CN1806050A - 通过溶液俘获快速纯化用于随后质谱分析的核酸的方法 - Google Patents

通过溶液俘获快速纯化用于随后质谱分析的核酸的方法 Download PDF

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CN1806050A
CN1806050A CNA2004800161879A CN200480016187A CN1806050A CN 1806050 A CN1806050 A CN 1806050A CN A2004800161879 A CNA2004800161879 A CN A2004800161879A CN 200480016187 A CN200480016187 A CN 200480016187A CN 1806050 A CN1806050 A CN 1806050A
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CN1806050B (zh
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S·A·霍弗斯塔德勒
L·L·卡敏斯
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Abstract

本发明提供了用于随后质谱分析的核酸的快速溶液俘获纯化方法，该方法相对于现有的方法是有效的和有成本效益的。本发明还提供了实施快速溶液俘获核酸的试剂盒，使得纯化的样品处于可以通过电雾化质谱分析的状态。

Description

通过溶液俘获快速纯化用于 随后质谱分析的核酸的方法
技术领域
本发明总地涉及通过质谱分析核酸的领域，并提供了用于该目的的方法和试剂盒。
背景技术
电雾化电离质谱(ESI-MS)已经成为生物聚合物分析的重要技术。该多电荷现象可以快速、准确和精确测量非常大质量生物聚合物的分子量、鉴定改变以及进行更详细的结构研究。
利用聚合酶链反应的特定DNA序列的扩增已经在许多特定学科中得到了广泛应用，包括微生物学、医学研究、法医检定和临床诊断。更常见的，使用传统生化技术如使用嵌入染料或荧光标记引物的标准凝胶电泳将PCR产物“按大小排列”。广泛用于多种基于PCR的诊断试剂盒中的TapmanTM试验能证实特定PCR产物的存在(或不存在)，但不提供扩增子大小的直接读数。所谓的“实时”PCR装置，在扩增循环中测量激光诱导的PCR产物的荧光，用于定量所给DNA模板和引物对的DNA扩增。这些方法对于相对小的扩增子(少于150个碱基对)的使用受到限制，因为成比例高的荧光背景，且不提供任何关于扩增子异质性或确切长度的信息。
和这些更传统的方法相比较，质谱作为在其上表征PCR产物的平台具有数个潜在优势，包括速度、灵敏度和质谱精确度。因为每个含有DNA的碱基的确切质量是已知具有高度精确性的，通过质谱获得的高精确度质量测量可以用来得出实验获得的质量测量不确定性中的碱基组成(J.Aaserud，Z.Guan，D.P.Little和F.W.McLafferty，Int.J.Mass Spectrom.Ion Processes，1997，167/168，705-712和D.C.Muddiman，G.A.Anderson，S.A.Hofstadler和R.D.Smith，Anal.Chem.1997，69，1543-1549)。使用核酸扩增和通过分子量分析确定提供信息扩增子的碱基组成的组合快速鉴定未知生物试剂的方法公开且要求于公开的美国专利申请20030027135，20030082539，20030124556，20030175696，20030175695，20030175697和20030190605和美国专利申请Nos.10/326,047，10/660,997，10/660,122和10/660,996中，所有都是共有的且在此以其整体引入作为参考。
已经使用MALDI(基质辅助的，激光解吸电离)和电雾化(ESI)质谱来电离PCR产物用于随后的质谱检测。虽然MALDI广泛用于分析短的(20-mer或更小的)寡核苷酸，但是应用于超过100bp的扩增子不太常见。由于电离过程的内在“柔软度”，ESI是大生物分子最广泛使用的电离技术之一，其可以使超过500bp的DNA得到电离而不离解。
