CN1439646A - 离析核酸的方法 - Google Patents

离析核酸的方法 Download PDF

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CN1439646A
CN1439646A CN03106636A CN03106636A CN1439646A CN 1439646 A CN1439646 A CN 1439646A CN 03106636 A CN03106636 A CN 03106636A CN 03106636 A CN03106636 A CN 03106636A CN 1439646 A CN1439646 A CN 1439646A
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nucleic acid
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J·克莱恩
T·沃尔特
H·哈尔蒂希
C·列斯尼亚克
M·梅恩伊格
M·里德林
H·施密特
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Abstract

本发明涉及一种离析核酸的方法，它包括使流体中含有天然形态核酸的试样与具有玻璃表面的磁性颗粒，在核酸能直接结合到玻璃表面上去的条件下接触，该玻璃表面基本上无孔或有直径小于10nm的孔，和从流体中分离结合的核酸。

Description

离析核酸的方法
本发明涉及具有玻璃表面的磁性颗粒，以及使用玻璃颗粒在离液序列高的盐存在条件下纯化生物物质，特别是核酸的方法。本发明还涉及离析这些生物物质的方法以及浓缩生物物质并将其从高浓度盐溶液转移到低浓度盐溶液的方法。
许多生物物质，特别是核酸，要从其自然环境中将它们离析出来存在着特殊的困难。一方面，它们常以非常低的浓度存在，另一方面，它们经常与许多其它固体和溶解物质一起存在，使它们难以被离析或测量。
因为这个原因，近年来提出了许多方法和材料用于把核酸从其自然环境中离析出来。例如在Proc.Natl.Acad.USA 76,615-691(1979)中提出了把琼脂糖凝胶中的核酸在碘化钠存在下结合到磨碎的火石玻璃上的方法。
在高氯酸钠存在条件下，在玻璃粉上纯化DNA质粒以去除细菌的方法。在Anal.Biochem.121，382-387(1982)上有介绍。
在DE-A 37 34 442中介绍了用醋酸通过噬菌体颗粒沉淀把单旋M13噬菌体的DNA离析到玻璃纤维过滤器上并用高氯酸裂解这些噬菌体颗粒。结合到玻璃纤维过滤器上的核酸经洗涤后用含薄荷醇缓冲液洗脱到三甲醇氨基甲烷/乙二胺四乙酸缓冲液中。
从λ噬菌体中纯化DNA的类似方法在Anal.Biochem.175，196-201(1988)有介绍。
按过去已知的方法，核酸必须在离液序列高的盐溶液中有选择地结合到玻璃表面并且把核酸与污染物如琼脂糖，蛋白质或细胞碎片分离。按照过去的方法，为把玻璃颗粒与污染物分离，如是颗粒则通过离心分离，如是液体则通过玻璃纤维过滤器滤出。但是这是一个卡关的步骤，它限制了这些方法用于处理大量样品。
通过加入盐和乙醇产生沉淀后，用磁性颗粒来固定核酸的方法在Anal.Biochem.201，166-169(1992)和PCT GB 91/00212有介绍。在这个方法中核酸随磁性颗粒被凝集。通过加磁场并进行一次洗涤把凝集物和原始溶剂分离。经一次洗涤后，核酸被溶于三甲醇氨基甲烷缓冲液中。但是此法有缺点，其中沉淀作用对核酸不是选择的。而是许多固体和溶解物质也被凝集出来。结果，这种方法不能用来去除可能存在的特定酶反应的大量抑制剂。
一种有磁性颗粒埋置其中的多孔玻璃在US-A-4,233,169中有介绍。
磁性多孔玻璃在市场上可以买到，它在多孔的特种玻璃基体中包容磁性颗粒，并带有含链酶抗生物素的包复层。这种产品可以用来离析生物物质，例如蛋白质或核酸，如果在络合物制备时对其作些修饰，则可以共价结合到生物素上。
本发明的任务是提供更好的材料来固定生物物质以及一种简单的方法来离析生物物质，特别是核酸，它也适用于常规诊断过程。
本发明主要涉及具有基本上无孔或孔径小于10nm的玻璃外表面的磁性颗粒。本发明还涉及具有玻璃表面的铁磁颗粒，一种离析生物物质特别是核酸的方法，以及一种制备磁性玻璃颗粒的方法。
按照专家的说法，颗粒是直径小的固体材料。这里谈到的颗粒也常称为色素。按照本发明，平均颗粒尺寸小于100μm的颗粒特别适用。更为适用的是平均颗粒尺寸10-60μm的颗粒。颗粒尺寸的分布最好较均匀。特别是不含尺寸＜10μm或＞60μm的颗粒。
会受磁体吸引的材料被认为是磁性的，例如铁磁或超顺磁材料。此外，被叫做软磁的那些材料也被认为是磁性的，如铁素体。按照本发明特别优选的是铁磁材料，特别是它们还没有被预磁化过。在这里预磁化意指与磁体接触，从而增加了剩磁。特别优选的是铁磁材料，如磁铁矿(Fe3O4)或Fe2O3。
