CN1703521B - 基因表达的定量 - Google Patents
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Abstract
本发明涉及测定样品中靶核酸的数量的方法,其中将使用的标准物设计成与目标基因或″靶核酸序列″相比具有一个碱基的差异。使用这种标准物与方法结合来″增加″标准物和测试的核酸样品中的差异,其中利用例如,正好在突变位点进行的碱基延伸反应而容许以相同的效率扩增标准物和靶核酸,从而有助于定量靶核酸。此后使用定量″增加″的标准物和靶核酸样品的方式来确定靶核酸的数量。在优选的实施方案中,该定量方式是质谱法。
Description
发明背景
在许多病理学的状况,例如不同的良性和恶性肿瘤、神经系统紊乱、心脏病和自身免疫紊乱中检测和定量差异表达的基因可对这些病理学状况的诊断、预后和治疗有用。基因表达的定量也可用于诊断感染性的疾病和在分子水平上追踪药物或毒素的作用。例如,基因表达数据可用于确定药物或毒素的药理学机制(Libutti et al.,Microarray technology and gene expression analysis for thestudy of angiogenesis.Expert Opin Biol Ther.2002 Jun;2(5):545-56)。
用于转录本检测和定量的方法传统上包括基于Northern-印迹杂交、核糖核酸酶保护分析、和逆转录酶聚合酶链式反应(RT-PCR)的方法。然而,除缺少灵敏性(除RT-PCR外)的缺点外,这些方法只可用于粗略地评估来自不同来源的样本间每种转录本的相对表达的变化。基于RT-PCR的不同方法是为了诊断目的的最合适的定量方法,因为它们是很灵敏的并且因此只需要合乎诊断测试所需的小的样本规模。
如果想要比较样品之间,甚至相同样品内的基因表达,需要绝对定量样品中转录本的拷贝数。然而,由于PCR反应固有的非线性特性,利用基于PCR的方法很难定量核酸的拷贝数。PCR扩增可从指数阶段变化到试剂耗尽或酶钝化的平台期阶段。经常地,必须分别确定PCR的指数阶段,其可能包括在不同时间点对PCR反应进行取样或利用不同稀释度的模板进行PCR。进一步地,由于模板间扩增效率的差异,不能在线性范围中直接比较不同PCR产物的起始数量。传统上在扩增完成后进行PCR产物的检测。一般地,通过琼脂糖凝胶电泳、用溴化乙啶染色、并且用紫外光显现来区别PCR反应产物等分的大小。备选地,可以用荧光染料或放射性的分子标记引物。比较样品间的条带强度容许定性地估计扩增的模板的相对起始浓度,但是这个方法不是定量的并且不能确定绝对的拷贝数。
已经描述了许多基于定量的RT-PCR的方法,包括利用PCR和互补的DNA(cDNA)陈列的RNA定量法(Shalon et al.,Genome Research6(7):639-45,1996;Bernard et al.,Nucleic Acids Research24(8):1435-42,1996)、基于引物延伸反应的固相迷你测序方法(美国专利号6,013,431,Suomalainen et al.Mol.Biotechnol.Jun;15(2):123-31,2000)、离子对高效液相色谱(Doris et al.J.Chromatogr.A May 8;806(1):47-60,1998)、和5′核酸酶分析或实时RT-PCR(Holland et al.Proc Natl Acad Sci USA88:7276 7280,1991)。
研制提供灵敏和精确的定量mRNA转录本的方法将是有用的,其可以是容易地自动化操作的,并且规模扩大至容许测试大量样品,以及克服与PCR扩增相关的问题。这种方法能够诊断不同的病理学状况,包括病毒、细菌和寄生虫,以及不同的良性和恶性肿瘤、神经系统紊乱、心脏病和自身免疫紊乱。这种方法也提供以诊断、预后和治疗为目的的定量令人感兴趣的转录本,并且最终便于基因组药理学的应用。这种方法也可提供对基因表达有影响的大量试剂的筛选。
