 1200
gtcctgaccg gcatctggca ctcggactcc caatcatact ggatcacact ggctcgggat 1260
gtgtaaagtc cagggcttct cacatttgat gacaccaaag ccgcctaaaa acaagagaga 1320
attaacaact acctacggcg gtctgatatt tgcccaagag atgccgcccc ataaaactcc 1380
tttacatctt tataacgttt ttattttgcg ttctccttca taacccacat ttaactcacc 1440
atagatgtaa tgtttaaaat tagttaccag ataaactctt acgcttccaa actttaaggt 1500
tccttcgaaa ccttctggta aaactgttgt tccacggaaa tgggaacgta acggatgagg 1560
caatcttcca cagccgcaca cagttgtgta tccaccgcta aacggtccca gtcatacatt 1620
caacgaccca cgcggagtca gaagctacca ccacacactg tcaaaatcac gcacacacag 1680
tgacggcccc ttgcccactc ggtcactcgc ccacaatctc tcgctagaga atcacacgca 1740
gatagcacac ccagcaccac agaccccagg aagcaaccca gggactcgaa cacacgaaca 1800
gcactcctcc gcgcactgcg caggcacgcc tgcgtccggc tcaccctgaa acatcgcgag 1860
atccggcttc aaggccgggc tgctgccttt acgcctaaag actatgtttc ccggaagaca 1920
ctgcggcgcc ggccctatca tggcgcagca tcggtgtgct ttgtgcgtct gcgccatctt 1980
ccggctgcgc acggcgaatc caccggtacc gtggtggaag cgcgccctgg gctgccgggg 2040
gcgcggccgc ggtggcactt ggacccgagg aggcggcagg tgagaggttc cggagctttc 2100
caggcgctct ggggtccagc aggagctggt gcccgggccg gttgggtctc aggcctgaga 2160
agaacgcaga cgtctcgcct catcgtcgct ctgtggcttt accggcgtga gactacattt 2220
cccgccggcc ctcgcggcgt gcgctttctg gcgccccctt tctgcttcca gcctcatagc 2280
ccaggtgcat ggacccctta ggtgggtgcg caggggtctc cgaccccctg aaattgcgga 2340
tgttttgcat tcactgttat gcgtgccttt tttttttttt aatctgagag gaaatcttgt 2400
ttcatgaggt tctcagagag ttacaagacc ccagaagact tagaaccgct agtttagaaa 2460
aacttatact tgggaacatt ttatatgttc aaattctatg tccagcaatg gggaaacact 2520
tgagtggatc acaggccatc ctttgtaaat aggatatcgt gcagttaata caaataatgc 2580
ttataaaagt ggtcataata aggaaaagct gcctattaca tcatattaag caacattcat 2640
aattatttca cttacacgtt acttagaacc aaatgtttgt aatgcagtag gcaccttata 2700
tactccttta cagagtactt cttgtttaac agtgagctca agaactggtt aaatgacaac 2760
actgcgaata cttgggtgtt ttttcttcca atttttttgt tttgttttgt ttttagcctg 2820
tgtgttagtg agtttgactt ctttttgctg aatgttctca aaggattcag tagttaatat 2880
ctttgcttgt cagtttttga tagtcatttt tgatagtcac ttggcatccc attatatggc 2940
tgtgccaaaa atgattcaac ctcttggcta cttgtggact tgcctactta cgtactaatt 3000
ttcagttctc ctatttagca aatattattg gacatgcagc tttgtgcact gctgtgtctc 3060
tatggaagga cacatttatg tgctgataat ggagcactgg gcgtgctgtg tgtaatttgt 3120
ctcttactgt tccttccaac tctatcccat ttgttgatgc aaggcatgct ccatgaagtg 3180
gcatgcacaa agcctcagtt tgggctgtag agctttttgt atatttggcg gttgggaaaa 3240
gagccaggta gactttcaaa ggtgttatat ttgctgcagg agagagttaa atggcctagt 3300
cctggaaagg ctttattcta ggctggggct atgcaaatga tgcaggcctt ctatgctccc 3360
tggcttgagg gaagccctcc ttgtccttga ggtaacatgt gggtctcagt cactagaaca 3420
gccctcagca tccctgtctt agacttgcat ttgcagcaaa acagaaccac tggatgttta 3480
ggggaaatgg gagggaaagg accactggtt cccctttgcc ttgcagcagc taaagctggt 3540
ttcctggggt tgggtgagga tccctttgcc tccccctcct tctcattttc ttggtgggca 3600
gagggcaagc aggtggggac ttgtaacttg gcattctagg aactgtgagg caagcctagc 3660
ctctcatgtt tgctctttcc tcccccagct ctaccgtcgc aggactctgc ccttccccaa 3720
gagaggaacc agaaggagga cctatcagcc cttgaaattc taaaagtcat gtcccttgtg 3780
agtttttaaa tcccataaat atttgctttc cttatcttga aactttccac ctgggttcca 3840
tccagaaatt tggttctcag actttcccat tttagggaat aaaaggacca ggcaggagtc 3900
tcctctccca ttgtcctctc ccctctgaca aatgcccaca gatttattct ctatagcatt 3960
ctgcatctcc tgatggtttt cgtctgctag gaggagaaac tcatttccct gtatgaaatg 4020
agagcatcct ttgtaagaac tgaagtttga agagatggaa tgaatgctta ggagcaatca 4080
catatagaaa agcaggggag gtggtgttgg ttggtttcag tttcagcgca tggtacagtg 4140
tcctgctttg ggaatgatta atgattggat atgtaattga ggtttggctg ggaatccatc 4200
aaaaatttga tgccagaatt aaagtctagt ttgacgttgg acagagcatt tgaatctggg 4260
acatcctcca ttagtttctt tagcttaaga ggaagaaaag tacagagctg aggatggtga 4320
gcgatgtagc aagtgagatg atcatctagg tgggtgagtg gtcagggctt ggaaaagcct 4380
tagagatctg atccaacctc gggatccaaa ataagaggat gtttgagtca gttgtttcac 4440
ttgatagaaa attgtgcact caataaaagt tatctccatg gtttttccca tgaagaaaaa 4500
tgcttgtgtt atgttccaac ataatgatga actttcatct ccaagaaatt cttgacttac 4560
atgaaaatcc tgtatctaag atagaaaaca tgtttccata attttaaaaa ataaaatatt 4620