颗粒的外表面意指邻接的表面，从表面向颗粒外面的环境画垂线，它不穿会过颗粒自己。
孔就是颗粒外表面的凹进处。该表面一直深达颗粒的这个位置，即在凹进处表面向颗粒相邻环境画的垂线至少穿过该颗粒一次。此外，孔深达颗粒内的深度大于孔的半径。
按照本发明的玻璃就是一种包含硅的非晶形材料。玻璃可以包含其它材料如下：
    B2O3                (0-30％)
    Al2O3               (0-20％)
    CaO                   (0-20％)
    BaO                   (0-10％)
    K2O                  (0-20％)
    Na2O                 (0-20％)
    MgO                   (0-18％)
    Pb2O3               (0-15％)
玻璃还可以包含较小百分数(0-5％)的许多其它氧化物如Mn2O3，TiO2，As2O3，Fe2O3，CuO，CoO等。由硼硅酸盐玻璃，火石玻璃或二氧化硅组成的表面被证实特别有效。根据核酸的收率，特别优选的是硼硅酸盐玻璃，其氧化硼含量超过25％。SiO2/B2O3成分为70/30的玻璃是特别优选的。按照本发明特别优选的是用溶胶凝胶法成型然后干燥和压缩的玻璃。这种方法的基本原理是已知的，并有有所报道，例如C.J.Brinker，G.W.Scherer“溶胶凝胶科学-溶胶凝胶工艺的物理化学”，AcadamicPress Inc.1990，溶胶凝胶光学，工艺和应用，Lisa C.Klein，Ed.，Kluwer Academic Publishers 1994，p.450 ff.，和DE-A-1941191，DE-A-3719339，DE-A-4117041和DE-A-4217432。但是到目前为止，磁性颗粒的原理尚未有报道。但是这种方法可以用来创造一种磁性颗粒，当用它们来离析生物物质特别是核酸时所具有的令人惊奇的特性是没有预计到的。在凝胶溶胶法中，构成骨架的成分，如SiO2，B2O3，Al2O3，TiO2，ZrO2，GeO2的醇盐与其它组分如在酒精溶液中的氧化物和盐化合，然后被水解。下面的方程描写了制备硼铝硅酸钠玻璃的方法： NaOH + B 2 O 3 + Al ( OR ) 3 + Si ( OR ) 4 → - ROH + H 2 O → NaOH + B 2 O 3 + ⟨ Al ( OH ) 3 ⟩ + ⟨ Si ( OH ) 4 ⟩ →
Figure A0310663600064
加入水以使起始的组分开始水解。因为碱性离子对硅酸酯的水解速度有催化作用，所以反应进行得相当快。一旦凝胶形成，就可以利用热处理过程进行干燥并密实以形成玻璃。
溶胶：色素的比例对本发明的磁性色素的收率有相当大的影响。比例受如下的原因限制，色素的份额必须小到所产生的浆状物仍能被泵送和喷雾。如果色素的份额太小，则粉末部分如非磁性材料变得太大并造成干扰。从色素的收率来讲，发现10-25克色素∶100毫升溶胶的比例是适用的。
为造备粉末，浆液最好通过管嘴喷雾，气溶胶在下降过程中就被干燥了。管嘴最好要加热以加速浆液的干燥。根据管嘴的尺寸，管嘴温度优选地为120-200℃。通过选用足够的蒸发速度但又避免过热可以找到折中方案。
为了收率的最佳化，密实温度应尽可能高。但是如果过高的话，颗粒会粘在一起并形成结块，它必须被筛掉。在过高的温度下对颗粒作进一步处理会造成丧失磁性。因此过高的温度应该不于考虑。
基本无孔的表面意指孔(如上所述)所占面积小于5％的表面，优选地小于2％，特别优选地小于1％。如果有孔存在，其直径最好小于10nm，特别优选地为小于1nm。
按照本发明特别优选的颗粒是含有带TiO2包层的云母核的颗粒，磁性颗粒固定在其上面。在这种设计中，形成的复合材料由玻璃层包围。核和磁性颗粒两者都是晶状无孔的。云母表面的未被磁性颗粒占据的空间由玻璃层包复，它要比磁性颗粒顶部要厚，结果基本上是一种无孔的玻璃表面。
磁性颗粒无孔性仅在外表面而不在颗粒内部。因此在颗粒内部可以有孔，只要表面基本上被无孔玻璃或带有直径小于10nm的玻璃包围。
令人惊奇地，本发明提供的磁性颗粒特别适用于把生物物质从试样中离析。特别是当长链核酸固定在上面时，它们不被破坏，或仅仅最少量地被破坏。此外，核的材料是天然的，因此不引起生态学的问题。此外，按照本发明的颗粒既便宜又容易制备。
本发明还涉及具有玻璃表面的铁磁颗粒。超顺磁颗粒早先已有报道。已经证实用玻璃表面包复的铁磁颗粒对离析生物物质表现出相当大的优越性。如果铁磁颗粒未与磁场接触，则重力是造成它们沉淀出来的唯一的力。摇晃溶液可使它们既快又容易地再悬浮起来。不利用磁场的沉淀过程最好进行得比生物物质在颗粒表面的固定要慢。对核酸来讲更是这样。利用磁场铁磁颗粒可以很容易地在试样流体中一个特定的位置收集起来。然后把流体与颗粒分离，因此也就是与固定其上的生物物质分离。