发明概述
本发明涉及测定样品中靶核酸的数量的方法,其中使用的标准物被设计成与目标基因或″靶核酸序列″相比具有一个碱基的差异。使用这种标准物与″增加″标准物与测试的核酸样品中的差异的方法的结合可,利用例如,正好在突变位点进行的碱基延伸反应,容许以相同的效率扩增标准和靶核酸,从而有助于定量靶核酸。此后使用定量″增加的″标准和靶核酸样品的手段来确定靶核酸的数量。在优选的实施方案中,定量手段是质谱法。
本发明的方法是灵敏的、精确的和高度可重复的,并且它也不依赖于PCR循环的数目,而极大地使分析简单化。本发明的方法是独特的,因为可同时测量相同基因的不同等位基因,可获得对基因表达的绝对定量,因此可直接比较来自不同实验的数据,并且可用于高-通量分析而几乎不需要为了PCR进行优化。另外,该方法容许精确地测定生物标本,例如人的液体(血清、血浆等)中的传染物,例如病毒、细菌和寄生虫的拷贝数。
本发明提供定量生物标本中一种靶基因/核酸或多种靶基因/核酸的数量的方法,包括将已知浓度的核酸标准加入生物标本中,其中该标准被设计成与靶核酸序列具有一个碱基的差异;例如利用聚合酶链式反应用的靶和标准核酸扩增样品,通过例如用磷酸酶(例如Shrimp碱性磷酸酶)处理扩增样品以除去过量的dNTPs,并因此通过例如靶和标准核酸样品中的不同碱基来增加标准和测试核酸之间的核酸差异。标准和靶核酸产生2种不同的产物,一般具有1个至2个碱基的差异,随后进行定量。可根据扩增样品中存在的标准物的数量来计算转录本的浓度。
例如,本发明能够检测,更重要的是定量传染物。它可容易地用于高通量的方法,其中在384-规格的硅芯片上可以定量大约100个传染物。
在一个优选的实施方案中,利用MALDI-TOF质谱法(例如,利用Sequenom′s MassArrayTM系统)根据″增加的″靶和标准核酸产物的不同质量进行定量,其中使用质谱中峰值的比率来计算标准物和靶核酸的比率。可以根据扩增前样品中使用/加入的标准的初始量来计算转录本的浓度。
在一个优选的实施方案中,利用引物延伸方法来增强标准和靶核酸之间的核酸差异。
在另一个实施方案中,利用荧光标记的dNTP/ddNTP进行碱基延伸来增强PCR后靶和标准中的核酸差异。
在另一实施方案中,利用不同染料标记的ddNTPs增强PCR后靶和标准中的核酸差异,其中将不同染料标记的ddNTPs差异性地掺入到引物延伸反应中的靶和标准核酸中。
在一个实施方案中,利用实时PCR来增强PCR后靶和标准中的核酸差异。
在另一个实施方案中,利用基于杂交的方法来增强PCR后靶和标准中的核酸差异,其中2种寡核苷酸特异于设计用于杂交的靶或标准。
在另一个实施方案中,利用热测序技术来增强PCR后靶和标准中的核酸差异。
在另一个实施方案中,利用使用人工的单核苷酸多态性(SNP)作为内参照的第三浪潮侵入分析(Third Wave Invader assay)来增强PCR后靶和标准中的核酸差异。在备选的实施方案中,当使用焦磷酸测序时,不需要预扩增。
在一个实施方案中,靶核酸是来自至少一种传染物的核酸。
在另一实施方案中,本发明提供一种试剂盒,其包括至少一个,优选几个不同的引物,该引物被设计成与不同小瓶中的至少一种,优选几种靶核酸相差一个核酸,或优选地与所有的标准核酸相差一个核酸,该标准核酸以已知的预定浓度存在于适于PCR的缓冲液中,或在直接的增强反应中如上所述增加标准物和相应靶核酸之间的差异。该试剂盒也包括说明反应条件和利用标准核酸测量靶核酸数量的手册。本发明预期的试剂盒包括但不限于:用于确定生物样品中传染物数量的试剂盒和确定在施用医药或药物后,或由于例如肿瘤的疾病状况而预期有增减的一种或多种转录本的数量的试剂盒。
附图简述
图1显示用于测量基因表达的实时竞争性PCR和质谱方法的流程图。为简单起见,只显示一个DNA链。在该流程图中也显示延伸的oligos一般为约20个碱基,而不是7个碱基。
