CN1957093B - 使用含有环氧基的聚合物涂层的塑料基片的pna芯片、制造该pna芯片的方法和使用该pna芯片检测单核苷酸多态性的方法 - Google Patents
使用含有环氧基的聚合物涂层的塑料基片的pna芯片、制造该pna芯片的方法和使用该pna芯片检测单核苷酸多态性的方法 Download PDFInfo
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- CN1957093B CN1957093B CN2005800161981A CN200580016198A CN1957093B CN 1957093 B CN1957093 B CN 1957093B CN 2005800161981 A CN2005800161981 A CN 2005800161981A CN 200580016198 A CN200580016198 A CN 200580016198A CN 1957093 B CN1957093 B CN 1957093B
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Abstract
本发明提供了一种PNA(肽核酸)芯片,其中,含有目的DNA序列的探针PNA固定在用含有环氧基的聚合物涂层的塑料基片上。因此,可以有效和节省成本的方式通过含有环氧基的聚合物层将单链PNA固定在透明塑料基片上。基于PNA/DNA杂交的荧光信号检测能够鉴定SNP(单核苷酸多态性)。
Description
技术领域
本发明涉及一种PNA(Peptide Nucleic Acid,肽核酸)芯片,并且更具体而言,本发明涉及一种PNA芯片,其中,包括目的DNA序列的探针PNA固定在用含有环氧基的聚合物涂层的塑料基片上。
背景技术
肽核酸(下文称为“PNA”)最初开发为基因靶向药物。由于PNA与互补DNA的良好的杂交性,已进行许多关于PNA的报道。PNA/DNA杂交基于PNA单链和互补DNA单链之间的强碱基配对。即,PNA最显著的性质是通过与互补DNA的稳定杂交的优异的DNA识别能力。PNA的结构与DNA的结构非常相似。PNA具有与DNA的带负电荷的糖-磷酸酯骨架不同的中性肽骨架和通过酰胺键连接的N-(2-氨乙基)甘氨酸重复单位。已知PNA中含有的四个核碱基占据与DNA的四个碱基相似的空间尺寸并且PNA与DNA的分子间距也几乎相同。与DNA不同,PNA不被核酸酶或蛋白酶降解,因此生物学上非常稳定。此外,由于带负电荷的DNA骨架,所以DNA双链体的热稳定性受盐浓度影响;由于PNA的中性骨架,所以PNA/DNA双链体的热稳定性基本上不受盐浓度的影响。PNA/DNA双链体的低盐浓度依赖性降低了PNA和DNA之间的静电排斥,因此增强了PNA/DNA双链体的热稳定性。由于PNA的这些许多优点,所以PNA在通过常规的DNA相关的方法不能达到的生物学上重要的或诊断用途中得到极大的关注。
已经以DNA固定技术相似的方式研究了PNA固定技术。大部分现行的DNA固定方法基于能与分析物杂交的单链DNA的固定。主要使用将DNA吸附到固体表面的方法(Nikiforov和Rogers,Anal Biochem.1995)。也已开发杂交法(Proudnikov等,Anal Biochem.1998;Rehman等,Nucleic Acids Res.1999)和复合物形成法(Nilsson等,Anal Biochem.1995)。光刻法是众所周知为固定主要化学合成的寡核苷酸的方法(Gerhold等,Trends Biochem Sci.1999)。通过硅烷单层(Rogers等,AnalBiochem.1999)、自组装单层(Higashi等,J Colloid Interface Sci.1999)等介导载体与掺入到寡核苷酸中的反应基之间的共价键。在上述方法中,通过如吸附的物理方法固定生物物质受到空间或结构的限制,并且由于非特异性吸附具有如高本底信号的检测限制。此外,由于能将环氧硅烷涂覆在玻璃基片上但不能涂覆在塑料基片上,所以基片的改进受到限制。