本发明提供的铁磁颗粒的玻璃表面可以是无孔或者有孔的。由于上面对本发明提供的磁性颗粒所述的原因，铁磁颗粒的外表面也是基本上无孔或者有直径小于10nm的孔。本发明提供的铁磁颗粒的尺寸在10-60μm，特别优选为20-50μm。特别优选的颗粒是其表面孔(如果有)的直径小于10nm，特别优选的为1nm。本发明铁磁颗粒的一个例子是上面所描写的由云母和磁性颗粒构成的复合材料，外面包着一层玻璃。
本发明还涉及离析生物物质的方法，此方法是通过：
-使流体中所包含生物物质的样品与本发明的磁性颗粒或本发明的铁
磁颗粒接触，其条件为生物物质结合到颗粒表面上去，和
-从流体中分离出生物物质。
生物物质意指带有特定基础或分子基础的物质。它们特别包括，细胞如病毒或细菌，以及离析的人类和动物的细胞如白细胞，和免疫活性低或高的分子化学化合物如半抗原，抗原，抗体和核酸。核酸如脱氧核糖核酸或核糖核酸是特别优选的。
本发明的试样包括医学试样如血，血清，漱口水，尿液，脑液，痰液，大便，活组织样品和骨髓样品。试样也可能是用于环境分析，食品分析或分子生物学研究，例如来自细菌培育，噬菌体溶菌液和扩增过程的产物如PCR。
本发明的颗粒有一个内核，外面包了一层玻璃表面。核可以是复合材料，或者是一个简单的铁核。核可以由结晶体，陶瓷或类似玻璃的结构构成，其中埋入了氧化铁。
所描述的方法可以用来离析天然或者修饰的生物物质。天然生物物质就是一种其结构与天然存在的生物物质相比没有不可逆的改变的物质。但是这并不意味试样的其它成分不可以被修饰。举例来说，如果细胞被离析，包围细胞的介质可以修饰，但不是细胞本身。如果核酸被离析，它们应该按它们天然的形式即非变性地切割或修饰，不要把它们通过和活性基团结合进行切割或修饰。因此，天然生物物质的概念特别是不包括生物素化的核酸。天然生物物质的例子有噬菌体脱氧核糖核酸或血液细胞核酸。
修饰的生物物质包括自然界不存在的物质，例如通过接上活性的，可探测的或有固定能力的基团修饰的核酸。这种例子有生物素化的核酸。
某些情况下，按照本发明试样可以不经予处理而用于本发明的离析过程中。但是，在许多情况下试样应使用适当的方法裂解，释放包含在试样中的生物物质。专家知道裂解试样的方法，它可以是化学的，酶的或物理的。也可以是这些方法的组合。例如，可以用超声，高压，利用剪切力，用碱，洗涤剂或离液序列高的盐溶液，或者利用蛋白酶或脂肪酶。
关于用裂解得到核酸的方法，特别要引用Sambrook et al.：MolecularCloning，A Laboratory Manual，2nd Addition，Cold Spring HarbourLaboratory Press，Cold Spring Harbour，NY and AuSubel et al.：CurrentProtocols in Molecular Biology 1987，J.Viley and Sons，NY。
除了要被离析的生物物质外，试样也可能包含流体中的其它成分如细胞残基，蛋白质，盐和其它不被离析的物质。优选地包含天然形式生物物质的试样，在靶生物质可结合到颗粒表面上的条件下与颗粒接触。这个条件依赖于有关的生物物质的类型，但这基本上是已知的。它们也依赖于生物物质结合到表面上去的方法。举例来说，如果是免疫相互作用结合，则必须选择适宜于免疫络合物形成的条件。如果使用修饰的核酸，结合可以通过体现修饰的核酸基团，即生物素与链酶抗生物素包复表面来进行。但是，特别是对于核酸，优选的是核酸与玻璃直接结合，因为除了其它原因之外，核酸不必要经过修饰，甚至天然核酸也可以被结合。天然核酸与玻璃颗粒结合的方法类似于早先报道过的方法。最好在离液序列高的盐中进行，其浓度在2-8mol/l，优选地4-6mol/l。离液序列高的盐可以是碘化钠，高氯酸钠，硫氰酸胍，异硫氰酸胍或氢氯化胍。其它化合物也是可能的。
为使试样和颗粒接触，将试样和颗粒混合并培育一段足够的时间使结合发生。专家通常熟悉用非磁性颗粒处理的方法所需的培育时间。通过在不同时间点测定固定到表面的生物物质的数量可以使这一步最佳化。对核酸来讲，培育时间在10秒到30分之间是合适的。
根据磁性颗粒的尺寸和类型，颗粒可以在培育阶段从流体中分离出来，也可以让悬浮体保持较长时间不动。如果颗粒很小和超顺磁的，则悬浮体保持较长时间不变。如果颗粒尺寸大，在培育阶段颗粒会慢慢从流体中分离出来。这种性质的结块特别在涉及铁磁颗粒时会形成。当铁磁颗粒没有予磁化，这是优选的，可以保证只有轻度分离。
固定作用最好不要借助降低被固定物质的溶解度而产生沉淀的方法来进行。相反，固定要基于生物特异的相互作用(俘获分子)或吸附。这就大大地防止非特异性的污染。
培育以后，把生物物质从流体中分离。一般使用磁场把结合在磁性颗粒上的物质分离。例如，磁性颗粒可以被吸引到进行培育的器壁。然后，含有未被结合到磁性颗粒上的试样成分的流体可以被去除。所用的去除方法取决于培育容器的类型。合适的方法包括通过移液管或抽吸器去除流体。
