CN1396954A - 一种固定核苷酸的支持相及其制备方法 - Google Patents
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- C—CHEMISTRY; METALLURGY
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/11—Compounds covalently bound to a solid support
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Abstract
一种固定核苷酸的支持相,能够在不损伤DNA末端的情况下实现对DNA的阐述,因此可用于分子生物学及生物化学等领域。此固定核苷酸的支持相是一种固定了寡核苷酸的经过化学修饰的基质,其特征在于此寡核苷酸具有限制性酶切位点。此支持相的制备是通过基质的化学修饰、单链寡核苷酸的固定、单链寡核苷酸与另一与之具有互补序列的寡核苷酸的杂交、以及与末端具有限制性酶切位点的寡核苷酸的连接等而实现。
Description
技术领域
本发明涉及一种固定核苷酸的支持相及其制备方法,该支持相能够用来固定核酸等,可应用于分子生物学及基因工程相关领域。
发明背景
基因分析广泛用于分子生物学领域和基因工程领域,近年来也被用于疾病诊断等医学领域。
在基因分析中,近年来发展了DNA芯片,以此进行基因分析的速度也越来越快。传统的DNA芯片技术是将高分子物质如聚赖氨酸涂于玻片或者有机硅基质表面,然后固定DNA。另一种方法是运用光蚀刻等半导体技术在玻璃基质表面合成寡核苷酸。
但是在将高分子物质如聚赖氨酸涂于玻片或者有机硅基质表面的方法中,DNA的固定状态是不稳定的,在后续的杂交和漂洗步骤中DNA容易脱落。而且,在采用半导体技术的DNA芯片中,存在由于加工步骤复杂而成本昂贵的问题。
为了解决上述问题,必须将DNA以高强度和高密度固定在固体基质表面。
还已知另一种固体基质,其表面经过了化学修饰。但是当目的DNA以酰胺键直接结合于这些经过了化学修饰的部位时,目的DNA的末端在例如这种化学键合的过程中存在被该处理损伤的危险。因此这种基质也需要改进。
本发明的目的之一就是提供用来固定核苷酸的、在分子生物学和基因工程等领域有用的支持相,由此可有效地进行DNA的阐明而其末端将不受损伤。
发明内容
本发明人发现,如果将含有预定的限制性酶切位点的寡核苷酸固定在经过化学修饰的基质上,使相应于待固定的DNA的该限制性酶切位点得以形成,则有可能稳定地固定。由此实现本发明。
因此,本发明用于固定核苷酸的支持相的特征在于,它是一种经过化学修饰的基质,寡核苷酸被固定在其上面,且该寡核苷酸具有限制性酶切位点。
这种情况下,优选该寡核苷酸中只有一条链与化学修饰的基质键合;还优选该寡核苷酸通过酰胺键与化学修饰的基质键合。
本发明用于固定核苷酸的支持相的制备方法的特征在于:将该基质进行化学修饰,将单链寡核苷酸固定在基质上,然后将含有与以上单链寡核苷酸互补的碱基序列的另一条单链寡核苷酸和所述以上单链寡核苷酸杂交,由此键合了末端含有限制性酶切位点的寡核苷酸。
这种情况下,优选基质的化学修饰包含氯化、氨化和羧化,还优选在基质被化学修饰后,末端在脱水缩合剂存在下被活化。进一步优选脱水缩合剂是碳二亚胺。
这种情况下,还优选基质的化学修饰是基质表面被氯化或者氨化,且产生的伯胺基与该活化二酯的一个酯基脱水缩合。还优选该活化二酯的酯基是N-羟基琥珀酰亚胺或对羟基琥珀酰亚胺。
这种情况下,还优选固定在基质上的单链寡核苷酸在其待被固定侧的末端具有1-10个带有伯胺的核苷酸。还优选固定在基质上的单链寡核苷酸在其待被固定侧的末端具有1-10个伯胺,之后有1-5个胸腺嘧啶或者鸟嘌呤。而且优选固定在基质上的单链寡核苷酸在其待被固定侧的末端具有1-5个腺嘌呤或者胞嘧啶,之后有1-5个胸腺嘧啶或者胞嘧啶。
实施本发明的最佳方式
根据本发明用于固定核苷酸的支持相的特征在于:它是这样一种基质,其上通过化学修饰固定了寡核苷酸,而且该寡核苷酸具有限制性酶切位点。
限制性酶切位点是指可以被限制性内切酶特异性切割的核酸序列。对于限制性内切酶没有特别限制,只要它是通常使用的。限制性内切酶的例子有:
AatI,AatIIAccI,AflII,AluI,Alw44I,ApaI,AseI,AvaI,BamHI,BanI,BanII,BanIII,BbrPI,BclI,BfrI,BglI,BglII,BsiWI,BsmI,BssHII,BstEII,BstXI,Cfr9I,Cfr10I,Cfr13I,CspI,Csp45I,DdeI,DraI,Eco47I,Eco47III,Eco52I,Eco81I,Eco105I,EcoRI,EcoRII,EcoRV,EcoT22I,EheI,FspI,HaeII,HaeIII,HhaI,HinlI,HincII,HindIII,HinfI,HpaI,HpaII,KpnI,MboII,MluI,MroI,MscI,MspI,MvaI,NaeI,NarI,NciI,NcoI,NheI,NotI,NruI,NspV,PacI,PpuMI,PstI,PvuI,PvuII,RsaI,SacI,SacII,SalI,Sau3AI,Sau96I,ScaI,ScrFI,SfiI,SmaI,SpeI,SphI,SrfI,SspI,TaqI,TspEI,XbaI和XhoI
从上述限制性内切酶中自由选择的限制性内切酶的切割位点称为限制性酶切位点。
由于寡核苷酸上需要有限制性酶切位点,因此必须是双链核酸,如DNA。
寡核苷酸可以是天然的,如来自高等动物,真菌,细菌或者病毒,当然也可以是经过结构改变和人工合成的。由于人工合成简单易行(后面还将提到),因此优选使用合成的寡核苷酸。一般地,寡核苷酸链中碱基的数目优选为10-50个。
用下述方法可以容易地制备本发明用于固定核苷酸的支持相:
(1)基质的化学修饰
在本发明用于固定核苷酸的支持相中,这种寡核苷酸固定在经过化学修饰的基质上。
基质的例子是玻璃;金刚石;金、银、铜、铝、钨、钼等金属;与上述玻璃、金刚石和金属等复合成层的陶瓷类产品;以及聚碳酸酯和氟树脂(fluorine resin)等塑料。
也可以使用其他材料,只要这些材料是化学稳定的材料,如石墨,金刚石样碳(diamond-like carbon)等。也可以用塑料和上述的金属、玻璃、陶瓷类、金刚石等物质的混合物或者成层产品。
出于导热性的考虑,上述物质中优选使用金刚石或部分使用金刚石的材料作为基质。金刚石具有良好的导热性,可以被迅速加热和冷却,这样大大缩短了像在PCR过程中重复加热和冷却的热循环时间。
优选本发明基质的导热率不低于0.1W/cm·K,优选不低于0.5W/cm·K,尤其优选不低于1.0W/cm·K。这是因为,若基质的导热率不低于1.0W/cm·K,在将DNA固定于本发明支持相的化学修饰部分以后进行PCR等操作时,加热和冷却的跟随特性(follow-up property)极佳。
作为金刚石基质的材料,可以使用任何合成金刚石、高压制作的金刚石和天然金刚石。在结构上可以是单晶也可以是多晶。从生产效率的角度,优选使用气相合成法如微波等离子体CVD(化学气相沉积)法制造的金刚石。
可以通过已知方法来形成以金刚石或其它物质为材料的基质。例子有微波等离子体CVD法,ECR CVD(电子回旋共振化学气相沉积,electric cyclotron resonance Chemical Vapor Deposit)法,ICP(感应耦合等离子体)法,直流喷镀法(direct current sputteringmethod),ECR(电子回旋共振)喷镀法(sputtering method),离子电镀(ion plating)法,电弧离子电镀(arc ion plating)法,EB(电子束)蒸汽淀积(electron beam vapor deposition)法和电阻加热蒸汽沉积(resistance heating vapor deposition)法等。
此外,制造基质的方法的一个例子是通过将金属粉末或者陶瓷粉末与作为粘合剂的树脂混合而键合和形成的产物。而且,制造基质的方法的一个例子是如下方法,即将金属粉末或者陶瓷粉末等材料用压模机压缩,并使此产品高温烧结。
优选有意将基质表面粗造化。其原因是,在这种粗糙表面,基质的表面积增大,由此便于固定大量的DNA等。基质的形状可以是板状,条状,球状,多边状,筒状,粉末状等任何形状。此外,基质也可以是金刚石和其它物质(如两相物质)的复合物。
通过用在末端具有极性基团的烃基取代基质的表面来进行化学修饰,这些极性基团的例子有羟基,羧基,环氧基,氨基,巯基或者异氰酸酯基(isocyanate group)等。这样使得多核苷酸能与基质表面发生键合。这种烃基的烃部分的碳原子数优选为0-12个,更优选为0-6个。实例有甲酸、乙酸和丙酸等单羧酸,草酸、丙二酸、琥珀酸、马来酸和延胡索酸等二羧酸,以及偏苯三酸等多元羧酸等。也可以使用由一种或者两种酸组成的酸酐。特别优选草酸和琥珀酸。
烃基优选通过氨基键合于基质表面。因为氨基键使得化学修饰作用更加容易且更加牢固。
这种化学修饰可以通过以下方式完成:紫外线照射使基质表面氯化,将基质置于氨气中用紫外线照射使其氨化,然后用适当的酰基氯或者二羧酸酐使其羧化。
(2)单链寡核苷酸的固定
上述步骤完成后,单链寡核苷酸(以下称为寡核苷酸A)通过酰胺键固定于经过了化学修饰的部位上。在固定之前如果将化学修饰的烃基末端活化,固定方法就很容易进行,因此优选进行这种活化。特别优选使用碳二亚胺为脱水缩合剂。