可通过点样、微阵列技术、光刻法或电子寻址制造生物分子或聚合物的阵列。点样是通过微机器人的三维运动将生物分子滴在目的位置,和微阵列技术是使用自来水笔样针的微阵列形成。光刻法通过选择性地将光照在目的位置以仅将生物分子粘着于表面目的位置,和电子寻址通过选择性地向微电极阵列施加电压进行,以仅将生物分子固定在预定电极上。在本发明中,通过非接触喷墨印刷点样制备PNA阵列。
在寻找现有技术的上述问题的解决方案时,本发明人发现在通用的塑料基片上掺入或涂覆含有环氧基的聚合物能有效和节省成本地固定PNA,并因此完成本发明。
发明内容
技术问题
本发明提供了一种能以有效和节省成本的方式在通用塑料基片上固定PNA的新型PNA芯片。
本发明也提供了一种有效地制造所述PNA芯片的方法。
本发明也提供了一种使用所述PNA芯片检测SNP(SingleNucleotide Polymorphism,单核苷酸多态性)的方法。
技术方案
根据本发明的一个技术方案,提供了一种PNA芯片,其中,含有目的DNA序列的探针PNA固定在用含有环氧基的聚合物涂层的塑料基片上。
在本发明中,所述塑料基片可以是可用聚合物涂覆的任一塑料基片。优选地,所述塑料基片是由选自包括聚甲基丙烯酸甲酯(PMMA)、聚碳酸酯(PC)、聚降冰片烯、COC(环烯烃共聚物)、氟化聚酰亚胺、聚苯乙烯(PS)、SBC(苯乙烯丁二烯共聚物)、ABS(丙烯腈丁二烯苯乙烯)、SAN(苯乙烯丙烯腈)和聚砜的组的材料制成的透明塑料基片。普通塑料基片相对于普通玻璃基片更便宜并且不需要单独的表面处理。此外,与易碎的玻璃基片不同,塑料基片是韧性的,并因此在运输、储藏和处理上是优异的。此外,由于无荧光发射,所以透明塑料基片能容易地检测信号。
在本发明中,含有环氧基的聚合物可以是具有环氧基的任一聚合物,但优选含有环氧基的丙烯酸单体和无环氧基的丙烯酸单体的共聚物。根据塑料基片的类型可以任意选择共聚物。特别是,通过调节含有环氧基的丙烯酸酯单体的重量比可以调节共聚物中环氧基的含量。此外,使用适当的单体可以增强共聚物对PNA的接近性。
在本发明的一个实施方式中,所述含有环氧基的聚合物是如由下式1表示的含有环氧基的丙烯酸酯单体和高粘性丙烯酸酯单体的共聚物:
<式1>
-[CR3R3-CR1R3-X]n-
其中,R1是含有环氧基的酯,R3是氢或烷基,和X是高粘性丙烯酸酯化合物。
在本发明中,高粘性丙烯酸酯单体可以是能增强丙烯酸酯的UV-固化涂覆的任一丙烯酸酯单体(参见T.Jaworek,MacromolSymp.,159,197,2000;Cliff Roffey, “Photogeneration of Reactive Species for UVCuring”,1997)。优选地,所述高粘性丙烯酸酯单体是在25℃具有8-6,000cp的粘性的丙烯酸酯单体,并且更优选为选自包括二季戊四醇羟基五丙烯酸酯(DPHA)、9-乙二醇二丙烯酸酯(9-EGDA)、季戊四醇三-四丙烯酸酯(PETA)、聚乙二醇400二丙烯酸酯、三丙二醇二丙烯酸酯、三羟甲基丙烷三丙烯酸酯和二季戊四醇六丙烯酸酯的组。
在本发明的另一实施方式中,所述含有环氧基的聚合物是如由下式2表示的含有环氧基的丙烯酸单体和能粘着于所述塑料基片的粘性丙烯酸酯衍生物的共聚物:
<式2>
-[CR3R3-CR1R3-CR3R3-CR2R3]n-
其中,R1是含有环氧基的酯,R2是烷基酯,和R3是氢或烷基。
在本发明中,所述粘性丙烯酸酯衍生物可以是与制成塑料基片的单体类型相似的任一丙烯酸酯衍生物,以使包括丙烯酸酯衍生物的聚合物具有与塑料基片相似的物理性质以保证聚合物容易地粘着于塑料基片上。优选地,所述粘性丙烯酸酯衍生物选自包括甲基丙烯酸甲酯(MMA)、丙烯酸乙酯、甲基丙烯酸乙酯(EMA)、丙烯酸正丙酯、甲基丙烯酸正丙酯、丙烯酸异丙酯和甲基丙烯酸异丙酯的组。当所述塑料基片由聚甲基丙烯酸甲酯(PMMA)制成时,由于PMMA与MMA的物理性质几乎相同,所以特别优选使用甲基丙烯酸甲酯(MMA)作为粘性丙烯酸酯衍生物。