tggggaaaat ccatcagaaa tattttttgt tgttattgtt attccaaaag atagaagtga 4680
taggtctcac cttgagaatt tattgtatgc caagtataga gtgggcagtg tgctttatta 4740
cactgtttct taacaataag gaaaactcca tttagcttga ttttaatttt catcccagac 4800
ctgtacacag agcattgatt tgcagttaaa aggaatgttt gagaacaatt tgatcattct 4860
gttttactca tccccatttc ttctgtcacc tttcacattc agtcccaccc ttcttgttca 4920
acaaaacaac cccccctccc gcaaagacct gccccatctc ctttcatccc actgtgaacc 4980
attgaaatac atatatatca 5000
<210>5
<211>27
<212>DNA
<213>人工序列
<220>
<223>正向引子
<400>5
gttttcgtcg tttttcgttc ggagatc 27
<210>6
<211>25
<212>DNA
<213>人工序列
<220>
<223>反向引子
<400>6
gctctcgctc tcgctattac tcgct 25
<210>7
<211>23
<212>DNA
<213>人工序列
<220>
<223>正向引子
<400>7
tgtgtatgcg cgttttttcg ttc 23
<210>8
<211>23
<212>DNA
<213>人工序列
<220>
<223>反向引子
<400>8
acctatatat ccgccgctcc gtc 23
<210>9
<211>24
<212>DNA
<213>人工序列
<220>
<223>正向引子
<400>9
cggcgttggg tatgtttagt agtc 24
<210>10
<211>29
<212>DNA
<213>人工序列
<220>
<223>反向引子
<400>10
aattacgaat aaaaaaaaca aaaacgctc 29
<210>11
<211>28
<212>DNA
<213>人工序列
<220>
<223>正向引子
<400>11
tgacggtttt ttgtttattc ggttattc 28
<210>12
<211>21
<212>DNA
<213>人工序列
<220>
<223>反向引子
<400>12
cgaacgcaaa cgtacctacg c 21
<210>13
<211>26
<212>DNA
<213>人工序列
<220>
<223>正向引子
<400>13
ggttaagttt ttttttyggy gtagtt 26
<210>14
<211>26
<212>DNA
<213>人工序列
<220>
<223>反向引子
<400>14
tactccctct acctcccrac tctctc 26
<210>15
<211>28
<212>DNA
<213>人工序列
<220>
<223>正向引子
<400>15
ttgtggygta gaggattatt agtttggt 28
<210>16
<211>22
<212>DNA
<213>人工序列
<220>
<223>反向引子
<400>16
ctaaaaacrc aacccatccc tc 22
<210>17
<211>27
<212>DNA
<213>人工序列
<220>
<223>正向引子
<400>17
ggaattttat tttgaaattt ttttgtt 27
<210>18
<211>33
<212>DNA
<213>人工序列
<220>
<223>反向引子
<400>18
ccccacttca aataaaatac tattaaaaaa aac 33
<210>19
<211>30
<212>DNA
<213>人工序列
<220>
<223>正向引子
<400>19
tagtgayggt tttttgttta ttyggttatt 30
<210>20
<211>26
<212>DNA
<213>人工序列
<220>
<223>反向引子
<400>20
taaacrtaaa aacaacaacc cracct 26
<210>21
<211>5000
<212>DNA
<213>未知
<220>
<223>DBC1经亚硫酸盐定序的序列
<400>21
ggtttttatt tgaagttgtt tgattaygtt gttttagatt gattttagaa ttgtattatt 60
agtagggtag ttgttgttta ttaatagtta tggaagtaga ttttagttgg gggatgatgg 120
ggaaataaag agaaagatag ggaygaataa aggaaggaat ggggtataga tagaaagaga 180
gagagagaaa agaaagaaag gaggagaggt aagagatagt gatttggggt tttatatygy 240
ggtgggtggg agaaggagtg tgtgtggtgg gygtattttt tygtggttag aattaaagtt 300
agaattgatt tggggaattt gtgtttaaga tttgaatttg gattagaagt taagggaaag 360
agaaagttgt ttaagaaaaa agggagaaat ttttattagg ygaaaataga aatygttgat 420
tgggtttgtg gttggagagt tgttygtggt gttgattttt ttttattggg attttagtga 480
aggttgagaa gttttgtatt ggttgttttt tttttttttt tttttttttt ttagttttga 540
ttttttgaga tttaggatta ttttggttag ttygatgttt tttttttttt tttttttttt 600
ttttttgtyg attaaataat ttttttygag tttttagttt attatttaaa tatttttttt 660
ttattttgtt ttaagatttt ttttttttta attagtttat tggtttttag attgttttag 720
gtttttttgg tggttatatt ttttttttat tattttttga attaaaaaat aaataaaaaa 780
tatataatgg taattataaa agtagataat atgtattgag tatttattag gtgttaggga 840
gtagtttaag tgtttttgta tatattaatt tatgagtagt ttaagtgttt ttgtatatat 900
taatttatta taattttata ttaattttat tagatgggta gatatattat tattttatta 960
aatagataag gaattgaggt ttaaggggta agtgatttta ttaagattag atagttatag 1020
gattttaatt tttttggagt ttgtattttt attgaatttg gtttttttat atattttaat 1080
attttttttt atgatttttt gtttttttat ttggtagttt taatgttttt tatttttgtt 1140
taatttttag gggattaggg atttgtattg ttgatttggt ttaaattaga agggaagtaa 1200
agtaaataaa tatattaaat gtgygttgtg tgggaattat tataygaggg ttttattttt 1260
tgttttagga agaggtttta tgttagtagt tttagtttgt atttaggttg attgtagagt 1320
attttgtttt ttatttttat gttttagttt ttgtatttty gttttttttt ygtttttggt 1380
ggtttttaga gaatttygtg tttttttagt tttttttttt tatattttgt ttaygtagag 1440
aagtttttgt tttattttgg gaggttaygt gggttttygt ttatatatyg agagaaataa 1500
atagtgttaa atatttatag agagaygygt agatataaay ggatttatay gggtaatttt 1560
ygagataaaa tttatattyg atggatttay gyggtygtgg aaatatttgt ygttttagaa 1620
atatttaggt attygygata tatatagtat agttaygttt aagggtatta ggatttyggg 1680
tttgygygta tgygyggttt ttttggatgt tygtgygtat agatataata ttttataygt 1740
