CN1202204A - 基于质谱的dna诊断 - Google Patents
基于质谱的dna诊断 Download PDFInfo
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- CN1202204A CN1202204A CN96193883.8A CN96193883A CN1202204A CN 1202204 A CN1202204 A CN 1202204A CN 96193883 A CN96193883 A CN 96193883A CN 1202204 A CN1202204 A CN 1202204A
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Abstract
本发明提供了基于质谱的、用以检测生物样品中特异DNA序列的快速而高度精确的方法。根据被检测的序列,这些方法可用于,例如,诊断遗传性疾病或染色体异常,诊断患某种疾病的倾向性,病原体的感染,或用于确定身份或遗传性。
Description
发明背景
所有活的有机体(如动物、植物和微生物)的遗传信息均编码于脱氧核糖核酸(DNA)。在人类,完整的基因组含有约100,000个基因,位于24条染色体上(《人类基因组》,T.Strachan著,BIOS科技出版社1992年出版)。每一个基因编码一个特异的蛋白,这个蛋白质通过转录和翻译表达后,在活细胞中行使特定的生化功能。DNA序列的变化,如突变,会产生活性改变或在某些情况下失去了生化活性的蛋白,这将会导致遗传性疾病。突变包括核苷酸缺失、插入或改变(即点突变)。点突变可能是“错义的”,导致蛋白质氨基酸序列的改变,或是无义的,编码一个终止密码子,结果导致一个截断的蛋白质。
目前已知超过3000种遗传性疾病(《人基因组突变》,D.N.Cooper和M.Krawczak著,BIOS出版社1993年版),包括血友病、地中海贫血症、假肥大性肌营养不良(DMD)、亨廷顿舞蹈病(HD)、早老性痴呆和囊性纤维变(CF)。除了导致遗传病的突变基因外,一些生殖缺陷是染色体异常如21三体(唐氏综合症)、13三体(Patau综合症)、18三体(爱德华综合症)、X单体(特纳综合症)和其它性染色体非整倍体如克莱恩费尔特综合症(XXY)的结果。另外,有越来越多的证据表明一些DNA序列能诱导个体产生下述的任何疾病,如糖尿病、动脉粥样硬化、肥胖、多种自身免疫疾病和癌症(如直肠、乳腺、卵巢、肺部癌症)。
病毒、细菌、真菌和其它感染性有机体含有特异的核酸序列,它们与宿主细胞的序列不同。因此,感染性有机体可根据其特异的DNA序列被检出并鉴别。
既然16核苷酸的序列甚至对人类基因组的大小在统计范围内是特异的,那么相对短一些的核酸序列可用于检测较高等生物正常和有缺陷的基因并检测感染性微生物(如细菌、真菌、原生生物和酵母)和病毒。DNA序列甚至可作为指纹检测同一物种的不同个体(Thompson,J.S.和M.W.Thompson编《医学遗传学》,W.B.Saunders Co.,Philadelphia,PA(1986))。
许多检测DNA的方法目前已被应用。例如,用凝胶电泳法通过比较一个扩增的核酸片段与已知的标准的迁移率来鉴定核酸序列,或者用与被检测序列互补的探针杂交来鉴定。但是,只有在核酸片段用敏感的报道官能团(如放射性(32P、35S)、萤光或化学发光)标记后才能实施鉴定。然而,放射性标记是危险的而且它们产生的信号随时间衰减。非同位素标记(如萤光)则灵敏度不够而且当用到高强度激光时信号衰减。另外,标记、电泳和随后的检测是费力、耗时而且易出错的方法。电泳尤其易出错,因为核酸的大小或分子量与其在胶中的迁移率没有直接的关系。已知序列特异性效应,二级结构和与胶质的相互作用都能引起人为假象。
通常,质谱通过分子在真空中离子化并使它们汽化飞起来提供一种给单个分子“称重”的方法。在电场和磁场的联合影响下,离子依其质量(m)和电荷(z)留下飞行轨迹。在低分子量的分子中,质谱长期以来就是通过确定母体分子离子的质量来分析和鉴定有机分子的常用的物理-有机方法。另外,通过母体分子离子与其它粒子(如氩原子)的碰撞,该分子离子以称作碰撞诱导裂解(CID)的方式片段化形成二次离子。片段化的方式/途径通常能推导出详细的结构信息。质谱法的许多应用在本领域,尤其在生命科学领域已为人熟知,并总结于《酶学方法》193卷:“质谱”(J.A.McCloskey编),1990年Academic Press,New York。
由于质谱在提供高检测灵敏度、质量测量的精确性,由CID与质谱-质谱联用结合所提供的详细结构信息和分析速度,以及直接将数据输入计算机等方面具有明显的分析优势,人们已对将质谱用于核酸的结构分析产生深厚的兴趣。最近总结这方面的综述包括K.H.Schram,《核酸成份的质谱、质谱在生物医学中的应用》(Mass Spectrometry ofNucleic Acid Components,Biomedical Applications of MassSpectrometry)1990年34卷203-287页;和P.F.Crain“核酸研究中的质谱技术(Mass Spectrometric Techniques in NucleicAcid Research)”,《质谱述评》,1990年9卷505-554页。
然而,核酸是非常极性化的生物多聚体,非常难以汽化。因而,质谱检测仅限于低分子量的合成的寡聚核苷酸,通过检测母体分子离子的质量并通过它来证实已知的寡聚核苷酸序列,或者用另一种方法,利用CID与质谱-质谱联用结合,特别是用来产生离子化和气化的快速原子轰击方法(FAB质谱)或等离子解吸方法(PD质谱)产生的二级离子(片段离子)来证实已知序列。例如,FAB对化学合成寡聚脱氧核苷酸的保护二聚体的分析的应用已详述(Kster等《生物医学环境中的质谱》,1987年14卷111-116页)。
两种较新的离子化/解吸附技术是电喷雾/离子喷雾(ES)和基质辅助激光解吸附/离子化(MALDI)。ES质谱已由Fenn等介绍(《物理化学杂志》(J.Phys.Chem.)1984年88卷4451-59页;PCT申请No.Wo90/14148)。目前的应用总结于最近的综述文章(R.D.Smith等,《分析化学》(Anal.Chem.)1990年62卷882-89页和B.Ardrey,“电子喷射质谱(Electrospray Mass Spectrometry)”,《欧洲谱学》(Spectroscopy Europe,1992年4卷10-18页)。一个40核苷酸(Covey等,“利用离子喷雾质谱确定蛋白质、寡聚核苷酸和多肽的分子量(Determination of Protein Oligonucleotide and PeptideMolecular Weights by Ionspray Mass Spectrometry);《质谱快讯》(Rapid Communications in Mass Spectrometry),1988年2卷249-256页)和一个21核苷酸(《酶学方法》(Methods inEnzymology)193卷,《质谱》(Mass Spectrometry)”,McCloskey编,425页,1990,Academic Press,New York)的分子量已发表。作为一个质量分析器,四极杆(quadrupole)最常用到。飞摩尔量级样品中分子量的确定是非常精确的,因为有许多离子峰存在,而它们均能用于质量计算。
与四极杆相比,当飞行时间(TOF)配置被用作质量分析器时,MALDI质谱术则更具吸引力。MALDI-TOF质谱术已由Hillenkamp等介绍(“基质辅助紫外激光解吸附/离子化:生物大分子质谱的新方法(Matrix-Assisted UV-Laser Desorption/Ionization:A NewApproach to Mass Spectrometry of Large Biomolecules)”,《生物学质谱》(Biological Mass Spectrometry)(Burlingame和McCloskey编),Elsevier Science Publishers,Amsterdam,49-60页,1990年)。由于在大多数情况下,这项技术中没有许多的分子离子峰产生,因而与ES质谱相比,质谱图原则上看起来简单些。
尽管分子量达410,000道尔顿的DNA分子已被解吸附和汽化(Williams等,“高分子量DNA的冰冻水溶液脉冲激光烧蚀法汽化作用(Volatilization of High Molecular Weight DNA by PulsedLaser Ablation of Frozen Aqueous Solutions)”,《科学》(Science),1989年246卷1585-87页),这项技术至今仅显示出非常低的分辨率(寡聚胸腺嘧啶脱氧核苷酸至18核苷酸,Huth-Fehre等,《质谱快讯》(Rapid Communications in Mass Spectrometry)1992年6卷209-13页;500核苷酸长的DNA片段,K.Tang等《质谱快讯》(Communications in Mass Spectrometry),1994年8卷727-730页;28碱基对双链DNA(Williams等,“冰冻含水基质中核酸激光烧蚀和离子化飞行时间质谱(Time of Flight MassSpectrometry of Nucleic Acids by Laser Ablation andIonization from a Frozen Aqueous Matrix)”,《质谱快讯》(RapidCommunications in Mass Spectrometry),1990年4卷348-351页)。
日本专利No.59-131909描述了一种装置,它能检测由电泳、液相色谱或高速凝胶过滤分离的核酸片段。通过往核酸中掺入DNA中通常不存在的原子如S、Br、I或Ag、Au、Pt、Os、Hg,质谱检测可以实现。发明概述
本发明提供了质谱法检测生物样品中特定的核酸序列,根据被检测的序列,本方法可用于,例如,诊断(如产前或产后)遗传性疾病或染色体异常、诊断患某种疾病(如肥胖、动脉粥样硬化、癌症)的倾向或病原体(如病毒、细菌、寄生虫或真菌)的感染,或提供与身份、遗传性或相容性(如HLA表型)相关的信息。
在第一个实施方案中,一个含待测(即靶)核酸序列的核酸分子首先固定到固体支持物上。例如,固定化可根据杂交完成,即靶核酸分子上区别于待测位点的部位与已固定在固体支持物上的捕获核酸分子杂交。另一种方法,靶核酸分子可直接与固体支持物结合而完成固定化。优选地,在靶核酸分子与支持物间有一个隔离物(如一个核酸分子)。一个与靶检测位点互补的检测核酸分子(如寡聚核苷酸或寡聚核苷酸模拟物)能与靶检测位点接触,形成二体,表明质谱可检测靶检测位点的存在。在优选的实施方案中,靶检测位点在检测前被扩增并对核酸分子进行修饰。在更优选的实施方案中,靶检测序列按一定方式排列以使多点同时检测(多路检测),除此之外,用寡聚核苷酸阵列(“DNA芯片”)并行处理。
在第二个实施方案中,靶核酸分子的固定化是选择性的而不是必要的步骤。替代地,一旦核酸分子从生物样品中获得,靶检测序列扩增后直接用质谱检测。在优选的实施方案中,靶检测位点和/或检测核苷酸在质谱检测前进行修饰。在另一优选的实施方案中,扩增的靶检测位点按一定方式排列以使多点同时检测(多路检测),除此之外,用寡聚核苷酸阵列(“DNA芯片”)并行处理。
在第三个实施方案中,从来自生物样本的核酸分子复制出的核酸分子能用一种或多种核酸酶(对DNA用脱氧核糖核酸酶,对RNA用核糖核酸酶)特异地消化且消化片段被捕获到带有相应互补序列的固体支持物上。被捕获的靶序列的杂交和实际分子量可提供基因上是否有和哪里有突变发生的信息。DNA核酸的序列可用质谱逐点分析。DNA也可用包括限制性内切酶的混合核酸酶消化。在优选的实施方案中,核酸片段在质谱检测前进行修饰。
在第四个实施方案中,至少一个3′端碱基与一等位基因(突变的或正常的)互补的引物与含有等位基因的靶核酸分子杂交。一种合适的聚合酶和一整套核苷三磷酸或其中一种核苷三磷酸用于独立的反应以使引物延伸的长度各不相同。只有当引物正确地退火(即无3′错配)和适当的(即互补)核苷酸掺入,引物才能延伸。产物可利用质谱确定的分子量位移来分析。
在第五个实施方案中,含有待测(即靶)核酸序列的核酸分子首先被固定到固相支持物上。固定化可通过如杂交的方式完成,即靶核酸分子上与靶检测位点不同的部分与已固定在固相支持物上的捕获核酸分子杂交。另一种方法,可直接将靶核酸分子与固相支持物结合而完成固定化。优选地,在靶核酸分子与支持物间有一间隔物(如一核酸分子)。与紧接着突变位点5′端的靶检测位点部位互补的一个核酸分子随即与靶核酸分子杂交。加入一整套双脱氧核苷酸或3′脱氧核苷三磷酸(如pppAdd、pppTdd、pppCdd和pppGdd)和一依赖于DNA的DNA聚合酶,使得只有一种与X互补的双脱氧核苷酸或3′-脱氧核苷三磷酸能掺入。杂交产物可用质谱检测。
在第六个实施方案中,靶核酸与一可与含一突变M的靶位点区杂交的互补寡聚核苷酸杂交。杂合二聚体随即用一个特异断裂未杂交区的试剂(如单链特异性内切酶)处理,于是表明突变存在的错配将导致靶核酸的断裂。两个断裂的产物可用质谱检测。
在第七个实施方案中,该方案建立在连接酶链反应(LCR)基础上,一靶核酸与一套连接体和一热稳定DNA连接酶杂交,使各连接体彼此共价连接形成连接产物。连接产物随后可用质谱检测并与已知值比较。如果反应以循环的方式进行,获得的连接产物可得到扩增更有利于小量靶核酸的检测。连接点处野生型和突变型引物的选择可得到点突变的检测。
本发明的方法提高了核酸质谱检测的精确性和可靠性。另外,该方法还考虑到严密的对照以防止错误的阴性或阳性的结果。本发明的方法避免了电泳步骤;标记和随后的标记检测。实际上,估计全过程,包括核酸分离、扩增和质谱图分析,仅需要2-3小时。因而本发明公开的方法比已有的DNA检测系统操作更快更便宜。另外,由于此处公开的方法使得核酸片段通过其特异的分子量(一确定的物理量)同时得到鉴定与检测,因而与目前应用的方法相比,本公开的方法也要精确且可靠得多。附图简述
图1A是一个显示应用质谱分析一包含在从生物样本中获得的靶核酸分子(T)中的靶检测位点(TDS)的示意图。一特异的捕获序列(C)通过一间隔物(S)与一固相支持物(SS)相接。该捕获序列可与靶核酸分子(T)上的互补序列,即靶捕获位点(TCS)特异杂交。间隔物(S)有利于不妨碍杂交。一与TDS互补的检测核酸序列(D)与TDS作用。D和TDS间的杂交可由质谱检测。
图1B是一个显示应用质谱分析直接与固相支持物连接的至少一个靶检测位点(这里指TDS1和TDS2)的方法图。含有靶检测位点(TDS1和TDS2)的靶序列(T)通过靶核酸分子(T)上合适的官能团(L′)与固相支持物上合适的官能团(L)之间形成可逆或不可逆的化学键而固定在固相支持物上。与靶检测位点(TDS1和TDS2)互补的检测核酸序列(这里指TDS1或TDS2)随后与TDS作用。TDS1和D1和/或TDS2和D2间的杂交可由其分子量的差异得到检测和区别。
图1C是一个显示检测靶(T)核酸分子上野生型(Dwt)和/或突变(Dmut)序列的方法图。如同图1A中所示,一特异的捕获序列(C)通过一间隔物(S)与固相支持物(SS)结合。另外,选择的捕获序列可特异地与待用杂交检测的靶序列(T)上的互补序列,即靶捕获位点(TCS)相互作用。但是,如果靶检测位点(TDS)上含有一个突变,X,该突变改变了分子量,突变的靶检测位点可通过质谱与野生型相区别。优选地,检测核酸分子(D)设计成在分子中间有一突变,因而当野生型检测寡聚核苷酸(Dwt)与靶检测序列作用时不能形成稳定的杂交体,例如作为对照。如果带有与突变位点配对碱基的突变检测寡聚核苷酸(Dmut)用于杂交时也就能检测到突变。如果从生物样品中获得的核酸分子对某一特定序列是杂合的(即同时含有Dwt和Dmut),Dwt和Dmut均与合适的链结合,那么质量差异将使Dwt和Dmut同时得到检测。