ESI中，在“气动雾化”过程中产生大的带电的微滴，其中迫使分析物溶液通过针，在针末端运用电势，该电势足以将形成的溶液分散成非常细喷雾的带电微滴，所有带电微滴具有相同的极性。溶剂蒸发，压缩微滴大小并提高微滴表面的电荷浓度。最后，在瑞利限度，库仑斥力克服了微滴的表面张力，微滴破裂。该“库仑斥力”形成一系列较小的、带较低电荷的微滴。重复压缩接着破裂的过程直至形成单个带电的分析物离子。电荷统计地分布在分析物可获得的电荷位点中，导致多电荷离子状态形成的可能。通过将干气流反电流引至雾化的离子来提高溶剂蒸发率，提高了多电荷的程度。降低毛细管直径并降低分析物溶液的流速，即在纳米雾化电离中，将产生m/z比例高于那些通过“常规”ESI产生的离子(即，其是较软的电离技术)，且更多地用于生物分析领域中。
不幸地，ESI需要相对干净的样品且众所周知地不可耐生化实验室中常用的阳离子盐、去污剂和许多缓冲液。
通常用于聚合酶链反应中的缓冲系统含有电雾化不相容试剂如50mM KCl，2mM MgCl2，10mM Tris-HCl，和200：M的四种脱氧核苷酸三磷酸(dNTP)中的每种。甚至相对低浓度(低于100：M)金属离子的存在能显著降低寡核苷酸的MS灵敏性，因为每种分子离子的信号随着多个盐加合物而展开。因此，除了除去去污剂和dNTP，PCR产物的有效ESI-MS需要在分析之前将盐浓度降低高于1000倍。
已经使用乙醇沉淀来将PCR产物脱盐，随后作为短的寡核苷酸用于MS分析，并除去盐而将样品浓缩(M.T.Krahmer，Y.A.Johnson，J.J.Walters，K.F.Fox，A.Fox和M.Nagpal，Electrospray Anal.Chem.1999，71，2893-2900；T.Tsuneyoshi，K.Ishikawa，Y.Koga，Y.Naito，S.Baba，H.Terunuma，R.Arakawa和D.J.Prockop RapidCommun.Mass Spectrom.1997，11，719-722；和D.C.Muddiman，D.S.Wunschel，C.L.Liu，L.Pasatolic，K.F.Fox，A.Fox，G.A.Anderson和R.D.Smith Anal.Chem.1996，68，3705-3712)。该方法中，将PCR产物从浓缩的醋酸铵溶液中沉淀出来，在5℃过夜或用冷(-20℃)乙醇处理10-15分钟。不幸地，单独的沉淀步骤通常不足以获得合适脱盐的PCR产物来获得高质量ESI质谱；因此，沉淀后通常为渗析步骤来进一步将样品脱盐(D.C.Muddiman，D.S.Wunschel，C.L.Liu，L.Pasatolic，K.F.Fox，A.Fox，G.A.Anderson和R.D.Smith Anal.Chem.1996，68，3705-3712)。虽然数个研究者已经成功使用这些方法来表征多个PCR产物，但是以稳固和完全自动化高处理量的方式使用这些方法的途径不明显。
商业DNA纯化试剂盒也可以和传统脱盐技术如微量渗析结合使用(S.Hahner，A.Schneider，A.Ingendoh和J.Mosner Nucleic AcidsRes.2000，28，e82/i-e82/viii；和A.P.Null，L.T.George和D.C.Muddiman.J.Am.Soc.Mass Spectrom.2002，13，338-344)。其它纯化技术，如凝胶电泳，接着高效液相色谱或液滴渗析，或使用膜或树脂的阳离子交换，也已经用来获得用于MS检测的高纯度的、脱盐的DNA(L.M.Benson，S.-S.Juliane，P.D.Rodringues，T.Andy，L.J.Maher III和S.Naylor，In：The 47th ASMS Conference on MassSpectrometry and Allied Topics，Dallas，Texas(1999)；C.G.Huber和M.R.Buchmeiser Anal.Chem.1998，70，5288-5295；H.Oberacher，W.Parson，R.Muehlmann和C.G.Huber Anal.Chem.2001，73，5109-5115；和C.J.Sciacchitano J.Liq.Chromatogr.Relat.Technol.1996，19，2165-2178)。不幸地，因为使用上述技术，实现快速和完全自动化高处理量的路径不明显。