除上述方法外,也可以以这种方式形成,即将基质表面变成氯化和氨化状态(非羧化),产生的伯胺基就可以很容易与含有酯基的活化二酯的一个酯基如N-羟基琥珀酰亚胺和对硝基酚脱水缩合。
虽然对寡核苷酸A的序列没有限制,但其未固定端(3’-末端)应设计成与下一个其他寡核苷酸杂交获得的双链寡核苷酸具有限制性酶切位点。
考虑到与化学修饰基质之间容易形成酰胺键,待固定侧优选具有1-10个带有伯胺的核苷酸碱基,比如腺嘌呤,胞嘧啶和鸟嘌呤。特别优选待固定侧(5’-末端)1-5个碱基是腺嘌呤或胞嘧啶。还优选腺嘌呤后面1-5个碱基是胸腺嘧啶或鸟嘌呤,以抑制后续杂交的趋异性。
(3)另一单链寡核苷酸与寡核苷酸A杂交
另一单链寡核苷酸(以下称为寡核苷酸B)应该与寡核苷酸A具有互补序列。
在本发明通过杂交获得的双链寡核苷酸中,与待固定侧相对的一端具有限制性酶切位点,因此寡核苷酸A和B必形成限制性酶切位点。杂交条件与通常使用的条件相同。
相应于待结合的DNA分子的限制性酶切位点形成后,如此制备的本发明用于固定核苷酸的支持相就能够进行结合。
下面通过实施例详细地举例说明本发明。
实施例1
带有EcoRI酶切位点的用于固定核苷酸的支持相的制备
(1)基质的化学修饰
将含有金刚石的基质置于氯气中用紫外线照射使基质表面氯化。之后置于氨气中用紫外线照射使其氨化,然后用酰氯在氯仿中回流使基质羧化,由此基质表面实现化学修饰。
(2)单链寡核苷酸的固定
将上述(1)制备的化学修饰基质浸在1,4-二氧六环溶液中15分钟(金刚石基质和二氧六环的比为1∶100μl),二氧六环溶液中溶有2.5mg/ml碳二亚胺和1.5mg/ml N-羟基琥珀酰亚胺,使末端羧基脱水缩合。该反应完成后,先用水冲洗产物,再用1,4-二氧六环溶液洗涤,然后干燥。
然后,具有序列表中SEQ ID NO:1所示序列的寡核苷酸A-1的5’-末端腺嘌呤通过酰胺键与基质活化部位键合。
(3)寡核苷酸B与寡核苷酸A杂交
制备具有与寡核苷酸A(序列:aaaggttttt tttttttttt tttg,参见SEQ ID NO:1)互补的序列的寡核苷酸B(序列:aattcaaaaaaaaaaaaaaa aaacctt,参见序列表中SEQ ID NO:2),并与在上述(2)中已经固定的寡核苷酸A杂交。
将寡核苷酸B溶于78μl无菌水中,再加入20μl的20×ssc(柠檬酸钠盐溶液)和2μl的SDS溶液,使总体积为100μl。在35℃条件下使上述固定有寡核苷酸A的基质浸泡在此溶液中10小时。
结果,制备了带有EcoRI酶切位点的本发明固定用支持相。
(4)带有限制性酶切位点的gDNA的制备
在具有下列组成的缓冲液中用EcoRI处理目的DNA(37℃ 1小时),反应后加热失活酶,琼脂糖凝胶电泳,回收具有EcoRI酶切位点的gDNA片断。缓冲液组成如下:EcoRI 2μl10×H缓冲液 4μl无菌水 34μl总体积 40μl
(5)用连接酶将DNA的限制性酶切部分与基质连接。
工业应用
本发明用于固定核苷酸的支持相通过连接酶将待固定的核酸(如DNA)固定于具有限制性酶切位点的寡核苷酸上。因此,与传统通过化学键直接固定的方法相比,DNA等核酸能被固定得更加牢固。
此外,根据本发明的制备方法,能够有效地产生DNA等核酸可以稳定地固定的用于固定核苷酸的支持相。
当本发明用于固定核苷酸的支持相以这种状态投放市场时,用户可以预先通过用相应的限制性内切酶切割DNA来制备限制性酶切位点,这样用户自己就可以容易地将该DNA稳定地固定。
序列表<110>TOYO KOHAN CO.,LTD.<120>一种固定核苷酸的支持相及其制备方法<130>1738PCT<150>JP 2000-019301<151>2000-01-27<160>2<210>1<211>24<212>DNA<213>人工序列<400>1aaaggttttt tttttttttt tttg 24<210>2<211>27<212>DNA<213>人工序列<400>2aattcaaaaa aaaaaaaaaa aaacctt 27
Claims (12)
1.用于固定核苷酸的支持相,其中经化学修饰的基质上固定了寡核苷酸,其特征在于所述寡核苷酸具有限制性酶切位点。
2.根据权利要求1的用于固定核苷酸的支持相,其中寡核苷酸的仅一条链键合至经化学修饰的基质上。
3.根据权利要求1或2的用于固定核苷酸的支持相,其中寡核苷酸通过酰胺键键合至经化学修饰的基质上。
4.制备用于固定核苷酸的支持相的方法,其中将基质进行化学修饰,在该基质上固定单链寡核苷酸,然后将另一具有与以上单链寡核苷酸互补的碱基序列的单链寡核苷酸与所述以上单链寡核苷酸杂交,由此键合在末端具有限制性酶切位点的寡核苷酸。