如果需要，可以用洗涤液净化磁性颗粒一次或多次。所用的洗涤液不会造成生物物质离开颗粒表面，而是尽可能彻底地洗掉不希望的污染物。洗涤步骤最好在洗涤液与颗粒培育时进行。这时颗粒最好又悬浮起来，即通过摇晃或施加磁场，此磁场与第一磁场不一样。被污染的洗涤液最好就和上面描写的试样一样在结合生物物质的那一步被分离。
在最后一步洗涤之后，磁性颗粒可以在真空中被初步干燥，或让流体蒸发。也可以用丙酮进行予处理。
如果需要，以这种方式净化的生物物质可以与磁性颗粒分离。这一步也取决于生物物质结合到磁性颗粒上的方式。如果生物物质是天然核酸，并且磁性颗粒是玻璃包复的颗粒，按照本发明核酸可以用低盐含量的洗脱缓冲液从颗粒上移去。这种性质的缓冲液可从DE 3724442和分析生物化学175，196-201(1988)中了解。低盐含量的洗脱缓冲液即特别是含量小于0.2mol/l的缓冲液。在一个特别优选的实施方案中，洗脱缓冲液含有三甲醇氨基甲烷。在另一个特殊的实施方案中，洗脱缓冲液是无离子水。
在另一个实施方案中，所描述的净化和离析过程在细胞(如病毒颗粒或原核或真核细胞)从体液或细胞组织免疫磁化分离以后进行。在这一步中，如在摇晃的同时样品与颗粒进行培育，抗体挨着细胞上的抗原被固定在颗粒上。这些颗粒可以是按照本发明的颗粒或者是市场上可买到的颗粒。(如Miltenyi Biotec GmbH，Bergisch Gladbach，Germany的MACS微粒)。在加上磁场后，用盐溶液进行一次或多次洗涤。获得结合有所希望的细胞的颗粒。然后，结合的细胞在盐缓冲液中再悬浮。在一个优选的实施方案中，这种盐缓冲液是一种离液序列高的盐溶液，以致包含在细胞中的核酸被从细胞中释放出来。
一种特别优越的把核酸从含细胞试样中离析出来的方法是把上述的细胞离析和上述的核酸-优选地以它们天然的形式-的离析，在本发明的磁性颗粒上结合起来。这个实施方案的优越性是潜在的简单性(单试管方法)，高灵敏度(在医药微生物学和肿瘤学上特别重要)，和易自动化。
使用本发明的方法离析的生物物质，需要时可以进一步被利用。例如，它们可以被用来作为许多酶反应的基质。当涉及核酸时，它们可以被用来确定序列结构，放射性或非放射性标记，它们包含的一个或多个序列结构的扩增，转录，与标记的示踪核酸杂化，转译或连接。本发明方法的优点是生物物质与流体非常容易分离。早先的方法中，使用离心分离来把玻璃颗粒与污染物分离，或者，当生物物质是结合到玻璃纤维过滤器上时，流体通过过滤器被去除。这是卡关的一步，它使处理大量试样很困难。
使用本发明的颗粒，生物物质可以更有效地与污染物分离。特别是按照本发明可在很大程度上去除某些酶反应的抑制剂。生物物质的收率相当高。没有观察到长链核酸的断裂。本发明的颗粒最好是能被较快磁化的颗粒。
图1表示核酸从包含细胞的试样中的离析。
图2表示按照本发明离析的核酸在琼脂糖凝胶中的分离。
图3描述在按照本发明离析后反应产物的分离和利用PCR的扩增。
图4给出显示例4结果的凝胶。
图1表示核酸从包含细胞的试样中的离析。包含细胞的试样(试片)经予处理成试样专有的形式，以致欲探测核酸的细胞就以合适的形式存在。例如，当使用已去除体液的试样时，需要添加试剂，如液化粘稠的试样如唾液。将结合到固相上，最好是微珠上的能探测和结合细胞的抗体加入到含有这种被处理试样的容器中。例如，细胞表面的抗原被证明是抗体适宜的配体。抗体的特性依赖于所作分析的特性。如果固相是容器壁，细胞就直接结合到壁上。如果固相由微珠所组成，则使用适当的分离方法，将其与流体分离。举例来说，可以利用过滤来进行。如果使用磁性微珠，在容器外壁加磁场就可以把它们分离。分离的细胞用液体洗涤以与包围细胞的介质一起把污染物(它们会干扰测量)去除。优选的条件是细胞既不从固相上分离也不被破坏。然后细胞被破坏，即裂解。例如，这可以通过用离液序列高的盐处理细胞来进行。其它的可能性包括加入蛋白酶和洗涤剂。
优选的实施方案中，本发明的颗粒被加到裂解混合物中。经适当的时间裂解后-这可通过吸附于表面的核酸来最佳化-颗粒与包含其它不需探测的细胞成分的流体分离。最好利用在容器壁上放一块磁体所产生的磁场来进行。
为了去除还可能存在的任何污染物，最好用一种不引起被探测的核酸从玻璃表面分离的流体进行洗涤。然后加入一种含有可将核酸从玻璃表面分离的试剂的洗脱缓冲液，以把核酸从玻璃表面分离。这种条件就是低盐条件。根据核酸进一步使用的打算，可以与颗粒分离并作进一步处理。分离步骤最好是通过加磁场以达颗粒相互分离。
