CN1950520B - 用干试剂分离dna的方法和装置 - Google Patents

用干试剂分离dna的方法和装置 Download PDF

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CN1950520B
CN1950520B CN200580013761XA CN200580013761A CN1950520B CN 1950520 B CN1950520 B CN 1950520B CN 200580013761X A CN200580013761X A CN 200580013761XA CN 200580013761 A CN200580013761 A CN 200580013761A CN 1950520 B CN1950520 B CN 1950520B
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沃尔特·冈布雷克特
彼得·波利卡
曼弗莱德·斯坦泽尔
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Abstract

本发明涉及一种在去除干扰后续PCR的成分情况下分离DNA的方法，其具有下列措施：所有物质和方法步骤完全整合在密闭的一次性使用的单元(所谓的药筒)中，该单元允许含DNA的样品的进入；为分离该释放出的DNA使用结合DNA的基底。特别是在应用通过白细胞的细胞分解从全血中分离DNA的方法时，用于实施细胞分解和其它反应所需的试剂以室温下稳定的形式储存，并且为分解白细胞和分离DNA，使干储存的裂解试剂溶于水溶液中，并与白细胞接触。在相关的装置中包括用于接受含DNA的生物容器和/或试剂的单元，因而至少提供一个微通道(5)以容纳试剂，而在微通道中试剂以具有可忽略蒸汽压的干混合物存在，其在室温下形成稳定物质。

