CN1474943A - 杂交支持体和固定杂交体的方法 - Google Patents
杂交支持体和固定杂交体的方法 Download PDFInfo
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- CN1474943A CN1474943A CNA018187129A CN01818712A CN1474943A CN 1474943 A CN1474943 A CN 1474943A CN A018187129 A CNA018187129 A CN A018187129A CN 01818712 A CN01818712 A CN 01818712A CN 1474943 A CN1474943 A CN 1474943A
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- C12N15/09—Recombinant DNA-technology
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
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- C12Q1/6813—Hybridisation assays
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Abstract
意图是为了提供在分子生物学、生物化学等领域有用的杂交支持体，借此可有效地阐明DNA而不损害DNA的末端部分。一种在其上固定有寡核苷酸、cDNA或gDNA的支持体，其是通过化学地修饰一种支持体，固定杂交的寡核苷酸、cDNA或gDNA，然后使其去杂交以得到具有固定于其上的寡核苷酸、并用于固定核苷酸的支持体而生产的。
Description
技术领域
本发明涉及一种能够在其上固定核酸且在分子生物学和基因工程相关的技术中有用的杂交支持体，以及一种固定寡核苷酸、cDNA或gDNA的杂交体的方法。
背景技术
基因分析在分子生物学和基因工程技术领域是有用的，目前它甚至用于医学领域以探测疾病等。
对于基因分析，最近已经开发了基因芯片以大大加快分析的速度。然而，常规的DNA芯片是通过将多聚体如聚赖氨酸置于玻璃载玻片或硅基质的表面并随后在其上固定DNA而制备的。除此之外，也采用的是根据半导体技术如光刻法在玻璃基质上合成寡核苷酸的方法。
然而，在将多聚体如聚赖氨酸置于玻璃载玻片或硅基质的表面以在其上固定DNA的方法中，DNA的固定是不稳定的，且该方法存在DNA在杂交步骤或洗涤步骤中剥落的问题。另外，根据半导体技术制作的DNA芯片存在的问题在于它们因为生产过程复杂而极其昂贵。
为了解决这些问题，必须将DNA密集且牢固地固定在固体支持体的表面。
另一方面，已知的是一种表面已被化学修饰的固体支持体。然而，当目的DNA通过酰胺键直接结合到化学修饰的部分时，DNA的末端部分可在化学键合等处理中被破坏。因此，期望改进该支持体。
本发明的一个目的是提供核苷酸固定的支持体，该支持体使得能够进行有效的DNA分析而不如上文所述损害DNA的末端部分，且在分子生物学和基因工程技术等领域是有用的。
发明内容
本发明者发现当将杂交的寡核苷酸固定在化学修饰的支持体上进行固定时，寡核苷酸在垂直于固定支持体表面的方向固定并使得易于读取单碱基多态性(monobasic polymorphism)的DNA信息。
特别地，权利要求1的发明提供了一种杂交支持体，其特征在于在其上固定了杂交的寡核苷酸、cDNA或gDNA。优选地，杂交的寡核苷酸、cDNA或gDNA包含至少两个连续的具伯胺的核苷酸。同样优选地，具伯胺的核苷酸是脱氧腺苷酸、脱氧胞苷酸或脱氧鸟苷酸。同样优选地，具伯胺的核苷酸存在于5’-末端一侧。同样优选地，寡核苷酸具有5’-突出端。cDNA可以通过用寡核苷酸作为引物而合成，该寡核苷酸在5’-末端一侧包含至少两个连续的具伯胺的核苷酸。gDNA特征在于它是用限制性元件进行切割以在5’-末端一侧包含至少两个连续的具伯胺的核苷酸。gDNA特征在于它是用限制性元件进行切割以在5’-突出端一侧包含至少两个连续的具伯胺的核苷酸。更进一步地，期望将脱氧腺苷酸、脱氧胞苷酸或脱氧鸟苷酸固定在杂交支持体的一面以进行固定。同样优选地，杂交的寡核苷酸、cDNA或gDNA在用于固定的末端含有1到10个具伯胺的核苷酸。