下面的实例将更详细解释本发明。实例1本发明磁性颗粒的制备使用六种溶胶。溶胶的制备如下：溶胶1(SiO2∶B2O3＝7∶3)：合成在250ml圆形烧瓶中进行，同时不停地搅动。86.6ml四乙基正硅酸盐+7ml无水非变性乙醇+14.1ml 0.15M HCl产生一种两相混合物。在室温情况下一直搅动到成为单相。一滴一滴加入+37.8ml硼酸三甲酯然后溶胶在50℃下放置2小时。加入+14.1ml 0.15M HCl溶胶2(SiO2∶B2O3＝4∶1)：合成在250ml圆形烧瓶中进行，同时不停地搅动。100.5ml四乙基正硅酸盐+7ml无水非变性乙醇+16.3ml 0.15M HCl产生一种两相混合物。在室温情况下一直搅动到成为单相。一滴一滴加入+25.6ml硼酸三甲酯然后溶胶在50℃下放置2小时。加入+16.3ml 0.15M HCl溶胶3(SiO2∶B2O3＝85∶15)：合成在250ml圆形烧瓶中进行，同时不停地搅动。107.8ml四乙基正硅酸盐+7ml无水非变性乙醇+17.5ml 0.15M HCl产生一种两相混合物。在室温情况下一直搅动到成为单相。一滴一滴加入+19.4ml硼酸三甲酯然后溶胶在50℃下放置2小时。加入+17.5ml 0.15M HCl溶胶4(SiO2∶B2O3＝4∶1；2Mol％P2O5)：合成在250ml圆形烧瓶中进行，同时不停地搅动。100.5ml四乙基正硅酸盐+7ml无水非变性乙醇+16.3ml 0.15M HCl产生一种两相混合物。在室温情况下一直搅动到成为单相。一滴一滴加入+25.6ml硼酸三甲酯然后溶胶在50℃下放置2小时。加入+16.3ml 0.15M HCl+1.63g P2O5溶胶5(SiO2∶B2O3＝4∶1，Mol％Al2O3)：合成在250ml圆形烧瓶中进行，同时不停地搅动。100.5ml四乙基正硅酸盐+7ml无水非变性乙醇+16.3ml 0.15M HCl产生一种两相混合物。在室温情况下一直搅动到成为单相。一滴一滴加入+25.6ml硼酸三甲酯然后溶胶在50℃下放置2小时。加入+16.3ml 0.15M HCl+3.06g Al2O3溶胶6(SiO2∶B2O3＝4∶1，Mol％ZrO2)：合成在250ml圆形烧瓶中进行，同时不停地搅动。100.5ml四乙基正硅酸盐+7ml无水非变性乙醇+16.3ml 0.15M HCl产生一种两相混合物。在室温情况下一直搅动到成为单相。一滴一滴加入+25.6ml硼酸三甲酯+5.15ml锆(IV)-丙基酸，70％(重量)1-丙醇溶液然后溶胶在50℃下放置2小时。加入+16.3ml 0.15M HCl再在50℃下放置2小时后，每150ml溶胶加入22.5g lriodin 600(黑云母)并搅动。然后用喷雾干燥器(B①chi190，Mini Spray Dryer)进行包复。喷雾干燥器管嘴温度为134℃。然后喷雾干燥过程得到的粉末要在氮的气氛(90l/h)下进行温度处理。温度以1k/min的速率增加，粉末在密实化温度中保持2小时。用溶胶1包复的情况，温度为750℃，用溶胶2包复为860℃。对所有其它包复过程温度为800℃。烧结处理以后，炉子就关掉，使粉末处于室温。使用50μm的筛子把结块筛掉。实例2GMP1，GMP2，GMP3和GMP4的制备GMP1，GMP2，GMP3和GMP4是从溶胶1(实例1)按实例1描述的过程在如下条件下制备的不同批色素：
参数 GMP1  GMP2  GMP3  GMP4
溶胶的老化(h)(30℃) 36  36  36  36
溶胶中色素百分数(g/100ml) 5  15  8  20
管嘴气流(％) 100  100  100  100
空气压力(bar) 6  6  6  3
管嘴温度(℃) 135  120  130  143
密实化温度(℃) 534  534  534  615
紧接的氧处理时间(1小时) (300℃) (300℃) (300℃) (400℃)
色素收率 低 高 中 高
脱氧核糖核酸收率 低 高 高 高
实例3使用磁性玻璃颗粒对人的全血PCR试样予处理核酸离析玻璃磁性颗粒3份(GMP2-4)，每份10mg放入微量试管。精确的试样重量在表1上给出。进行三次重复测定。
用移液管把40μl蛋白酶K(20mg/ml，由低压冻干制成)放入200μl解冻的全血中并立即混合。下一步加入200μl结合缓冲液(6M胍-HCl，10mM三甲醇氨基甲烷-HCl，10mM尿素，30％Triton X-100，pH4.4)并混合，然后在70℃下培育10分钟。加入200μl异丙醇，然后制剂在vortex混合器中混合10秒。试样在室温放置20分钟，然后再一次混合10秒钟。磁分离这一步在Boehringer Mannheim(ID#1 641 794)的磁性颗粒分离器中进行至少30秒。按下面描述取出上清液并进行分析。
磁性颗粒用500μl洗涤缓冲液(20mM NaCl，10mM三甲醇氨基甲烷-HCl，pH7.5(25°)，80％乙醇)混合10秒钟进行洗涤，在室温中放置1分钟，然后混合10秒钟。然后用磁性颗粒分离器把它们吸引到容器壁。弃去上清液。重复洗涤过程直到清洗液没有颜色为止(总共四次)。然后核酸经洗脱三次，每次用预热到70℃的200μl洗脱缓冲液(10mM三甲醇氨基甲烷-HCl，pH8.5)，然后混合10秒钟，在室温放置10分钟，再混合10分钟。上清液的制备
对在第一次结合到磁性玻璃颗粒上以后得到的上清液中的核酸含量进行如下研究：上清液放入滤管(Boehringer Mannheim ID#1744003，如在高纯PCR产品净化包内提供的)，并在微量台式离心机以8000rpm离心分离1小时。弃去流出的物质，过滤管用500μl洗涤缓冲液洗涤两次(如上述进行离心分离)。过滤管经离心分离近干，然后用2×200μl预热到70℃的洗脱缓冲液进行洗脱，并再次离心。结果