tttagattta ygaaattttt tayggtttag ttttagttta ttygggtygt ttttttttyg 1800
aggyggtttt tygttttttt ttttttygtt tttttttttt tttygtttaa agatgtataa 1860
aatattttty ggaagtaatt tyggygttta gttttttttt tttygttttt yggtygtygt 1920
ttttttattt attttyggty gtygtyggtt aagttttttt ttyggygtag ttyggtttty 1980
gtygttttty gttyggagat ygyggygtat ttggattttt ttttttatty gttagtygtt 2040
tygttttygg attttayggt tgtaaattga tttggygygy ggggaggagg agagygtagg 2100
ygagygaatt ygygagagag ggagagagyg agygagtaat agygagagyg agagygagag 2160
agtygggagg tagagggagt agtgatygtt tttyggagty gggatttatg tttgttttyg 2220
ggattagyga aggggatttt ayggttgagt atgagttagg ttgttaggag ttaggtattt 2280
ttaygtttgt agttttygyg tygtgttygg aatgygagtt gtaygtaggg tttttttaag 2340
tttttatyga gtygaataaa aagygttttt tygtagtttt tygttaaaga yggatattga 2400
ttttaggtaa ggyggygtyg ggtgtagygt ttygtagttt ygttgttttt ggattyggtt 2460
tygggtygta ttyggggygt tttygygttt ttttgttttt ttttttatyg gtatttttgt 2520
tygtttttta tttggtygtt tygtygtttt taagtttttt ttagttttag ggagggggtt 2580
tttgtgtttg gggtttaaag ggttaattgy gggtttgagt gagtggygtg tgtgttttyg 2640
ygygttttyg aygtgtgtat tatggtggga atttgatgtg gtgttagtgt gtttgygtgt 2700
gyggygtygt ttgygtttga tgygtgtgtt ygtggtgtgt gtgtgtgtty gtgtgtgtaa 2760
gggaggggtg aagagagaga ggttttataa tttatttayg gygygatgtg tgtgtgtatg 2820
tttgtaygta tgtgtttgta tgtgtgtttt ygtgtgtttt tttaattagg tttttttagt 2880
ttatayggaa tgggattttt attataggat taygtagtta tygggaaatt ygttgtggat 2940
ttttttttgg ggttttgggt ttggggtttg gggaggatta tggggttgta gatggtattt 3000
tatttagttt aatgttggta ygtttgaagg aaaatttttt taaayggtgg aattttgtta 3060
tattgggatt tatagtttaa tatgaaagag atatggttaa ttttygaggt aaatgagayg 3120
ttgttatttt aaattttata ttgggtagat tttaagattt tgatgggaat ttggagatat 3180
tggatagtgg gtgatagaga aagggggaag ttagyggtgg gttttttatt tggggtttgg 3240
aaagtttggg atagggattt attttgtatt ttygtttgta attaatagag ttttttyggt 3300
ttttgttggt tttggggagg atttggtaag tttgtatgga ttttttttag gggaaaggag 3360
aaagygtttt yggggatttg tttatttatt atagttgtaa agggttttag aggaaatttt 3420
atttggggyg gttgtggatg atatggaagg agatggatgg ggtatttttt taagatagat 3480
tygttttttt ttttttattt taggygggga agtttttaga gagttttttt ttaaatatgt 3540
ttttttatgg tttttttgga gttaggggga tattgagaga aggtagagag gtggagaggg 3600
agagagagag agtaagagyg agagagaggg agatagagag agagagtgtt ttagtattga 3660
gggagattta taatttgaaa aggggttgtg agtgtggaat ttatgataga atgtggtagt 3720
aattgattta aattgttgty ggtttgtatt tygygttttt tatttggttt taaaatattg 3780
ttaagggtag gggggyggtg gaggaatttt agttaggaag gtagtttggg ttaaggtttt 3840
tttttttttt ttttttattt tttgggaggt tttttaggta ggttttttgg ttattttggt 3900
ttggtattgt tgggtttggg ttttggggtt agttattgat ttgattttta gttgtttttt 3960
aaaggtttgt tttatttagt aaaaatgttg agttttattg ttgttagtat ggtttttaga 4020
ttyggtagtt gttatttttt taaggtaagy ggtagtygtt ttgtttttag gtagggaggt 4080
taggagaaga taagggttgt gaygttggga tatagttttt tgtttagttt agtayggagg 4140
ggtygttgaa aagttttttt aggggaaaaa agtagaaata tatttggttt aaaaaatgta 4200
tatatatatt ttaggaagat atatatatat atatatatat taattatata tatatatata 4260
tatatatatg tgtttatatt ttggggagag ataaaagtaa atatgtattt gtaggaattt 4320
gttttagtgg gtgggtttta tttgtaggta tttgtagata tattattttt tgtttagagg 4380
ttgatatttt aggttgaagt ttttatatag ttatagagaa ttttttttag gaaatttatg 4440
ttagggtaga gttttttgga ttattttggg tagggggtat tagggttaag gaggagattt 4500
atttttggga attgttttat tttttttttt ttttaattta tagaygatyg aaattggaag 4560
gaatattaaa gagtagaaag gtaggatttt gtggtttaaa gagggaatga gaatttttta 4620
atatttagta gtgggttagt ggtatgttta ggattagaat ttaggtttat taattagtta 4680
gattaatatt ttttttattt tttttagtaa tatatatttt aggattgtat attgttttta 4740
tttttatttt gttaatttgt tattatgatt tttattaaga taatygtttg ttttttttta 4800
ttaaatttta tatttttaga gatgtatttt agggtttttt tatttggaaa tatggttttg 4860
aagagaaaag atttaatgtt ttaattatta ttttygaaag atatatatat atatatatat 4920
atatatatat atatatatat atatatatat atatataygg tggtgggggg aggattgaga 4980
gagagataga gagagaatat 5000
<210>22
<211>5000
<212>DNA
<213>未知
<220>
<223>PDE8B经亚硫酸盐定序的序列
<400>22
agaataattt tgaagtatta ttttattttt agaatttttt ttgtagggat gtttgaggtt 60