Jiang和Hofstadler已经发展和报道了纯化并脱盐PCR产物的单步实验方案，其使用可购得的装填阴离子交换树脂的移液管尖端(Y.Jiang和S.A.Hofsadler Anal.Biochem.2003，316，50-57)。该实验方案产生了ES I-MS-相容样品，且仅需要10∶1的粗制PCR产物。然而，当用于高体积和高处理量方法如上述用于鉴定未知生物试剂的方法中时，该方法是成本过高的。估算使用可购得的ZipTipTM AX(Millipore Corp.Bedford，MA)的零售成本为每平板加样孔$1.77。
仍然需要快速、有效和非成本过高的纯化用于质谱的核酸的方法。本发明满足了该需要。
如那些从扩增反应获得的核酸的溶液俘获使提取和纯化这些用于随后质谱分析的分析物的快速、有成本效益的方法成为可能。因为核酸和阴离子交换介质在溶液中，通过涡旋或其它混合方法来实现核酸的有效俘获。这排除了将介质装填成柱子形式的需要，柱子形式需要核酸溶液穿过柱子多次来获得核酸的高水平回收。虽然较长的柱子需要较少的通过次数，但是显著的背压成为了问题。因此，将阴离子交换树脂装填于柱子或移液管尖端形式的处理增加了相关方法的成本。因此通过消除装填、平衡和测试柱子的需要，使用溶液俘获来纯化用于质谱分析的PCR产物实质性地降低了样品制备相关的成本。使用装填阴离子交换树脂的移液管尖端通过ZipTipTM AX(Millipore Corp.Bedford，MA)作为实例的通用方法的零售成本约为每个移液管尖端(对于每个样品)$1.77。PCR产物溶液俘获的估算成本为每个样品$0.10，并考虑阴离子交换树脂和滤板的组合。此外，PCR产物溶液俘获纯化所需的时间约为每96孔平板10分钟，和之前使用ZipTipTM AX移液管尖端的方法相比，其需要约20分钟。
发明内容
本发明特别涉及纯化溶液的溶液俘获方法，该溶液含有一种或多种用于随后电雾化质谱分析或任何其它分析的核酸，该方法通过将阴离子交换树脂加入溶液中并混合来产生阴离子交换树脂在溶液中的悬浮液，其中核酸和阴离子交换树脂结合，从溶液中分离阴离子交换树脂，用一种或多种洗涤缓冲液洗涤阴离子交换树脂来除去一种或多种污染物而保留结合的核酸，用洗脱缓冲液从离子交换树脂洗脱核酸，且任选地，通过电雾化质谱分析核酸。
阴离子交换树脂可以具有强阴离子交换官能团如季胺或弱阴离子交换官能团如聚乙烯亚胺、带电荷的芳族胺、二乙基氨甲基或二乙基氨乙基。这样的弱阴离子交换树脂含有pKa值为9.0或更大的官能团。
本发明进一步涉及用于纯化核酸的试剂盒，该试剂盒包括含有多个孔的滤板或含有多个管的管架、阴离子交换树脂、至少一种阴离子交换洗涤缓冲液和ESI-MS-相容洗脱缓冲液。
附图说明
图1是流程图，列出了本发明的步骤，从阴离子交换树脂加入核酸样品中并混合(100)开始。然后从溶液中分离树脂(110)并用合适的洗涤缓冲液洗涤来从树脂中除去污染物(120)，其后，通过电雾化电离(ESI)-相容洗脱缓冲液将核酸从树脂中洗脱，其使得通过ESI-质谱分析核酸的最后步骤(140)成为可能。
图2是通过ZipTipsTM(顶端的画面)纯化获得的和通过本发明溶液俘获纯化方法获得的纯化PCR产物的ESI-MS质谱比较。该比较表明了通过溶液俘获方法的纯化和以前确立的使用ZipTipsTM的方法同样有效。
实施方案的描述
例如，用于质谱分析的核酸的溶液俘获纯化方法的一个实施方案概述于图1中。除了本领域技术人员已知的质谱以外，在此所述的方法可以用于其它类型的分析中。该方法包括以下步骤：将阴离子交换树脂加入核酸样品中并混合(100)，从溶液中分离阴离子交换树脂(110)，洗涤阴离子交换树脂来除去污染物(120)，从阴离子交换树脂中洗脱核酸(无污染物)(130)，和任选地，通过ESI质谱分析核酸。