权利要求11的发明是固定寡核苷酸、cDNA或gDNA杂交体的方法，它包含化学修饰支持体以进行杂交，在其上固定杂交的寡核苷酸、cDNA或gDNA，然后将没有固定在支持体上的互补寡核苷酸、cDNA或gDNA去杂交(dehybridizing)。优选地，对杂交支持体的化学修饰包含对支持体表面依次的氯化、氨基化和羧基化。
实现本发明的最佳模式
在本发明的杂交支持体上，通过化学修饰固定了杂交的寡核苷酸、cDNA或gDNA，且该支持体特征在于寡核苷酸、cDNA或gDNA是垂直地固定在支持体上的。该支持体从而使得易于读取单碱基多态性的cDNA信息。
在将杂交的寡核苷酸固定在支持体上之后，使互补的寡核苷酸去杂交，然后读取保持在支持体上的寡核苷酸信息。
在那种作用下，该寡核苷酸不可避免地是双链的核酸如DNA等。该寡核苷酸包括那些衍生自高等动物、真菌、细菌、病毒等的天然的寡核苷酸以及那些通过人工修饰其结构而衍生自天然寡核苷酸的修饰的寡核苷酸和合成的寡核苷酸。优选地，该寡核苷酸可根据下面提到的方法以简化的方式合成。
期望构成寡核苷酸的碱基数目为10到50。
本发明的杂交支持体可根据下面提到的方法以简化的方式制作。
(1)杂交支持体的化学修饰：
在本发明中，如上所述的寡核苷酸是固定在化学修饰的杂交支持体上的。
杂交支持体包括玻璃、金刚石；金属如金、银、铜、铝、钨、钼；上面提到的玻璃、金刚石或金属与陶瓷制品的薄片制品；以及塑料如聚碳酸酯、氟树脂。
任何其他的材料只要它们是化学稳定的就可以使用，如石墨、金刚石样的碳等。上面提到的材料的混合物或薄片制品也是可用的。例如用金刚石样的碳包被的玻璃载玻片在此处就是可用的。
在那些材料中，优选的是金刚石或金刚石样的碳，这是因为在其上的DNA固定密度是极其高的。
金刚石可以是任何合成的金刚石、高压浇铸的金刚石或天然的金刚石等。其结构可具有单晶体或多晶体的任意一种形式。从其生产力的观点看，那些通过气相合成的金刚石是优选的，如通过微波等离子体辅助的CVD生产的。
金刚石或金刚石样的碳可用任何已知的方法形成。例如，这些方法包括微波等离子体辅助的CVD、ECRCVD、IPC、DC溅射法、ECR溅射法、离子电镀法、电弧离子电镀法、EB蒸汽沉积、电阻加热蒸汽沉积等。关于金属粉末、陶瓷粉末等，可以通过使用压力机将这些材料压进新鲜压块(green compact)中，且可在高温下使其烧结。
优选地，有意使支持体的表面变粗糙。粗糙的表面由于其表面积增加而有利于固定大量的DNA等。支持体的形状没有特别地定义，可以为扁平的、纱状的、球形的、多角形的、粉状的等。
包含金刚石或金刚石样的碳的杂交支持体可为金刚石或金刚石样的碳与任何其他物质的复合材料(例如一种二相的复合材料)。
化学修饰是为了使多核苷酸结合到修饰的支持体上的，且是通过用烃基基团取代固体支持体的表面而获得的，该烃基基团在其末端有一个功能基团如羟基、羧基、环氧基或氨基。优选地，该烃基基团的烃基部分具有0到12个碳原子，更优选地为0到6个碳原子。其实施例为单羧酸如甲酸、乙酸、丙酸；二元羧酸如草酸、丙二酸、琥珀酸、马来酸、延胡索酸；和多元羧酸如偏苯三酸。在这些羧酸中，草酸和琥珀酸是优选的。
优选地，烃基基团是通过酰胺键结合到固体支持体的表面的。该酰胺键结合促进并增强了化学修饰。
该化学修饰可通过用UV射线照射杂交支持体的表面以使其氯化，在氨气中进一步用UV射线照射以使其氨基化，且其后用适当的酰基氯或多元羧酸酸酐使其羧基化来获得。
(2)杂交的寡核苷酸的固定：
其次，将杂交的寡核苷酸(在下文中称为寡核苷酸H)通过酰胺键固定在化学修饰的部分。将含有寡核苷酸H(参见序列列表中的SEQ ID NO 5)的液体点样在杂交支持体上并借此使在寡核苷酸的5’-末端含有的伯胺结合到支持体上。在固定之前，化学修饰的烃基基团末端是优选地用脱水缩合剂活化的，因为这可以促进固定。具体而言地，碳二亚胺是优选地作为脱水缩合剂的。
除方法之外，其上用于固定寡核苷酸的支持体表面可被氯化和氨基化(但不羧基化)，并且这样形成的初级氨基基团可通过与活化的二酯的一个酯基脱水而缩合，该二酯是通过用N-羟基琥珀酰亚胺对二元羧酸进行预活化而制备的。
考虑到在通过酰胺键结合到化学修饰的杂交支持体上的容易程度，期望固定的侧面有1到10个碱基的伯胺如脱氧腺苷酸、脱氧胞苷酸或脱氧鸟苷酸。由于寡核苷酸H是以垂直于支持体表面的方向固定在杂交支持体上的，因此其DNA信息易于读取。
更优选地，期望位于固定侧面(5’-末端)的1到20个碱基是脱氧腺苷酸、脱氧胞苷酸或脱氧鸟苷酸。
(3)寡核苷酸H是去杂交的。
将在其上固定了寡核苷酸的杂交支持体浸在约96℃的热水中，然后用水洗涤以去杂交。实施例
实施例1(在其上固定了EcoRI-切割的gDNA的杂交支持体)：
(1)杂交支持体的化学修饰：
