CN1281462A - 核酸的分离 - Google Patents

核酸的分离 Download PDF

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CN1281462A
CN1281462A CN98811893A CN98811893A CN1281462A CN 1281462 A CN1281462 A CN 1281462A CN 98811893 A CN98811893 A CN 98811893A CN 98811893 A CN98811893 A CN 98811893A CN 1281462 A CN1281462 A CN 1281462A
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M·J·贝克
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Abstract

一种从血液中提取核酸的方法,该方法包括:将血细胞,最好于细胞裂解之后,在一个pH值时与活化固相接触以固定核酸,然后在电荷逆转或变为电中性时在较高pH值下取出核酸。固相可以是用作为结合剂的组氨酸活化的玻珠。血液样品借助气压被抽吸到含有玻珠的柱子上,随即玻珠被流化,从而改善接触,并避免堵塞。

Description

核酸的分离
本发明涉及一种从生物材料，特别是从血液中提取核酸和其他生物分子的方法。
很多情况下需要进行大量的DNA分析，这就需要快速、安全、高产量的分离和纯化DNA及其它核酸的方法。
用于DNA鉴定或分析的样品可以来自多种生物材料，比如，动植物的细胞、排泄物和组织等；也可以从土壤、食料和水等物质中获得。
现有提取DNA的方法包括酚/氯仿法、盐析法、离液盐和有机硅树脂法、亲和树脂法、离子交换层析法以及磁珠法。方法见US专利5057426、4923978，EP专利0512767A1和EP专利0515484B、WO95/13368、WO97/10331和WO96/18731。这些专利和专利申请公开了先将核酸吸附到固体支持物上，然后进行核酸分离的方法。以前的方法都是用某些易燃、可燃或有毒的溶剂分离核酸。
血液是一种用于DNA分析最丰富的样品来源，因为血液样品可由众多原因而常规获得。然而，当应用已有的DNA提取方法时，由于血液的粘稠和富含蛋白酶的性质，处理含有相对少量DNA的大体积血液非常困难，因此难于自动化完成。迄今为止，核酸提取只能依靠应用危险试剂和缓慢的操作步骤来实行部分的自动化。
现在我发明了一种用于从血液和其它生物材料中提取核酸和其它生物分子的改进方法。
本发明提供了一种从生物材料中提取生物分子的方法，该方法包括：首先用能够结合生物分子的固相在第一pH值接触生物材料，然后在第二pH值通过用一种洗脱剂洗脱将已结合到固相的生物分子提取出来。
本方法特别适用于血液材料，也可以广泛用于其它材料，例如质粒和载体的分离，以及植物DNA的提取。
最好将血液中的细胞裂解以释放出核酸分子。可以使用已知的裂解剂和方法，例如与离子型和非离子型去污剂、低渗盐溶液、蛋白酶、离液剂、溶剂接触，改变pH值或加热。分离核酸所采用的裂解细胞的方法见WO96/00228。