一些实施方案中，将强阳离子交换官能团如季胺用作阴离子交换树脂的官能团。另外的强阴离子交换官能团是本领域技术人员已知的。
其它实施方案中，弱阴离子交换官能团是合适的阴离子交换官能团，如聚乙烯亚胺、带电荷的芳族胺、二乙基氨甲基或二乙基氨乙基，例如用作阴离子交换树脂的官能团。这样的官能团具有9.0或更大的pKa值。弱阴离子交换树脂的商品包括但不限于：Baker PEI，BakerDEAM，Dionex ProPacTM WAX，Millipore PEI，Applied Biosystem PorosTMPI。
一些实施方案中，将阴离子交换树脂混入核酸溶液中通过重复用移液管吸取、涡旋、超声处理、振荡，或形成阴离子交换树脂在含有核酸的溶液中的悬浮液的任何其它方法来实现。
一些实施方案中，将干阴离子交换树脂直接加入核酸溶液中或包含在混合之前加入了核酸溶液的微管中或微滤板的孔中。其它实施方案中，阴离子交换树脂是预水合的并直接加入核酸溶液中或包含在混合之前加入了核酸溶液的微管中或微滤板的孔中。
一些实施方案中，通过过滤将含有结合核酸的阴离子交换树脂从溶液中分离出来。例如，使用能高处理量纯化多个样品的96孔或348孔规格的滤板或在装有滤器的任何其它容器或多个容器中来实现过滤。也可以使用其它孔规格的板。用于过滤的膜包括但不限于由以下材料构成的那些：聚四氟乙烯(PTFE)、聚二氟乙烯(PVDF)、聚丙烯、聚乙烯、玻璃纤维、聚碳酸酯和聚砜。可以通过真空、离心或用流体或气体的正压置换，或实现阴离子交换树脂从溶液中分离的任何其它方法来完成过滤。过滤的方法是本领域技术人员公知的。
一些实施方案中，阴离子交换树脂含有和磁珠连接的阴离子交换官能团。通过消除对离心、真空或正压置换的需要，这样的配置能够简化分离步骤(110)，离心、真空或正压置换需要从液体处理机工作台除去板或微管。替代的是，可以激活磁场来压缩磁珠树脂，使得液体可以通过液体处理机吸出。使用磁珠来实现生物分子分离的方法是本领域技术人员公知的。
一些实施方案中，洗涤含有结合核酸的阴离子交换树脂来除去一种或多种污染物。污染物包括但不限于：蛋白质如逆转录酶和限制酶、聚合物、盐、缓冲添加剂，或扩增反应中各种成分中的任一种如聚合酶，核苷酸三磷酸或其任意组合。根据核酸溶液中污染物的组成，可以使用多于一种的洗涤缓冲液来除去污染物。可以使用约20mM至约500mM NH4OAc毫摩尔范围的醋酸铵水溶液或用约20mM至约500mMNH4CO3来实现阴离子交换树脂的洗涤。用约10％至约50％的甲醇，约20％至约50％的甲醇，或约10％至约30％的甲醇用作最终的洗涤步骤。甲醇可以由本领域技术人员已知的其它合适的醇来替代。
一些实施方案中，使用约pH9或更大的碱性pH的ESI相容溶液来完成核酸从阴离子交换树脂的洗脱，如约2％至约8％的氢氧化铵水溶液或约10mM至约50mM，或25mM哌啶，约10mM至约50mM，或25mM咪唑和约30％甲醇或其它合适醇的水溶液。如在此所定义的，ESI相容溶液是不具有对电雾化(ESI)源有害影响的溶液。
如在此所用的术语“约”意思是以±10％来改变术语。因此，例如，“约”10mM意思是9至11mM。
另一实施方案中，本发明还提供了通过本发明的溶液俘获方法来纯化核酸的试剂盒。一些实施方案中，该试剂盒可以包括足够量的阴离子交换树脂。一些实施方案中，阴离子交换树脂是弱阴离子交换树脂，如以下可购得的弱阴离子交换树脂之一：Baker聚乙烯亚胺树脂、Baker二乙基氨甲基树脂、Dionex ProPacTM WAX、Millipore聚乙烯亚胺和Applied Biosystems POROSTM PI。
一些实施方案中，试剂盒可以含有滤板如96孔或384孔滤板，或含有多个微量过滤管的微管架。
一些实施方案中，干阴离子交换树脂是预装填在滤板或微管架的孔中的，且可以是预水合的或是干(粉)的形式。
试剂盒还可以包括含有多个孔的滤板或含有多个管的管架、阴离子交换树脂、至少一种阴离子交换洗涤缓冲液和ESI-MS-相容洗脱缓冲液。
一个实施方案中，试剂盒可以包括含有预水合阴离子交换树脂或干阴离子交换树脂的96孔或384孔板、第二个96或384孔样品混合板、96或384孔滤板、树脂处理缓冲液、一种或多种洗涤缓冲液和ESI-相容洗脱缓冲液。