将金刚石芯片在氯气中用UV射线照射以使其表面氯化，进一步在氨气中用UV射线照射以使其氨基化。然后，将其浸在含有重量比为10％的酰基氯的二噁烷中进行羧基化以获得化学修饰的芯片。
(2)用EcoRI对λ噬菌体DNA的切割：
将λ噬菌体DNA用EcoRI切割以回收21-kbp的片段。
将在(1)中获得的化学修饰的芯片用氢化氨腈(hydrogencyanamide)和N-羟基琥珀酰亚胺进行活化和酯化，然后浸入前面获得的λ噬菌体21-kbp片段的水溶液中(浓度，1μg/μl)，借此使gDNA固定在支持体上。
通过PCR证实了那个片段的500-bp片段的扩增，这支持gDNA在与芯片垂直的方向上固定。
(3)杂交的gDNA的去杂交：
将在(2)中获得的芯片浸入96℃的热水中并洗涤以进行去杂交。
(4)对λ噬菌体21-kbp片段的Cy3标记：
将在(2)中获得的21-kbp的λ噬菌体通过使用Label ITTM(由PanVera提供)而进行Cy3-标记，并形成水溶液(1μg/μl)。将该溶液在96℃加热5分钟，借此将双链结构转变为单链结构。将在(3)中去杂交的芯片浸入到结果所得的溶液中以进行杂交，并且用荧光扫描仪测量其中的荧光亮度。结果，21-kbp的λ噬菌体是以30fmol/mm2的程度固定的。
实施例2(用固定了寡核苷酸的杂交支持体对单碱基突变的探测)：
(1)杂交支持体的化学修饰：
将用软金刚石包被的玻璃载玻片在氯气中用UV射线照射以使其表面氯化，然后将其进一步在氨气中用UV射线照射以使其氨基化。然后，将其浸在含有重量比为10％的酰基氯的1，4-二噁烷中进行羧基化以获得化学修饰的芯片。
(2)寡核苷酸的固定：
利用点样器，将被制备得具有浓度为10pmol/μl的寡核苷酸溶液A和B固定在由(1)获得的杂交支持体上。寡核苷酸A(参见序列表中的SEQ ID NO 1)与寡核苷酸A’(参见序列表中的SEQ ID NO2)进行杂交，而寡核苷酸B(参见序列表中的SEQ ID NO 3)与寡核苷酸B’(参见序列表中的SEQ ID NO 4)进行杂交，均在4℃杂交5小时，然后将它们用作样品。
(3)杂交：
将在(2)中获得的固定了寡核苷酸的杂交支持体置于96℃的热水中，然后将具有浓度为1pmol/μl的寡核苷酸溶液A(未杂交的)添加于其上，并且这是用玻璃盖盖上的。随即将其在5℃杂交12小时。
(4)荧光观察：
将在(3)中获得的支持体洗涤并干燥，然后用荧光扫描仪进行观察。结果，寡核苷酸A固定的斑点显示了荧光，但寡核苷酸B固定的斑点没有显示。
工业适用性
在本发明的杂交支持体上，将杂交的寡核苷酸以垂直于支持体的方向固定。因此，该支持体使得能够容易且准确地读取DNA信息等。在读取之前，将支持体去杂交并因此得到更准确的信息。
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Claims (12)
1.一种杂交支持体，其特征在于杂交的寡核苷酸、cDNA或gDNA是固定在其上的。
2.权利要求1中所述的杂交支持体，其中杂交的寡核苷酸、cDNA或gDNA包含至少两个连续的具伯胺的核苷酸。
3.权利要求1或2中所述的杂交支持体，其中具伯胺的核苷酸是脱氧腺苷酸、脱氧胞苷酸或脱氧鸟苷酸。
4.权利要求1到3中所述的杂交支持体，其中具伯胺的核苷酸位于5’-末端一侧。
5.权利要求1到4中所述的杂交支持体，其中的寡核苷酸具有5’-突出端。
6.权利要求1或2中所述的杂交支持体，其中的cDNA是通过用寡核苷酸作为引物而合成的，该寡核苷酸在5’-末端一侧包含至少两个连续的具伯胺的核苷酸。
7.权利要求1或2中所述的杂交支持体，其中的gDNA特征在于它是用限制性内切酶进行切割以在5’-末端一侧包含至少两个连续的具伯胺的核苷酸。
8.权利要求1、2或7中所述的杂交支持体，其中的gDNA特征在于它是用限制性内切酶进行切割以在5’-突出端一侧包含至少两个连续的具伯胺的核苷酸。
9.权利要求1到8中所述的杂交支持体，其中的脱氧腺苷酸、脱氧胞苷酸或脱氧鸟苷酸是固定在杂交支持体的面上的。
10.权利要求9中所述的杂交支持体，其中固定在杂交支持体上的杂交的寡核苷酸、cDNA或gDNA在用于固定的末端含有1到10个具伯胺的核苷酸。
11.一种固定杂交体的方法，包含化学修饰杂交支持体，在其上固定杂交的寡核苷酸、cDNA或gDNA，然后将没有固定在支持体上的互补寡核苷酸、cDNA或gDNA去杂交。
12.权利要求11中所述的固定杂交体的方法，其中对杂交支持体的化学修饰包含对支持体表面的氯化、氨基化和羧基化。