图2是一显示利用相应的检测寡聚核苷酸在一靶序列上同时检测几个突变的方法图。检测寡聚核苷酸D1、D2和D3间分子量的差异必须足够大,使得同时检测(多路检测)成为可能。这可通过序列本身(组成或长度)或通过在检测寡聚核苷酸上引入质量修饰官能团M1-M3来达至目的。
图3是一显示另一多路检测方式的图。在这个实施方案中,差别在于使用了不同的特异捕获序列,它们位置特异地固定在一台面上(如“芯片排列”)。当不同的靶序列T1-Tn存在时,它们的靶捕获位点TCS1-TCSn将与互补的固定化的捕获序列C1-Cn相互作用。通过使用有合适的质量差异的检测寡聚核苷酸D1-Dn,可得到检测。D1-Dn的质量差异可通过它们的序列或通过质量修饰官能团M1-Mn获得。
图4是一流程图,其中应用PCR扩增将一预设计的靶捕获位点(TCS)掺入到靶序列中。只有一条链被捕获,而另一链被除去(如利用生物素与包被着链霉抗生物素蛋白磁珠间相互作用)。如果生物素与引物1结合则另一链可被TCS正确标记上。检测如上述,通过质谱检测特异的检测寡聚核苷酸D与相应的靶检测位点TDS的相互作用。
图5是一显示扩增(这里指连接酶链反应(LCR))产物如何制备及用质谱检测的示意图。可通过在引物(P1和P4)上接一质量修饰官能团(M1和M2)来获得质量差异。质谱检测可直接完成(即没有使用固定化和靶捕获位点(TCS))。多LCR反应能在提供有序排列的捕获序列(C)的条件下并行进行。这个程式可使连接产物分离并通过质谱逐点进行鉴定或如果质量差异足够的话,可进行多路检测。
图6A是一显示通过转录扩增法扩增所得核酸分子的质谱分析示意图。一个RNA分子通过其TCS序列被捕获,于是野生型和突变的靶检测位点可用上述方法利用合适的检测寡聚核苷酸(D)得到检测。
图6B是一显示利用质量修饰检测寡聚核苷酸M1-D1和M2-D2,用同时方式多路检测同一RNA上两个不同(突变的)位点的示意图。
图6C是一利用质量修饰的双脱氧核糖核酸或3′-脱氧核苷三磷酸和一RNA依赖的DNA聚合酶检测特异突变的不同多路检测法的示意图。另一方面中,DNA依赖的RNA聚合酶和核糖核苷三磷酸也可使用。这个方法可同时检测所有四种碱基在突变(X)位点的可能性。
图7A是一显示用质谱分析一从生物样品中获得的靶核酸分子(T)中的靶检测位点(TDS)的方法的示意图。一个特异的捕获序列(C)通过一间隔物(S)与固相支持物(SS)相接。选用的该捕获序列可与T上的已知为靶捕获位点(TCS)的互补序列特异性杂交。一个与TDS部分互补的核酸分子杂交到TDS中一突变(X)位点5′端的TDS上。一整套双脱氧核苷酸或3′-脱氧核苷三磷酸(如pppAdd、pppTdd、pppCdd、pppGdd)和一DNA依赖的DNA聚合物的加入使得仅有一种与X互补的双脱氧核苷酸或3′-脱氧核糖核苷三磷酸能掺入进去。
图7B是一利用质谱分析以检测一核酸分子中潜在突变位点(M)处突变存在的方法的示意图。这个程式使得一双链靶核酸分子中等位基因(A)和(B)能同时分析,因而可提供一诊断纯合正常、纯合突变或杂合的方法。等位基因A和B均与相应互补的寡聚核苷酸((C)和(D))杂交,杂交区中含有M。每个杂交二聚体随后用单链特异性内切酶处理,于是显示突变存在的M点的错配将导致(C)和/或(D)的断裂,这能用质谱检测到。
图8是一显示如何利用在相反位置有两个不同启动子(例SP6和T7启动子)的转录载体来制备用于检测的靶DNA的两条链的示意图。这个程式尤其适用于检测杂合的靶检测位点(TDS)。利用SP6或T7RNA聚合酶,两条链均能各自或同时转录。利用合适的质量差异的检测寡核苷酸,两条RNA能被特异地捕获并同时被检测。这可直接在溶液中完成或通过许多靶序列在顺序排列的特异的固定化捕获序列上并行处理而完成。
图9是一个显示如图6、7和8所述制备的RNA如何用一种或多种核酸酶特异地消化以及消化片段如何被捕捉到带有相应互补序列的固相载体上的示意图。被捕获的靶序列的杂交情况和实际分子量可提供基因上是否有或哪里有突变存在的信息。排列可用质谱逐点分析。利用包括限制性内切酶在内的混合核酸酶,DNA也能同样地被消化。与野生型片段分子量相比,特异的各个片段不同的分子量可被用于检测突变。
图10A显示了一个下面实施例1中描述的实验的结果图谱。小图i)显示了26聚体杂交前的吸收。小图ii)显示了杂交后离心滤液的吸收。小图iii)显示了用50mM柠檬酸铵第一次洗涤后的结果。小图iv)显示了50mM柠檬酸铵第二次洗涤后的结果。
图10B显示了得自下面实施例1中实验经过三次洗涤/离心步骤后的谱图。
图10C显示了得自下面实施例1中实验的谱图,显示了杂交的26聚核苷酸成功地从磁珠上解吸附。
图11显示了得自下面实施例1中实验的谱图,显示了杂交的40聚核苷酸成功的解吸附,检测效率说明比40聚核苷酸长得多的片段也能解吸附。
图12显示了得自下面实施例2中实验的谱图,显示了一个18聚核苷酸和一19聚核苷酸用电喷雾质谱解吸附及其差别,混合物(上面),得自18聚核苷酸的峰(中间),得自19聚核苷酸的峰(底部)。
图13是实施例3所述囊性纤维变突变ΔF508检测方法的图示。
图14是一ΔF508纯合正常的DNA延伸产物的质谱图。
图15是一ΔF508杂合突变的DNA延伸产物的质谱图。
图16是一ΔF508纯合正常的DNA延伸产物的质谱图。
图17是一ΔF508纯合突变的DNA延伸产物的质变图。
图18是一ΔF508杂合突变的DNA延伸产物的质谱图。
图19是载脂蛋白E基因型分析不同方法的图示。
图20显示了正常载脂蛋白E(由E3等位基因编码)的核酸序列和由E2和E4等位基因编码的其它异构体。
图21A显示了载脂蛋白E不同基因型的限制性图谱。
图21B显示了不同基因型载脂蛋白E在3.5%MetPhor琼脂糖凝胶中获得的限制性图谱。
图21C显示了不同基因型载脂蛋白E在12%聚丙烯酰胺凝胶中获得的限制性图谱。
图22A是一列出了载脂蛋白E的E2、E3和E4等位基因限制性酶切后得到的91、83、72、48和35碱基对片段的分子量的表。
图22B是一纯合E4载脂蛋白E基因型限制性产物的质谱图。
图23A是一纯合E3载脂蛋白E基因型限制性产物的质谱图。
图23B是一E3/E4载脂蛋白E基因型限制性产物的质谱图。
图24是一加入10%(5μl)每个PCR产物的7.5%聚丙烯酰胺凝胶的放射自显影照片。样品M:AluI消化的pBR322;样品1:血清学分析HBV阳性;样品2:HBV阳性;样品3:没有经血清学分析,但转氨酶水平升高,显示有肝病;样品4:HBV阴性;样品5:血清学分析HBV阳性;样品6:HBV阴性;(-)阴性对照;(+)阳性对照)。用溴化乙锭染色。
图25A是样品1的质谱图,样品1为HBV阳性。20754 Da处的信号代表HBV相关的PCR产物(67核苷酸,计算质量为20735 Da)。10390 Da处的质量信号代表[M+2H]2+信号(计算值:10378 Da)。
图25B是样品3的质谱图,样品3用血清学、PCR和点杂交方法检测为HBV阴性。PCR的产物为痕量水平。然而,可明显地检测到20751Da处的信号(计算值:20735 Da)。10397 Da处的质谱信号代表[M+2H]2+分子离子(计算值:10376 Da)。
图25C是样品4的质谱图,样品4为HBV阴性但CMV阳性。如预期,没有HBV特异信号产生。
图26显示了带有用于连接酶链反应(LCR)的互补寡聚核苷酸的结合位点的一部分大肠杆菌IacI基因。这里显示的是野生型序列。突变体在bp 191处含一点突变,该位点也是一连接位点(凸露的)。突变为C换成T(相应地G换成A)。这就导致寡聚体A中T-G错配(相应地寡聚体B中A-C错配)。
图27是一用溴化乙锭染色的7.5%的聚丙烯酰胺凝胶。M:链长标准(MspI消化的pUC 19DNA)。泳道1:野生型为模板的LCR。泳道2:突变体为模板的LCR。泳道3:无模板的(对照)LCR。只有在含野生型模板的阳性反应中才能产生连接产物(50bp)。
图28是两个有意选择的合并的阳性LCR的HPLC色谱图。
图29显示了同样条件下但以突变体为模板的HPLC色谱图。连接产物中的小信号是由于连接物的无模板连接或在(G-T,A-C)错配的连接。其“假阳性”信号明显低于图28示出的以野生型为模板的连接产物的信号。连接体的分解由于两个寡聚核苷酸为5′-磷酸化而导致“双峰”。
图30a中绘出了MALDI-TOF-MS分析Pfu DNA连接酶溶液的复杂信号图。b中显示了一未纯化LCR的MALDI-TOF谱图。质量信号67569 Da可能代表Pfu DNA连接酶。
图31显示了两有意选择合并的阳性LCR的MALDI-TOF质谱图(a)。7523 Da处信号代表未连接的寡聚体A(计算值:7521 Da)而15449 Da处信号代表连接产物(计算值:15450 Da)。3774 Da处信号为寡聚体A的[M+2H]2+信号。低于2000 Da质量范围的信号归因于基质离子。该图谱与图2a中泳道1和图2b中色谱图相对应。在b中,显示了两有意选择合并的阴性LCR(突变体模板)的质谱图。7517 Da处的信号代表寡聚体A(计算值:7521 Da)。在C中显示的是两有意选择合并的对照反应(以鲑精DNA为模板)的质谱图。在2000 Da左右质量范围的信号归因于Tween 20。
图32显示了两有意选择合并的LCR中获得的质谱图,其中只有鲑精DNA作为阴性对照,如预期的那样,只有寡聚体A能被检测到。
图33显示了两个有意选择合并的阳性LCR的质谱图(a)。用文中描述的超滤和链霉抗生物素蛋白DynaBeads结合使其得以纯化。15448 Da处的信号代表连接产物(计算值15450 Da)。7527 Da处信号代表寡聚体A(计算值:7521 Da)。3761 Da处信号代表寡聚体A的[M+2H]2+信号,而5140 Da处的信号为连接产物的[M+3H]2+信号。在b中显示的是两个有意选择合并的阴性LCR(无模板)的质谱图。7514Da处的信号代表寡聚体A的信号(计算值:7521 Da)。
图34是一在反应混合物中使用ddTTP(A)或ddCTP(B)所得突变检测引物b的寡聚碱基延伸的流程图。理论质量计算值在括号中给出。显示的序列是带有最常见囊性纤维变突变ΔF508和较罕见突变ΔI507以及Ile506Ser的CFTR基因外显子10的部分序列。
图35是一直接从沉淀的用于突变检测的寡聚碱基延伸的引物中记录的MALDI-TOF-MS谱。每一部分(ddTTP或ddCTP)上面的谱线表示了没有进一步延伸反应的退火的引物(CF508)。诊断的模板在每个谱下标出而且观察的/预期的分子质量在括号中注明。
图36显示了pRFc1 DNA序列的一部分以及19聚体引物和两个18聚体反向引物的序列。pRFc1序列用作未修饰的和含7-脱氮嘌呤的99聚体和200聚体核酸PCR扩增的模板。
图37显示了M13mp18RFI DNA的部分核苷酸序列,它用于未修饰的和含7-脱氮嘌呤的103聚体核酸的PCR扩增。也显示了用于PCR的17聚体引物的核苷酸序列。
图38显示了用于MALDI-TOF MS分析的纯化且浓缩的PCR产物的聚丙烯酰胺凝胶电泳的结果。M:链长标记物,泳道1:含7-脱氮嘌呤的99聚体PCR产物,泳道2:未修饰的99聚体,泳道3:含7脱氮嘌呤的103聚体,泳道4:未修饰的103聚体PCR产物。
图39:用5′-[32P]标记引物1和4进行PCR反应的聚丙烯酰胺凝胶电泳的放射自显影照片。泳道1和2:未修饰和7-脱氮嘌呤修饰的103聚体PCR产物(53321和23520计数)。泳道3和4:未修饰和7-脱氮嘌呤修饰的200聚体(71123和39582计数)和泳道5和6:未修饰和7-脱氮嘌呤修饰的99聚体(173216和94400计数)。
图40a)未修饰的103聚体PCR产物的MALDI-TOF质谱图(12个单轰击的叠加)。两条单链(31768u和31759u)质量的计算平均值为31763u。质量分辨率:18。b)含7-脱氮嘌呤103聚体PCR产物的MALDI-TOF质谱图(三个单轰击的叠加)。两条单链(31727u和31719u)计算质量的平均值为31723u。质量分辨率:67。
图41:a)未修饰的99聚体PCR产物的MALDI-TOF质谱图(12个单轰击的叠加)。两条单链计算质量值为30261u和30794u。b)含7-脱氮嘌呤99聚体PCR产物的MALDI-TOF质谱图(12个单轰击的叠加)。两条单链计算质量值:30224u和30750u。
图42:a)未修饰200聚体PCR产物的MALDI-TOF质谱图(30个单轰击的叠加)。两条单链(61873u和61595u)计算质量的平均值为61734u。质量分辨率:28。b)含7-脱氮嘌呤的200聚体PCR产物的MALDI-TOF质谱图(30个单轰击的叠加)。两条单链(61772u和61514u)计算质量的平均值为61643u。质量分辨率:39。
图43:a)利用核糖标记的引物,含7-脱氮嘌呤的100聚体PCR产物的MALDI-TOF质谱图。两条单链(30529u和31095u)计算质量的平均值为30812u。b)引物水解断裂后PCR产物的MALDI-TOF质谱图。两条单链(25104u和25229u)计算质量的平均值为25167u。断裂引物(5437u和5918u)的平均值为5677u。
图44A-D显示了从通过3′生物素化固定在链霉抗生物素蛋白珠上的39聚体模板上(SEQ.ID.No.13)获得的4个序列梯度的MALDI-TOF质谱图。在测序中用到了一14聚体引物(SEQ.ID.No.14)。
图45显示了通过3′生物素化固定在链霉抗生物素蛋白珠上的78聚体模板(SEQ.ID.No.15)的固相测序的一MALDI-TOF质谱图。一18聚体引物(SEQ.ID.No.16)和ddGTP用于测序。
图46显示了一带有单链突出的双链DNA探针捕获特异DNA模板并用作固相测序引物的示意图。
图47A-D显示了从5′萤光标记的23聚体(SEQ.ID.No.19)中获得的MALDI-TOF质谱图,该23聚体与一3′生物素化的18聚体(SEQ.ID.No.20)退火,留下一5碱基突出,用以捕获一15聚体模板(SEQ.ID.No.21)。
图48显示了一图35所述反应中同样的产物但用常规DNA测序仪所得的堆积萤光图。发明详述
概括地说,本发明提供了质谱法以检测生物样品中特异的核酸序列。这里用到的术语“生物样品”指任何从任何生命源(如人、动物、植物、细菌、真菌、原生生物、病毒)获得的物质。为了在本发明中应用,生物样品中须含有核酸分子。本发明中可用的合适的生物样品的例子包括:固体材料(如组织、细胞团、活体解剖样品)和生物液(如尿、血、唾液、羊水、漱口液)。
可以用许多方法从一特定的生物样品中分离核酸分子,这些方法在本领域已为人熟知,可根据特定的生物样品选择合适的特定分离方法。例如冻融和碱裂解法可用于从固体材料中获得核酸分子;加热和碱裂解法可用于从尿中获取核酸分子;蛋白酶K抽提可用于从血中获取核酸分子(Rolff.A.等《PCR:临床诊断与研究》,Springer(1994))。
为了得到合适量的核酸分子用于质谱,扩增可能是必须的。可用于本发明的合适的扩增方法的实例包括:克隆(Sambrook等,《分子克隆:实验室手册》(Molecular Cloning:A Laboratory Manual,ColdSpring Harbor Laboratory Press,1989)、聚合酶链反应(PCR)(C.R.Newton和A.Graham,《PCR》,BIOS Publishers,(1994)),连接酶链反应(LCR)(Wiedmann M等,(1994)《PCR方法应用》(PCR Methods Appl.),卷3,57-64页;F.Barany,《美国国家科学院学报》(Proc.Natl.Acad.Sci.USA),1991年88卷189-93页)、链置换扩增(SDA)(G.Terrance Walker等,《核酸研究)》(Nucleic Acids Res.),1994年22卷2670-77页)和变更方法如RT-PCR(Higuchi等,《生物/技术》(Bio/Technology)1993年11卷1026-1030页)、等位基因特异扩增(ASA)和基于转录的方法。
为了方便质谱分析,含有待测序列的核酸分子可固定到固相支持物上。合适的固相支持物的实例包括球珠(如硅胶、可控多孔玻璃、磁珠、Sephadex/Sepharose,纤维素)、平台表面或嵌片(如玻璃纤维滤器、玻璃表面、金属表面(钢、金、银、铝、铜和硅)、毛细管、塑料(如聚乙烯、聚丙烯、聚酰胺、聚二氧乙二烯膜或微量滴定板));或者用包括球珠或平台表面相似材料做成的钉或梳子、或将球珠置入平台表面的凹点中如垫片(例如硅垫片)。