一个实施方案中，核酸溶液是制得的用于鉴定未知生物试剂的PCR产物并包含于自动液体处理机台面上的96孔样品板的单个孔中。液体处理机是与发现药物和高处理量筛选相关的许多实验室处理的基石。液体处理机的分配和吸气功能用来进行溶剂/试剂添加、稀释、平板复制固结、再分配和其它基于微板的任务，且通常使用可任意处理的移液管尖端来转移液体。将液体处理机编程来进行该实施方案的各种液体处理任务是本领域普通技术人员技能范围内的而不需要过度的实验。
将液体处理机编程来将预定体积的阴离子交换树脂悬浮液转移并混入含有PCR产物的孔中。可以通过液体处理机将树脂悬浮液装入树脂源容器如96孔板中并转移至PCR产物板。通过反复分配和吸取PCR-树脂混合物由液体处理机进行混合并在该阶段将核酸结合至树脂。接着，液体处理机将PCR产物-树脂混合物从96孔板转移至96或384孔滤板。在此阶段，可以将滤板从液体处理机台面除去，且在将滤板返回至液体处理机台面之前，通过离心或正压置换将树脂从液体中分离出来。
然后通过液体处理机将洗涤溶液移吸至滤板用合适的洗涤溶液如约100mM NH4HCO3来一次或多次洗涤含有结合核酸的树脂，接着离心、真空或正压置换，随后用约20％至约50％甲醇洗涤一次或多次，然后将含有树脂和结合核酸的滤板返回至液体处理机台面。
最后，使用ESI相容洗脱缓冲液如约25mM哌啶、约25mM咪唑和约50％甲醇的水溶液将核酸从树脂中洗脱出来。该ESI相容缓冲液还可以任选地含有在随后的ESI质谱分析过程中用于校准ESI质谱的内标物。
为了能更有效地理解在此公开的本发明，以下提供了实施例。应当理解这些实施例仅仅是为了说明的目的，并不是以任何方式来限定本发明。贯穿这些实施例，根据Maniatis等的分子克隆实验指南(Molecular Cloning：ALaboratory Manual)，第2版，Cold SpringHarbor Press(1989)中所述的方法进行分子克隆反应和其它标准重组DNA技术，使用可购得的试剂，除非另外指出。
具体实施方式
               实施例1：核酸分离和PCR
一个实施方案中，在通过质谱BCS测定之前，从生物体中分离核酸并使用标准方法通过PCR扩增。例如，通过去污剂裂解细菌细胞、离心和乙醇沉淀来分离核酸。核酸分离方法描述于，例如，最新分子生物学方案(Current Protocols in Molecular Biology)(Ausubel等)和分子克隆实验指南(Sambrook等)。然后使用标准方法如PCR扩增核酸，使用和核酸保守区结合的引物，核酸保守区含有如下所述的插入可变序列。
概括的基因组DNA样品制备方案：使用Supor-200 0.2μM膜注射器滤器(VWR International)来过滤粗制样品。将样品转移至预先装有0.45g 0.7mm氧化锆珠子的微量离心管中，接着加入350μl ATL缓冲液(Qiagen，Valencia，CA)。将样品在Retsch Vibration Mill(Retsch)中以19l/s的频率接受珠子撞击10分钟。离心后，将样品转移至S-嵌段板(Qiagen)上并用BioRobot 8000核酸分离自动装置(Qiagen)完成DNA分离。
拭子取样方案：将Allegiance S/P牌的培养拭子和收集/运输系统用来收集样品。干燥后，将拭子放入17×100mm的培养管(VWRInternational)中并用Qiagen Mdx自动装置和Qiagen QIAamp DNABlood BioRobox Mdx基因组制备试剂盒(Qiagen，Valencia，CA)自动化进行基因组核酸分离。
                   实施例2：质谱