                上清液1∶8                       第1次洗脱液1∶8
              260      280nm     收率    260/28    260      280        收率260/28
              nm                          0        nm       nm                  0GMP/2 12mg   1    0,021    0,013     1,7μg   1,6      0,171    0,164    13,7μg    1,0
  10mg   2    0,045    0,035     3,7μg   1,3      0,137    0,138    11,0μg    1,0
  9mg    3    0,036    0,027     2,9μg   1,3      0,153    0,164    12,2μg    0,9GMP/3 10mg   1    0,050    0,042     4,0μg   1,2      0,245    0,246    19,6μg    0,9
  10mg   2    0,033    0,022     2,6μg   1,5      0,397    0,398    31,8μg    1,0
  10mg   3    0,042    0,030     3,4μg   1,4      0278     0,282    22,2μg    0,9GMP/4 10mg   1    0,065    0,056     0,7μg   1,2      0,135    0,142    11,0μg    1,0
  11mg   2    0,071    0,142     2,4μg   0,5      0,140    0,142    11,2μg    1,0
  10mg   3    0,066    0,051     1,7μg   1,3      0,130    0,130    10,4μg    1,0第2次洗脱液1∶8    第3次洗脱液1∶4
              260      280nm     收率     260/     260      280      收率       260/     ∑
              nm                          280      nm       nm                  280    洗脱液GMP/2        1    0,099    0,101     7,9μg   1,0      0,057    0,062    2,3μg     0,9    23,9μg
         2    0,078    0,076     6,2μg   1,0      0,041    0,049    1,6μg     0,8    18,8μg
         3    0,103    0,112     8,2μg   0,9      丢失GMP/3        1    0,147    0,147     11,8μg  1,0      0,084    0,098    3,4μg     0,9    34,8μg
         2    0,256    0,252     20,5μg  1,0      0,042    0,043    1,7μg     1,0    54,0μg
         3    0,147    0,143     11.8μg  1,0      0,073    0,093    2,9μg     0,8    36,9μgGMP/4        1    0,106    0,108     8,5μg   1,0      0,083    0,098    3.3μg     0,8    22,8μg
         2    0,111    0,114     8,9μg   1,0      0,054    0,063    2,2μg     0,9    22,3μg