tggttaattt ttttttttgt ttaagtttgt ttttttttat tttttttttt tatttttttt 120
tataaatgtt gattataaga gtatttttta aaaagttttt ttttgtttgt taaatattat 180
tttagaattt gtttttagga ggatttaatt tgtgtagaaa ttttatgagt aatttgtttg 240
tttattaaaa aatatagtat agattttttt taggttagtt ggagatggag tttttttgta 300
ttaaaaaaag agagttaaat tgagaagaga agtaaaggta gaagaataag gttagatttt 360
ttttgtataa ttgaatttgg gattgggaat gatattttgt ttttttatga aatttagaaa 420
ttgttttttg aagtttttat ttagttatat ttaagagtgg tttaggaaga aaaaggaaaa 480
taagaatgaa aaaaaaataa aaaataaatt ttaaaattgg atattttttt gtgttttgta 540
ttaagttttt atgttattgt ttttgttygt ataagtaatt ttagataaaa ttttttttta 600
gattatttaa tttatttttt ygtgttattg tgaaaatygg gtaggaatta ttgygggaat 660
tagtgattga atgggtagtg gtgtttygta gttgtaattt agagtttata agggaaggat 720
gaatagtagt tattttttta aaggtatttt tttttttttt tagtatagtt tagtttgtgt 780
atgttttatt aaggagaatg agggaaggtt ttgttgattg tttagtttag agtgtgagga 840
atagtagttg atatgggygg ttggayggga tagaaattag tatgaatatt tagtgatatt 900
taaggaagag gtgaaaatgg gggagaaagt taagaattat tagatagttt ttttttgttt 960
ttgttttgga ttttagggga aygtatgtgt ttaagggtgt tttttttygt agggtttttt 1020
attttttggt ataaattttt aaaygatgag tagatatgga atgggttttt tttgaagatt 1080
gggttattat ttaatttagt tttyggagta ggyggtgggg ttgggggaag aggayggaat 1140
tatgaggttt ttttattatt tttygggtgt gttgagaggt aagtaaatgy ggtgggtggg 1200
tttgggttgt agggtttttg ttatttyggg aggggttttt tggtgtttag ttttgggatg 1260
tgtgaaaatg tgttggtgaa aagygagagg tttatatagt tttttagggg aagaggggtt 1320
ggggygttgg gggyggygty gggatgagtg tagaagagay gaggtttttg gatagyggag 1380
gaggagggga gggygtygag gygyggtgtt agttgtygyg tataggggtt tygyggygga 1440
gtygagtygy gggtaygttt tgttttgtyg ggagagttty gggagyggyg ggaggggygg 1500
aggggygtag tggggttygg gyggttgygg tygyggagty ggggtatttg aggaggaagg 1560
agggtgggag ygagggaggg aggggayggg ygtagatyga aagtggggaa agaaggtgta 1620
ggtaggyggg taggygggyg ggygttttgg tttagggtyg ygggtgyggg agttyggyga 1680
ggtygagttg ggyggyggyg ggggtygygt ygagggagga ggggaaggyg gaggygyggg 1740
gagygtgttt ggggygtygy ggyggggagg gtggyggtyg ttggtgygyg yggggygttg 1800
tgtatgygyg ttttttygtt yggggaggaa gatggtttaa aagggaaagt tggggtgayg 1860
ygygyggttt tyggaggtty ggyggggggt atygyggtta gttygaygga gyggyggata 1920
tataggtygg ggggygygta gttygggygt ygtygyggty gtttttttat tgtaggtggt 1980
agygggtgyg ttgggtttyg gyggtygygg gygygggygg gygygygggg gagttyggty 2040
gagggatggg ttgygttttt agtatttatg tttygtagag yggygtgatt tattgtyggg 2100
attyggayga gtttagttyg tttygttaga ttattagygt gtygtagggt tyggyggtat 2160
ttttgttygg ttttttygtt tagatygayg tygtygaygt tattttttyg agtygygygt 2220
ygggattttt tagygtagtt ygygttygta gggttygtat ygagttgggt agyggtagta 2280
gygygggttt ygtagtttty gtygygatta ttagtagggg tyggaggygt tattgttgta 2340
gtagygtyga ggtygagatt tagatttgtt atattagygt gaaggtaaat gtttygygtt 2400
ggtataygty gtgggggtyg ttygtttygt yggyggggtt ygtaygggta gggggtttyg 2460
gyggagttgg gtgatygtga ggyggttggt ttggagaggt tgttattaag gaggagttta 2520
ttttttattt gtggagatga tgggagttta ggaaatgtgg ttagaaaaag gtttttggag 2580
gggttttgga agygttttta gttggttttg ggggattggg yggggaaggg agygtagaag 2640
gaagtaggtg ggttggtttg tttttttttg agggtaggaa ggttgtggtt tggtttatgt 2700
aggaagaggg gtggggatta ttgagagtat tyggtggtta gttttgttga atgaaatttg 2760
agtattgagt tggatttgyg tgttttgtag gtgattggtg tagttgtagt attaggatag 2820
atagtgtttt atatttygat ttttatttgg gattattagt taggttggag ttttagtata 2880
ggaatygagy gtagggattt gtgaatgaat gagtgttygt gttttaagag atgtgggaay 2940
ggagtagagt ggaatttgtt gtttgttatt gtaaygtttt tttgggttgg ttgtatttta 3000
gatagaattg agagaatygg gttatgagtt yggagtgtta gtagagttat ygtgagggga 3060
ygtggttttt agtgtagtat agttgtttga ggatgatttt gtatatataa ttgatttttt 3120
tagagagtgg gattttgagg aagtggaaaa aattgtttag atggttaaag tagtygttaa 3180
atatttattt tttaaagata gaagaaaaat aattatttaa atagtgtttt ttygtatgtt 3240
tttaaagtat ygttaaattt aagaggtttt ttattgtgta aatttgggta ttgggttttt 3300
ttttttttta gtaaataaga taataatggt atattttatt gtatagagag aaaaaaatat 3360
ttttaatgtt agatagaatt atagttttta tttggttaat aggttattaa gaattatttt 3420
atggattatt tgttaaagat atatttgtat ttttaatagt taaaaatttg gtttttattg 3480