图2显示相同浓度相同oligo的峰面积分布。使用0.3μm的Oligo47954(5′-ATGGCCACAGTTGTATCA-3′),并且将15nL点样到用3-羟基吡啶甲酸(HPA)预点样的硅芯片上。相同芯片的不同位置、相同浓度斑点的oligos的绝对峰面积显示平均峰面积为12395(任意数)和标准偏差为3737的适度变异性。
图3A和3B显示质谱中峰面积比与oligo的浓度比准确相关。Courtesy of Kal Tang(Sequenom)。利用MassArrayTM(Sequenom)分析4.5nL的不同比率的2种oligo混合物溶液(1∶1、1∶2、1∶5、1∶10、1∶20)。图3A显示质谱,而图3B显示质谱中实际浓度比率对信号强度(峰面积)比率的折线图。
图4A-4E显示以不同比率混合的仅有一个碱基不同的2种DNA模板的质谱。在图4A中比率是1∶1;在图4B中比率是3∶1;在图4C中比率是10∶1;在图4D中比率是1∶3;而在图4E中比率是1∶10,但是总浓度固定(2×10-7μg/μL)。通过PCR(30个循环)、碱基延伸(40个循环)来扩增模板,然后点样到用3-羟基吡啶甲酸(HPA)预点样的硅芯片上,并且用MALDI-TOF分析。
图5显示推定的DNA浓度比和测量的DNA浓度比(由峰面积比表示)之间的相关性。PCR扩增分别是20、30和40个循环,结果是不依赖 于PCR-循环。各个数据点重复4次(n=4)并显示误差线。
图6A-6H显示利用实时竞争性PCR和质谱的基因表达(GAPDH、HMBS和CXCR4)分析。
发明详述
本发明涉及测量样品中基因表达或核酸量的新方法。这个方法组合了竞争性的PCR(聚合酶链式反应)、碱基延伸以及此后进行的测量。该方法可用于直接测量特异性基因的拷贝数,或比较来自不同样品的特异性基因的上调或下调。
将已知浓度的标准核酸(DNA或RNA)加入RNA样品(用于RNA标准)或逆转录产物(用于DNA标准)中。然后通过PCR扩增包括标准物的逆转录产物。将该标准物设计成与目标基因,即靶核酸相比具有一个碱基突变的差异。因此,在PCR中以相同的效率扩增标准物和靶核酸。可利用例如正好在突变位点处进行的碱基延伸反应来鉴定这2种核酸。
因此通过多种方式中的任何一种,优选通过质谱(MALDI-TOF,或矩阵辅助的激光解吸电离-飞行时间)测量PCR产物的数量。来自标准物和目标基因的产物之间的峰面积比代表了标准物和目标基因的比率。既然已知标准的浓度,可以计算目标基因的浓度。
在至少下列的方面中,本发明的方法是独特的。首先,可选择基因的自然突变来构建标准物。因此,不仅可以测量基因的表达水平,而且可以确定表达基因的基因型。第二,PCR中单一点突变的使用确保了几乎等同的扩增。这消除了由其他竞争性PCR方法中的差异扩增产生的问题,在这些竞争性的PCR中,所述的标准物一般与基因是不同长度的。
在优选的实施方案中,碱基延伸和MALDI-TOF MS检测的组合也消除了来自常规的检测方法,例如凝胶电泳遇到的形成异源双链的问题。来自标准物的延伸产物也在MALDI-TOF MS中起内标准的作用。因此,当加入到反应中的标准物的数量是已知的时,可定量地测量核酸的数量。
这个方法具有至少下列优点。首先,这个方法在PCR中几乎不需要优化。第二,这个方法不依赖于PCR循环的数目。第三,该方法是高度精确的、灵敏的和可重复的。第四,该方法可用于高通量的基因表达分析,其中在一个384-硅芯片上可以测量至少50-100个、甚至达到 至少1000个基因的表达。
如下列实施例所示,通过这个方法分析在人的培养细胞中GAPDH、HMBS和CXCR4的表达可产生与其他方法一致的结果。
如在此所使用的,术语″生物样品″是指从任何来源(例如人、动物、植物、细菌、真菌、原生生物、病毒)获得的任何生物材料。为了用于本发明,该生物样品应包含核酸分子。用于本发明的合适的生物样品的实例包括:固体材料(例如组织、细胞沉淀颗粒、活检样品)和生物液体(例如尿液、血液、唾液、羊水、漱口剂)。
可利用本领域已知的许多方法中的任何一种从特定的生物样品分离核酸分子,其中选择适于特定的生物样品的特定分离方法。