固定化可利用杂交法来完成,例如已固定到固相支持物上的捕获核酸序列与一含待测核酸序列的核酸分子中的互补核酸序列杂交(图1A)。因而互补的核酸分子间的杂交不会被支持物妨碍,捕获核酸分子在固相支持物和捕获核酸序列间可含一至少5核苷酸长的间隔区。在激光脉冲影响下形成的二聚体可被断裂且解吸附可被激发。固相支持物结合的碱基序列可用天然的寡聚核糖核酸或寡聚脱氧核糖核酸及其类似物(如巯基修饰的磷酸二酯或磷酸三酯骨架)或使用寡核苷酸模拟物如PNA类似物(参见Nielsen等,《科学》(Science),1991年254卷1497页)提供,它们可降低碱基序列对酶促降解的敏感性,因而提高固相支持物结合的捕获碱基序列的稳定性。
另一种方法,靶检测位点可通过靶核酸分子(T)上合适的官能团(L′)与捕获分子上合适的官能团(L)间可逆或不可逆的化学键直接连接到固相支持物上(图1B)。可逆的连接可使得它在质谱条件下断裂(即可光解的键如电荷转移复合物或相对稳定的有机自由基间形成的不稳定化学键)。而且,连接可通过与季胺基团的L′形成,在这样的情况下,优选地,固相支持物表面带负电荷,因而可排斥带负电荷的核酸骨架从而有利于质谱分析所需的解吸附。解吸附可通过激光脉冲产生的热量和/或依靠L′,通过与L′生色团共轭的激光能量的特异吸收作用而完成。
根据实施例的方式,L-L′化学性质可能是二硫键形式(化学可分解的,例如用巯基乙醇或二硫苏糖醇)、生物素/链霉抗生物素蛋白系统、三苯甲基酯的异双功能衍生物(Kster等,“用于合成的生物分子修饰的多功能酸不稳定生接头(A Versatlie Acid-Labile Linker forModification of Synthetic Biomolecules)”,《四面体通讯》(TetrahedronLetters),1990年31卷7095页),它能在温和的酸性条件及在质谱条件下断裂,用肼/乙酸盐缓冲液在几乎中性条件下可降解的乙酰丙酰基团,可被内肽酶如胰蛋白酶切开的精氨酸-精氨酸或赖氨酸-赖氨酸键或可被焦磷酸酶切开的焦磷酸键或寡聚脱氧核酸间的核糖核苷酸键,它可被如核酸酶或碱切开。
官能团L和L′,也可形成一电荷转移复合物,因而形成一暂时的L-L′连接。由于在许多情况下“电荷转移带”可被紫外/可见分光光度计检测(参见R.Foster著《有机电荷转移复合物》,Academic Press,1969),激光能量可被调至电荷转移波长相应的能量,因而从固相支持物上特异的解吸附可被触发。本领域的技术人员可考虑到用几种方法来达到此目的,而且供体官能团既可在固相支持物上,也可偶联到待测核酸分子上,或反之亦然。
在另一方法中,可逆的L-L′连接可通过均裂形成的相对稳定的自由基形成。在激光脉冲的影响下,解吸附(上文讨论的)以及离子化作用将在自由基位置形成。本领域的技术人员会认识到也可选择其它一些有机自由基,考虑到它们之间均裂化学键所需的解结合能量,可选用相应的激光波长(参见C.Wentrup著《活性分子》(ReactiveMolecules)”,John Wiley & Sons,1984)。
利用在如PCR(图4)、LCR(图5)或转录扩增(图6)的扩增反应中使用合适的引物,一锚定的官能团L′也可掺入到靶捕获序列(TCS)上。
在质谱分析前,“修饰”核酸分子可能是有用的,例如可降低汽化所需的激光能量和/或将片段化作用降至最低程度。修饰优选适用于固定化的靶检测位点。修饰的一个实例是核酸分子中磷酸二酯骨架的修饰(如阳离子交换),这可消除由于每核苷酸单位上结合的阳离子的不均一性导致的峰加宽现象。将核酸分子与烷基化试剂如烷基碘、碘乙酰胺、β-碘乙醇或2,3-环氧乙烷-1-丙醇反应,核酸分子中的单硫磷酸二酯键可转换为磷酸三酯键。同样地,利用氯化三烷基硅可将磷酸二酯键转换为无电荷的衍生物。进一步的修饰包括掺入降低脱嘌呤(MS中的片段化作用)敏感性的核苷酸如N7-或N9-脱氮嘌呤核苷酸或RNA元件,或使用寡聚核苷酸三酯或掺入烷基化的硫代磷酸酯官能团或掺入寡聚核苷酸模拟物如PNA。
对于某些应用,同时检测位于一特异被捕获核酸片段(在矩阵上的一个点上)上超过一个(突变的)位点或用不同固相支持物上矩阵排列寡聚核苷酸或寡聚核苷酸模拟物来实行并行检测可能是有用的。“多路检测”可由一些不同的方法来获得。例如,通过使用相应的检测(探针)分子(如寡聚核苷酸或寡聚核苷酸模拟物),在一靶序列上的几个突变可被同时检测。然而,检测核苷酸D1、D2和D3的分子量差异必须足够大,同时检测(多路检测)才有可能。这种差异可通过序列本身(组成或长度)或在检测寡聚核苷酸中引入质量修饰官能团M1-M3来获得(图2)。
质量修饰部分可加到以下位置,如加到寡聚核苷酸的5′端(M1),加到核酸基(或碱基)上(M2,M7),或加到磷酸骨架上(M3),和加到核苷(多核苷)的2′位置上(M4,M6)或/和加到3′末端上(M5)。质量修饰部分的实例包括,如卤素元素、叠氮化物或XR类型物,其中X是连接基团而R是质量修饰官能团。质量修饰官能团可在寡聚核苷酸分子上引入确定的质量增量。
这里质量修饰部分,M,可接到核酸碱基上,M2(如果含C7-脱氮核苷酸也加到C-7上,M7),接到三磷酸基的α磷酸上,M3,或接到核苷三磷酸中糖环的2′位置上,M4和M6。而且,加入质量修饰官能团后可导致影响链终止,如将其加到核苷三磷酸中糖环的3′位置上,M5。对于本领域的技术人员来说,很明显有很多结合方法可同样地达到本发明的目的。同样地,本领域的技术人员也可考虑到用官能团与结合位点的许多不同选择与组合以相似的方式对链延伸的核苷三磷酸加以质量修饰。
在不限制本发明范围的情况下,质量修饰物M可作为XR中的X,而用寡聚/多聚乙二醇衍生物作为R。在这种情况下质量修饰的增幅为44,即m从0变到4将产生五个不同的质量修饰体,可将45(m=0),89(m=1),133(m=2),177(m=3),和221(m=4)质量单位加到核酸分子(如检测寡聚核苷酸(D)或核苷三磷酸(图6(C)))上。寡聚多聚乙二醇也可被低级烷基如甲基、乙基、丙基、异丙基、叔丁基等烷基化。连接官能团X的选择也作了说明。质量修饰复合物中可用到的其它化学物质,这方面的例子,最近描述于F.Eckstein编,《寡核苷酸及其类似物:实用方法》(Oligonucleotides andAnalogues.A Practical Approach),IRL Press、Oxford,1991。
在另一个实施方案中,不同于寡聚/多聚乙二醇的其它一些质量修饰官能团R,可被选择并通过合适的连接化学物X固定住。用卤素元素如F、Cl、Br和/或I,或拟卤素如SCN、NCS取代H,或使用不同的烷基、芳基、芳烷基如甲基、乙基、丙基、异丙基、叔丁基、己基、苯基、取代苯基、苯甲基或如CH2F、CHF2、CF3、Si(CH3)3、Si(CH3)2(C2H5)、Si(CH3)(C2H5)2、Si(C2H5)3等功能基,可得到简单的质量修饰物。通过核酸分子(如检测物(D))或核苷三磷酸附着同形或异形肽可获得另一质量修饰物。一个在产生具有57质量增幅的质量修饰物中有用的例子是加入寡聚甘氨酸,如可获得74(r=1,m=0),131(r=1,m=2),188(r=1,m=3),245(r=1,m=4)的质量修饰物。简单的寡聚胺也可被用到,如获得74(r=1,m=0),88(r=2,m=0),102(r=3,m=0),116(r=4,m=0)等质量修饰物。对于本领域的技术人员来说,很显然除上面提到的这些外,还有许多可能性。
这里用到的上标0-i表示i+1个质量差异的核苷酸、引物或标记物。在某些情况下,上标0可表示一特定反应物的未修饰状态,而上标i可表示该反应的第i质量修饰态。例如,如果核酸的不止一种状态需要同时检测,那么i+1个不同的质量修饰检测寡聚核苷酸(D0,D1,…Di)可被用于利用质谱将质量修饰寡聚核苷酸(D)的不同状态与其它的区分开来。
不同的质量修饰检测寡聚核苷酸可被用于同时检测所有可能的变异/突变(图6B)。另一种方法,通过设计并放置一检测寡聚核苷酸,作为DNA/RNA聚合酶的引物,突变位点所有四种碱基的突变均可检测到(图6C)。例如,质量修饰物可在扩增过程中被掺入。
图3显示一不同的多路检测程式,在这个程式中,通过使用位点特异地固定在平台表面的不同的特异捕获序列(如“嵌片矩阵”)可完成鉴别。如果存在不同的靶序列T1-Tn,它们的靶捕获位点TCS1-TCSn将特异地与互补的被固定化的捕获序列C1-Cn相互作用。使用合适的质量差异检测寡聚核苷酸D1-Dn可完成检测,其质量差异可通过其自身的序列或质量修饰官能团M1-Mn获得。
本发明中用到的优选的质谱检测方式是基质辅助激光解吸附离子化(MALDI),电喷雾(ES),离子回旋共振(ICR)和傅立叶变换。对于ES,溶于水或可汽化缓冲液的样品连续或不连续喷射到一常压离子化界面(API)并随后通过一四极杆(quadrupole)进行质量分析。使用ES质谱时多离子峰的产生可提高质量确定的精确性。利用一质谱-质谱联用四极杆(quadrupole)组合,甚至可得到有关特异结构更详细的信息。
在MALDI质谱中,可用到不同的质量分析器,如质谱领域中熟知的以单或3四极杆(quadrupole)方式(质谱-质谱联用)的磁扇形/磁偏转装置,傅立叶变换和飞行时间(TOF)组合。对于解吸附/离子化过程,可使用多种基质/激光组合。离子阱和反射器(reflectron)组合也常用到。
上面所描述的质谱方法可用于,例如诊断目前已知的或待鉴定的超过3000种遗传病(如血友病、地中海贫血症、假肥大性肌营养不良(DMD)、亨廷顿舞蹈病(HD)、早老性痴呆和囊性纤维变(CF))中的任何一种。
下面的实施例3提供了用于检测囊性纤维变跨膜传导调节基因(CFTR)中的突变(ΔF508)的质谱方法,与野生型CFTR基因相比,突变体只有3个碱基对(900 Da)不同。如实施例3中进一步描述的,检测是以单管竞争性寡聚核苷酸单链碱基延伸(COSBE)反应为基础,该反应使用了一对3′端碱基与正常或突变等位基因互补的引物。根据杂交和聚合酶及一碱基下游的核苷三磷酸的加入,只有能正确退火(即无3′错配)的引物才能延伸;产物可通过利用基质辅助激光解吸附离子化飞行时间质谱确定的分子量位移来分析。对于囊性纤维变ΔF508多态性,28聚体“正常”(N)和30聚体“突变”(M)引物对N和M纯合子相应地产生29和31聚体,而对杂合子则两者都产生。由于引物和产物分子量相对较低(<10 kDa)且它们间质量差异至少有一个约300 Da的单核苷酸单位,因而低分辨率的装置就适用于这样的检测。
除了突变基因将导致遗传性疾病之外,某些生殖缺陷是染色体异常如21三体(唐氏综合症)、13三体(Patau综合症)、18三体(爱德华综合症)、X单体(特纳综合症)和其它一些性染色体非整倍体如克莱恩费尔特综合症(XXY)的结果。
另外,越来越多的证据表明某些DNA序列可诱发个体产生许多疾病,如糖尿病、动脉粥样硬化、肥胖、多种自身免疫疾病和癌症(如直肠、乳腺、卵巢、肺部癌症)、染色体异常(产前或产后);或为疾病(如肥胖、动脉粥样硬化、癌)的诱因。同样,“DNA指纹”检测,例如多态性(如“小卫星序列”),被用于检测身份与遗传性(如父本或母本)。
下面的实施例4提供了一种质谱方法,用于检测人载脂蛋白E三种不同的异构体,它们由E2、E3和E4等位基因编码,这里用合适的限制性内切酶消化合所得片段的分子量,可用于检测突变的存在。
依据生物样品,对遗传病、染色体非整倍体或遗传倾向的诊断可在产前或产后进行。
病毒、细菌、真菌和其它感染性有机体含有宿主细胞中所含序列不同的独特的核酸序列。对感染有机体来说非常特异的核酸序列的检测与定量对诊断和传染的监控是非常重要的。能引起疾病并能传染人与动物,已能由公开的方法检测的病毒的实例包括:反转录病毒科(例如人免疫缺陷病毒、如HIV-1(也称HTLV-III、LAV或HTLV-III/LAV,见Ratner,L.等,《自然》(Nature),1985年313卷227-284页;Wain Hobson,S.等,《细胞》(Cell),1985年40卷9-17页);HIV-2(见Guyader等,《自然》(Nature),1987年328卷662-669页;欧洲专利出版物No.0269520;Chakraborti等,《自然》(Nature),1987年328卷543-547页;和欧洲专利申请No.0655501);和其它分离物,如HIV-LP(国际出版物No.WO94/00562,题目为“一种新的人免疫缺陷病毒”));微小RNA病毒科(例如脊髓灰质炎病毒,甲肝病毒(Gust,I.D.等,国际病毒学(Intervirology),1983年20卷1-7页),肠道病毒,人柯萨奇病毒,鼻病毒,埃可病毒);嵌杯病毒科(例如引起胃肠炎的病毒株);外衣病毒科(例如马脑炎病毒,风疹病毒);黄热病毒科(例如登革热病毒,脑炎病毒、黄热病毒);冠状病毒科(如冠状病毒);棒状病毒科(例如疱疹性口炎病毒,狂犬病毒);丝状病毒科(例如埃博拉病毒);副粘液病毒科(如副流感病毒,流行性腮腺炎病毒,麻疹病毒,呼吸合胞体病毒);正粘液病毒科(例如流感病毒);崩格病毒(例如汉坦病毒,崩格病毒,静脉病毒和内罗病毒);沙粒病毒科(出血热病毒);呼吸道肠道病毒科(例如呼吸道肠道病毒,眼病毒和轮状病毒);伯恩纳病毒科;肝DNA病毒科(乙肝病毒);细小病毒科(细小病毒);乳多空病毒科(乳头瘤病毒,多瘤病毒);腺病毒科(大多数腺病毒);疱疹病毒科(单纯性疱疹病毒(HSV)1和2,带状疱疹病毒,巨细胞病毒(CMV),疱疹病毒);痘病毒科(天花病毒,牛痘病毒,痘病毒);和虹膜病毒科(例如非洲猪热病毒);和未分类的病毒(例如海绵状脑病病原体,γ肝炎病原体(认为是乙型肝炎病毒的缺陷型卫星病毒),非甲非乙肝炎病原体(类型1=内传染的;类型2=旁传染的(即丙型肝炎;诺沃克及相关病毒,和星状病毒)。
感染性细菌的实例包括:幽门螺旋菌,布氏疏螺旋体,嗜肺军团菌,分枝杆菌属种(例如,结核分枝杆菌,鸟分枝杆菌,胞内分枝杆菌,堪萨斯氏分枝杆菌,戈特氏分枝杆菌),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,化脓链球菌(A族链球菌),无乳链球菌(B族链球菌),链球菌属(草绿色链球菌),粪链球菌,牛链球菌,链球菌属(嫌气链球菌种类),肺炎链球菌,病原弯曲杆菌属种,肠球菌属种,流感嗜血杆菌,炭疽芽孢杆菌,白喉棒状杆菌,棒状杆菌属种,猪红斑丹毒丝菌,产气荚膜梭状芽孢杆菌,破伤风梭状芽孢杆菌,产气肠杆菌,肺炎杆菌,出血败血性巴斯德氏菌,类杆菌属种,核粒梭杆菌,念珠状链杆菌,梅毒密螺旋体,极细密螺旋体,钩端螺旋体属,及以色列放线菌。
感染性真菌包括:新型隐球酵母,荚膜组织胞浆菌,粗球孢菌,皮炎芽生菌,沙眼衣原体,白色假丝酵母。其它感染性生物(如原生生物)包括:恶性疟原虫和鼠弓形体。
下面的实施例5提供了一种用于检测血液样品中乙型肝炎病毒(HBV)DNA的嵌套PCR并基于质谱的方法。同样地,其它一些血液病毒(如HIV-1,HIV-2,丙型肝炎病毒(HCV),甲型肝炎病毒(HAV)和其它一些肝炎病毒(如非甲非乙肝炎,庚型肝炎,戊型肝炎病毒),巨细胞病毒和单纯性疱疹病毒(HSV))也可用这里描述的方法独自或联合检出。
既然大约16核苷酸的序列在统计学范围内是特异的(甚至对人基因组大小的基因组),那么相对短一些的核酸序列可用于检测较高等有机体的正常或缺陷基因并检测感染性微生物(如细菌、真菌、原生生物和酵母)和病毒。DNA序列甚至可作为指纹图来检测同一物种的不同个体。(Thompson,J.S.和M.W.Thompson,编,《医学遗传学》(Genetics in Medicine),W.B.Saunders Co.Philadelphia,PA(1986)。
一种检测靶(T)核酸分子野生型(Dwt)和/或突变体(Dmut)序列的方法如图1C所示。一特异的捕获序列(C)通过一隔离物(S)与一固相支持物(SS)相接。另外,所选的捕获序列可与靶序列(T)上的一段互补序列特异地相互作用,靶捕获位点(TCS)可通过杂交而被检测。然而,如果靶检测位点(TDS)含有一个突变X,该突变将提高或降低分子量,突变的TDS可通过质谱与野生型区别开来。例如,如果插入一个腺苷(dA),那么Dwt和Dmut间分子量的差异约为314道尔顿。