         3    0,135    0,141     10,8μg  1,0      0,077    0,095    3,1μg     0,8    24,3μg表1：使用磁性玻璃颗粒和200μl血的核酸收率洗脱液和试样上清液的分析在50μl洗脱液中和用过滤管制备的上清液中分别加入10μl试样缓冲液。45μl的制剂在0.8％的琼脂糖凝胶中用120V的电泳分离90分钟。不同稀释度的洗脱液和制备的上清液用Uvikon710(Kontron)光谱仪在260和280nm处进行光谱测定。对两等份5μl的洗脱液使用ExpandTM Long Template PCR(BoehringerMannheim ID#1681834)用人类tPA基因(预计产物长度15kb)的特定引物进行了重复测定。
混合物1         每批          混合物2             每批dNTP，每批100mM     lμl     ExpandTM缓冲液，10X      5μl引物1，200ng/ml     1μl     ExpandTM聚合酶           0.75 μl引物2，225ng/ml     1μl     两次蒸馏，H2O            19.25μl两次蒸馏H2O        17μl
                20μl                              25μl将混合物1与5μl洗脱液放入一个薄壁PCR管中，然后加入混合物2。该制剂进行初步混合后用一层30μl的矿物油覆盖。该制剂用一台PerkinElmer thermal cycler 9600按如下设定参数进行扩增：2分           92℃10秒          92℃30秒          65℃          10循环12分          68℃10秒          92℃30秒          65℃          20循环12分+         68℃每循环20秒7分           68℃然后          7℃
将10μl试样缓冲液加到含50μl制剂的PCR管中，45μl的混合物在0.8％的琼脂糖凝胶中进行120v的电泳分离90分钟。第1次洗脱液颜色还轻微带黄并且被细的磁性颗粒轻微污染。在琼脂糖凝胶(图2)中洗脱物的分析表明收率的重复性很好。磁性颗粒GMP2-4表明没有显著的不同。洗脱液1(上)和洗脱液2(下)几乎含有相同浓度的核酸(用凝胶来估计)。洗脱液3中的核酸浓度低。上清液也含低浓度的核酸。除个别外，ExpandTM PCR对所有的试样都得到非常好的特定的扩增产物(表2)。当使用磁性玻璃微粒时，核酸从人血试样中离析，然后在PCR步骤中产生特定的扩增。
                 15kb ExpandTMPCR       人类tPA基因
                   第1次洗脱液           第2次洗脱液GMP/2    1                不适用        +         +
     2      +           +           +         +
     3      +           +                   不适用GMP/3    1      +           +           +         +
     2     (+)          +           +         +
     3      -          (+)          +         +GMP/4    1      +           +           +         +
     2      +           +           +        (+)*
     3      +           +                   不适用K，BM Control DNA表2：ExpandTMPCR的结果
 *第3次洗脱液图3表示在PCR扩增后含反应产物的凝胶。MWM III是克分子量标记(洗脱液1，上；洗脱液2，下)。实例4把DNA长度标准结合到磁性玻璃颗粒上1.磁性玻璃颗粒的准备将12mg GMP4玻璃磁性颗粒放入微量试管。2.裂解和结合900μl裂解缓冲液(4.6M GuSCN，45mM三甲醇氨基甲烷，20mM EDTA，pH7.3)和100μl DNA试样，其中加入Boehringer Mannheimm(Cat.No.528552)的DNA长度标准III作为标样，并在1.5ml的微量试管中与12mg磁性玻璃颗粒混合2-10秒直到得到一个均匀的悬浮液为止。该溶液在室温培育20分钟，并每5分钟混合一次。在磁性颗粒分离器中至少进行15秒磁分离。用移液管去掉上清液。3.洗涤和干燥用移动磁场的方法洗涤磁性玻璃颗粒，用洗涤缓冲液(5.2M GuSCN，50mM三甲醇氨基甲烷，pH6.5)洗涤两次，用70％预冷的乙醇洗两次，再用丙酮洗一次，用移液管加入800μl溶液，混合2秒钟，在室温中放置1分钟，加上磁场，然后通过移液管去掉上清液。去除丙酮，颗粒在敞口的加热装置内于56℃下干燥10分钟。4.DNA洗脱DNA用4×50μl洗脱缓冲液(10mM三甲醇氨基甲烷-HCl，1mM EDTA，pH8.0)在56℃下不停地摇晃地培育10分钟来进行洗脱。然后含有DNA的上清液用移液管移到新的微量试管中。5.洗脱液分析五分之一体积的洗脱液与试样缓冲液混合，在1％的琼脂糖凝胶中在90V下进行DNA分离。为了测定收率，把DNA长度标准III的一系列稀释液加到同一种包含有试样预计数量DNA的凝胶中。用扫描法对琼脂糖凝胶的Polaroid照片进行定量评定。以标准稀释系列用作定标。使用磁性玻璃颗粒的DNA的收率在表1中给出。