所用的质谱仪是Bruker Daltonics(Billerica，MA)Apex II 70e电雾化电离傅里叶转换离子回旋加速共振质谱仪(ESI-FTICR-MS)，该质谱仪使用活性屏蔽的7特斯拉超导磁铁。在1.1GHz Pentium II数据站上运行Bruker’s Xmass软件来进行脉冲序列控制和数据采集的所有方面。使用通过数据站启动的CTC HTS PAL自动取样器(LEAPTechnologies，Carrboro，NC)从96孔微量滴定板直接提取20μL样品等分试样。将样品以75μL/小时的流速直接注入ESI源中。在使用离轴的、接地的电雾化探针的改良分析(Branford，CT)源中通过电雾化电离形成离子，该电雾化探针放置于离玻璃退溶毛细管金属化末端约1.5cm。在数据采集过程中玻璃毛细管的大气压端相对于ESI针偏离6000V。使用干N2/O2反向电流来帮助去溶剂化过程。在注入捕获离子的小室之前，离子聚集于外部离子存储器中，离子在小室中得到质谱分析，该存储器由仅有射频六极器(rf-only hexapole)、分离锥(skimmer cone)和辅助门电极构成。
以连续运行循环模式进行频谱采集，借此离子在六极器离子存储器中累积，同时在捕获离子小室中检测离子。将离子转移至捕获离子小室中的1.2ms转移事件后，对离子进行对应于8000-500m/z的1.6ms线性调频脉冲激发。在500-5000的m/z范围内采集数据(1M数据点跨越225K Hz的带宽)。每个频谱是32个瞬变值叠加的结果。瞬变值是数量模式傅立叶转换之前和使用内部质量标准校准之后的零负荷一瞬。ICR-2LS软件包(G.A.Anderson，J.E.Bruce(PacificNorthwest National Laboratory，Richland，WA，1995)用来将质谱去卷积并使用用于DNA的改良“平均”拟合程序(M.W.Senko，S.C.Beu，F.W.McLafferty，J.Am.Soc.Mass Spectrum.1995，6，229)计算单一同位素种类的质量。使用该方法，计算出单一同位素的分子量。
实施例3：使用可购得的ZipTipsTM半自动化纯化PCR混合物的方法
为了预处理ZipTipsTM AX(Millipore Corp.Bedford，MA)，将以下步骤编程，通过EvolutionTM P3液体处理机(Perkin Elmer)来进行，使用从96孔板(Marshall Bioscience)单个孔中的储液中取出的流体：装填ZipTipsTM AX架；用15∶1的10％ NH4OH/50％甲醇洗涤ZipTipsTM AX；用15∶1的水洗涤ZipTipsTM AX 8次，用15∶1的100mMNH4OAc洗涤ZipTipsTM AX。
为了纯化PCR混合物，使用BioHitTM多通道移液管将20∶1的粗制PCR产物转移至MJ Research板的单个孔中。向96孔板的单个孔中填装300∶1的40mM NH4HCO3。向96孔板的单个孔中填装300∶1的20％甲醇。向MJ研究板中填装10∶1的4％ NH4OH。向两个存储器中填装去离子水。将所有板和存储器以预先排列的次序放置于EvolutionTM P3(EP3)移液装置的台面上。将以下的步骤编程，通过EvolutionTM P3移液装置来进行：将20∶1的空气吸入EP3 P50头部；将预处理的ZipTipsTM AX架装入EP3 P50头部；从ZipTipsTM AX分配20∶1的NH4HCO3；通过将PCR溶液吸取/分配18次将PCR产物装入ZipTipsTM AX；用15∶1的40mM NH4HCO3洗涤含有结合核酸的ZipTipsTM AX 8次；用15∶1的20％甲醇洗涤含有结合核酸的ZipTipsTM AX 24次；通过用15∶1的4％NH4OH吸取/分配18次将纯化的核酸从ZipTipsTM AX中洗脱出来。为了ESI-MS分析的最后制备，用含有50mM哌啶和50mM咪唑的70％甲醇将每个样品以1∶1的体积比稀释。
实施例4：使用溶液俘获半自动化纯化PCR混合物的方法
为了预处理ProPacTM WAX弱阴离子交换树脂，成批进行以下步骤：
用下列溶液中的每种连续洗涤三次(缓冲液与树脂的体积比为10∶1)：(1)1.0M富马酸/50％甲醇(2)20％甲醇(3)10％ NH4OH(4)20％甲醇(5)40mM NH4HCO3(6)100mM NH4OAc。将树脂保存于4℃的20mMNH4OAc/50％甲醇中。
用250∶1的NH4OH漂洗两次和用100∶1的NH4HCO3漂洗两次来预处理Corning 384孔玻璃纤维滤板。