优选地,检测核酸(D)如下设计,突变在分子的中央且侧翼序列足够短,因而当野生型检测寡聚核苷酸(Dwt)与突变型靶检测序列作用作为对照时,不能形成稳定的杂交体。当在突变位点带有配对碱基的突变型检测寡聚核苷酸(Dmut)用于杂交时,也可检测突变。如果一个生物样品中获得的核酸在某一特定序列上是杂合的(即含有Dwt和Dmut),Dwt和Dmut都将结合到合适的链上,它们间的质量差异将使Dwt和Dmut被同时检测。
本发明的方法使用了靶序列上已知的序列信息和已知的突变位点。然而新的突变也能被检测。例如,如图8所示,从生物样品中获得的一核酸分子的转录产物可用一种或多种核酸酶特异地消化,其消化片段被携有相应互补核酸序列的固相支持物捕获。被捕获的靶序列的杂交和分子量检测将能提供基因中是否有且哪里有突变存在的信息。另一种方u 法,DNA可被一种或多种特异的核酸内切酶断裂形成片段混合物,通过比较野生型和突变型片段混合物的分子量,可检测突变。
本发明将用下面的实施例进一步详细说明,但它们不应被认作是任何方式的限制。所有引用的参考文献(包括文献、授权专利、出版的专利申请(包括国际专利申请出版号WO 94/16101,题目为“质谱法DNA测序(DNA Sequencing by Mass Spectrometry)”,H.Koester;和国际专利申请出版号WO 94/21822,题目为“通过外切核酸酶降解的质谱法DNA测序(DNA Sequencing by Mass Spectrometry ViaExonuclease Degradation)”,H.Koester),和共同未决的专利申请,(包括美国专利申请流水号08/406,199,题目为“基于质谱的DNA诊断(DNA Diagnostics Based on Mass Spectrometry)”,H.Koester))的内容,在本申请中均有引用,特此引入作为参考。实施例1 直接连在固相支持物上的寡聚核苷酸的MALDI-TOF解吸附
1g CPG(可控多孔玻璃)用3-(乙氧硅烷基)-环氧丙烷活化,在聚合物表面形成OH基。利用β-氰乙基-磷酰胺(Kster等,《核酸研究》(Nucleic Acids Res.)1994年12卷4539页)和TAC N-保护基团(Kster等,《四面体》(Tetrahedron)1981年37卷362页),在DNA合成仪(Milligen.Model7500)上用13mg OH-CPG的标准寡聚核苷酸合成3′-T5-50聚体寡聚核苷酸,其中50个核苷酸与一“假想的”50聚体序列互补。T5作为间隔物。根据含约10μmol 55聚体/g CPG的DMT基CPG的测定,用甲醇中的饱和氨水于室温处理2小时完成脱保护。这个55聚体作为模板与一26聚体(带有5′-DMT基团)和一40聚体(无DMT基团)杂交。反应体积为100μl且含有约1nmolCPG结合的55聚体作为模板,等摩尔量的寡聚核苷酸(26聚体或40聚体),反应体系另含20mM Tris-HCl,pH7.5,10mM MgCl2和25mM NaCl。反应混合物于65℃加热10′并在30′内冷却至37℃(退火)。通过离心和随后进行的再次使用100μl冰冷的50mM柠檬酸铵的三次洗涤/离心步骤,没有杂交到聚合物结合的模板上的寡聚核苷酸被除去。多孔玻璃珠在空气中晾干并与基质溶液(3-羟基吡啶甲酸/溶于乙腈/水1∶1的10mM柠檬酸铵)混合,并用MALDI-TOF质谱分析,结果见图10和11。实施例2 18聚体和19聚体的电喷雾(ES)解吸附与区分
利用一电喷雾质谱仪同时分析浓度为50pmole/μl,溶于2-丙醇/10mM碳酸铵(1/9,v/v)的DNA片段。
利用电喷雾质谱的一18聚体和一19聚体的成功的解吸附和分辨如图12所示。实施例3 利用一步双脱氧延伸和MALDI-TOF质谱分析检测囊性纤
维变突变ΔF508材料与方法
PCR扩增和链的固定化:在标准PCR条件(30循环:1′@95℃,1′@55℃,2′@72℃),用外显子10特异的引物进行扩增,反向引物用生物素进行5′标记并用柱纯化(Oligpurification Cartridge,Cruachem)。扩增后PCR产物用柱分离纯化(Qiagen Quickspin)并用标准方法将其固定在链霉抗生物素蛋白包被的磁珠(DynabeadsDynal,Norway)上;DNA用0.1M NaOH变性并用0.1M NaOH,1×B+W缓冲液和TE溶液洗涤以除去未生物素化的有义链。
COSBE条件:含连接的反义链的磁珠重悬浮于18μl反应混合物1(2μl 10×Taq缓冲液,1μl(1单位)Taq聚合酶,2μl 2mMdGTP和13μl H2O)并在加入反应混合液2(100ng 每一COSBE引物)前于80℃温育5′。温度降至60℃并将混合物温育5′的退火/延伸期;随后用25mM乙酸三乙胺(TEAA)洗涤磁珠,再后用50mM柠檬酸铵洗涤。
引物序列:所有的引物都是用常规的磷酸酰胺化合物在PerseptiveBiosystems Expedite 8900 DNA合成仪上合成(Sinha等,1984年,《核酸研究》(Nucleic Acids Res)12卷4539页)。COSBE引物(两者在3′端前均含一个故意错配的碱基)是那些用于预先的ARMS研究(Ferrie等1992,《美国人类遗传杂志》(Am J Hum Genet)51卷251-262页)的引物,两者区别在于前者在正常的5′端被除去两个碱基:外显子10 PCR(正向):5′-BIO-GCA AGT GAA TCC TGA GCG TG-3′(SEQ.ID.No.1)外显子10 PCR(反向):5′-GTG TGA AGG GTT CAT ATG C-3′(SEQ.ID.No.2)COSBEΔF508-N 5′-ATC TAT ATT CAT CAT AGG AAA CAC CACA-3′(28聚体)(SEQ.ID.No.3)COSBEΔF508-M 5′-GTA TCT ATA TTC ATC ATA GGA AAC ACCATT-3′(30聚体)(SEQ.ID.No.4)
质谱:洗涤后,磁珠重悬浮于1μl 18Mohm/cm H2O,300nl每种基质(Wu等,1993)溶液(0.7M 3-羟基吡啶甲酸,溶于1∶1H2O∶CH3CN的0.7M 2碱式柠檬酸铵)并将悬浮的磁珠(Tang等,1995年,《质谱快讯》(Rapid Commun Mass Spectrom),8卷727-730页)在一样品靶上混合并晾干。加至20个样品点到探测靶盘上并引入一未修饰的Thermo Bioanalysis(以前的Finnigan)Virsions2000 MALDI-TOF的离子源区,在靶和转换倍增电极上相应用5和20kv以反射方式操作。根据原子组成可计算出理论平均分子量(Mr(calc))。卖方提供的软件利用外部校正确定峰的质量中心;从这些值中减去1.08Da,对携带电荷的质子质量加以校正,得到测量Mr(exp)值。
模式:与结合的模板退火后,N和M引物(分别为8508.6和9148.0Da)被引入了dGTP,只有在可变(V)位置有正确的Watson-Crick碱基配对的引物才能被聚合酶延伸。因此,当V与N的3′端碱基配对,N则延伸为8837.9 Da产物(N+1)。同样,当V正确地配对到M末端,M则延伸为9477.3 Da M+1产物。
结果:
图14-18显示了COSBE反应产物具代表性的质谱图。当PCR产物在生物素反义链结合前被纯化后,可得到更好的结果。实施例4 利用质谱检测人载脂蛋白E异构体
载脂蛋白E(ApoE),为脂蛋白的一蛋白组分,在脂代谢中起着重要作用。例如,它与胆固醇转运、脂蛋白颗粒代谢及许多脂水解酶的免疫调节和活化都有关系。
人ApoE(由E2、E3和E4等位基因编码)有三种常见的异构体。最常见的是E3等位基因。E2等位基因被证明能降低血浆中的胆固醇水平,因而对动脉粥样硬化的形成具有保护作用。最后,E4异构体与胆固醇水平的提高呈正相关,可能诱发动脉粥样硬化。因此,不同个体中apoE等位基因的鉴定是心血管疾病发展风险的一个重要的决定因素。
如图19所示,编码载脂蛋白E的DNA样品可从患者处获得,并扩增(如用PCR);而PCR产物可用一种合适的酶(如CfoI)消化。获得的限制性消化产物可用多种方法分析。如图20所示,载脂蛋白E的三种异构体(E2、E3和E4)具有不同的核酸序列,因而也具有不同的分子量值。
如图21A-C所示,不同的载脂蛋白E基因型在3.5%Metphor琼脂糖凝胶或12%聚丙烯酰胺凝胶中展现出不同的限制性方式。如图22和23所示,不同的载脂蛋白E基因型也可用质谱精确快速地确定。实施例5 血清样品中乙型肝炎病毒的检测材料与方法
样品制备
根据标准方法用酚/氯仿抽提并最后用乙醇沉淀DNA。
第一PCR
每个反应中用到5μl从血清中制备的DNA,并用到15pmol每种引物和2单位Taq DNA聚合酶(Perkin Elmer,Weiterstadt,Germany)。每种dNTP的终浓度为200μM,反应终体积为50μl。10×PCR缓冲液(Perkin Elmer,Weiterstadt Germany)包含100mM Tris-HCl,pH8.3,500mM KCl,15mM MgCl2,0.01%明胶(w/v)。引物序列:引物1:5′-GCTTTGGGGCATGGACATTGACCCGTATAA-3′(SEQ.ID.No.5)引物2:5′-CTGACTACTAATTCCCTGGATGCTGGGTCT-3′(SEQ.ID.No.6)嵌套PCR
每个反应用1μl第一反应液或用第一PCR的1∶10稀释液作为模板。在50μl终体系中含100pmod每种引物,2.5u Pfu(exo-)DNA聚合酶(Stratagene,Heidelberg,Germany),终浓度为200μM的每种dNTP和5μl 10×Pfu缓冲液(200mM Tris-HCl,pH8.75,100mMKCl,100mM(NH4)2SO4,20mM MgSO4,1% Triton X-100,1mg/ml BSA)(Stratagene,Heidelberg,Germany)。该反应在热循环仪(OmmiGene,MWG-Biotech,Ebersberg,Germany)中进行,用以下程序:92℃1分钟,60℃1分钟,72℃1分钟,共20循环。寡聚脱氧核苷酸序列为(购自MWG-Biotech,Ebersberg,Germany,HPLC-纯化):HBV 13:5′-TTGCCTGAGTGCAGTATGGT-3′(SEQ.ID.No.7)HBV 15bio:生物素-5′-AGCTCTATATCGGGAAGCCT-3′(SEQ.ID.No.8)PCR产物的纯化:
为了记录每个图谱,用到了一个PCR,50μl(如上述进行)。纯化按下面的程序进行:用厂商提供的方法,用8000rpm离心20分钟,在Ultrafree-MC滤器(Millipore,Eschborn,Germany)上进行超滤。25μl(10μg/ml)链霉抗生物素蛋白Dynabeads(Dynal,Hamburg,Germany)用厂商说明制备并重悬浮于25μl B/W缓冲液(10mMTris-HCl,pH7.5,1mM EDTA,2M NaCl)。这个悬浮液加入仍在滤器中的PCR样品中。混合物在环境温度下温和摇晃15分钟。将悬浮液转移至一1.5ml Eppendorf管中,利用一磁粒收集器MPC(Dynal,Hamburg,Germany)的帮助除去上清。磁珠用50μl 0.7M柠檬酸铵溶液,pH8.0洗涤2次(每次用MPC除去上清)。在90℃用甲酰胺可将DNA从磁珠上切下来。上清液在真空离心干燥器中干燥1小时并重悬于4μl超纯水(MilliQ UF加Millipore,Eschborn,Germany)中。这个制备物可用于MALDI-TOF MS分析。MALDI-TOF MS:
半微升样品加入样品池,迅速与0.5μl基质溶液混合(0.7M 3-羟基吡啶甲酸50%乙腈,70mM柠檬酸铵)。在环境温度下将混合物干燥并置入质谱仪中。所有图谱利用装备有一反射器(5keV离子源,20keV后段加速)和一337nm氮激光的Finnigan MAT Vision 2000(Finnigan,MAT,Bremen,Germany)以正离子方式记录下来。用一40聚体和100聚体的混合物进行校准。每个样品用不同的激光能量测量。在阴性样品中,既不用较低的也不用较高的能量即能检测PCR产物。对于阳性样品,PCR产物用不同的激光能量,在样品点的不同位置被检出。结果:
利用一与编码HBV核心抗原(HBVcAg)的HBV基因组的C区互补的寡核苷酸序列,一嵌套PCR系统被用于血液样品中HBV DNA的检测(引物1:起始于基因组图的1763位点,引物2起始于其互补链基因组图的2032位点)。按标准方法从患者血清中分离DNA。用第一套引物和来自这些制备物的DNA进行第一PCR。如果在样品中有HBVDNA存在,则会产生一269bp的DNA片段。
在第二个反应中,用到了与在第一PCR中产生的PCR片段中某一区域互补的引物。如果在第一PCR中产生了与HBV相关的PCR产物,那么在此嵌套PCR中将产生一67bp的DNA片段(见图25A)。用于检测的嵌套PCR系统的使用,提高了灵敏度,并可作为外部PCR的特异的对照(Rolfs.A.等,《PCR:临床诊断与研究》(PCR:ClinicalDiagnostics and Research),Springer,Heidelberg,1992)。另一个优点是,尽管纯化损失不可避免,但第二PCR产生的片段的量足以保证一没有问题的检测。
在固定到链霉抗生物素蛋白Dynabeads前,样品用超滤纯化以除去引物。由于空间原因,较短的引物将以更高的产量固定到磁珠上,所以必须进行纯化。固定化直接在超滤膜上进行,以避免由于对膜非特异的吸附而导致物质损失。固定化后,磁珠用柠檬酸铵洗涤以进行阳离子交换(Pieles,U.等,1993年,《核酸研究》(Nucleic Acids Res),21卷3191-3196页)。用25%的氨水将固定化的DNA从磁珠上切下来,氨水可用非常短的时间将DNA从磁珠上切下来,而又不引入钠阳离子。
在不知道血清学分析结果的情况下进行嵌套PCR和MALDI-TOF分析。由于不知道病毒的滴度,第一PCR中的每个样品分别用未稀释和1∶10的稀释液作为模板。
样品1来自慢性活动性HBV感染的患者,其用HBs-和HBe-抗原试验为阳性,但用点杂交分析为阴性。样品2是一来自活性HBV感染并伴有严重病毒血症的患者的血清,该患者用点杂交分析为HBV阳性。样品3是一灭活的血清,因而无法进行血清学分析,但检测到显示肝脏疾病的转氨酶水平升高。在放射自显影照片分析(图24)中,这个样品的第一PCR是阴性的,然而有许多HBV感染的证据。这个样品有意用MALDI-TOF分析,因为已证实纯化后非常低水平的PCR产物也能被检测。样品4来自HBV感染治愈后的患者。样品5和6选自慢性活动性HBV感染的患者。
图24显示了嵌套PCR反应PAGE分析的结果。在样品1,2,3,5和6中,一PCR产物明显地展现出来。在样品4中没有PCR产物产生,根据血清学分析,这为确实的HBV阴性。阴性和阳性对照分别用+和-表示。如果使用未稀释的模板,在泳道2,5,6和+中将可见扩增假象,如果用1∶10稀释液作模板,这些假象将不再产生。在样品3中,只有当模板未稀释时才可检测到PCR产物。除上面讨论的样品3外,PAGE分析的结果与用血清学分析所得的结果一致。
图25A显示了用上述方法产生和纯化的来自样品1的嵌套PCR产物的质谱图。20754 Da处的信号代表单链PCR产物(计算值:20735 Da,从磁珠上切下的PCR产物两链的平均值)。计算值和获得的质量差异为19 Da(0.09%)。如图25A所示,样品1产生了大量的PCR产物,形成明确的检测。