当生物材料是血液时，样品可以任意用水或其它溶剂稀释，以便于操作和进一步加工。
样品可以稀释多达十倍使用，一般情况下更高的稀释倍数可能更好。本发明的特点是可以使用高倍稀释的血液。
与血液接触的固相可由对核酸有天然亲和力的材料制成，或者由表面经过某种试剂处理的材料制成，这种试剂使核酸能够结合在固相上或者增加固相对核酸的亲和力。合适的材料包括可控孔度玻璃、多糖(琼脂糖或纤维素)、其它类型硅石/玻璃、陶瓷材料、多孔塑料材料，例如多孔塑料塞(它在一个单独的模制部分中，或是一个标准管中的插入体)、聚苯乙烯珠和顺磁珠等。材料的尺寸和孔隙度没有严格要求，根据不同的用途可以有不同的选择。
处理固相表面或者衍生化加工固相的合适方法包括用一种能够在固相表面导入电荷，例如正电荷，或者给固相加上亲水或疏水表面，例如羟基、硝基、自反应基团、染料和其它芳香族化合物的物质处理固相表面。
在本发明的优选实施方案中，固相可以使DNA在一个pH值时优先于血液样品中的杂质结合到其上，而在不同的pH值时由于洗脱剂的接触，已经结合到固相的核酸将被释放出来。本系统可以与掺入了组氨酸或多聚组氨酸的固相一起使用，所述固相在较低的pH值时，例如pH值小于6时将结合核酸，而在pH值升高时，例如大于8时已结合的核酸被释放出来。或者，核酸在基本中性的pH值时结合到胺化表面，而在非常高的pH值时被释放出来。
在本发明的另一实施方案中，可以向塑料器皿中掺入结合剂，例如在平板的一个孔中，使结合剂被加入到其表面，然将血液样品与表面接触，使核酸结合到表面上。血液样品被取出后，用洗脱剂处理器皿表面，释放出被结合的核酸。当接触表面是多孔平板中一个孔部分时，整个系统适用于快速大规模取样和提取技术。
适用的结合剂包括使用带正电荷的固相的电荷开关型离子交换树脂，该固相可以通过改变pH值到高于其pKa而逆转成带负电荷或变成中性，例如，核苷酸、多胺、咪唑基团和其它具有合适pKa值的类似试剂。
而且，核酸也可以应用掺入到固相的多种嵌入型化合物，例如放线菌素D、溴化乙锭等，通过嵌入反应而结合。
在本发明的另一实施方案中，可修饰塑料表面使其含有官能团。该塑料可以是可用于容纳样品的任何一种塑料制品，例如聚丙烯。官能团可以带正电荷或负电荷，从而在正确的缓冲液中结合核酸。
或者，官能团也可以是能够和其他配基或聚合物共价偶联的化学基团。
当在塑料模具中，例如平板的孔中或PCR管中使用塑料时，塑料表面的性质可以通过在模制化合物(moulding compound)中，例如在注入型模制化合物中包含或加入合适的化学品而改性以用于本发明。
当在PCR管中或在深孔平板中使用塑料表面时，这些管或孔可以用来分离和固定少量的DNA或RNA，得到可用于进行下一步PCR反应或进行其它基因分析和操作的纯化模版。
当塑料是聚丙烯材料时，例如薄壁PCR管，聚丙烯表面可以用氧化剂进行氧化而改性，例如用高锰酸钾和硫酸，以造成羧化表面(COOH基团)。这样的PCR管可以改善从溶液中或粗提样品中，例如血液中，进行DNA的分离。通过调整pH值、介电常数、溶解度或离子强度，可以将DNA或RNA固定在管壁上，并洗除杂质，直接用于PCR或其它分析技术。