为了将PCR产物核酸结合至树脂，将以下步骤编程，通过EvolutionTM P3液体处理机来进行：将0.05至10∶1的预处理ProPacTMWAX弱阴离子交换树脂(1∶60稀释度的30∶1)加入96孔板中50∶1的PCR反应混合物中(80∶1总体积)；通过吸取/分配混合溶液2.5分钟；并将溶液转移至预处理的Corning 384-孔玻璃纤维滤板中。接着该步骤为离心，从树脂除去液体，并且是手动或在机械臂控制下进行的。
然后通过用200∶1的100mM NH4OAc，200∶1的40mM NH4HCO3漂洗三次来洗涤含有核酸的树脂，通过约15秒钟离心来除去缓冲液，接着用20％甲醇漂洗三次约15秒钟。最后的漂洗之后为延长的离心步骤(1-2分钟)。
通过加入40∶1的洗脱/电雾化缓冲液(25mM哌啶/25mM咪唑/35％甲醇和50nM用于校准质谱信号的内标物寡核苷酸)，接着通过离心从384孔滤板洗脱至384孔收集板中来完成核酸从树脂中的洗脱。该条件下洗脱的核酸能经受ESI-MS的分析(参见图2)。使用液体处理机纯化单个96孔板中的样品所需的时间约为5分钟。
实施例5：ZipTipsTM纯化方法和溶液俘获方法的比较
为了研究本发明溶液俘获方法的效率，将用溶液俘获方法纯化产物获得的ESI-MS分析结果(实施例4)和实施例3中概述的ZipTipsTM方法所获得的结果相比较。
分离炭疽杆菌(Bacillus anthracis)的DNA并通过PCR扩增，使用扩增残基756-872的炭疽杆菌lef基因片段的引物对。图2中所示的为通过ZipTipsTM(顶端画面)纯化获得的纯化PCR产物和通过本发明溶液俘获纯化方法获得的纯化PCR产物的ESI-MS质谱比较。该比较表明了通过溶液俘获方法纯化和以前确立的使用ZipTipsTM的方法同样有效。然而，通过溶液俘获纯化显示出显著的成本节约且更有效。如Jiang和Hofstadler所述的，使用ZipTipsTM的PCR产物的纯化“在少于20分钟内产生ESI-相容样品(96孔板)。”本发明的溶液俘获方法在约5分钟内产生ESI-相容样品。使用装填阴离子交换树脂的移液管尖端通过ZipTipsTM AX(Millipore，Bedford，MA)示例的通用方法的零售成本约为每个移液管尖端(对于每个样品)$1.77。PCR产物溶液俘获的估算成本为每个样品$0.10，并考虑了阴离子交换树脂和滤板的结合。
除了那些在此所述的，本发明的各种改变，从前面的描述对本领域普通技术人员而言是显而易见的。这样的改变也落入所附权利要求的范围内。本申请中所引用的每篇参考文献在此以其整体引入作为参考。2003年5月13日提交的美国临时申请No.60/470,547在此以其整体引入作为参考。

Claims (42)

1.一种含有核酸溶液的纯化方法，包括：
将所述溶液和阴离子交换树脂混合来产生所述阴离子交换树脂在所述溶液中的悬浮液，其中所述核酸和所述阴离子交换树脂结合；
从所述溶液中分离所述阴离子交换树脂；
用一种或多种洗涤缓冲液洗涤所述阴离子交换树脂来除去一种或多种污染物，而保留结合的核酸；
用洗脱缓冲液从所述离子交换树脂洗脱所述核酸。
2.权利要求1的方法，进一步包括通过质谱分析所述核酸。
3.权利要求1的方法，其中所述混合、洗涤和洗脱步骤通过液体处理机来进行。
4.权利要求1的方法，其中所述混合、洗涤和洗脱步骤由自动装置来进行。
5.权利要求1的方法，其中所述阴离子交换树脂是强阴离子交换树脂。
6.权利要求5的方法，其中所述强阴离子交换树脂的官能团是季胺。
7.权利要求1的方法，其中所述阴离子交换树脂是弱阴离子交换树脂。
8.权利要求7的方法，其中所述弱阴离子交换树脂的官能团包括聚乙烯亚胺、带电荷的芳族胺、二乙基氨甲基或二乙基氨乙基。
9.权利要求7的方法，其中所述弱阴离子交换树脂含有以下商品中的一种：Baker聚乙烯亚胺树脂、Baker二乙基氨甲基树脂、DionexProPacTMWAX、Millipore聚乙烯亚胺和Applied Biosystems POROSTMPI。
10.权利要求1的方法，其中通过移吸、涡旋、超声处理或振荡来实现所述混合。
11.权利要求1的方法，其中通过过滤来实现所述分离步骤。
12.权利要求11的方法，其中所述过滤在96孔板、384孔板或微量过滤管中进行。