病毒、细菌、真菌和其他感染性的生物体包含与宿主细胞中含有的序列不同的独特的核酸序列。检测或定量特异于感染性生物体的核酸序列对于诊断或监测感染是很重要的。感染人和动物,并且可通过已公开的方法检测的致病病毒的实例包括:逆转录病毒科(例如,人类免疫缺陷性病毒、例如HIV-1(也称为HTLV-III、LAV或HTLV-III/LAV、参见Ratner,L.et al.,Nature,Vol.313,Pp.227-284(1985);Wain Hobson,S.et al,Cell,Vol.40:Pp.9-17(1985));HIV-2(参见Guyader et al.,Nature,Vol.328,Pp.662-669(1987);欧洲专利公开号0 269 520;和其他分离物,例如HIV-LP(国际公开号WO94/00562,题目为“新的人免疫缺陷病毒”;小RNA病毒科(例如,脊髓灰质炎病毒、甲型肝炎病毒、(Gust,I.D.等人,Intervirology,Vol.20,Pp1-7(1983);肠道病毒属、人柯萨奇病毒、鼻病毒、艾柯病毒);环状病毒科(例如,导致肠胃炎的毒株);披盖病毒科(马脑炎病毒、风疹病毒)、黄病毒科(登革热病毒、脑炎病毒、黄热病毒);冠状病毒科(例如,冠状病毒);棒状病毒科(例如水泡性口炎病毒、狂犬病毒);纤丝病毒科(例如埃波拉病毒);副黏病毒科(副流感病毒、腮腺炎病毒、麻疹病毒、呼吸道合胞病毒);正黏病毒科(流感病毒);Bungaviridae(例如,Hantaan viruses、bunga病毒、phleboviruses和Nario病毒);沙粒病毒科(出血热病毒);呼肠孤病毒科(呼肠孤病毒、环状病毒和轮状病毒);双RNA病毒科;嗜肝DNA病毒科(乙型肝炎病毒);细小病毒科(细小病毒);乳多空病毒科(乳头状瘤病毒、多瘤病毒);腺病毒科(大多数腺病 毒);疱疹病毒科(1和2型单纯性疱疹病毒(HSV)、水痘带状疱疹病毒、巨细胞病毒(CMV)、疱疹病毒);痘病病毒(天花病毒、牛痘病毒、痘病毒);虹彩病毒(例如,非洲猪瘟病毒);以及未分类的病毒(例如,海绵状脑病的病原体、丁型肝炎的病原体(认为是乙型肝炎病毒的缺陷型卫星)、非甲非乙型肝炎的病原体(1类=内部传递的;2类=非肠道传递的)(即丙型肝炎);Norwalk及相关病毒,以及星形病毒)。传染性细菌的实例包括:幽门螺杆菌、布氏疏螺旋体、嗜肺军团菌、分枝杆菌属(例如结核分枝杆菌、贪食分枝杆菌、胞内分枝杆菌、堪萨斯分枝杆菌、戈登分枝杆菌)、金黄色葡萄球菌、淋病奈瑟氏球菌、脑膜炎奈瑟氏球菌、单核细胞增生利斯特氏菌、酿脓链球菌(A群链球菌)、无乳链球菌(B群链球菌)、链球菌(绿色群)、粪链球菌、牛链球菌、链球菌(厌氧种)、肺炎链球菌、致病的弯曲杆菌属、肠球菌属、流感嗜血菌、炭疽芽孢杆菌、白喉棒杆菌、棒杆菌属、猪红斑丹毒丝菌、产气荚膜梭菌、破伤风梭菌、产气肠杆菌、肺炎克雷伯氏菌、多杀巴斯德氏菌、拟杆菌属、具核梭杆菌、念珠状链杆菌、彻白密螺旋体、极细密螺旋体、钩端螺旋体和衣氏放线菌。
这种技术可直接用于开发高通量和精确的基因表达分析。其也可用于开发用于诊断疾病的测量至少大约2、5、10、25、50-100以及达到至少1000个基因的临床诊断芯片。
用于“增强”其中已经根据本发明的方法增强了标准物和靶核酸之间碱基差异的PCR产物的方法包括但不限于PYROSEQUENCINGTM、实时PCR、基于杂交的技术、第三浪潮侵入分析、基于荧光的PCR技术、固相迷你侧序。随后可利用例如MALDI-TOF质谱法(MS),通过增强的靶和标准核酸之间的质量差异定量“增强的”PCR产物。
本发明所使用的术语“增强”意在涵盖了使得靶和标准核酸包括质量差异的不同技术。由于标准物和靶核酸优选地只具有一个碱基的差异,因此可对其加以鉴别,并且利用标记核酸的引物延伸技术扩增或增强该差异。备选地,可利用等位基因特异的杂交探针或不同产物的酶切割,类似于INVADER分析而产生质量差异。