羧基基团还可以通过与阴离子基团，例如咪唑、多聚组氨酸、或其它强或弱的离子交换剂共价偶联而被改性，以便凭借电荷的相互作用来结合核酸。这种管可以改善从溶液中或粗提样品中，例如血液中，进行DNA的分离。同样可以通过调整pH值、介电常数或离子强度，将DNA或RNA固定在管壁上，并洗除杂质，直接用于PCR或其它分析技术。
核酸可以在低盐溶液中洗脱，以备用于PCR或其它分析。
通过在混合/搅拌装置中与固相混合，让血液样品经过固相，而使血液样品与固相接触。或者固相可以是顺磁性的并在磁场中操作。尽管本发明特别适用于从血液中分离核酸，但是也可以用于多种生物分子，特别是那些需要除掉细胞壁碎片或不溶性颗粒的生物分子。
在本发明的优选实施方案中，固相在柱中呈颗粒状，血液凭借加在柱上的压差而通过柱，血液样品借助气压流过柱，而颗粒状的固体材料被流化，以加强混合和接触率，并减少堵塞。
本发明的方法适于以多孔方式进行，其中从不同的样品里基本上同时进行一系列的提取，这将便于提取过程的自动化，以完成快速、大量的提取和进行组合化学反应。在一系列标准孔中进行大产量提取，例如在8×12个孔中在同一时间自动处理大量样品。
本发明在实施例中进行说明。
实施例1
从全血中提取核酸
用glutaldehyde将多聚组氨酸共价偶联到100(m玻璃珠上，即将1克胺化玻珠与0．01％(v/v)glutaldehyde在含有20mg多聚组氨酸，pH值8的0.1M碳酸氢钠溶液中混合从而制备电荷开关型离子交换剂。温育过夜后，彻底洗涤玻珠以去除非共价结合的物质，贮存于含有0.1％(v/v)Tween 20，pH 5的10mM MES中。
将100(m衍生化玻珠约300mg加入1ml封住两端的塑料柱中。将血液样品与同体积的含有1％Tween 20、蛋白酶(200(g／ml)和1mMEDTA的10mM MES(pH5)混合。消化完全后，将血液吸入含有玻珠的柱中，DNA随即被固定，杂质蛋白通过柱子流出而被废弃。
含有被固定DNA的玻珠用含有10mM MES pH5的缓冲液洗涤，该MES含有1％Tween20和1mM EDTA。重复洗涤过程，直到洗涤液无色。
洗涤之后，空气干燥玻珠，用少量10mM Tris HCl，pH8.5洗脱DNA，然后收集到无菌管中以备进一步分析实验。这样，即从血液中分离出了DNA。
针对不同的生物分子，缓冲液等可做适当改变。
实施例2
1克羧化顺磁珠用50mM咪唑缓冲液(pH6)洗涤后，与溶于50ml 50mM咪唑缓冲液(pH6)中的100mg多聚组氨酸混合。加入化学偶联剂(EDC)直终浓度为5mg／ml，混合过夜。顺磁珠用含0.5M氯化钠、1％Tween20、100mM Tris HCl pH8的水溶液洗涤，贮存于10mM MES，0.1％Tween20 pH5。
为了从血液中提取DNA，将1mg顺磁珠与稀释于10％Tween20，25 mMMES，1mM EDTA pH5中的血液混合。用磁铁分离出顺磁珠，重悬于1mM MES，0.1％Tween20中洗涤。为了洗脱DNA，将顺磁珠重悬于10mM Tris HClpH8.5，用磁铁将其分离出来，而DNA留存于溶液中。