13.权利要求11的方法，其中使用以下的一种膜来实现所述过滤：聚四氟乙烯、聚乙烯二氟乙烯、聚丙烯、聚乙烯、玻璃纤维、聚碳酸酯或聚砜。
14.权利要求11的方法，其中通过真空、离心或用流体或气体的正压置换来实现所述过滤。
15.权利要求1的方法，其中所述阴离子交换树脂包括结合到磁珠上的阴离子交换官能团。
16.权利要求15的方法，其中通过应用磁场来压缩所述磁珠并吸取所述溶液来实现所述分离步骤。
17.权利要求1的方法，其中所述洗涤缓冲液含有醋酸铵。
18.权利要求1的方法，其中所述洗涤缓冲液含有碳酸氢铵。
19.权利要求1的方法，其中所述洗涤缓冲液含有甲醇。
20.权利要求1的方法，其中所述洗脱缓冲液含有氢氧化铵。
21.权利要求1的方法，其中所述洗脱缓冲液和电雾化电离(ESI)相容。
22.权利要求1的方法，其中所述洗脱缓冲液含有哌啶、咪唑和甲醇的组合。
23.权利要求1的方法，其中所述核酸溶液为核酸扩增反应的产物。
24.权利要求23的方法，其中通过聚合酶链反应、连接酶链反应或链置换扩增来实现所述扩增反应。
25.权利要求1的方法，其中所述污染物包括以下的任何一种：缓冲盐、稳定剂、金属离子、蛋白质和脱氧核苷酸三磷酸。
26.一种含有用于随后电雾化质谱分析的核酸溶液的纯化方法，包括：
将弱阴离子交换树脂加入所述溶液中并混合来产生所述弱阴离子交换树脂在所述溶液中的悬浮液，其中所述核酸和所述弱阴离子交换树脂结合；
从所述溶液中分离所述弱阴离子交换树脂；
用一种或多种洗涤缓冲液洗涤所述弱阴离子交换树脂来除去污染物，而保留结合的核酸；和
用洗脱缓冲液从所述弱离子交换树脂洗脱所述核酸。
27.权利要求26的方法，进一步包括通过质谱分析所述核酸。
28.权利要求26的方法，其中所述弱阴离子交换树脂的官能团包括聚乙烯亚胺、带电荷的芳族胺、二乙基氨甲基或二乙基氨乙基。
29.权利要求26的方法，其中所述弱阴离子交换树脂含有以下商品中的一种：Baker聚乙烯亚胺树脂、Baker二乙基氨甲基树脂、DionexProPacTMWAX、Millipore聚乙烯亚胺和Applied Biosystems POROSTMPI。
30.一种纯化用于随后电雾化质谱分析的核酸溶液的方法，包括：
将ProPacTMWAX弱阴离子交换树脂加入所述溶液中并混合来产生所述弱阴离子交换树脂在所述溶液中的悬浮液，其中所述核酸和所述弱阴离子交换树脂结合；
从所述溶液中分离所述弱阴离子交换树脂；
用一种或多种洗涤缓冲液洗涤所述弱阴离子交换树脂来除去污染物，而保留结合的核酸；和
用含有哌啶、咪唑和甲醇的洗脱缓冲液从所述弱离子交换树脂洗脱所述核酸。
31.权利要求30的方法，进一步包括通过质谱分析所述核酸。
32.一种用于纯化核酸的试剂盒，包括：
含有阴离子交换树脂的树脂源板；
样品混合板；
滤板或含有多个微量过滤管的微管架；
一种或多种树脂洗涤溶液；和
ESI-MI-相容洗脱缓冲液。
33.权利要求32的试剂盒，其中所述滤板为96孔板或384孔板。
34.权利要求32的试剂盒，其中所述阴离子交换树脂含有弱阴离子交换官能团。
35.权利要求32的试剂盒，其中所述阴离子交换树脂包括ProPacTMWAX。
36.权利要求32的试剂盒，其中所述阴离子交换树脂含有结合到磁珠上的弱阴离子交换官能团。
37.权利要求32的试剂盒，其中所述弱阴离子交换树脂为ProPacTMWAX。
38.权利要求32的试剂盒，其中所述一种或多种洗涤溶液含有约50mM至约200mM的醋酸铵或约50mM至约200mM的碳酸氢铵。
39.权利要求32的试剂盒，其中所述至少一种阴离子交换洗涤缓冲液含有约20％至约50％的甲醇。
40.权利要求32的试剂盒，其中所述ESI-MS相容洗脱缓冲液含有约2％至约6％的氢氧化铵。
41.权利要求32的试剂盒，其中所述ESI-MS相容洗脱缓冲液含有约10mM至约50mM的咪唑，约10mM至约50mM的哌啶和约20％至约50％的甲醇。
42.权利要求32的试剂盒，其中所述ESI-MS相容洗脱缓冲液含有约10mM至约50mM的咪唑，约10mM至约50mM的哌啶和约20％至约50％的甲醇。
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