图25B显示了一从样品3获得的质谱图。如图24中所示,这个样品中产生的PCR产物的量明显低于样品1中的量。然而,该PCR产物用20751 Da(计算值20735)的质量明确显示出来。质量差异为16 Da(0.08%)。图25C所绘的质谱图从HBV阴性(也为图24所示)的样品4中获得。如所预期的没有检测到对应于PCR产物的信号。图25所示的所有样品均用MALDI-TOF MS分析,由此所有HBV阳性样品中检测到了PCR产物,而在HBV阴性样品中则未检测到。这个结果用几次不同的实验得到重复。实施例6 利用MALDI-TOF质谱的连接酶链反应产物的分析材料与方法
寡聚脱氧核苷酸
除生物素化的一个寡聚核苷酸外,其余所有的均用β-氰乙基磷酰胺法(Sinha,N.D.等,1984年《核酸研究》(Nucleic AcidsRes.)12卷4539-4577页)在一MilliGen 7500 DNA合成仪(Millipore,Bedford,MA,USA)上以0.2μmol规模合成。寡聚脱氧核苷酸用RP-HPLC纯化并用标准方法脱保护。生物素化的寡聚脱氧核苷酸从Biometra,Gottingen,Germany购买(HPLC纯)。所用寡聚核苷酸的序列及计算质量:寡聚脱氧核苷酸A:5′-p-TTGTGCCACGCGGTTGGGAATGTA(7521 Da)(SEQ.ID.No.9)寡聚脱氧核苷酸B:5′-p-AGCAACGACTGTTTGCCCGCCAGTTG(7948 Da)(SEQ.ID.No.10)寡聚脱氧核苷酸C:5′-bio-TACATTCCCAACCGCGTGGCACAAC(7960 Da)(SEQ.ID.No.11)寡聚脱氧核苷酸D:5′-p-AACTGGCGGGCAAACAGTCGTTGCT(7708 Da)(SEQ.ID.No.12)
寡聚核苷酸A和D的5′磷酸化
这可用多核苷酸激酶(Boehringer,Mannheim,German)依据公开的方法进行,未纯化的5′-磷酸化的寡聚核苷酸用于LCR。
连接酶链反应
LCR用Pfu DNA连接酶和含两个不同pBluescript KII噬菌粒的连接酶链反应试剂盒(Stratagene,Heidelberg,Germany)进行。其中一个噬菌粒携带大肠杆菌lacI基因的野生型,另一个含该基因的突变体,即lacI基因的191 bp处含一单点突变。
下面的LCR条件用于每个反应:100pg模板DNA(0.74fmol),以500pg超声处理的鲑精DNA作为载体,25ng(3.3pmol)每一5′-磷酸化的寡聚核苷酸,20ng(2.5pmol)每一未磷酸化的寡聚核苷酸,4U Pfu DNA连接酶,以Pfu DNA连接酶反应缓冲液作为缓冲液(Stratagene,Heidelberg,Germany),终体积20μl。在一对照实验中一化学合成的单链(SS)50体用作模板(1fmol),在这个实验中寡聚体C也被生物素化。所有反应在一热循环仪(OmniGene,MWG-Biotech,Ebersberg,Germany)上完成,反应程序:92℃4分钟,60℃2分钟和25循环的92℃20秒,60℃40秒。除HPLC分析外,用到了生物素化的连接体C。在一对照实验中,生物素化和非生物素化的寡聚核苷酸显示了同样的凝胶电泳结果。反应物在7.5%聚丙烯酰胺凝胶上分析。连接产物1(寡聚体A和B)的计算质量:15450 Da,连接产物2(寡聚体C和D)的计算质量:15387 Da。
SMART-HPLC
用一Pharmacia Mono Q,PC 1.65/5柱,在SMART-系统(Pharmacia,Freiburg,Germany)上进行离子交换HPLC(IEHPLC)。洗脱液为缓冲液A(25mM Tris-HCl,1mM EDTA和0.3M NaCl,pH8.0)和缓冲液B(同A,但用1M NaCl)。开始以50μl/min流速用100%A洗脱5分钟,在30分钟内使用0-70%的B液梯度洗脱,随后在2分钟内提高至100%B,并继续用100%B洗5分钟。以野生型或突变体模板进行的两个合并的LCR样品(40μl)被加样至色谱仪。
MALDI-TOF-MS样品制备
固定化DNA的制备:为了每个图谱的记录,两个LCR(如上述进行)合并并用2×B/W缓冲液(10mM Tris-HCl,pH 7.5,1mMEDTA,2M NaCl)1∶1稀释。往样品中加入5μl链霉抗生物素蛋白DynaBeads(Dynal,Hamburg,Germany),反应物于环境温度下温和摇晃15分钟使之结合。利用一磁粒收集器MPC,(Dynal,Hamburg,Germany)除去上清,并将磁珠用50μl 0.7M柠檬酸铵溶液(pH8.0)洗涤两次(每次用MPC除去上清)。将磁珠悬浮于1μl超纯水(MilliQ,Millipore,Bedford,MA,USA)中。该悬浮液直接用于下述的MALDI-TOF-MS分析。
超滤和链抗生物素蛋白DynaBeads的结合:为了记录图谱,两个LCR(如上述进行)合并并用2×B/W缓冲液1∶1稀释,并用5000 NMWLUltrafree-MC滤器(Millipore,Eschborn,Germany)根据厂商的说明浓缩。浓缩后样品用300μl 1×B/W缓冲液洗涤并加入链抗生物素蛋白DynaBeads。磁珠用上述方法用300μl 1×B/W缓冲液在Ultrafree-MC滤器上洗涤一次。磁珠用30至50μl 1×B/W缓冲液重悬浮并移至一1.5ml Eppendorf管中。除去上清并将磁珠用50μl 0.7M柠檬酸铵(pH8.0)洗涤二次。最后,磁珠用30μl丙酮洗涤一次并悬浮于1μl超纯水。固定到磁珠上后的连接混合物如下述用于MALDS-TOF-MS分析。
MALDI-TOF-MS
带有固定化DNA的链抗生物素蛋白包被的磁珠悬液注入样品池中,迅速与0.5μl基质溶液(溶于50%乙腈的0.7M 3-羟基吡啶甲酸,70mM柠檬酸铵)混合。混和液在环境温度下干燥并置入质谱仪中。利用装备有一反射器(5keV离子源,20keV后段加速)和一氮激光(337nm)的Finnigan MAT Vision 2000(Finnigan MAT,Bermen,Germany),所有谱均以正离子方式记录下来。对于Pfu DNA连接酶分析,0.5μl溶液与1μl基质溶液在样品池中混合并按上述方法制备。对于未纯化的LCR分析,1μl LCR与1ml基质溶液混合。结果与讨论
大肠杆菌lacI基因作为一简单的模型系统以调查MALDI-TOF-MS作为连接酶链反应产物的检测方法是否合适。这个模板系统中含有一克隆于pBluescript KII噬菌粒的大肠杆菌lacI野生型基因和一克隆于同种噬菌粒,于191 bp处带有一单点突变(C变为T)的大肠杆菌lacI基因。四种不同的寡聚核苷酸均被使用,而只有当大肠杆菌lacI野生型基因存在时,它们才能被连接(图26)。
利用Pfu DNA连接酶,LCR条件被优化,在每个阳性反应中至少可获得1pmol连接产物。连接反应用聚丙烯酰胺凝胶电泳(PAGE)和SMART系统上的HPLC分析(图27,28,29)。图27显示了一用野生型模板(泳道1)的阳性LCR,一用突变模板的阴性LCR(泳道1和2)和一含酶、寡聚核苷酸但不含模板的阴性对照的PAGE。凝胶电泳清楚地说明了只有在用野生型模板的反应中才有连接产物(50bp)产生,而无论是带有点突变的模板,还是用鲑精DNA的对照反应,都不能产生扩增产物。在图28中,HPLC用于分析在同样条件下进行的使用野生型模板的两合并的LCR。连接产物被清楚地显示。图29显示了一HPLC的结果,其中分析了使用突变模板的两合并的阴性LCR。这些色谱图证实了图27所示的结果,而且综合的结果清楚地证明,只有用到野生型模板时,该系统才能产生明显量的连接产物。
为了确定LCR实验中用到的不同化合物的保存时间,进行了适当的对照实验。这些化合物包括四种寡聚核苷酸(A、B、C和D),一合成的双链50聚体(与连接产物序列相同),野生型模板DNA,超声处理的鲑精DNA和连接缓冲液中的Pfu DNA连接酶。
为了检测在LCR反应用MALDI-TOF-MS分析前应使用哪种纯化方法,部分未纯化的LCR(图30A)和部分酶贮存液(图30B)用MALDI-TOF-MS分析,证明合适的样品制备物是绝对必需的,因为未纯化的LCR中所有的信号均与Pfu DNA连接酶的MALDI-TOF-MS分析中所得的信号一致。寡聚A和连接产物的计算质量值分别为7521Da和15450 Da,图30的数据表明酶溶液产生的质量信号干扰了所预期的连接体和产物的信号从而导致不可能得到明确的信号。而且,该图谱还显示了酶贮存缓冲液中去污剂Tween 20以不利的方式影响了被分析物/基质的结晶化行为的信号。
在一纯化方式中用到了链抗生物素蛋白包被的磁珠。如最近的一论文所述,通过Watson-Crick碱基配对方式固定到与磁珠共价结合的互补DNA片段上的DNA的直接解吸附作用是可能的,并且只是未生物素化的链将解吸附(Tang,K等,1995年,《核酸研究》(Nucleic AcidsRes.)23卷3126-3131页)。这个用到固定化双链DNA的方法可确使只有未生物化链才可解吸附。如果分析未固定的双链DNA,两条链都将解吸附(Tang K.等,1994年,《质谱快讯》(Rapid Comm.MassSpectrom)7卷183-186页),将依两链的质量差异导致信号变宽。因此,对LCR用这种方法纯化,当寡聚体C5′端被生物素化且固定到链抗生物素蛋白包被的磁珠上时,只有未连接的寡聚核苷酸A,计算质量为7521 Da,和寡聚体A和寡聚体B的连接产物(计算质量:15450 Da)将被解吸附。这可得到简单且明确的LCR连接体和产物的鉴定。
图31A显示了从用链抗生物素蛋白DynaBeads纯化且直接从磁珠上解吸附的两合并的LCR(按上述方法进行)中获得的MALDI-TOF质谱图,表明所用的纯化方法是有效的(与图30相比)。可检测到一代表未连接的寡聚体A的信号和一对应于连接产物的信号。计算所得和实验所得的质量值明显是一致的,这将获得一明确的峰定位和连接产物精确的检测。相反,从用突变模板的两合并LCR得到的图谱中,只有寡聚体A而没有连接产物可被检测到(图31B)。当在无特异模板时进行连接反应,进一步证明了LCR条件的特异性和选择性以及MALDI-TOF检测的灵敏性。图32显示了从一只有鲑精DNA作为阴性对照的两合并的LCR中获得质谱图,如预期的,只能检测到寡聚体A。
图31A所示的结果与图27凝胶中泳道1一致,图31B所示图谱等价于图27中泳道2,而图32所示图谱对应于图27的泳道3。该结果与图28和29中的HPLC分析一致。虽然凝胶电泳(图27)和HPLC(图28和29)都显示连接产物量多于或几乎等于连接体的量,但MALDI-TOF质谱分析所得连接产物的信号则较小(图31A)。
连接产物信号的较低强度可能归因于24和50聚体间不同的解吸附/离子化效率。由于50碱基对的双链比起24碱基对的双链其Tm值明显更高,因而更多的24聚体可解吸附。信号强度的降低也可能是较长寡聚核苷酸更大程度片段化的结果。
如果不用链抗生物素蛋白DynaBeads纯化,图32显示了Tween 20在2000 Da附近的记录。粘稠性物质可负面影响结晶化过程因而不利于质谱分析。Tween 20和甘油是酶贮存缓冲液中的物质,因而在质谱分析前必须完全除去。由于这个原因,研究了一改进的纯化方法,该方法在用DynaBeads处理前还包括一附加的超滤步骤。确实,这种样品纯化明显提高了MALDI-TOF质谱的效能。
图33显示了分别从两合并的阳性(33A)和阴性(33B)LCR中获得的图谱。阳性反应中用到了一化学合成的单链50聚体作为模板,其序列与寡聚体C和D的连接产物一致。寡聚体C被5′生物素化。因而不能检测到模板。如所预期的,只有寡聚体A和B连接产物(计算质量15450Da)才能从固定化并连接的寡聚体C和D上解吸附。这个新产生的DNA片段由图33A中15448 Da的质量信号代表。与图32A相比,这个图谱清楚地说明这种样品制备方法产生的信号提高了分辨率和强度。实施例7 引物固相寡聚碱基延伸和MALDI-TOF质谱分析检测突变
概述
固相寡聚碱基延伸法可检测扩增的DNA中的点突变和小的缺失及插入。该方法的基础是检测引物的延伸,该检测引物与一亲和捕获的扩增模板上可变核苷酸位置邻近退火,延伸反应中使用了DNA聚合酶,三种dNTP的混合物,和另一种双脱氧核苷酸。得到的产物用MALDI-TOF质谱评价和分析而不经标记过程。下面实验的目的即以快速而可靠的方式来检测突变和野生型的等位基因。
实验描述
本方法在一寡聚核苷酸延伸步骤前使用了一单检测引物,以得到对突变型或野生型等位基因特异的一些碱基所形成的长度不等的产物。它们很容易用MALDI-TOF质谱分析。本方法用CFTR基因外显子10的实例来描述。该基因的外显子10在许多人种中产生最常见的突变(ΔF508),其纯合状态即产生囊性纤维变的临床症状。材料与方法
基因组DNA
基因组DNA从健康个体、ΔF508突变纯合或杂合的个体和一1506S突变杂合的个体中获得。野生型和突变型等位基因用标准的Sanger测序法证实。
CFTR基因外显子10的PCR扩增
PCR扩增的引物为CFEx10-F(5-GCAAGTGAATCCTGAGCGTG-3′(SEQ.ID.No.13),位于内含子9并生物素化)和CFEx10-R(5′-GTGTGAAGGGCGTG-3′(SEQ.ID.No.14),位于内含子10)。所用引物浓度为8pmol。Taq聚合酶及10X缓冲液购自Boehringer-Mannheim,dNTPs从Pharmacia获得。总反应体积为50μl。PCR循环条件为开始95℃5分钟,随后94℃1分钟,53℃45秒,72℃30秒,40个循环。最后于72℃延伸5分钟。
PCR产物的纯化
扩增产物利用Qiagen′s PCR纯化试剂盒(No.28106)按厂商的说明进行纯化。纯化的产物用50μl TE缓冲液(10mM Tris-HCl,1mM EDTA,pH7.5)从柱中洗脱。
双链DNA的亲和捕获及变性
10μl纯化的PCR产物试样转移至一链抗生物素蛋白包被的微量板(No.1645684 Boehringer-Mannheim or No.95029262 Labsystems)的一孔中。随后加入10μl温育缓冲液(80mM磷酸钠,400mM NaCl,0.4%Tween 20,pH7.5)和30μl水。室温下温育1小时后,孔用洗涤缓冲液(40mM Tris,1mM EDTA,50mM NaCl,0.1%Tween20,pH8.5)200μl洗涤三次。为了变性双链DNA,孔用100μl 50mMNaOH溶液处理3分钟。最后,孔用200μl洗涤缓冲液洗涤三次。
寡聚碱基延伸反应
25pmol检测引物(CF508:5′CTATATTCATCATAGGAAACACCA-3′(SEQ.ID.No.15)在50μl退火缓冲液(20mM Tris,10mMKCl,10mM(NH4)2SO4,2mM MgSO4,1%Triton X-100,pH8.75)中于50℃退火10分钟。孔用200μl洗涤缓冲液洗涤三次,用200μlTE缓冲液洗涤一次。延伸反应使用了从USB获得的DNA测序试剂盒(No.70770)中的一些成份和从Pharmacia得到的dNTPs或ddNTPs。总反应体积为45μl,包括21μl水,6μl测序酶缓冲液,3μl 10mMDTT溶液,4.5μl 0.5mM三种dNTPs,4.5μl 2mM另一种ddNTP,5.5μl甘油酶稀释缓冲液,0.25μl测序酶2.0,和0.25μl焦磷酸酶。反应物在冰上加入随即于室温温育15分钟和于37℃15分钟。最后,孔用200μl洗涤缓冲液洗涤三次并用60μl70mM柠檬酸铵洗涤一次。
延伸引物的变性及沉淀
延伸的引物于80℃在50μl 10%DMSO(二甲基亚砜)水溶液中变性10分钟。为了沉淀,10μl乙酸铵(pH6.5),0.5μl糖原(10mg/ml水溶液,Sigma No.G1765),和100μl无水乙醇加入上清中并于室温放置1小时。13,000×g离心10分钟后,DNA沉淀用70%乙醇洗涤并重悬于1μl 18Mohm/cm水中。
样品制备和MALDI-TOF质谱分析
0.3μl每种基质溶液(0.7M 3-羟基吡啶甲酸,0.07M溶于1∶1H2O∶CH3CN的二碱式柠檬酸铵)和悬浮的DNA/糖原沉淀在一样品靶上混合并在空气中干燥以制备样品。在一探测靶盘上点至20个样品,将其置入一未修饰的热生物分析(以前为Finnigan)Visions 2000 MALDI-TOF的离子源区,该仪器在靶及转换倍增电极上分别以5和20kv反射模式操作。理论平均分子质量(Mr(计算值))通过原子组成计算而得。报道的实验Mr(Mr(实验))值为单质子化形式的分子量,用外标校正确定。结果