在一个实施方案中,通过为合成所必需的测序的PYROSEQUENCINGTM(Uppsala,瑞典)来增强只有一个碱基不同的PCR产物。首先将设计为直接邻近于靶和标准物之间的不同核酸的测序引 物与单标准的、PCR扩增的、包括靶和标准PCT产物的DNA模板杂交,并且与酶、DNA聚合酶、ATP硫酸化酶、荧光素酶和腺苷三磷酸双磷酸酶以及底物、磷酸硫酸化5′腺苷酸(APS)和荧光素孵育。然后将4种三磷酸脱氧核苷酸(dNTP)中的一种,例如相应于存在于标准模板中的核苷酸加入反应中。DNA聚合酶催化dNTP掺入到标准的DNA链中。各个掺入事件伴随有与掺入核苷酸的数量等摩尔数量的焦磷酸(PPi)的释放。因此,ATP硫酸化酶在存在磷酸硫酸化5′腺苷酸时定量地将PPi转化为ATP。这个ATP将荧光素的荧光素酶-介导的转化驱动为产生与ATP的数量成比例的数量的可见光的氧化荧光素。通过电荷耦合装置(CCD)照相机检测在荧光素酶-催化反应中产生的光并且视为PYROGRAMTM中的峰值。各个光信号与掺入的核苷酸的数目成比例,并且容许确定标准核酸序列的数量。此后,腺苷三磷酸双磷酸酶,一种降解核苷酸的酶连续地降解未掺入的dNTPs和过量的ATP。当完成降解作用时,加入相应于存在于待确定数量的靶模板中的dNTP的另一种dNTP。最后,一次加入一种dNTPs。使用脱氧腺苷α-硫代三磷酸(dATPaS)作为天然脱氧腺苷三磷酸(dATP)的替代物,因为其通过DNA聚合酶而不是由荧光素酶辨别来被有效地使用。因为加入PCR中的标准物的数量是已知的,可从掺入的dNTPs的比率来计算靶的数量。关于反应条件的详细信息,参见例如美国专利号6,210,891,其在此全部引入作为参考。
可用于增加PCR产物的标准物和靶核酸之间的碱基差异的方法的另一个实例是实时PCR。所有的实时PCR系统依赖于荧光报告子的检测和定量,荧光报告子信号的增加与反应中PCR产物的数量成正比。根据本发明的有用的实时PCR方法的实例包括,TaqMan 和分子信标,两者都是用于定量的依赖于荧光共振能量传递(FRET)的杂交探针。TaqMan探针是在5′碱基上一般包含荧光染料,而在3′碱基上一般定位有淬灭染料的寡核苷酸。当照射时,激发的荧光染料将能量转移到附近的淬灭染料分子上而不是发荧光,从而产生非荧光的底物。设计TaqMan探针能够与PCR产物内部的区域杂交(ABI 7700(TaqMan ),AppliedBioSystems,Foster City,CA)。因此,设计2种不同的引物,一种与靶杂交,而另一种与标准的核酸模板杂交。因此容许引物与实时PCR反应中的相应核酸杂交。在PCR期间,当聚合酶复制其上结合有TaqMan 探针的模板时,该聚合酶的5′核酸外切酶活性切割探针,因此这使荧光染料和淬灭染料分离开来而不再发生FRET。在每个循环中荧光增加,并与探针裂解的速率成比例。
分子信标也包含荧光和淬灭染料,但是只有当淬灭染料直接邻近于荧光染料时才发生FRET。设计分子信标使其具有发夹同时游离在溶液中,使得荧光染料和淬灭染料更为接近。因此,设计2种不同的分子信标,一种识别靶而另一种识别标准的核酸。当分子信标与靶和标准核酸杂交时,荧光染料和淬灭剂分离开来,FRET不再发生,因此荧光染料在照射下发光。不同于TaqMan探针,分子信标被设计成在扩增反应期间保持完整,并且在每个循环中必须与靶再结合来用于信号测量。TaqMan探针和分子信标容许测量相同样品中的多种DNA种类(多重PCR),因为具有不同发射光谱的荧光染料可能结合不同的探针,例如将不同的染料用于制备标准探针和靶探针。多重PCR提供共-扩增的内对照,并且容许单试管、同质分析中的等位基因鉴别。(Ambion Inc,Austin,TX,TechNotes 8(1)-2001年2月,Real-time PCR goesprime time)。