5.根据权利要求4的制备用于固定核苷酸的支持相的方法,其中基质的化学修饰包含基质表面的氯化,氨化和羧化。
6.根据权利要求5的制备用于固定核苷酸的支持相的方法,其中基质被化学修饰,然后在脱水缩合剂存在下活化其末端。
7.根据权利要求6的制备用于固定核苷酸的支持相的方法,其中脱水缩合剂为碳二亚胺和/或N-羟基琥珀酰亚胺。
8.根据权利要求4的制备用于固定核苷酸的支持相的方法,其中基质的化学修饰按下述方式进行:基质表面被氯化和氨化,形成的伯胺基与活化过的二酯中的一个酯基脱水和缩合。
9.根据权利要求8的制备用于固定核苷酸的支持相的方法,其中活化过的二酯中的酯基是N-羟基琥珀酰亚胺或对羟基琥珀酰亚胺。
10.根据权利要求4的制备用于固定核苷酸的支持相的方法,其中待固定于基质上的单链寡核苷酸在待固定侧末端具有1-10个带有伯胺的核苷酸。
11.根据权利要求10的制备用于固定核苷酸的支持相的方法,其中待固定于基质上的单链寡核苷酸在待固定侧末端具有带有1-10个伯胺的核苷酸和然后的1-5个胸腺嘧啶或鸟嘌呤。
12.根据权利要求10或11的制备用于固定核苷酸的支持相的方法,其中待固定于基质上的单链寡核苷酸在待固定侧末端具有1-5个腺嘌呤或胞嘧啶和然后的1-5个胸腺嘧啶或鸟嘌呤。
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- 2000-01-27 JP JP2000019301A patent/JP2001204463A/ja active Pending
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2001
- 2001-01-24 AU AU2001227098A patent/AU2001227098A1/en not_active Abandoned
- 2001-01-24 US US10/182,434 patent/US20030190633A1/en not_active Abandoned
- 2001-01-24 CN CN01804171A patent/CN1396954A/zh active Pending
- 2001-01-24 KR KR1020027009564A patent/KR100695057B1/ko not_active IP Right Cessation
- 2001-01-24 EP EP01901529A patent/EP1256626A4/en not_active Withdrawn
- 2001-01-24 WO PCT/JP2001/000443 patent/WO2001055365A1/ja not_active Application Discontinuation
-
2005
- 2005-09-30 US US11/239,418 patent/US20060024741A1/en not_active Abandoned
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2013097290A1 (zh) * | 2011-12-31 | 2013-07-04 | 北京唯尚立德生物科技有限公司 | 一种转录激活子样效应因子的固相合成方法 |
CN111094318A (zh) * | 2017-09-11 | 2020-05-01 | 生命技术股份公司 | 用于将核酸偶联至官能化的载体的方法和组合物 |
CN111094318B (zh) * | 2017-09-11 | 2024-01-09 | 生命技术股份公司 | 用于将核酸偶联至官能化的载体的方法和组合物 |
Also Published As
Publication number | Publication date |
---|---|
EP1256626A1 (en) | 2002-11-13 |
WO2001055365A1 (fr) | 2001-08-02 |
KR20020079806A (ko) | 2002-10-19 |
US20060024741A1 (en) | 2006-02-02 |
JP2001204463A (ja) | 2001-07-31 |
US20030190633A1 (en) | 2003-10-09 |
EP1256626A4 (en) | 2003-06-18 |
AU2001227098A1 (en) | 2001-08-07 |
KR100695057B1 (ko) | 2007-03-14 |
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