本实验的目的是建立一种对突变检测快速可靠的方法,该方法将能高质高效地对遗传病进行诊断且不需十分苛刻的严格性。为此,某种DNA测序(一突变检测引物的寡聚碱基延伸)与基质辅助激光解吸附离子化(MALDI)质谱(MS)所得小测序产物的评价相结合。飞行时间(TOF)反射方式被选为可能的质量测量系统。为验证这一假说,用CFTR基因的外显子10进行了检验,该序列的一些突变将产生高加索地区人最常见单基因遗传病囊性纤维变的临床症状。
图34列出的序列给出了带有CFTR基因外显子10的野生型和不同突变体的理论计算分子质量的预期的短测序产物。在短测序产物的制备中使用了ddTTP(图34A)或ddCTP(图34B),从而在新生链中引入一特定序列相关的终止信号。健康的、突变杂合的和突变纯合的个体的MALDI-TOF-MS谱在图34中给出。所有样品均用标准的Sanger测序法证实,与质谱分析相比,它们之间没有差别。不同分子质量的实验检测的精确度在预期范围减去21.8和加上87.1道尔顿(Da)的范围内。这是在每种情况下都允许的结果的确切分析。这种方法的另一个优点是对ΔI507突变的明确检测。在ddTTP反应中,将检测到野生型等位基因,而在ddCTP反应中,揭示了三个碱基对的缺失。
上述的方法非常适合DNA的单点突变或微小损伤的检测。突变检测引物的细心选择将打开多路检测的窗口,从而得到大量而高质的遗传诊断方法而不需要在相当的等位基因特异方法中必需的苛刻的严格性。由于遗传信息的唯一性,突变检测引物的寡聚碱基延伸可应用于每个疾病基因或基因组的多态性区,如串联重复的不同数目(VNTR)或其它的单核苷酸多态性(如载脂蛋白E基因)。实施例8 用基质辅助激光解吸附/离子化飞行时间(MALDI-TOF)
质谱检测含7-脱氮嘌呤基团的聚合酶链反应产物材料与方法
PCR扩增
下面的寡聚脱氧核苷酸引物或是用标准的磷酰胺化学法(Sinha,ND.等,1983年,《四面体通讯》(Tetrahedron lett)24卷5843-5846页;Sinha,N.D.等,1984年,《核酸研究》(Nucleic AcidsRes.)12卷4539-4557页)在一Milligen 7500 DNA合成仪(Millipore,Bedford,MA,USA)上以200nmol量合成或购自MWG-Biotech(Ebersberg,Germany,引物3)和Biometra(Goettingen,Germany,引物6-7)。引物1:5′-GTCACCCTCGACCTGCAG(SEQ.ID.No.16)引物2:5′-TTGTAAAACGACGGCCAGT(SEQ.ID.No.17)引物3:5′-CTTCCACCGCGATGTTGA(SEQ.ID.No.18)引物4:5′-CAGGAAACAGCTATGAC(SEQ.ID.No.19)引物5:5′-GTAAAACGACGGCCAGT(SEQ.ID.No.20)引物6:5′-GTCACCCTCGACCTGCAgC(g:RiboG)(SEQ.ID.No.21)引物7:5′-GTTGTAAAACGAGGGCCAgT(g:RiboG)(SEQ.ID.No.22)
99聚体和200聚体DNA链(修饰的和未修饰的)以及核糖-和7-脱氮-修饰的100聚体在100μl体积反应液中从pRFc1 DNA(10ng,由汉堡大学S.Feyerabend惠赠)中扩增,反应液包含10mmol/L KCl,10mmol/L(NH4)2SO4,20mmol/L Tris HCl(pH=8.8),2mmol/L MgSO4,(exo(-)Pseduococcus furiosus(Pfu)-缓冲液,Pharmacia,Freiburg,Germany),0.2mmol/L每种dNTP(Pharmacia,Freiburg,Germany),1μmol/L每种引物和1单位exo(-)Pfu DNA聚合酶(Stratagene,Heidelberg,Germany)。
对99聚体用到了引物1和2,对200聚体用到了引物1和3,对100聚体用到了引物6和7。为了获得7-脱氮嘌呤修饰的核酸,在PCR扩增中,dATP和dGTP被7-脱氮-dATP和7-脱氮-dGTP替换。反应在热循环仪(OmniGene,MWG-Biotech,Ebersberg,Germany)上进行,用以下循环:95℃变性1分钟,51℃退火1分钟,72℃延伸1分钟,对所有的PCR反应循环均为30。最后一循环完成后,反应液在72℃继续延伸10分钟。
103聚体DNA链(修饰的和未修饰的)在100μl反应液中从M13mp18RFI DNA(100ng,Pharmacia,Freiburg,Germany)中进行扩增,使用引物4和5,其它浓度不变。反应按下述循环进行:95℃变性1分钟,40℃退火1分钟,72℃延伸1分钟。未修饰的103聚体经30循环而修饰的103聚体经40循环后,样品继续于72℃保温10分钟。
5′-[32P]-标记的PCR引物的合成
引物1和4用T4多核苷酸激酶(Epicentre Technologies)和(γ-32P)-ATP、(BLU/NGG/502A,Dupont,Germany)按厂商提供的方法进行5′[32P]标记。反应中引物1和4的10%被标记的引物替换,而其它反应条件不变。扩增的DNA用10%聚丙烯酰胺凝胶电泳分离。切下合适的条带并于Packard TRICARB 460C液闪仪(Packard,CT,USA)上计数。
从核糖修饰的PCR产物上分离引物
用Ultrafree-MC过滤装置(30,000 NMWL)纯化扩增的DNA,然后重溶于100μl 0.2mol/L NaOH并于95℃加热25分钟。随后溶液用HCl(1mol/L)酸化并用Ultrafree-MC过滤装置(10,000 NMWL)用下述方法进一步纯化以便进行MALDI-TOF分析。
PCR产物的纯化
所有的样品均用Ultrafree-MC 30000 NMWL装置(Millipore,Eschborn,Germany)按厂商的说明进行纯化和浓缩。冷冻干燥后,PCR产物重溶于5μl(对200聚体为3μl)超纯水。这个待分析溶液直接用于MALDO-TOF测量。
MALDI-TOF MS
等分的0.5μl待分析溶液与0.5μl基质溶液(0.7mol/L 3-HPA和0.07mol/L溶于乙腈/水(1∶1 v/v)的柠檬酸铵)在一平台状金属样品支持物上混合。在环境温度下干燥后样品放入质谱仪中进行分析。所用的MALDI-TOF质谱仪为Finnigan MAT Vision 2000(Finnigan MAT,Bremen,Germany)。用5keV离子源和20keV后段加速,图谱以正离子反射方式记录下来。该设备装备有一氮激光(337nm波长)。系统的真空度在分析器区为3-4×10-8hPa,在离子源区为1-4×10-7hPa。修饰和未修饰的DNA样品的质谱图用同样的相对激光能量获得,用合成的寡聚脱氧核苷酸(7-至50-聚体)混合液进行外部样正。结果与讨论
含7-脱氮嘌呤核苷酸的核酸的PCR酶促合成
为了证实短PCR产物快速且不用凝胶的MALDI-TOF分析的可行性和调查在MALDI-TOF条件下核酸中7-脱氮嘌呤修饰的效果,两种不同的引物-模板系统用于合成DNA片段。图36和37展示了其序列。103聚体PCR产物的两条单链具有几乎相等的质量(Δm=8u)而99聚体两条单链的质量差异为526u。
考虑到用于DNA化学合成的7-脱氮嘌呤核苷酸结构单元比常规核苷酸几乎贵160倍(产品信息,Glen Research Corporation,Sterling,VA),且它们在标准的β-氰磷酰胺化学法中的应用很重要(产品信息,Glen Research Corporation,Sterling,VA;Schneider,K和B.T.Chait,1995年,《核酸研究》(Nucleic Acids Res.)23卷1570页),7-脱氮嘌呤修饰的引物的价格将很高。因此,为了提高本方法的应用范围,所有的PCR均用通常能购得的未修饰的寡聚核苷酸引物,在聚合酶链反应中用c7-dATP和c7-dGTP代替dATP和dGTP将使99聚体和103聚体产物含约80%的7-脱氮嘌呤修饰的核苷酸,而200聚体产物则含约90%。表I列出了所有PCR产物的碱基组成。
表I:
99聚体、103聚体和200聚体PCR扩增产物的碱基组成
(未修饰和7-脱氮嘌呤修饰)
1“s”和“a”分别表示双链PCR产物的有义和反义链。2指以7 脱氮嘌呤修饰的核苷酸占总嘌呤核苷酸的百分比表示的相对修饰。
DNA片段1 | C T A G c7-脱氮A c7-脱氮 G相对修饰2 |
200聚体s修饰的200聚体s200聚体a修饰的200聚体a103聚体s修饰的103聚体s103聚体a修饰的103聚体a99聚体s修饰的99聚体s99聚体a修饰的99聚体a | 54 34 56 56 - - -54 34 6 5 50 51 90%56 56 34 54 - - -56 56 3 4 31 50 92%28 23 24 28 - - -28 23 6 5 18 23 79%28 24 23 28 - - -28 24 7 4 16 24 78%34 21 24 20 - - -34 21 6 5 18 15 75%20 24 21 34 - - -20 24 3 4 18 30 87% |
然而,仍需确定80-90%的7-脱氮嘌呤修饰是否足以用精确的质谱检测。因此确定在酶促扩增步骤中是否所有的嘌呤核苷酸均可被取代是非常重要的。因为有报道如果使用Taq DNA聚合酶,在PCR中c7-dATP不能完全取代dATP(Seela,F.和A.Roelling,1992年,《核酸研究》(Nucleic Acids Res.)20卷55-61页),所以这不是一个小问题。庆幸的是我们发现exo(-)Pfu DNA聚合酶在未修饰的嘌呤三磷酸不存在时确实能接受c7-dATP和c7-dGTP。但是,掺入效率较低导致低产量的PCR产物。溴化乙锭通过与双链DNA堆积的碱基相互作用而对其染色。因此溴化乙锭染色的凝胶中较低强度的带可能是假象,因为修饰的DNA链不一定需要给出与未修饰的DNA链相同的带强度。
为了证实这些结果,用[32P]标记的引物重复了PCR。放射自显影照片(图39)清楚地显示了修饰的PCR产物较低的产量。将带从胶上切下并计数。对所有的PCR产物,修饰的核酸的产量与相应的未修饰的扩增产物相比,只有约50%。进一步的实验表明,exo(-)DeepVent和Vent DNA聚合酶也能在PCR中掺入c7-dATP和c7-dGTP。综观所有的实验,证明exo(-)Pfu DNA聚合酶最佳,它在扩增中给出的副产物最少。使用所有三种聚合酶,发现那些用c7-dATP和c7-dGTP代替了对等物的PCR更少有副反应,得到的更纯的PCR产物。扩增中副产物的减少可解释为,由于引物与PCR扩增合成的含7-脱氮嘌呤的模板形成的复合物稳定性降低,而减少了引物的错配。含7-脱氮嘌呤的DNA双螺旋的熔点降低已有报道(Mizusawa,S.等,1986年,《核酸研究》(Nucleic Acids Res.)14卷1319-1324页)。除上面提到的三种聚合酶(exo(-)Deep Vent DNA聚合酶。VentDNA聚合酶和exo(-)(Pfu)DNA聚合酶)外,估计其它的聚合酶,如大肠杆菌DNA聚合酶Klenow大片段、测序酶、Taq DNA聚合酶和U Ampli Taq DNA聚合酶也能使用。另外,当RNA作为模板时,必须使用RNA聚合酶,如SP6或T7 RNA聚合酶。
修饰和未修饰的PCR产物的MALDI-TOF质谱
用MALDI-TOF MS分析了99聚体、103聚体和200聚体PCR产物。根据过去的经验,已知脱嘌呤化程度依赖于分析中解吸附和离子化所用激光的能量。既然要研究脱嘌呤造成的片段化中7-脱氮嘌呤修饰的影响,所有的谱均在同一相对激光能量下测量。
图40a和40b给出了修饰和未修饰的103聚体核酸的质谱图。在修饰的103聚体例子中,片段化导致了加宽的(M+H)+信号。峰的最大值移向较低的质量,以致于所给出的质量代表(M+H)+信号与片段化离子信号的平均值,而不是(M+H)+信号本身。虽然修饰的103聚体中仍含有约20%的来自寡聚核苷酸引物的A和G,但显示出较少的片段化作用,其特征在于更多窄而对称的信号。尤其是脱嘌呤造成的峰向较低质量拖尾的现象明显减少。因此,测量质量和计算质量间的差异显著减少,虽然仍低于预期的质量。对于未修饰的样品,观察到一31670的(M+H)+信号,其与计算质量的差异为97u或0.3%。而对于修饰的样品,这个质量差异减小到了10u或0.03%(检测值31713u,计算值31723u)。这个观察结果也被两条信号链的(M+H)+信号质量分辨率的明显提高所证实(m/Δ=67,于此相对照未修饰的样品为18,Δm=半峰宽,fwhm)。由于两条单链的质量差异较小(8u),它们各自的信号不能分辨。
在99碱基对DNA片段的结果中,对含7-脱氮嘌呤DNA而言,质量分辨率提高的影响更加明显。未修饰的样品中的两条单链,甚至在由于嘌呤和嘧啶的不同分布而致的PCR产物两条链间质量差异很大(526u)时,也不能分辨(图41a)。与此相反,修饰的DNA显示出对应于两条单链的不同的两个峰(图41b),这使得对于检测分子量,本发明方法与凝胶电泳相比具有优越性,甚至更为奥妙。虽然不能获得基线分辨率,但每个质量可以0.1%的精度给出,对轻链(计算质量30224u)Δm=27u而对重链(计算质量30750u)Δm=14u。这再次证明了含7-脱氮嘌呤的样品半峰宽显著降低。
在99聚体和103聚体的例子中,含7-脱氮嘌呤的核酸虽然仍含约20%未修饰的嘌呤核苷酸,但似乎给出较高的灵敏度。为了得到(M+H)+信号在相似强度下可比较的信噪比,未修饰的99聚体需要20次激光轰击,与此对照修饰的需12次,103聚体未修饰的样品需12次轰击而含7-脱氮嘌呤核苷酸的PCR产物只需3次。
比较未修饰和修饰的200体扩增物的质谱,再次发现含7-脱氮嘌呤的样品提高了质量分辨率以及提高了信号强度(图42a和42b)。虽然在修饰的样品的图谱中单链的信号占主要地位,但对于未修饰的样品,DNA双螺旋和单链二聚体却给出最强的信号。
完全7-脱氮嘌呤修饰的核酸可通过在PCR中使用修饰的引物或将未修饰的引物从部分修饰的PCR产物上切下来的方法获得。由于如上述的修饰的引物往往带来不利,因而用一核糖修饰的引物合成了一100聚体。用本实验室早期建立的方法(Koester,H.等,《动物生理化学》(Z.Physiol.Chem.),359卷1570-1589页),用NaOH水解切除引物。图10a和10b显示了PCR产物在引物切除前后的质谱图。图10b表明水解是成功的,水解的PCR产物以及两个释放出来的引物都能检测出来,同时也测出了残留的未切开的100聚体的微小信号。这个方法尤其适用于非常短的PCR产物的MALDI-TOF分析,因为随扩增序列长度的降低,来自引物的未修饰的嘌呤所占比例也随之增高。
7-脱氮嘌呤修饰的核酸的这些显著的优点可解释为,其具有更有效的解吸附和/或离子化作用,提高了离子的稳定性和/或降低了双链嘌呤修饰的核酸的变性能量。N-7被甲基替换后导致失去一个氢键的受体,由于非Watson-Crick碱基配对,这将影响核酸形成二级结构的能力(Seela,F.和A.Kehne,1987年,《生物化学》(Biochemistry),26卷2232-2238页),这可能是在MALDI过程中更好解吸附的原因。除此之外,7-脱氮嘌呤的芳香基团具有较低的电子密度,从而削弱了Watson-Crick碱基配对,导致双链的熔点降低(Mizusawa,S.等,1986年,《核酸研究》(Nucleic Acids Res.)14卷1319-1324页)。这个结果可以降低MALDI过程中双螺旋变性所需的能量。这些方面以及可能携带一正电荷的N-7氮原子位点的失去,使得7-脱氮嘌呤修饰的核酸极性降低并可能提高解吸附的效率。