gatgaaggta gtttatagtg gaggagtgag gagaaggata atatgttttg gttttaattt 3540
ggaagtttaa aagtttttta atgaatttat ttttttttta atagtatttg attgtttaat 3600
atgaagtttg attgatgttt gtttgtggtt gtagtgtttt tttggtagta attataatta 3660
ttattatagt aaaaatatyg tttattgagt atttattatg tgttaggagt aagttttttt 3720
ttttaattat tgaatgttaa tagtgtgatt ttagtatagt atgtttgaag attagagttt 3780
aggatttatg atagtataaa tttattagtt gtttattygt tatagaggta tgtgttgaay 3840
gttatatttt gtttttgtta agtttttttt tttttttttt ttttgtttat ataaagttag 3900
ttttaggagt aatttttagg gatgaaaatg ttaaatttgg tgaaaatatt aagttgggat 3960
attattagta aagattattt ttttgtttga yggttgaaga tttgaagtag tagtttaaaa 4020
ttgtgatagt ttatgaattt tgtgatattt tttttggttg ttttagagaa attgtgaatt 4080
tttgtttttt agaatattta taattttagt ttagtatttg gtaattttag ttattttatg 4140
tttgtttagt agaagaaaga atattgaatg aatttatttt gtatattttt aatatttttt 4200
ttaagaggtt gtttagagat taaatgaagt aaaaattgaa gggataatga gttttaatta 4260
ttagtagttt gtataaattt gtattaattt tattgttatt tggtttaagt attaaaaggt 4320
ttgatttggg taggagttta atttattttg ttttattaag aagattttta aataagagtt 4380
taatttattt taatttatta ttgggaatgt tggaaatgaa atttttggtt tattgattta 4440
atattatttt gtattgtgtg gattttagaa agtatatatt tgaagtgaaa tttgagatta 4500
gagttattta tgtaaatttt atagaattat tattatagaa ttgttttaat atatttaata 4560
gtattttttt aggtttgtga ttyggtgttg tattgttttt ttttaatygt aaagtttata 4620
tgttttgtgt ttttttgttg ttgagataat tttataaaaa ttttatattt atttttttgt 4680
agattaagta tttttttttt gtygtaagag tttgagatag ttgttaggta ygtggtggtg 4740
ttttattttt ttttaaagta tataggtgga aaaataaaat ttttattatt tggttttgtt 4800
tatagtggtg gaaggttgta atagttttta tttttttttt ttggttattt tatattaatt 4860
atgaagtgta tttttattat ttatttggtt tttggtattt tttagagtgt aaaaatgtgt 4920
gtttgatttt tttttttttt ttttttagta ataatttatt ttattaagtt tttagaatga 4980
tgaggtatta tgatgttaaa 5000
<210>23
<211>5000
<212>DNA
<213>未知
<220>
<223>PTPRR经亚硫酸盐定序的序列
<400>23
aagaaaaata gatttattgt aagtgggata taattatttt tttttaattt ttagttttgt 60
agtattttta gtaatagatg ttagatgaga agagttattt ttttaagttt tatagaatgt 120
taaataaatt attttaaagg ttgatttagt aagaatttta aagagttaat tgttagtatt 180
gaattttgtt aattattatt tttatttatt tatttttgag tatttatttt gtatgaggta 240
ttgttttagg gagttgggat atattaataa agttattaaa gttttttaat attggagttt 300
aaattttgtt ttgygggggg gagtagggaa atagatatta aataatagat tttataagta 360
attattttgg tttgttagaa agtgataygg gaaaagtaaa ttggaggata gtgaattagg 420
aataggagaa gaggttgtag tttagagtgg tttgggatga aatgatagtg ttatagaaat 480
ggagtgtgtt ataygtatgg gggagtaggg ggtggagtay gttgttagag aggggttaga 540
tatggagata tgtttttatt tttagtagag aaggtttgtt aagatattaa aagtatattt 600
tattgttttt tttttttatt tgttggagtg ttttttggtt ttgtttttta gagttgaaga 660
tgatataaat tataaaagaa ataaatggta tygtttgggt tttttgattt tataaggttt 720
tatttaattt aagttttggt ggatagttta ttttttatta gttgttttaa tttatagagg 780
atggagaaag aaagtattga atgagatgtg tagtttaatt taatgggtat ttgttagttt 840
taattatttg gttaggataa ttttatttaa gttaatggaa tataaaattt agtagttttt 900
atggagattt taatgtgttg tagaataaat tatagataaa tgaattgttt gaaagaataa 960
tatgaggtay ggttaaataa gtgttgtgga agagaaagat tattgtaatt tagagttagt 1020
tagagttagg gttttttgga aaagtgagat taatattata ttttygaaaa ttagtgtggt 1080
ggtggtgtaa gtggaggaaa atgggaaaga ggtaatagtg tttataaaga tatagaggta 1140
gaaagggtag aggttttggg atagttaaaa gagaagttta gttgagggat tttttgattt 1200
gattagtagt aattagttat gattaataag gttttttata ttttaaagat ttagatggag 1260
ggataaaaaa ttatttaatg gtagattgtg gtagtttttt tagagatata gagttgggty 1320
ggatgagttt aggtattgay gtgatttatt attttttatt ttaaagagta aaagggaaat 1380
taaagttaat tatttttayg aaataaaaag gtgttttttt gtgttttaat tatatggata 1440
tattttatta gtttaaaagt atttttttat ttttttttgt tattgtgagg atttgagtta 1500
gaagaaagtt taaatatagt tattgagttg gaaagagtgg aaagagaagt aaagaggggg 1560
aagttgtagg aaggaygaag ttatttttaa gatatatggt tattgtttat attaagtaag 1620
ttgttttggg aaygtttttt tygagtagtt agaatgttta gtagtggaag atatttttat 1680
ttttgtaggy gagttttggg aagttggtta atttgtaaat gttaattttt agtagtgagt 1740
tyggtttayg tgtaaattaa gatttgggga aagagtaggg tgggtggtat ggttgataat 1800
gttattagtt tttttttttg atttttgtgg tygtgttttt atttattttt atttagttat 1860