Description

用干试剂分离DNA的方法和装置 
本发明涉及一种用干试剂分离DNA的方法。此外本发明还涉及一种用于实施该方法的相关装置。 
在核酸分析要回答如全血的白细胞中人类基因的问题时，在样品制备步骤中首先必需破裂细胞，并接着分离释放出的DNA。这时必须去除可能阻碍后续PCR的血液成分如血红蛋白、免疫球蛋白和乳铁蛋白。 
在实验室这种操作步骤是按熟知的现有技术进行的。除其它方法外，细胞还可用碱性溶液(NaOH)破裂(所谓裂解)，并接着将DNA结合到涂有硅胶的磁珠上。通过施加磁场固定载有DNA的磁珠并洗涤之。其后如此分离出的DNA可以有珠或无珠地用于PCR(“聚合酶链反应”)。 
在现有技术中，该结合DNA的磁珠以悬浮体混入细胞裂解缓冲液中。所有的操作步骤例如均在1.5ml反应容器(所谓的“Eppendorf”容器，即微量离心管)中进行。将如10μl的全血加到给定体积的珠悬浮物中(例如，200μl)。由此血细胞分解并释出DNA。该磁珠结合DNA并形成一种DNA/珠-复合体。接着该DNA/珠-复合体通过磁场固定在“Eppendorf管”的容器壁上，以致可进行洗涤步骤以去除阻碍PCR的物质。最后可进行PCR。 
实施上述方法是依赖于现有的实验室设备如“Eppendorf”容器(管)、管支承设备包括磁体、移液管设备、用于试剂的冷却容器，并必须由经培训过的人员在遵守安全规定(传染危险、废物处置等)下进行。部分对健康有危害的物质(如NaOH)的各种容积和高准确的计量必需通过移液管进行。这些操作步骤也是很耗时的。 
由US 2002/0022261A1已知一种适于微型遗传分析的体系和相关的操作方法，其中应用一种具有至少一个入口和一个多孔性输送道的药筒，该药筒应有结合DNA的特性。这时进行细胞分解，为此需要时在容器壁上提供有试剂。此外，该壁的结构区可配置有结合DNA的材料，需要时还有磁珠。总之其中给出了一系列用于进行基因分析测量的各种建议，但仍未实现连续的方法。此外，由EP 0723549 B1已知一种用于分离DNA的混合物，该混合物特别是含硅胶、玻璃细粒和至少一种离液序列高的盐的混合物。 
此外，在WO 99/33559A中描述了一种用于分离一种流体样品的分析 物的设备，该设备是在样品流路径中包含分解腔的药筒，其中借助于过滤器滤出样品的细胞并使其分解。将如此分离的分析物送入该设备中的各传感器室进行分析。 
基于最近现有技术，本发明的目的在于，将DNA分离完全地实施在整合的微型药筒中，并提供一种相关的装置。 
本发明的目的是通过权利要求1的措施实现的。相关设备示于权利要求13。该方法和相关装置的扩展方案列于从属权利要求中。 
在本发明范围内，权利要求2包括含DNA的生物容器(biologische 
Figure G200580013761X01D00021
)如细胞、细菌或病毒的分解方法的设计。由此可在特别有利的和实用的实践中以DNA信息检测全血样品。 
本发明除了基于上述现有技术外还基于WO 02/072262 A1中的“分析设备”附图。其中描述了干式储存的、在“嵌片”的微通道或微空腔中的、在室温下稳定的试剂，该试剂在使用前加水而变成溶液。该干试剂按预定剂量提供，以在溶解后产生定量的分析介质。对此，在本发明中涉及一种生物容器的细胞分解，例如从全血样品中分离DNA以进行其后的PCR和/或分析。由此以合适的方法提供样品中分离出的DNA。 
与至今应用的实验室方法相比，用本发明方法具有下列优点： 
·所有物质和方法完全整合在一个密封的一次性使用的药筒中； 
·确保该试剂以安全、对健康无害及在室温下可稳定储存的方式储存； 
·除注入血液样品外，无需手工操作步骤； 
·与有害健康的物质无直接接触，即血液和试剂废物留在药筒中；该药筒可小量并低成本批量生产。 
本发明的装置具有下列特征： 
·存在至少一个微通道或微空腔。 
·在微通道或微空腔中，该裂解介质特别在包含成膜剂时与用于DNA的基底一起安置在通道的壁上。 
引入到微通道或微空腔中的裂解试剂具有下列特性： 
·适于白细胞和/或其它细胞、细菌、病毒的溶解特性； 
·具有可忽略的蒸汽压的固体或液体； 
·在室温下稳定； 
·在微通道壁上或微空腔壁上的优良粘附性。 
本发明中重要的是，在连贯的方法链中释放出含于样品中的DNA，收集分离出的DNA并以合适的方法送到PCR或测定的地点。 
本发明的其它详情和优点由下面按实施例的附图描述并结合权利要求 给出。 
附图简介
图1示出分析设备(药筒)的俯视图 
图2-图6各为图1中沿线II-II的截面图，用于说明由全血中的DNA分离，其中图2-6各包括不同的功能阶段。 
下面的描述是阐明含DNA的样品。这类样品可以是液体中DNA的溶液，但是也可以是生物容器的含DNA悬浮物。在本文中例如细胞、细菌或病毒均视为生物容器(biologische 
Figure G200580013761X01D00031
)。为释放DNA必需分解该生物容器。如果相应于人类基因目的使用全血样品，则需对DNA位于其中的白细胞进行细胞分解。 
图1中示出分析设备图，下文中也作为“药筒”，且可作为集中的或分散的测量设备。特别是形成嵌片(“嵌片实验室”)类的分析设备，该嵌片含有用于处理和分析测量样品的所有介质。本设备按规定操作所需要的相关控制设备/读出设备在本文中未给出。 