由于作为质子受体的N-7的缺乏以及7-脱氮嘌呤核苷中C-N键极化作用的降低,溶液中水解过程后的脱嘌呤作用可以避免。虽然反应在溶液和在气相条件下的直接相关性是未知的,但修饰的核酸的脱嘌呤作用造成的片断化作用降低在MALDI过程中是能预知的。脱嘌呤化作用可能伴随着电荷的丢失,这将降低带电物质的总产量,或者可能产生能降低未片段化分子离子信号强度的带电荷的片段化产物。
由于含7-脱氮嘌呤样品降低了片段化作用而导致的灵敏度提高及(M+H)+信号在较低质量端谱峰拖尾现象的减弱表明N-7原子实际上在MALDI-TOF过程的脱嘌呤机制中是非常重要的。总之,含7-脱氮嘌呤的核酸显示出MALDI-TOF条件下显著提高的离子稳定性和灵敏度,从而提供了更高的质量精度和质量分辨率。实施例9:固相测序和质谱检测材料和方法
寡聚核苷酸以未纯化的形式购自Operon Technologios(Alameda.CA)。测序反应用测序酶2.0版(Amersham,Arlington Heights,Illinois)测序试剂盒中的试剂在一固相表面上进行。
39聚体靶序列测序
测序复合物5′-TCTGGCCTGGTGCAGGGCCTATTGTAGTTGTGACGTACA-(Ab)a-3′(DNA11683)(SEQ.ID.No.23)
3′TCAACACTGCATGT-5′(PNA16/DNA)
(SEQ.ID.No.24)
为了进行固相DNA测序,对模板链DNA 11683采用末端脱氧核苷酸转移酶进行3′生物素化。含60pmol DNA11683,1.3nmol生物素14-dATP(GIBCO BRL,Grand Island,NY),30单位末端转移酶(Amersham,Arlington Heights,Illinois)和1×反应缓冲液(与酶一起提供)的反应液于37℃温育1小时,然后于70℃10分钟加热灭活末端转移酶而终止反应。所得产物经一TE-10自旋柱(Clonetech)脱盐。不止一分子的生物素-14-dATP能加到DNA 11683的3′端上。生物素化的DNA 11683与0.3mg Dynal链抗生物素蛋白磁珠在30μl1×结合与洗涤缓冲液中于环境温度下温育30分钟。然后磁珠用TE洗涤两次并重溶于30μl TE,其中10μl(含0.1mg磁珠)用于测序反应。
将从上面步骤中得到的0.1mg磁珠重悬浮于含2μl得自测序酶试剂盒的5×测序酶缓冲液(200mM Tris-HCl,pH7.5,100mMMgCl2和250mM NaCl)和5pmol相应的引物PNA16/DNA的10μl体积中。退火混合物加热到70℃并在20-30分钟时间里缓慢冷却至室温。随后加入1μl 0.1M二巯苏糖醇溶液,1μl锰缓冲液(0.15M异柠檬酸钠和0.1M MnCl2)和2μl稀释的测序酶(3.25单位)。将反应混合物分为3μl的四等份,与终止混合物(每种含3μl适当的终止混合物:溶于50mM NaCl的32μM c7dATP,32μM dCTP,32μM c7dGTP,32μM dTTP和3.2μM四种ddNTP中的一种)混合。反应混合物于37℃温育2分钟。延伸完成后,沉淀磁珠并弃去上清。得到的磁珠洗涤两次并重悬于TE,4℃保存。
78聚体靶序列测序
测序复合物:5′-AAGATCTGACCAGGGATTCGGTTAGCGTGACTGCTGCTGCTGCTGCTGCTGC
TGGATGATCCGACGCATCAGATCTGG-(Ab)n-3(SEQ.ID.NO.25)(TNR.PLASM2)
3′-CTACTAGGCTGCGTAGTC-5′ (CM1)(SEQ.ID.NO.26)
靶序列TNR.PLASM2生物素化和测序均按与前面所述(39聚体靶序列测序)相似的方法进行。
用部分双螺旋探针对一15聚体靶序列测序
测序复合物5′-F-GATGATCCGACGCATCACAGCTC3′(SEQ.ID.No.27)3′-b-CTACTAGGCTGCGTAGTGTCGAGAACCTTGGCT3′(SEQ.ID.No.28)
60pmol CM1B3B与0.3溶于30μl 1M NaCl和TE(1×结合与洗涤缓冲液)的磁珠于室温温育30分钟,将CM1B3B固定到带有链抗生物素蛋白的Dynabeads M280(Dynal,Norway)上。磁珠洗涤两次并重溶于30μl TE,其中10或20μl(含0.1或0.2mg磁珠)用于测序反应。
在一9μl含2μl来自测序酶试剂盒的5×测序酶缓冲液(200mMTris-HCl,pH7.5,100mM MgCl2和250mM NaCl)的体系中,前步所得的磁珠的相应量与10pmol DF11a5F(或者,0.2mg磁珠与20pmol DF11a5F)退火形成双螺旋。退火混合物加热至65℃后在20-30分钟内缓慢冷却至37℃。随之将双螺旋引物与1μl体积的10pmolTS10(0.2mg磁珠用20pmol TS10)混合,得到的混合物进一步于37℃加热5分钟,室温放置5-10分钟。然后加入1μl 0.1M二硫苏糖醇溶液,1μl锰缓冲液(0.15M异柠檬酸钠和0.1M MnCl2),和2μl稀释的测序酶(3.25单位)。反应混合物分成各3μl的四等份,与终止混合物(每种含4μl合适的终止混合物:溶于50mM NaCl的16μM dATP,16μM dCTP,16μM dGTP,16μM dTTP和1.6μM四种ddNTP中的一种)混合。反应混合物于室温放置5分钟后,37℃加热5分钟。延伸完毕后,沉淀磁珠并弃去上清。将磁珠重悬于20μl TE并于4℃保存。从每管中取出2μl(从20μl中取出)与8μl甲酰胺混合,得到的样品于90-95℃变性5分钟,取2μl(从总共10μl中取出)用于ALF DNA测序仪(Pharmacia,Piscataway,NJ),使用含7M脲和0.6×TBE的10%聚丙烯酰胺凝胶。剩余的样品用于MALDI-TOF MS分析。
MALDI样品制备与装置
在MALDI分析前,载有测序梯度的磁珠用50mM柠檬酸铵洗涤两次并重悬于0.5μl纯水。将悬液加到质谱仪的样品靶上,并加入0.5μl饱和基质溶液(50%乙腈中3-羟吡啶酸(HPA)∶柠檬酸铵=10∶1摩尔比)。混合液在质谱分析前进行干燥。
反射TOFMS质谱仪(Vision 2000,Finnigan MAT,Bremen,Germany)用于分析。5kV用于离子源而20kV用于后段加速。所有的图谱均采用正离子方式,并使用氮激光。通常,每个图谱均取100次以上轰击的平均值并使用标准25点平滑。结果与讨论
传统的固相测序
在传统的测序方法中,一条引物直接与模板退火并随后按Sanger双脱氧测序法延伸并终止。正常情况下,用到生物素化的引物,而测序梯度被包被有链抗生物素蛋白的磁珠捕获。洗涤后,产物用EDTA和甲酰胺从磁珠上洗脱下来。然而,我们以前的发现表明,双螺旋上只有退火链解吸附而固定化链仍保留在磁珠上。因此,固定模板并退火引物是有利的。测序反应完成并洗涤后,带有固定化模板和退火的测序梯度的磁珠可直接放在质谱仪的靶上与基质混合。在MALDI中,只有退火的测序梯度将被解吸附和离子化,而固定化的模板仍保留在靶上。
一39聚体模板(SEQ.ID.No.23)首先通过加入生物素-14dTAP和末端转移酶而在3′端生物素化,酶可将不止一个生物素-14-dATP分子加到模板上,但由于模板被固定化且在MALDI过程中仍保留在磁珠上,因此生物素-14-dATP的数量并不影响质谱图。一14聚体引物(SEQ.ID.No.29)被用于固相测序。四个测序梯度的MALDI-TOF质谱图如图34所示,预期的理论值在表II中列出。
表II1. 5′-TCTGGCCTGGTGCAGGGCCTATTGTAGTTGTGACGTACA-(Ab)n-3′2. 3′-TCAACACTGCATGT-5′3. 3′-ATCAACACTGCATGT-5′4. 3′-CATCAACACTGCATGT-5′5. 3′-ACATCAACACTGCATGT-5′6. 3′-AACATCAACACTGCATGT-5′7. 3′-TAACATCAACACTGCATGT-5′8. 3′-ATAACATCAACACTGCATGT-5′9. 3′-GATAACATCAACACTGCATGT-5′10. 3′-GGATAACATCAACACTGCATGT-5′11. 3′-CGGATAACATCAACACTGCATGT-5′12. 3′-CCGGATAACATCAACACTGCATGT-5′13. 3′-CCCGGATAACATCAACACTGCATGT-5′14. 3′-TCCCGGATAACATCAACACTGCATGT-5′15. 3′-GTCCCGGATAACATCAACACTGCATGT-5′16. 3′-CGTCCCGGATAACATCAACACTGCATGT-5′17. 3′-ACGTCCCGGATAACATCAACACTGCATGT-5′18. 3′-CACGTCCCGGATAACATCAACACTGCATGT-5′19. 3′-CCACGTCCCGGATAACATCAACACTGCATGT-5′20. 3′-ACCACGTCCCGGATAACATCAACACTGCATGT-5′21. 3′-GACCACGTCCCGGATAACATCAACACTGCATGT-5′22. 3′-GGACCACGTCCCGGATAACATCAACACTGCATGT-5′23. 3′-CGGACCACGTCCCGGATAACATCAACACTGCATGT-5′24. 3′-CCGGACCACGTCCCGGATAACATCAACACTGCATGT-5′25. 3′-ACCGGACCACGTCCCGGATAACATCAACACTGCATGT-5′26. 3′-GACCGGACCACGTCCCGGATAACATCAACACTGCATGT-5′27. 3′-AGACCGGACCACGTCCCGGATAACATCAACACTGCATGT-5′
表II(续)
A-反应 C-反应 G-反应 T-反应1.2. 4223.8 4223.8 4223.8 4223.83. 4521.14. 4809.25. 5122.46. 5434.67. 5737.88. 6051.19. 6379.210. 6704.411. 6995.612. 7284.813. 7574.014. 7878.215. 8207.416. 8495.617. 8808.818. 9097.019. 9386.220. 9699.421. 10027.622. 10355.823. 10644.024. 10933.225. 11246.426. 11574.627. 11886.8
测序反应产生了一相对均一的梯度,而且全长序列很容易确定。在所有反应中均出现的5150附近的一个峰未被鉴定。一种可能的解释是,一小部分模板形成了某种二级结构,如袢环,这阻碍了测序酶的延伸反应。错误的掺入是不重要的,因为这些峰的强度较测序梯度的峰要低得多。虽然7-脱氮嘌呤被用于测序反应,这将稳定N-糖苷键以防止脱嘌呤,但由于引物不被7-脱氮嘌呤取代,故仍可观察到少量碱基损失。全长的梯度,3′端为ddA,在A反应中表现出的表观质量为11899.8,但是,强度更大的122峰出现在所有四个反应,可能是测序酶加入了一个额外的核苷酸的结果。
同样的技术可用于更长DNA片段的测序。一含CTG重复序列的78聚体模板(SEQ.ID.No.25)通过利用末端转移酶加入生物素-14-dATP而3′生物素化。一18聚体引物(SEQ.ID.No.26)在紧靠CTG重复序列的外侧退火,从而在引物延伸后该重复序列可立即被测序。四种反应液洗涤后用MALDI-TOF MS分析。图35显示了G-反应的一个例子,预期的测序梯度如表III所示,并给出了每一梯度成份的理论质量值。除了最后一个成份(理论值20577.4)不能从背景中区别出来外,所有的测序峰均能很好地分辨。两个相邻的测序峰(62聚体和63聚体)也能分开表明这个测序分析能用于更长的模板。同样,在这个谱中可看到测序酶加入了额外的核苷酸。这种加入是非模板特异的且在所有4个反应中出现,这使其很容易辨别。与引物峰相比,在长模板的情况下,测序峰的强度要低得多。测序反应也许需要进一步优化。
表III
5′-AAGATCTGACCAGGGATTCGGTTAGCGTGACTGCTGCTGCTGCTGCTGGATGATCCGACGCATCAGATCTGG-(Ab)n-3′1. 3′-CTACTAGGCTGCGTAGTC-5′2. 3′-CCTACTAGGCTGCGTAGTC-5′3. 3′-ACCTACTAGGCTGCGTAGTC-5′4. 3′-GACCTACTAGGCTGCGTAGTC-5′5. 3′-CGACCTACTAGGCTGCGTAGTC-5′6. 3′-ACGACCTACTAGGCTGCGTAGTC-5′7. 3′-GACGACCTACTAGGCTGCGTAGTC-5′8. 3′-CGACGACCTACTAGGCTGCGTAGTC-5′9. 3′-ACGACGACCTACTAGGCTGCGTAGTC-5′10. 3′-GACGACGACCTACTAGGCTGCGTAGTC-5′11. 3′-CGACGACGACCTACTAGGCTGCGTAGTC-5′12. 3′-ACGACGACGACCTACTAGGCTGCGTAGTC-5′13. 3′-GACGACGACGACCTACTAGGCTGCGTAGTC-5′14. 3′-CGACGACGACGACCTACTAGGCTGCGTAGTC-5′15. 3′-ACGACGACGACGACCTACTAGGCTGCGTAGTC-5′16. 3′-GACGACGACGACGACCTACTAGGCTGCGTAGTC-5′17. 3′-CGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′18. 3′-ACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′19. 3′-GACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′20. 3′-CGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′21. 3′-ACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′22. 3′-GACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′23. 3′-CGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′24. 3′-ACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′25. 3′-GACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′26. 3′-TGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′27. 3′-CTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′28. 3′-ACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′29. 3′-CACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′30. 3′-GCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′31. 3′-CGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′32. 3′-TCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′