用于增加靶和标准核酸之间差异的另一方法是例如用于固相“迷你”测序(Hultman,et al.,1988,Nucl.Acid.Res.,17,4937-4946;Syvanen et al.,1990,Genomics,8,684-692)的引物延伸方法。在原始的报道中,测量放射性标记的核苷酸的掺入并用于分析人脱脂蛋白E基因的三等位多态性。可变核苷酸的检测方法是基于检测步骤中引物的延伸和可检测的三磷酸核苷的掺入。通过从紧邻于可变核苷酸的区域选择检测步骤的引物,可在掺入几乎只有一个三磷酸核苷后检测到这个变化。加入相配于可变核苷酸的标记的三磷酸核苷,并且测量掺入到检测步骤引物中的标记。使该检测步骤的引物退火至靶核酸的拷贝,将包含包括至少一个标记的或修饰的三磷酸核苷的一种或多种三磷酸核苷的溶液在促进引物延伸的条件下与聚合剂一起加入。可使用标记的脱氧核糖核苷三磷酸(dNTPs)或端链双脱氧核糖核苷三磷酸(ddNTPs),并且标记优选为染料,包括荧光染料。固相“迷你”测序方法在例如美国专利号6,013,431和Wartiovaara和Syvanen,Quantitative analysis of human DNA sequences by PCRand solid-phase minisequencing.Mol Biotechnol 2000Jun;15 (2)123-131中有描述。
另一个增加PCR产物中靶和标准核酸之间差异的方法是利用荧光标记的dNTP/ddNTPs。除在固相“迷你”测序方法中使用荧光标记之外,可使用标准的核酸测序凝胶来检测掺入PCR扩增产物的荧光标记的数量。设计测序引物使其能退火至紧接于从模板辨别标准物的碱基上。利用以荧光染料标记的端链双脱氧核糖核苷三磷酸(ddNTPs)进行引物延伸反应,将一个结合ddNTP的标记加入标准核酸中,而另一个结合ddNTP的标记加入靶核酸中。此后利用荧光检测核酸测序仪中的变性凝胶或利用毛细管凝胶电泳分离引物延伸产物,掺入到标准物和靶核酸中的荧光标记的数量导致荧光峰,从峰的大小确定该数量。标准的荧光测序方法是本领域技术人员已知的(例如,参见Amersham LifeSciences,Uppsala,瑞典,和Applied Biosystems,Foster City,CACity,CA)。
备选地,可使用INVADER 分析(Third Wave Technologies,Inc(Madison,WI))。这个分析一般是基于多种酶的结构特异的核酸酶活性,使用这种酶切割靶依赖的裂解结构,由此指明样品中存在特异的核酸序列或其特异的变化(参见,例如美国专利号6,458,535)。例如,INVADER 操作系统(OS)提供用于检测和定量DNA和RNA的方法。INVADER OS是以″完全匹配″的酶-底物反应为基础的。INVADES OS使用专有的CLEAVASE 酶(Third Wave Technologies,Inc(Madison,WI)),其识别并且只切割在INVADER 过程期间形成的特异结构。INVADER OS依赖于由INVADERQ 过程产生的信号的线性扩增,而不是依赖于靶的指数扩增。这就容许定量靶的浓度。
在INVADER 过程中,使2种短的DNA探针与靶杂交以形成被CLEAVASE 酶辨别的结构。然后该酶切割一个探针而释放短的DNA″片状结构(flap)″。各个释放的片状结构结合荧光-标记的探针而形成另一个断裂结构。当CLEAVASE 酶切割标记的探针时,该探针发射可检测的荧光信号。
定量的优选方法是MALDI-TOF MS。利用MALDI-TOF MS定量的方法的细节如下在实施例中给出。
本发明也预期包括至少一种、优选几种不同引物的试剂盒,设计该引物使其与在单独的小瓶或试管中的至少一种,优选与几种靶核酸 相差一个核酸,或优选地与一组在相同小瓶或试管中的具有已知量的干燥的标准核酸、包括至少2种不同的标准物的组合标准物有一个核酸不同,并且含有在合适的缓冲液,例如PBS中稀释样品至已知浓度,以用于定量反应的说明书。备选地,在合适的缓冲液,例如PBS中以已知的浓度预先稀释标准物。合适的缓冲液可以是适于储备核酸或适于例如PCR或直接的增强反应,以如上所述增加标准物和相应靶核酸之间的差异,或该缓冲液刚好适于储备样品并且提供单独的稀释缓冲液而更适于后继的PCR、增强和定量反应。在优选的实施方案中,将所有的标准核酸组合在有缓冲液的一个试管或小瓶中,使得只向包含靶核酸的核酸样品加入一个标准混合物。