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| CN1474943A true CN1474943A (zh) | 2004-02-11 |
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| US5237016A (en) * | 1989-01-05 | 1993-08-17 | Siska Diagnostics, Inc. | End-attachment of oligonucleotides to polyacrylamide solid supports for capture and detection of nucleic acids |
| US5106730A (en) * | 1989-07-24 | 1992-04-21 | Microprobe Corporation | Lactam-containing compositions and methods useful for the hybridization of nucleic acids |
| KR100289793B1 (ko) * | 1992-01-29 | 2001-12-01 | 테츠오 토미나가 | 폴리누클레오티드고정지지체 |
| CA2140763A1 (en) * | 1992-07-28 | 1994-02-03 | Masato Mitsuhashi | Gene detection system |
| EP0998347A1 (en) * | 1997-07-22 | 2000-05-10 | Rapigene, Inc. | Polyethylenimine-based biomolecule arrays |
| US6391655B1 (en) * | 1997-07-30 | 2002-05-21 | Corning Incorporated | Oxidized styrenic polymers for DNA binding |
| KR20010022409A (ko) * | 1997-07-31 | 2001-03-15 | 고사이 아끼오 | 아크릴계 수지 필름 및 그의 적층 필름 |
| US5858653A (en) * | 1997-09-30 | 1999-01-12 | Surmodics, Inc. | Reagent and method for attaching target molecules to a surface |
| US6465182B1 (en) * | 1999-04-29 | 2002-10-15 | The Regents Of The University Of California | Comparative fluorescence hybridization to oligonucleotide microarrays |
| WO2000068368A1 (fr) * | 1999-05-10 | 2000-11-16 | Toyo Kohan Co.,Ltd. | Procedes de construction de banque d'adn et support sur lequel ladite banque d'adn est immobilisee |
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- 2001-11-09 CN CNA018187129A patent/CN1474943A/zh active Pending
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| EP1343008A1 (en) | 2003-09-10 |
| EP1343008A4 (en) | 2007-08-22 |
| WO2002039111A1 (fr) | 2002-05-16 |
| AU2002212738A1 (en) | 2002-05-21 |
| KR20030051806A (ko) | 2003-06-25 |
| KR100509217B1 (ko) | 2005-08-18 |
| JPWO2002039111A1 (ja) | 2004-03-18 |
| US20040132031A1 (en) | 2004-07-08 |
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