attttatttt yggatttgtg atggaygttg ggtttttagt aattatagta agtgtttttt 1920
tygtattttt ttttttttta tttttatttt tatttttaat tattatttta gygatggagt 1980
ttattttgtt ttaagtygty gttaagatty ggagaagygg aattttattt tgaaattttt 2040
ttgtttygtg agggtyggyg ttgggtatgt ttagtagtyg yggygttgtt gttgggttgt 2100
tgggttggyg yggagtttat tttgtygttt tygttttggt ttttgggygt ttagaaggtt 2160
aggtatttgt ygtttttgag ygtttttgtt ttttttatty gtaatttttt attgtttttt 2220
tttttttttt tttttaggga ggttgaagtt ggtgttggtt tttgtyggyg ttatagattg 2280
attgttttgt aaattttagt ygaggatttg aatttyggag attagaagat ttttggyggt 2340
ggtttttttt aatagtattt tatttgaagt ggggtygtgg tggagttttt tttttatttt 2400
ttaatgtaaa tattatgygg agagtagttt gtttttttgy gttgtgtttg ttttttaatt 2460
tttaygttgt aggtaagggg ttgttaggtt tagagtygga gttgtgtatg agatgggaaa 2520
ttgtatatgt ttaaggattt taggaaaagt ttgttttgyg aagaaagttt ttttaaaaag 2580
gtaaaatagg atagtatttg atagggaagg ggtgagggag tygtgtattt gttggaaaat 2640
tgaggtygaa aatttaattt aaaattagtt tttgattttt ttttatttta taygaaaata 2700
gtgaatattt ttaatatgaa tataggtttg tttttttttt ygtttttttt tygtttagat 2760
tttttgtttt tgtttttagg ttatgtattt aattgttaat tttttaggag agggataaga 2820
tattttgtta gatgtaattt tttttattgt agyggttatt atatttataa ggttttttgg 2880
tttagygagy gtttatagga aggtattggt tgtaatttga tggattatat tttygtttaa 2940
aagatygaaa tatatggttt atattagtga tgttgtatta ggtttttttg gtttttgttt 3000
ttgttattyg aattttttaa gttaataaga ggaagaaaag gttttaaata gaattttttt 3060
gtttttttta gtatygtggt tagygttygt tggtatatta aaaaaaaaaa aaaaaaaaaa 3120
aaaaaaaaaa ataattatag ttttaaagtt gataattttg ggttaggtat tgtttttygg 3180
atagatttga taaggtgtaa ttaagagtaa tagtgagatt gtttttaaat ataaatagat 3240
ttgaggtatt agaattaata ttaagtttyg tgaggaagta aattttggga agtatttgag 3300
agatattata tgtggtgttt ttgttttttg tatatatagt atttttattt aatgtagata 3360
gatttgttag gttgaatttt ttattattgt tatatatata tgaattaaaa taaaatatgg 3420
tagggagatt ttagaaattt tattgtgata gtagttgttt atgtagtagt tttgtaataa 3480
ttagagayga ggtatttttt ttgagtgtgt tttttattta attatatata tttatttatt 3540
tttttaagta atatttgttg agtgtatatt atgttttagg ggtaatgagt aaaaataatt 3600
gttttgaaat tataattttt gtagtatttt ttttaaaaga gtgggtgaat tttatttttt 3660
gtgtataata gtaggagtga aatatggtat ttttttattt tagttaaygt tattatttaa 3720
atgtattatg aattttttga gtaaataatg tataggatta ttaaagttat tattaaatat 3780
atatgttaat tgttataatt atgatgttat attgtggagt ttttaatagt tttttggtat 3840
attttgagaa gtagaaaata gttatgattt gattattaaa ttatttgtaa atagttgtag 3900
tgaattattt ygttattata tataattttt taatgttgaa atgtgggatg aaaatatata 3960
tattgggttg agttttattt atagtattgt ttaataatag attgatataa atatttatta 4020
tagaaaaatt ttaattttta ttaaggtagt aatgatttag ttggtttgtt aataatatta 4080
gtgtagttat ttttttaaaa ttaatatata aaaaagttta aagattatat ttgatatatt 4140
ttgagtaatt atattttaaa agttataaat aaattgaagt atgtgaaaaa tatgttttga 4200
tttattgtag atgaatatty gggatgagaa aaatgaggat ttttgtttat tagatgagaa 4260
atttagaagt ttgtagtttt agattttatt tggagttttg ttaaagggta tattattaaa 4320
gttaatttaa tttattttta ttttaatttt ttaatttgta aaataaagat aaaaagtaag 4380
attaattaat taattttaat ttaataagaa aatgtattta aggatatttt tatattttaa 4440
ttgtaagata ttatttattg tatttatatt taatatagta aaatatattg gtgttttatt 4500
atagtagatt gtaagtttaa tattatagtt tttttgttaa ttttatttta aatgtatttt 4560
aattttaaat agtaatttgt gggttataag ttgttattag attaaaattt attatagtta 4620
tttaataata ttgttagggt tttttattag aattaaaata aatatagttt tttggtttaa 4680
gtttttatat atttggagat taagaaaata agtggatggg atgataaaga ttaagaaatt 4740
aagatattaa tttttatttg ttgaaggtgt gagtttgtta tttttatgga agattttttt 4800
tttagaatag aggttgattt tataaagaag ggaaatgaat tttatatatt taggtttttt 4860
taattgtaaa gttaattgat ttattttggt taygtttagg gtattagtaa ttgttttatg 4920
ttggtggaat tgatgatatt ttataatgtt ttagagatgg gaggaatgtg ttaaygggat 4980
gtaagtgttt tttttagtta 5000
<210>24
<211>5000
<212>DNA
<213>未知
<220>
<223>ZNF582经亚硫酸盐定序的序列
<400>24
gaygattttg aatgattttt gttttttagt atttatattt ttgtgtagtt tttatttatt 60
tagatttata gttgattttg tgattaagag tataatggtg tatgatttta gaggtttggt 120
ttagagttag aagtggttat tttattattt tttaaatttt tttttggtag attaatgaat 180