表III(续)33. 3′-ATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′34. 3′-AATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′35. 3′-CAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′36. 3′-CCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′37. 3′-GCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′38. 3′-AGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′39. 3′-AAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′40. 3′-TAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′41. 3′-CTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′42. 3′-CCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′43. 3′-CCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′44. 3′-TCCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′45. 3′-GTCCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′46. 3′-GGTCCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′47. 3′-TGGTCCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′48. 3′-CTGGTCCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′49. 3′-ACTGGTCCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′50. 3′-GACTGGTCCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′51. 3′-AGACTGGTCCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′52. 3′-TAGACTGGTCCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′53. 3′-CTAGACTGGTCCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′54. 3′-TCTAGACTGGTCCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′55. 3′-TTCTAGACTGGTCCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′
表III(续)
ddATP ddCTP ddGTP ddTTP1. 5491.6 5491.6 5491.6 5491.62. 5764.83. 6078.04. 6407.25. 6696.46. 7009.67. 7338.88. 7628.09. 7941.210. 8270.411. 8559.612. 8872.813. 9202.014. 9491.215. 9804.416. 10133.617. 10422.8818. 10736.019. 11065.220. 11354.421. 11667.622. 11996.823. 12286.024. 12599.225. 12928.426. 13232.627. 13521.828. 13835.029. 14124.230. 14453.431. 14742.632. 15046.833. 15360.034. 15673.235. 15962.436. 16251.637. 16580.838. 16894.039. 17207.240. 17511.441. 17800.642. 18089.843. 18379.044. 18683.245. 19012.446. 19341.6
表III(续)
47. 19645.8
48. 19935.0
49. 20248.2
50. 20577.4
51. 20890.6
52. 21194.4
53. 21484.0
54. 21788.2
55. 22092.4
双螺旋DNA探针用于捕获和作为引物的测序
具有单链突出的双螺旋DNA探针也被证明可捕获特异的DNA模板并作为固相测序的引物。该流程如图46所示。双螺旋探针和单链模板间堆积的相互作用使得只有5个碱基突出就足以进行捕获。根据这个原理,一5′荧光标记的23聚体(5′-GAT GAT CCG ACG CAT CAC AGCTC)(SEQ.ID.No.29)与一3′生物素化的18聚体(5′-GTG ATG CGTCGG ATC ATC)(SEQ.ID.No.30)退火,留下一5碱基突出。一15聚体模板(5′-TCG GTT CCA AGA GCT)(SEQ.ID.No.31)被双螺旋捕获且利用5碱基突出的延伸进行测序反应。反应物的MALDI-TOF质谱图如图47A-D所示。虽然相对强度低,但所有测序峰均能分辨。每个反应中最后一个峰都是由于测序酶在全长的延伸产物上非特异地加上了一个核苷酸的结果。作为比较,相同的产物在一传统的DNA测序仪上进行测序,结果的堆积荧光图如图48所示。从图中可看到,质谱图与荧光图具有相同的特征,与23聚体引物相比,测序峰的强度要低得多。
MALDI-TOF质谱作为一项检测手段的改良
通过使用皮升瓶技术(picoliter vial technique)可使样品分布更加均匀并可能提高信号强度。实际中,样品可放在正方形开口为100μm的小凹瓶(pit)中。用于固相测序的磁珠其直径小于10μm,因而它们能很好地填入微升瓶(microliter vial)中。基质和含“甜斑”(“SweetSpot”)的DNA的微晶将被限制在瓶中。由于激光斑点的直径约100μm,因而它将完全覆盖瓶口。因此,寻找甜斑(Sweet Spot)是不需要的而且高重复频率的激光(如>10Hz)可用于获取图谱。一篇较早的报道指出这个方法可将肽和蛋白的检测灵敏度在传统的MALDI样品制备技术的基础上提高几个数量级。
为了使测序范围超过100碱基,MALDI对DNA的分辨率需进一步提高。目前,用3-HPA/柠檬酸铵作为基质,用5kV离子源和20kV后段加速的反射TOF质谱仪,图33(73聚体)穿透峰的分辨率已超过了200(FWHM),对此例而言这足以进行序列测定。这个分辨率也是超过70聚体范围MALDI解吸附DNA离子的最高报道。延迟提取技术(delayed extraction technigue)的使用可进一步提高分辨率。
所有上面引用的参考文献和著作在此引入作为参考。等效方法
本领域的技术人员,用常规的实验方法,就能知道或弄清与这里描述的特定方法等效的许多方法。这些等效的方法被认为在本发明的范围之内并为下面的权利要求所包括。
Claims (49)
1.一种检测生物样品中靶核酸序列的方法,包括以下步骤:
a)从生物样品中获得核酸分子;
b)将核酸分子固定到一固相支持物上,产生一固定化的核酸分子;
c)将一检测寡聚核苷酸与固定化的核酸分子杂交并除去未杂交的检
测寡聚核苷酸;
d)将步骤c)的产物离子化并挥发;和
e)用质谱对检测寡聚核苷酸进行检测,其中检测到检测寡聚核苷酸
表明生物样品中靶核酸序列的存在。
2.权利要求1的方法,其中步骤b)用一预先固定在固相支持物上的互补的捕获核酸分子与一靶核酸序列上的互补的特异序列的杂交完成固定化。
3.权利要求1的方法,其中步骤b)通过将靶核酸序列直接结合到固相支持物上完成固定化。
4.权利要求1的方法,其中在步骤b)前扩增靶核酸序列。
5.权利要求4的方法,其中靶核酸序列用选自以下的方法扩增,这些方法包括:克隆,基于转录的扩增,聚合酶链反应(PCR);连接酶链反应(LCR),和链置换扩增(SDA)。
6.权利要求1的方法,其中固相支持物选自:玻璃珠,平台表面,钉,梳子和垫片。
7.权利要求6的方法,其中步骤b)通过一预先固定在固相支持物上的多个互补捕获核酸分子形成的阵列和核酸分子上不同于靶核酸序列的部分杂交完成固定化。
8.权利要求7的方法,其中互补的捕获核酸分子为寡聚核苷酸或寡聚核苷酸模拟物。
9.权利要求1的方法,其中固定化是可逆的。
10.权利要求1的方法,其中质谱仪选自:基质辅助激光解吸附/离子化飞行时间(MALDI-TOF),电喷雾(ES),离子回旋共振(ICR),傅立叶变换及其组合。
11.权利要求1的方法,其中在步骤d)前样品被修饰。
12.权利要求11的方法,其中样品用质量不同的至少两个检测寡聚核苷酸或寡聚核苷酸模拟物修饰,以同时检测和区别至少两种不同的靶核酸序列。
13.权利要求12的方法,其中质量差异通过至少两种寡聚核苷酸长度和序列的不同来实现。
14.权利要求12的方法,其中质量差异通过在检测寡聚核苷酸的碱基、糖或磷酸部分引入质量修饰官能团来获得。
15.权利要求12的方法,其中质量差异通过阳离子交换或磷酸二酯键上电荷的去除而实现。
16.权利要求1的方法,其中在质谱检测前,从生物样品中获取的核酸分子用质量修饰的脱氧核苷三磷酸和RNA依赖的DNA聚合酶复制成DNA。
17.权利要求1的方法,其中在质谱检测前,从生物样品中获取的核酸分子用质量修饰的核糖核苷三磷酸和DNA依赖的RNA聚合酶复制成RNA。
18.权利要求1的方法,其中靶核酸序列为DNA指纹或与疾病有关,这些疾病包括:遗传病,染色体异常,遗传诱因,病毒感染,真菌感染,细菌感染和原生生物感染。
19.一种检测生物样品中靶核酸序列的方法,包括以下步骤:
a)从生物样品中获得含靶核酸序列的核酸分子;
b)用适当的扩增方法扩增靶核酸序列,从而获得扩增的靶核酸序
列;
c)用检测寡聚核苷酸与核酸分子杂交,并除去未杂交的检测寡聚核
苷酸;
d)离子化并挥发步骤c)的产物;和
e)用质谱对检测寡聚核苷酸进行检测,其中检测到检测寡聚核苷酸
表明生物样品中存在靶核酸序列。
20.权利要求19的方法,其中靶核酸用选自以下的一种扩增方法扩增,这些方法包括:克隆,基于转录的扩增,聚合酶链反应(PCR);连接酶链反应(LCR),和链置换扩增(SDA)。
21.权利要求19的方法,其中质谱仪选自:基质辅助激光解吸附/离子化,飞行时间(MALDI-TOF),电喷雾(ES),离子回旋共振(ICR),傅立叶变换及其组合。
22.权利要求19的方法,其中在步骤d)前样品被修饰。
23.权利要求22的方法,其中样品用质量差异修饰。
24.权利要求23的方法,其中质量差异通过在用于扩增的引物上加上质量修饰官能团获得。
25.权利要求23的方法,其中质量差异通过阳离子交换或磷酸二酯键上电荷的去除实现。
26.权利要求19的方法,其中核酸分子为DNA。
27.权利要求19的方法,其中核酸分子为RNA。
28.权利要求19的方法,其中在步骤d)前,扩增的靶核酸分子固定到固相支持物上以产生固定化的靶核酸序列。
29.权利要求28的方法,其中固定化通过一预先固定在固相支持物上的互补的捕获核酸分子与靶核酸序列的杂交完成。
30.权利要求28的方法,其中固相支持物选自:玻璃珠,平台表面,钉,梳子和垫片。
31.权利要求28的方法,其中固定化是可逆的。
32.权利要求19的方法,其中靶核酸序列为DNA指纹或与疾病有关,这些疾病包括:遗传病,染色体异常,遗传诱因,病毒感染,真菌感染,细菌感染和原生生物感染。
33.一种检测生物样品中靶核酸序列的方法,包括以下步骤:
a)从生物样品中获取靶核酸序列;
b)复制靶核酸序列,从而得到复制的靶核酸分子;
c)用至少一种合适的核酸酶特异地消化复制的核酸分子,从而产生
消化的片段;
d)将消化片段固定在含互补的捕获核酸序列的固相支持物上产生固
定化的片段;和
e)用质谱分析固定化的片段,其中固定化片段的杂交和分子量确定
提供了靶核酸序列的信息。
34.权利要求33的方法,其中固相支持物选自:玻璃珠,平台表面,钉,梳子和垫片。
35.权利要求33的方法,其中互补的捕获核酸序列为寡聚核苷酸或寡聚核苷酸模拟物。
36.权利要求33的方法,其中固定化是可逆的。
37.权利要求33的方法,其中质谱仪选自:基质辅助激光解吸附/离子化,飞行时间(MALDI-TOF),电喷雾(ES),离子回旋共振(ICR),傅立叶变换及其组合。
38.权利要求33的方法,其中在步骤e)前样品进行修饰。
39.权利要求38的方法,其中样品用质量差异修饰。
40.权利要求38的方法,其中质量差异通过在检测寡聚核苷酸的碱基、糖或磷酸部分引入质量修饰官能团获得。
41.权利要求39的方法,其中质量差异通过阳离子交换或磷酸二酯键上电荷的去除获得。
42.权利要求33的方法,其中在步骤a)后,用质量修饰的脱氧核苷和/或双脱氧核苷三磷酸和RNA依赖的DNA聚合酶将靶核酸序列复制成DNA。
43.权利要求33的方法,其中在步骤a)后,用质量修饰的核糖核苷和/或3′-脱氧核苷三磷酸和DNA依赖的RNA聚合酶将靶核酸序列复制成RNA。
44.权利要求33的方法,其中步骤a)后,用质量修饰的脱氧核糖核苷和/或双脱氧核苷三磷酸和DNA依赖的DNA聚合酶将靶核酸序列复制成DNA。
45.权利要求33的方法,其中靶核酸序列为DNA指纹或与疾病有关,这些疾病包括:遗传病,染色体异常,遗传诱因,病毒感染,真菌感染,细菌感染或原生生物感染。
46.一种检测生物样品中靶核酸序列的方法,包括以下步骤:
a)从生物样品中获取含靶核酸序列的核酸分子;
b)将靶核酸序列与至少一种引物接触,所说的引物3′端碱基与靶核
酸序列互补;
c)将步骤b)的产物与一合适的聚合酶作用,随后顺序加入四种核
苷三磷酸的一种;
d)离子化并汽化步骤c)的产物;和
e)用质谱检测步骤d)的产物,其中产物的分子量表明靶核酸序列上紧接着引物3′端的突变存在与否。
47.一种检测生物样品中靶核苷酸的方法,包括以下步骤:
a)获取含靶核苷酸的核酸分子;
b)将核酸分子固定在固相支持物上,产生固定化的核酸分子;
c)将固定化的核酸分子与一寡聚核苷酸引物杂交,该引物与该核酸分子上紧挨着靶核苷酸5′端的位点互补;
d)将步骤c)的产物与一整套双脱氧核苷或3′脱氧核苷三磷酸和一种DNA依赖的DNA聚合酶作用,从而只有与靶核苷酸互补的双脱氧核苷或3′-脱氧核苷三磷酸在引物上延伸;
e)离子化并汽化步骤d)的产物;和
f)用质谱检测引物,确定靶核苷酸的种类。
48.一种检测核酸分子上突变的方法,包括以下步骤:
a)获得核酸分子;
b)用寡聚核苷酸探针与该核酸分子杂交,从而形成突变位点的错配;
c)将步骤b)的产物与一单链特异的内切核酸酶作用;
d)离子化并汽化步骤c)的产物;和
e)用质谱检测获得的产物,其中一个以上片段的存在,表明核酸分子上含一突变。
49.一种检测生物样品中靶核酸序列的方法,包括以下步骤:
a)从生物样品中获取含靶核酸序列的核酸;
b)将靶核酸序列与一套连接体和热稳定DNA连接酶进行至少一次杂交,从而形成连接产物;
c)离子化并汽化步骤b)的产物;和
d)用质谱检测连接产物并比较所得质量值与已知值,从而确定靶核酸序列。
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EP (5) | EP0815261B3 (zh) |
JP (2) | JP3948746B2 (zh) |
CN (1) | CN1202204A (zh) |
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DK (1) | DK0815261T3 (zh) |
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