Claims (35)

1．一种从生物材料中提取生物分子的方法，该方法包括将生物材料在第一pH值与能结合所述生物分子的固相接触，然后在第二pH值用洗脱液通过洗脱将已结合到固相上的生物分子提取出来。
2．根据权利要求1的方法，其中所述生物分子包括核酸。
3．根据权利要求1或2的方法，其中所述生物材料是血液。
4．根据权利要求1、2或3的方法，其中所述生物分子在pH小于6时与固相接触，DNA结合于固相上，而在pH大于8时将DNA从固相中释放出来。
5．根据权利要求1、2或3的方法，其中所述生物分子在基本中性的pH时与固相接触，DNA结合于固相上，而在pH大于10时将DNA从固相中释放出来。
6．根据权利要求3到5中任意一项的方法，其中血液中的细胞通过裂解释放出核酸。
7．根据权利要求6的方法，其中细胞通过与离子或非离子型去污剂、低渗盐溶液、蛋白酶或离液剂接触，或者通过改变pH值或加热而被裂解。
8．根据权利要求3到7中任意一项的方法，其中血液用水或其它稀释剂稀释以便于操作和倾注。
9．根据权利要求8的方法，其中血液稀释度高达10倍。
10．根据前述任一权利要求的方法，其中与生物材料接触的固相由对核酸有天然亲和力的材料制成。
11．根据前述任一权利要求的方法，其中与生物材料接触的固相是由表面经过能使核酸结合其上或增强其对核酸的亲和力的试剂处理过的材料制成。
12．根据权利要求11的方法，其中固体材料的表面用能使固相表面带正电荷或者具有亲水性或疏水性的物质进行处理。
13．根据权利要求11的方法，其中使用的结合试剂是采用带正电荷的固相的电荷开关型离子交换树脂，可以通过改变pH值至其pKa值之上或之下而逆转或中和电性。
14．根据权利要求13的方法，其中结合试剂可以是核苷酸、多胺，或含有咪唑部分的化合物。
15．根据权利要求11的方法，其中利用掺入到固相中的嵌入型化合物，通过嵌入而结合核酸。
16．根据权利要求15的方法，其中染料是放线菌素D或溴化乙锭。
17．根据权利要求11的方法，其中固相材料表面用组氨酸或多聚组氨酸处理。
18．根据前述任一权利要求的方法，其中固相是可控孔度玻璃、多糖、陶瓷材料或多孔塑料材料。
19．根据前述任一权利要求的方法，其中固相包含聚苯乙烯珠或顺磁材料珠。
20．根据前述任一权利要求的方法，其中固相包含经过改性而具有官能团的塑料表面。
21．根据权利要求20的方法，其中塑料是聚丙烯。
22．根据权利要求21的方法，其中聚丙烯表面通过氧化剂氧化而被改性，形成羧化表面。
23．根据权利要求22的方法，其中羧基通过共价偶联阴离子型基团，例如咪唑、多聚组氨酸、或者任何强或弱的离子交换剂而被进一步改性，使核酸借助电荷相互作用而结合。
24．根据权利要求11到23中任意一项的方法，其中所述官能团带正电荷或负电荷以结合核酸。
25．根据权利要求11到23中任意一项的方法，其中所述官能团是能够与其它配基或聚合物共价偶联的化学基团。
26．根据权利要求20到25中任意一项的方法，其中固体材料是多孔塑料塞，它是一个单独的模制部分，或是容器中的插入体。
27．根据权利要求20到25中任意一项的方法，其中塑料处于塑料模具中。
28．根据权利要求27的方法，其中固体材料是聚合酶链式反应(PCR)管。
29．根据权利要求27的方法，其中固体材料是深孔平板。
30．根据权利要求26到29中任意一项的方法，其中管或孔用于分离和固定少量DNA或RNA，得到可用于下一步PCR反应或进行其它基因分析和操作的纯模版。
31．根据前述任一权利要求的方法，其中固相包括颗粒状固体材料，通过使血液样品在混合/搅拌装置中与固相材料混合，使血液样品通过固相，而使血液样品与固相接触，或在可磁化支持物上操作固相。
32．根据前述任一权利要求的方法，其中容器是多孔平板中的孔，可基本上同时对不同的样品进行一系列的DNA提取。
33．根据前述任一权利要求的方法，其中固相在柱中为颗粒状，血液凭借加在柱上的压差而通过柱。
34．根据权利要求33的方法，其中血液样品借助气压流过柱，而颗粒状的固体材料被流化。
35．根据前述任一权利要求的方法，其中所述生物分子通过用低盐缓冲液或水洗脱而取出。
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AU755342B2 (en) 2002-12-12
WO1999029703A3 (en) 1999-08-26
WO1999029703A2 (en) 1999-06-17
PT1036082E (pt) 2002-10-31
JP2010162037A (ja) 2010-07-29
NO20002540L (no) 2000-07-07
EP1036082A2 (en) 2000-09-20
CN1230440C (zh) 2005-12-07
KR20050088164A (ko) 2005-09-01
KR20010032806A (ko) 2001-04-25
DE69805649T2 (de) 2002-12-05
AU1344799A (en) 1999-06-28
US20030130499A1 (en) 2003-07-10
ATE218140T1 (de) 2002-06-15
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ATE386044T1 (de) 2008-03-15
CA2318306A1 (en) 1999-06-17
EP1036082B1 (en) 2002-05-29
JP2004501054A (ja) 2004-01-15
DE69805649D1 (de) 2002-07-04
EP1234832B1 (en) 2008-02-13
ES2177093T3 (es) 2002-12-01
EP1234832A2 (en) 2002-08-28
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HK1034520A1 (en) 2001-10-26

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