该试剂盒优选也包括说明反应条件和利用标准核酸或其混合物测量靶核酸的数量并且给出所有标准物的详细浓度和缓冲液种类的手册。本发明预期的试剂盒包括但不限于用于确定生物样品中传染物数量的试剂盒和确定在施用医药或药物后,或由于例如肿瘤的疾病状况而预期有增减的一种或多种转录本的数量的试剂盒。
实施例
MALDI-TOF MS是定量的
在MassArray系统中绝对信号(通过质谱中的峰面积测量的)在MALDI-TOF MS试验中是相对稳定的(图2)。这对于精确的定量分析是够好的。然而,利用类似序列的oligo作为内对照,我们可以精确地测量oligo的浓度(图3)。
在2个DNA混合物系统中不依赖于PCR循环数的实时竞争性的PCR操作。
在这个实验中,以恒定的总浓度为2*10-7μg/μL的不同比率(10∶1、3∶1、1∶1、1∶3、1∶10)混合仅仅只有一个核苷酸不同的2种DNAs。用HotStart DNA聚合酶进行PCR扩增,然后通过Shrimp碱性磷酸酶(SAP)处理以除去过量的dNTPs。然后,用ThermoSequenase与适当ddNTP/dNTP的混合物(一般为3种不同的ddNTP和一种dNTP)进行碱基延伸实验。通过MALDI-TOF检测延伸产物,并且用RT(实时)软件(Sequenom Inc.)分析峰面积。图4显示来自5种不同比率的模板混合物的质谱。图5显示质谱中峰面积比和为了分析而预确定的DNA模板比率之间的相关性。
对另一对2种DNAs重复相同的实验并且获得了与上述相似的结果。这些原始数据明显地显示,至少在这个简单的人工系统中,与质谱法鉴定联合的实时竞争性的PCR是测量基因表达的潜在的精确方法。测量的峰面积比与达到100倍范围的已知DNA浓度比成线性相关(R2>0.999)。可以轻易地延伸在标准DNA的100-倍离析下的3个梯度至动态范围为106,而这足以用于大多数的实际应用。
测试用于人类基因表达的实时竞争性的PCR
通过这个实时竞争性的PCR和MALDI-TOF方法分析培养细胞中GAPDH、HMBS和CXCR4的表达。向cDNA样品中分别加入增加浓度的每种基因的竞争物。通过实时竞争性的PCR和MALDI-TOF MS测量内源基因及其竞争物的频率。既然我们已知竞争物的浓度,就可以计算目标基因的表达水平。
扩大高通量基因表达分析的规模
微阵列是用于以小的群体/条件规模(一般不多于50)筛选数以万计的基因的理想(至少目前是)方法。一般通过一些在对照和样品之间显著不同的统计标准选择几百个基因。例如,Golub等人报道利用38个骨髓样品的微阵列分析,选择能够共同鉴别急性淋巴母细胞性白血病(ALL)和急性的骨髓性白血病(AML)的50个基因。来自小样本规模的(38个样品)的大的统计自由度和大的基因数目规模(6817个),连同微阵列方法的低精确度,使得人们明显怀疑在更大的患者样本规模上这个预测值(50个基因)能够进行得有多好。从经济学上来说,在数百个患者样本的规模上用微阵列测试这个是不可行的。在我们的方法中,我们可以在384个芯片上轻易地测量大约100个基因的表达,并且可以测试数以百计的患者样品。微阵列是高通量基因数目方式的,而我们的方法是高通量患者数目方式的,其可使得这两种方法高度互补。
我们也可使用这个方法来研究基因表达的化学计量。这里科学的假定是,作为功能单元工作密切的基因(或其产物,蛋白质)同样将具有相似的表达水平。已经使用质谱法分析了蛋白质复合物(Gavinetal.,Ho et al.)。我们可以分析相同复合物中这些基因的mRNA表达,并且评估这些组合的化学计量。
计算
第一个问题是PCR oligo设计。对于其他的RT-PCR方法,例如实时PCR,如果对于你的目标基因扩增是非特异性的,则它可能是失败的,因为它将导致对表达水平明显的估计不足。并且更坏的是,非特异性的扩增可能依赖于样品。在我们的情况中,既然我们在目标基因的相同反应中总是具有内标准,这个问题应该是不严重的。如上所述,对避免非特异性的扩增来说它仍然是很重要的。设计扩增oligos中的另一个问题出现在倍增PCR中。此外应该注意避免引物-引物之间的相互作用。