详述之，该设备由塑料制成的嵌片体1组成，其具有流体的入口和出口。特别是有引入水的入口2(“Port(孔)”)和引入测量样品如血液的入口3(“Port(孔)”)。重要的是通过合适的液流设备2-10，特别是不同几何形状的通道和空腔以传输和汇集测量样品和溶液介质。 
详述之，该液流设备除上述水入口和样品入口2、3外还包括两个试剂通道4、4’以及有出口6的流道5、废物收集通道8和其它液流通道9。用10表示用于样品处理的流道5中的集中混合区。 
在混合段10中处理该样品之后，并分离出含于测量样品中的DNA后，收集释放出的DNA，并送入PCR腔20以进行PCR(聚合酶链反应)。 
图1装置中特别用干试剂进行PCR的方法详述于本申请人的具有同样申请优先权的同时申请并名称为“借助用干试剂的PCR的扩增DNA的方法和装置”中。 
该嵌片1(“药筒”)除包含用于PCR的介质外，还包含具有入口32和出口31的探测模块30以及相关的用于传感器信号读出的电路连接。此外，还有用于接纳探测用试剂的手段如通道4和4’以及用于接纳废物如血和用过的试剂溶液的手段如通道8和9。因此保证了整合性，这样无有害健康的物质 向外排出。 
由图2-6可看出，图1的具有水入口2和血液入口3的塑料嵌片1包含穿流通道5，通过该通道，可将作为冲洗液的水和作为测量样品的例如血或DNA溶液或细胞悬浮液/细菌悬浮液/病毒悬浮液引入其它封闭的体系中。经出口6可将废物转送到废物通道8或废物空腔中。穿流通道5的面对塑料嵌片1的一侧通过胶粘膜7所覆盖。在穿流通道5的中心区形成混合段10，在混合段10中特别地制备用于从全血中DNA分离的测量样品。 
从全血中的DNA分离是通过白细胞的分解而实现。为此，该细胞用裂解试剂进行化学破裂，并将其中所含的DNA释出和收集。特别是利用已知的前述方法即经所谓的磁珠，它至少暂时结合细胞分解后的DNA，并通过外磁场浓集。 
特别是由图2可看出，在塑料嵌片1的穿流通道5的区域10中安置水溶性的储存稳定的干物质11。本文中储存稳定性意指该固体在室温下可储存几个月并还保持对样品起分解作用的特性。 
在图2和3中，该干物质11具有在穿流通道壁上的优良粘附性。例如这可通过加入成膜剂来实现。详述之，在混合段10中呈大面积地安置包括已知的裂解试剂与磁珠13的混合物11。此外，磁铁15象征性地表明应用磁性以收集其上结合有DNA的磁珠13。 
由图3和图4看出，在图2的装置中经移液管尖17将全血12加入到测量体系中。加入样品后，该入口3用胶粘膜18封闭。 
经入口2引入水或缓冲溶液。水或缓冲溶液沿混合段与血液样品12混合，以同样方式溶解或悬浮包括磁珠13的裂解试剂11，并发生作用。为此该白细胞的细胞壁首先通过裂解试剂而破裂，接着将这时释放出的DNA结合在磁珠的表面。 
图4是通过水/缓冲溶液稀释该血液样品，并同时溶解该裂解试剂，该裂解试剂用于确保白细胞分解。以同样方式还使存在于通道壁上混合物11的磁珠进入或悬浮于溶液中，并经磁珠13结合DNA。由此下列4个重要的方法步骤可在单一过程中实现： 
·样品稀释， 
·试剂溶解磁珠悬浮， 
·细胞分解， 
·DNA结合。 
最后按图5进行说明。这时可收集因磁珠13而可受磁性作用的DNA，而PCR阻碍物质由水或缓冲溶液洗去，并经出口6以废物排出。 
作为上述措施的结果，图6中通过磁珠13结合的DNA在磁铁的磁极上的浓集。该DNA可用于分析，这时首先特别要进行PCR。 
另外，作为结合DNA的介质除了可以应用与磁场组合使用的、移动的即悬浮的磁珠之外，也可使用不移动的即位置固定的结合DNA的介质，其定位在细胞分解通道的出口。在此实施方案中，由细胞释放出的DNA在离开细胞分解通道时，通过溢流越过位置固定的结合DNA的介质而由液体流中引出，并结合在结合DNA的介质上。作为结合DNA的介质可使用如具有结合DNA的捕集分子的水凝胶或类似物质。 
通过本发明的方法和相关装置特别可以以一步法实现简单而快速的DNA分离，这仅需要最小的微流体介质、试剂和方法步骤。 
接着可对经分离的DNA进行PCR。采用PCR可将DNA浓度提高到分析可检测值的水平。这时可将PCR组合到分析过程中。该分析在相应于图1的探测模块30中进行，有关这方面这里不再详述。 
总之在本发明中实现了下列措施： 
·在微通道或微空腔中设置的基底具有结合DNA的特性。为此应用例如结合DNA的磁珠； 
·该裂解试剂和磁珠特别地一同包含在单一的干基体中； 
·有适于全血样品、细胞悬浮物/细菌悬浮物/病毒悬浮物的入口； 
·有加水的手段。例如可以是连接到外水源上的输入口； 
·需要时存在用于计量加入如调节所确定的离子强度用的盐的手段； 
·需要时存在用于稀释血液样品的手段(通道和空腔)； 
·需要时存在其它手段，特别是通道和空腔，以用于制备所确定的盐溶液/缓冲溶液以洗涤所结合的DNA，如DNA/磁珠-复合体； 
·存在以血或血/水混合物、血/缓冲液混合物通流具有裂解试剂/珠试剂涂敷过的微通道或微空腔的手段，所述手段优选在药筒外部； 
·存在产生磁场以例如在微通道的出口处固定DNA/磁珠-复合体的手段，该手段优选存在于药筒的外部。 
由此确保包括样品制备的整个分析过程是在封闭的体系中完成，该体系是由环境相容的塑料制成的一次性药筒构成的。 