标  标准  标准准  中    亮度编  DNA   (测号  数量  量)[ng]  [相对单位] 试  色素/微  试样亮  凝胶   试样   收率样     珠     度(测   中     中    [％]编    类型    量)[相  DNA   DNA号           对单位]  计算  计算总量  总量
 1  200    652  175    563  150    514  125    445  100    376  75     257  50     178  25     99  10     4  1    GMP4      45    139    695   69,52    GMP4      39    120    600   60,0
表1：使用磁性玻璃颗粒时DNA长度标准III的收率用作定量评定基础的琼脂糖凝胶在图4中给出。它是1％溴化乙锭-染色的琼脂糖凝胶。第1列到第10列相当于DNA长度标准III的一稀释系列液。它们是1：1 g DNA，2：200ng DNA，3：175ng DNA，4：150ng DNA，5：125ng DNA，6：100 DNA，7：75ng DNA，8：50ng DNA，9：25ngDNA，10：10ng DNA。第11和第12列相应于从磁性玻璃颗粒洗脱得到的DNA，并加入200ngDNA长度标准。
序列表(1)总信息(i)申请人：(A)名字：Boehringer Mannheim GmbH(B)街道：Sandhoferstr.116(C)城市：Mannheim(D)国家：DE(E)邮政编码：68298(F)：0621 759 4348(G)：0621 759 4457(ii)发明名称：磁性色素(iii)序列数：2(iv)计算机可读形式：(A)数据载体：软盘(B)计算机：IBM PC兼容(C)操作系统：PC-DOS/MS-DOS(D)软件：Patentln Release #1.0，Version #1.30(EPA)(2)序列识别号#1的信息：(i)序列识别：(A)长度：34碱基对(B)类型：核苷酸(C)螺旋体类型：单(D)结构：线性(ii)分子类型：其它核酸(A)描述：/deSc＝“oligodeoxyribonucleotide”(iii)假设：无(iv)序列描述：序列识别号：1：
ACTGTGCTTCTTGACCCATGGCAGAAGCGCCTTC 34(2)序列识别号#2的信息：(i)序列识别：(A)长度：34碱基对(B)类型：核苷酸(C)螺旋体类型：单(D)结构：线性(ii)分子类型：其它核酸(A)描述：/dese＝“oligodeoxyribonucleotide”(iii)假设：无(iv)序列描述：序列识别号：2：
CCTTCACTGTCTGCCTAACTCCTTCGTGTGTTCC 34

Claims (11)

1.一种离析核酸的方法，它包括
将流体中含有天然形态核酸的试样与具有玻璃表面的磁性颗粒在核酸能直接结合到玻璃表面上去的条件下接触，和
从流体中分离结合的核酸。
2.权利要求1的方法，其特征在于磁性颗粒在与试样接触时并未预磁化。
3.权利要求1或2的方法，其中所述具有玻璃表面的磁性颗粒的玻璃表面含有硼的氧化物。
4.权利要求1-3之任一项的方法，其特征在于所述具有玻璃表面的磁性颗粒的玻璃表面基本上不含孔或只含直径小于10nm的孔。
5.权利要求1或4的方法，其特征在于所述具有玻璃表面的磁性颗粒的玻璃表面含有直径小于100μm的孔。
6.权利要求1-5之任一项的方法，其特征在于所述玻璃表面所含的所有孔的直径小于1nm。
7.权利要求1-6之任一项的方法，其特征在于所述颗粒含有以云母为芯和固定其上的磁性颗粒的复合材料，所述复合材料被埋入一玻璃层中。
8.权利要求1-7之任一项的方法，其特征在于所述具有玻璃表面的磁性颗粒由如下方法制造：
提供一种磁心并通过在磁心表面沉积含有网络形成组分的醇化物的醇溶液形成的溶胶，将所述磁心包封在基本上无孔的玻璃表面中；
用喷雾干燥法将所述溶胶层转化为凝胶层；此后
将所述凝胶层致密化。
9.权利要求1-3之任一项的方法，其特征在于该分离是中磁铁帮助下进行的。
10.铁磁性颗粒在离析天然形式的核酸中的应用。
11.具有玻璃表面的铁磁性颗粒在离析天然形式的核酸中的应用。
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