计算机应用和统计技术也可应用于分析波谱。在MALDI-TOF实验中,通过激光束击中相同样品斑点的5个不同位置。并且,如果我们对每种样品进行4次重复,我们将具有20个数据点;足以应用统计模型例如正态分布来更精确地计算峰比率。另一个问题是标准化。已经使用多种管家基因(GAPDH、β-肌动蛋白、亲环蛋白、18srRNA)。使用这些基因的组合来进行标准化可能是更好的。
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Claims (10)
1.一种定量测定生物样品中几种靶核酸序列的数量的方法,包括下列步骤:
a)通过在包含几种靶核酸的标本中加入已知量的几种标准核酸来制备样品,其中该几种标准核酸具有与其相应的靶核酸序列相比有一个碱基不同的核苷酸序列;
b)扩增步骤a)的样品;
c)将扩增后的样品用进一步的方法进行处理以增强该标准和该靶核酸序列之间在所述一个碱基不同的位点处的差异,以使该标准和靶核酸序列之间由所述一个碱基不同引致的区别得以检测;以及
d)使用质谱法定量测量步骤c)增强的靶核酸和增强的标准核酸的数量,其方法是测量所述几种增强的靶核酸中每种靶核酸和几种增加的标准核酸中每种标准核酸之间的比率,以测量存在于样品中的所述几种靶核酸序列中每种靶核酸序列的数量。
2.权利要求1的方法,其中所述几种靶核酸序列来自于传染物。
3.权利要求1的方法,其中所述几种靶核酸是mRNA转录本。
4.权利要求1的方法,其中利用在所述一个碱基不同的位点处的引物延伸进行步骤c)。
5.权利要求1的方法,其中利用在所述一个碱基不同的位点处特异性裂解的酶进行步骤c)。
6.权利要求1的方法,其中利用在所述一个碱基不同的位点处的等位基因特异的杂交进行步骤c)。
7.权利要求1、4、5和6中任一项的方法,其中利用MALDI-TOF质谱法进行步骤d)的定量检测。
8.权利要求1的方法,其中使用至少5种靶核酸。
9.权利要求1的方法,其中使用至少10种靶核酸。
10.权利要求1的方法,其中使用至少25种靶核酸。
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附图2. |
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AU2003270397B2 (en) | 2009-07-16 |
US20120028838A1 (en) | 2012-02-02 |
EP1546385B1 (en) | 2013-04-17 |
WO2004022721A3 (en) | 2004-09-16 |
HK1085244A1 (en) | 2006-08-18 |
JP2005537804A (ja) | 2005-12-15 |
WO2004022721A2 (en) | 2004-03-18 |
CA2497988A1 (en) | 2004-03-18 |
CN102344960B (zh) | 2014-06-18 |
CN1703521A (zh) | 2005-11-30 |
EP1546385A4 (en) | 2006-12-20 |
JP2010268812A (ja) | 2010-12-02 |
AU2003270397A1 (en) | 2004-03-29 |
CA2497988C (en) | 2011-03-29 |
CN102344960A (zh) | 2012-02-08 |
US8034567B2 (en) | 2011-10-11 |
EP1546385A2 (en) | 2005-06-29 |
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