gtgagaaagt aaaaatggta tagaagaaag ttagtttgag agatggttgg ttttgtattt 240
gtttggtttt tttttttttt tttttttata agtaaatatt gaygtatgta tatatagata 300
tatttaatat agtgggatgt taaagggaaa ttagagttat tygttttttt ttaagattag 360
agaatatagg aaagatgatg attttttgta tttatttttt ttttttgaat tygtaatatt 420
gttttgaatt ataaataagg ttaagtagga taaatttata attgtttttt taattgttyg 480
tttataagtg tgaaatttaa ttatttttgt tgaagttgtt ttttaatatt agtaattagt 540
aataattaat ttttgagttt tgtttatatt attaaattat tttatttgat ttttaggata 600
aagttgtaag tttagtatta tatttttttt tttatggata aaaaaaaaaa aataaaataa 660
aatygagata ggtaygaaga aattaataaa tggttgaaat gttatttgaa tttatatttg 720
tttaatttta aaagtgtgtt ttttattata ttatygtatt tataaggaaa atgtagtttt 780
tatttttttt tgaagaatgt gtaaagaggt aatatggtgt gttttggaaa aagtattttt 840
gtagaaagaa agtattagtt ttagttatat tagtattttt atttagtagt agtagtatta 900
ttttatatat tagttatttt tttttaataa tttaatgaat tagatgttat aattattttt 960
atttttaaat gtggagatyg aggtatattt ttaagattgg agtttaggta ttttggtttt 1020
agttttagag atggttaagg gtttatattt ttaattattt attatattat agagtttatt 1080
aggtttgagg gaaataggat taaattaaaa gagttattta ggattttagt ttttatttaa 1140
ggataaattg ttttatttyg gatagggaga gttttygtat tttgagattt agtataatag 1200
gttttgatyg gtatttggta ttyggatttt taattatatt ggattatatt ggttygggat 1260
gtgtaaagtt tagggttttt tatatttgat gatattaaag tygtttaaaa ataagagaga 1320
attaataatt atttayggyg gtttgatatt tgtttaagag atgtygtttt ataaaatttt 1380
tttatatttt tataaygttt ttattttgyg ttttttttta taatttatat ttaatttatt 1440
atagatgtaa tgtttaaaat tagttattag ataaattttt aygtttttaa attttaaggt 1500
tttttygaaa ttttttggta aaattgttgt tttayggaaa tgggaaygta ayggatgagg 1560
taatttttta tagtygtata tagttgtgta tttatygtta aayggtttta gttatatatt 1620
taaygattta ygyggagtta gaagttatta ttatatattg ttaaaattay gtatatatag 1680
tgayggtttt ttgtttatty ggttattygt ttataatttt tygttagaga attataygta 1740
gatagtatat ttagtattat agattttagg aagtaattta gggattygaa tataygaata 1800
gtatttttty gygtattgyg taggtaygtt tgygttyggt ttattttgaa atatygygag 1860
attyggtttt aaggtygggt tgttgttttt aygtttaaag attatgtttt tyggaagata 1920
ttgyggygty ggttttatta tggygtagta tyggtgtgtt ttgtgygttt gygttatttt 1980
tyggttgygt ayggygaatt tatyggtaty gtggtggaag ygygttttgg gttgtygggg 2040
gygyggtygy ggtggtattt ggattygagg aggyggtagg tgagaggttt yggagttttt 2100
taggygtttt ggggtttagt aggagttggt gttygggtyg gttgggtttt aggtttgaga 2160
agaaygtaga ygtttygttt tatygtygtt ttgtggtttt atyggygtga gattatattt 2220
ttygtyggtt ttygyggygt gygttttttg gygttttttt tttgttttta gttttatagt 2280
ttaggtgtat ggatttttta ggtgggtgyg taggggtttt ygattttttg aaattgygga 2340
tgttttgtat ttattgttat gygtgttttt tttttttttt aatttgagag gaaattttgt 2400
tttatgaggt ttttagagag ttataagatt ttagaagatt tagaatygtt agtttagaaa 2460
aatttatatt tgggaatatt ttatatgttt aaattttatg tttagtaatg gggaaatatt 2520
tgagtggatt ataggttatt ttttgtaaat aggatatygt gtagttaata taaataatgt 2580
ttataaaagt ggttataata aggaaaagtt gtttattata ttatattaag taatatttat 2640
aattatttta tttataygtt atttagaatt aaatgtttgt aatgtagtag gtattttata 2700
tattttttta tagagtattt tttgtttaat agtgagttta agaattggtt aaatgataat 2760
attgygaata tttgggtgtt ttttttttta atttttttgt tttgttttgt ttttagtttg 2820
tgtgttagtg agtttgattt ttttttgttg aatgttttta aaggatttag tagttaatat 2880
ttttgtttgt tagtttttga tagttatttt tgatagttat ttggtatttt attatatggt 2940
tgtgttaaaa atgatttaat tttttggtta tttgtggatt tgtttattta ygtattaatt 3000
tttagttttt ttatttagta aatattattg gatatgtagt tttgtgtatt gttgtgtttt 3060
tatggaagga tatatttatg tgttgataat ggagtattgg gygtgttgtg tgtaatttgt 3120
tttttattgt tttttttaat tttattttat ttgttgatgt aaggtatgtt ttatgaagtg 3180
gtatgtataa agttttagtt tgggttgtag agttttttgt atatttggyg gttgggaaaa 3240
gagttaggta gatttttaaa ggtgttatat ttgttgtagg agagagttaa atggtttagt 3300
tttggaaagg ttttatttta ggttggggtt atgtaaatga tgtaggtttt ttatgttttt 3360
tggtttgagg gaagtttttt ttgtttttga ggtaatatgt gggttttagt tattagaata 3420
gtttttagta tttttgtttt agatttgtat ttgtagtaaa atagaattat tggatgttta 3480
ggggaaatgg gagggaaagg attattggtt tttttttgtt ttgtagtagt taaagttggt 3540
tttttggggt tgggtgagga tttttttgtt tttttttttt ttttattttt ttggtgggta 3600