Claims (25)

1.一种在去除干扰后续PCR的成分情况下分离DNA的方法，其具有下列措施：
·所有物质和方法步骤完全整合在密封的具有一次性使用单元的分析单元(所谓的药筒)中，该单元允许含DNA的样品进入，
·应用干试剂释放DNA，
·应用结合DNA的基底以分离该释放出的DNA，
·收集分离出的DNA，并将其输送到后续PCR的位置，
其中进行含DNA的生物容器，特别是细胞、细菌或病毒的分解；为此用于实施分解和其它的方法步骤所需的试剂以在室温下稳定的形式储存在该单元(药筒)中，其特征在于：
·为分解含DNA的生物容器，使在分解通道的壁上存在的包括裂解试剂和结合DNA的基底的干基体与所述生物容器相接触，所述生物容器特别是细胞、细菌或病毒相接触，
·与裂解试剂相接触而被释放出来的DNA直接、同时结合在结合DNA的基底上，
·收集其上结合了DNA的基底，并将其输送到PCR的位置。
2.权利要求1的方法，其特征在于，作为含DNA的容器使用的是白细胞。
3.权利要求2的方法，其特征在于，该干储存的并且储存稳定的裂解试剂借助于水而与白细胞接触。
4.权利要求1的方法，其特征在于，样品的稀释、干试剂的溶解或悬浮、生物容器的分解和DNA的结合是在单一的方法步骤中实现的。
5.权利要求1的方法，其特征在于，作为结合DNA的基底使用的是所谓的磁珠。
6.权利要求1的方法，其特征在于，作为结合DNA的基底使用的是固定在细胞分解通道出口处的、含DNA捕集物质的水凝胶。
7.权利要求6的方法，其特征在于，该释放出的DNA通过磁珠结合，并输送到收集位置或反应位置。
8.权利要求5的方法，其特征在于，通过施加磁场收集该磁珠。
9.权利要求8的方法，其特征在于，通过位置固定的磁场收集流动的磁珠。
10.权利要求1的方法，其特征在于，在反应位置通过与干储存的水溶性的试剂的热循环来进行该PCR。
11.权利要求1的方法，其特征在于，稀释加入到该单元中的血样品，并通过试剂通道泵入PCR的反应通道。
12.权利要求11的方法，其特征在于，经DNA分离后，残余的血、血成分和试剂废物保留在该单元中，以致不可能发生与有害健康的物质的直接接触。
13.上述权利要求之一的方法，其特征在于，应用一次性产品作为实施反应的单元，该单元可以少量并低成本的批量制备。
14.一种实施权利要求1或权利要求2-13之一的方法的装置，其具有接受样品和/或试剂的单元，其中有至少一个微通道(5)以接受试剂，在微通道(5)中的试剂存在有包括裂解试剂和结合DNA的基底的干基体，所述干基体为具有可忽略蒸汽压的混合物，其在室温下形成稳定物质，并且所述包括裂解试剂和结合DNA的基底的混合物通过加入成膜剂作为层(11)大面积地结合在微通道(5)的壁上。
15.权利要求14的装置，其特征在于，该含DNA的生物容器特别是细胞、细菌、病毒。
16.权利要求15的装置，其特征在于，为实施生物容器的分解，在微通道(5)中的具有可忽略蒸汽压的干物质是裂解试剂，其具有对白细胞的特异性质。
17.权利要求16的装置，其特征在于，用于裂解物质的通道(5)通到PCR空腔(20)内。
18.权利要求14的装置，其特征在于，该基底是结合DNA的所谓磁珠(10)。
19.权利要求14-18之一的装置，其特征在于，存在一种用于全血样品的入口(3)。
20.权利要求14-19之一的装置，其特征在于，存在用于加入水的手段(2)。
21.权利要求14-20之一的装置，其特征在于，存在用于调节确定的离子强度的干缓冲物质。
22.权利要求14-21之一的装置，其特征在于，存在用于混合血液样品和水或缓冲溶液的手段。
23.权利要求21或22的装置，其特征在于，存在使具有血、血/水混合物或血/缓冲液混合物流过用分解试剂/珠试剂涂敷过的微通道(5)或微空腔(20)的手段。
24.权利要求14-23之一的装置，其特征在于，存在用于产生磁场以在PCR空腔(20)中固定DNA/磁珠-复合体的手段。
25.权利要求14-24之一的装置，其特征在于，存在用于热循环的手段。
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US20070219366A1 (en) 2007-09-20
DE102004021780B4 (de) 2008-10-02
DE502005004947D1 (de) 2008-09-18
US8088576B2 (en) 2012-01-03
EP1740722B1 (de) 2008-08-06
WO2005106024A1 (de) 2005-11-10
EP1740722A1 (de) 2007-01-10
CN1950520A (zh) 2007-04-18

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