gagggtaagt aggtggggat ttgtaatttg gtattttagg aattgtgagg taagtttagt 3660
tttttatgtt tgtttttttt ttttttagtt ttatygtygt aggattttgt ttttttttaa 3720
gagaggaatt agaaggagga tttattagtt tttgaaattt taaaagttat gttttttgtg 3780
agtttttaaa ttttataaat atttgttttt tttattttga aattttttat ttgggtttta 3840
tttagaaatt tggtttttag atttttttat tttagggaat aaaaggatta ggtaggagtt 3900
ttttttttta ttgttttttt ttttttgata aatgtttata gatttatttt ttatagtatt 3960
ttgtattttt tgatggtttt ygtttgttag gaggagaaat ttattttttt gtatgaaatg 4020
agagtatttt ttgtaagaat tgaagtttga agagatggaa tgaatgttta ggagtaatta 4080
tatatagaaa agtaggggag gtggtgttgg ttggttttag ttttagygta tggtatagtg 4140
ttttgttttg ggaatgatta atgattggat atgtaattga ggtttggttg ggaatttatt 4200
aaaaatttga tgttagaatt aaagtttagt ttgaygttgg atagagtatt tgaatttggg 4260
atatttttta ttagtttttt tagtttaaga ggaagaaaag tatagagttg aggatggtga 4320
gygatgtagt aagtgagatg attatttagg tgggtgagtg gttagggttt ggaaaagttt 4380
tagagatttg atttaattty gggatttaaa ataagaggat gtttgagtta gttgttttat 4440
ttgatagaaa attgtgtatt taataaaagt tatttttatg gtttttttta tgaagaaaaa 4500
tgtttgtgtt atgttttaat ataatgatga atttttattt ttaagaaatt tttgatttat 4560
atgaaaattt tgtatttaag atagaaaata tgtttttata attttaaaaa ataaaatatt 4620
tggggaaaat ttattagaaa tattttttgt tgttattgtt attttaaaag atagaagtga 4680
taggttttat tttgagaatt tattgtatgt taagtataga gtgggtagtg tgttttatta 4740
tattgttttt taataataag gaaaatttta tttagtttga ttttaatttt tattttagat 4800
ttgtatatag agtattgatt tgtagttaaa aggaatgttt gagaataatt tgattatttt 4860
gttttattta tttttatttt ttttgttatt ttttatattt agttttattt tttttgttta 4920
ataaaataat ttttttttty gtaaagattt gttttatttt tttttatttt attgtgaatt 4980
attgaaatat atatatatta 5000
Claims (20)
1.一种目标基因在制备用于子宫颈癌筛检试剂中的应用,其中所述子宫颈癌筛检试剂用于检测受测检体内目标基因的CpG序列甲基化的状态,所述目标基因为DBC1、PDE8B、PTPRR或ZNF582基因中的至少一个。
2.根据权利要求1所述的应用,其特征在于所述受测检体为子宫颈抹片、血液、尿液、子宫颈上皮细胞、手术后的癌症组织的离体样本。
3.根据权利要求1所述的应用,其特征在于所述受测检体为异常的子宫颈抹片。
4.根据权利要求1所述的应用,其特征在于所述受测检体为人类乳突病毒检验呈阳性的子宫颈细胞检体。
5.根据权利要求1所述的应用,其特征在于所述目标基因的CpG序列甲基化状态检测方法为甲基化特异性聚合酶连锁反应、亚硫酸盐定序、微阵列、质谱仪分析、变性高效液相色谱、焦磷酸定序。
6.根据权利要求1所述的应用,其特征在于所述目标基因DBC1的核苷酸序列如SEQ ID No: 1所示,目标基因PDE8B的核苷酸序列如SEQ ID No: 2所示,目标基因PTPRR的核苷酸序列如SEQ ID No: 3所示,目标基因ZNF582的核苷酸序列如SEQ ID No: 4所示。
7.如权利要求6所述的应用,其用于辨认目标基因甲基化序列的亚硫酸盐定序所使用的引物对序列为: 针对目标基因DBC1的引物对如SEQ ID No: 13、14所示;针对目标基因的PDE8B的引物对如SEQ ID No: 15、16所示;针对目标基因PTPRR的引物对如SEQ ID No: 17、18所示;针对目标基因的ZNF582的引物对如SEQ ID No: 19、20所示。
8.如权利要求1所述的应用,其特征在于用于辨认目标基因甲基化序列的甲基化特异性聚合酶连锁反应所使用的引物对序列为:针对目标基因DBC1的引物对如SEQ ID No: 5、6所示;针对目标基因PDE8B的引物对如SEQ ID No: 7、8所示;针对目标基因PTPRR的引物对如SEQ ID No: 9、10所示;针对目标基因ZNF582的引物对如SEQ ID No: 11、12所示。
9.一种目标基因在制备用于卵巢癌筛检试剂中的应用,其中所述卵巢癌筛检试剂用于检测受测检体内目标基因的CpG序列甲基化的状态,所述目标基因为DBC1、PTPRR或ZNF582基因的至少一个。
10.根据权利要求9所述的应用,其特征在于所述受测检体为卵巢癌组织、腹水、血液、尿液、手术后的癌症组织的离体样本。
11.根据权利要求9所述的应用,其特征在于所述目标基因的CpG序列甲基化状态检测方法为甲基化特异性聚合酶连锁反应、亚硫酸盐定序、微阵列、质谱仪分析、变性高效液相色谱、焦磷酸定序。
12.根据权利要求9所述的应用,其特征在于所述目标基因DBC1的核苷酸序列如SEQ ID No: 1所示,目标基因PTPRR的核苷酸序列如SEQ ID No: 3所示,目标基因ZNF582的核苷酸序列如SEQ ID No: 4所示。
13.如权利要求12所述的应用,其用于辨认目标基因甲基化序列的亚硫酸盐定序所使用的引物对序列为: 针对目标基因DBC1的引物对如SEQ ID No: 13、14所示;针对目标基因PTPRR的引物对如SEQ ID No: 17、18所示;针对目标基因ZNF582的引物对如SEQ ID No: 19、20所示。
14.如权利要求9所述的应用,其特征在于用于辨认目标基因的甲基化序列的甲基化特异性聚合酶连锁反应所使用的引物对序列为:针对目标基因DBC1的引物对如SEQ ID No: 5、6所示;针对目标基因PTPRR的引物对如SEQ ID No: 9、10所示;针对目标基因ZNF582的引物对如SEQ ID No: 11、12所示。
15.一种目标基因在制备用于大肠癌筛检试剂中的应用,其中所述大肠癌筛检试剂用于检测受测检体内目标基因的CpG序列甲基化的状态,所述目标基因为DBC1、PDE8B、PTPRR 或ZNF582基因的至少一个。
16.根据权利要求15所述的应用,其特征在于所述受测检体为腹水、血液、尿液、手术后的癌症组织的离体样本。
17.根据权利要求15所述的应用,其特征在于所述目标基因的CpG序列甲基化状态检测方法为甲基化特异性聚合酶连锁反应、亚硫酸盐定序、微阵列、质谱仪分析、变性高效液相色谱、焦磷酸定序。
18.根据权利要求15所述的应用,其特征在于所述目标基因DBC1的核苷酸序列如SEQ ID No: 1所示,目标基因PDE8B的核苷酸序列如SEQ ID No: 2所示,目标基因PTPRR的核苷酸序列如SEQ ID No: 3所示,目标基因ZNF582的核苷酸序列如SEQ ID No: 4所示。
19.如权利要求18所述的应用,其用于辨认目标基因甲基化序列的亚硫酸盐定序所使用的引物对序列为: 针对目标基因DBC1的引物对如SEQ ID No: 13、14所示;针对目标基因PDE8B的引物对如SEQ ID No: 15、16所示;针对目标基因PTPRR的引物对如SEQ ID No: 17、18所示;针对目标基因ZNF582的引物对是SEQ ID No: 19、20所示。
20.如权利要求15所述的应用,其特征在于用于辨认目标基因的甲基化序列的甲基化特异性聚合酶连锁反应所使用的引物对序列为:针对目标基因DBC1的引物对如SEQ ID No: 5、6所示;针对目标基因PDE8B的引物对如SEQ ID No: 7、8所示;针对目标基因PTPRR的引物对如SEQ ID No: 9、10所示;针对目标基因ZNF582的引物对如SEQ ID No: 11、12所示。
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