在本发明中,含有环氧基的聚合物中环氧基的含量,即,含有环氧基的聚合物中含有环氧基的丙烯酸酯单体的含量可以为0.1~100wt%,优选10wt%或更高,并且更优选20~30wt%。如果环氧基的含量低于10wt%,由于低环氧基密度,在狭窄区域内积累PNA是困难的,所以降低了荧光信号。另一方面,由于PNA的固定率与环氧基的密度不成正比,所以不优选环氧基的含量高于40wt%。这可能因为相对提高涂覆溶液中含有的环氧基促进埋藏表面环氧基,因此防止了表面环氧基与PNA的有效反应。
在本发明中,探针PNA的胺端基可以直接与涂覆在塑料基片上的含有环氧基的聚合物的环氧基结合。然而,优选向探针PNA的胺端基加入含有胺基和存在或不存在醚的C5-8羧酸连接剂(linker)。该连接剂增强了探针PNA的空间方向性,因此优化了PNA/DNA杂交。
根据本发明的另一技术方案,提供了一种制造PNA芯片的方法,该方法包括:以10~90∶80~5∶1~10的比例混和含有环氧基的丙烯酸酯单体、高粘性丙烯酸酯单体和光引发剂;在塑料基片上涂覆该混合物;通过UV固化该混合物;和在塑料基片上点样探针PNA印刷溶液。
根据本发明的又一技术方案,提供了一种制造PNA芯片的方法,该方法包括:以10~99∶1~89∶0.1~0.5的比例混和含有环氧基的丙烯酸酯单体、具有与塑料基片相似物理性质的丙烯酸酯衍生物和自由基引发剂;自由基聚合该混合物;在塑料基片上涂覆通过在溶剂中溶解所得的聚合物获得的溶液;和在塑料基片上点样探针PNA印刷溶液。
在本发明中,可以向探针PNA印刷溶液中加入适当浓度、优选0.01~1.0M、更优选0.05~0.5M的碱,以有效地进行探针PNA的胺基和聚合物的环氧基之间的亲核取代反应,以因此辅助探针PNA的固定。该碱可以是如氢氧化钠、氢氧化钾、碳酸钠、碳酸钾的普通碱或路易斯酸。
根据本发明在所述自由基引发剂存在下制造PNA芯片的方法可另外包括在点样探针PNA印刷溶液之前,将涂覆聚合物的塑料基片在30%或更高的湿度条件下保存4小时或更长时间。如果在低于30%的湿度条件下保存涂覆聚合物的塑料基片,可降低所述环氧基和胺基之间的反应效率,因此导致信号降低。优选在50~95%的湿度条件中保存涂覆聚合物的塑料基片。将涂覆聚合物的塑料基片保存4小时或更长时间以蒸发有机溶剂。
根据本发明的又一技术方案,提供了一种检测SNP的方法,该方法包括:将含有靶DNA的反应样品施加在上述PNA芯片上;杂交探针PNA与靶DNA;洗涤PNA芯片以除去非特异性反应产物;和基于PNA/DNA杂交检测荧光信号。
现将参照显示本发明的示例性实施方式的附图更加全面地描述本发明。本发明提供了一种如由下式1或2表示的用于生物分子固定的含有环氧基的聚合物材料:
<式1>
-[CR3R3-CR1R3-X]n-
<式2>
-[CR3R3-CR1R3-CR3R3-CR2R3]n-
其中,R1是C3-12含有环氧基的酯,R2是C2-16烷基酯,R3是氢或C1-16烷基,X是高粘性丙烯酸酯化合物,和n取决于单体的浓度和反应持续时间但可以是10~2,000。
使用丙烯酸酯化合物,通过UV固化(参见T.Jaworek,MacromolSymp.,159,197,2000;Cliff Roffey,“Photogeneration of Reactive Speciesfor UV Curing”,1997)或自由基聚合(参见Bevington,J.C.,在“Comprehensive Polymer Science”第3卷,65,1989;Tedder,J.M.,Angew.Chem.,Iht.Ed.Engl.,21,401,1982)合成所述聚合物材料。
在所述聚合物材料的合成中,可以使用两种或多种丙烯酸酯单体。两种或多种所述丙烯酸酯单体的不同组合能适当地调节环氧基的含量,因此提供具有适当环氧基含量的聚合物材料。
在通过UV固化形成聚合物涂层的情况下,使用含有环氧基的丙烯酸酯单体和至少一种高粘性丙烯酸酯单体。所述丙烯酸酯单体可以是具有两个或多个乙烯基的单体。本发明提供了UV固化涂覆溶液的组成、丙烯酸酯单体的组成比例和用于UV固化的有效涂覆方法。
在通过自由基聚合形成聚合物涂层的情况下,使用含有环氧基的丙烯酸酯单体、与塑料基片具有相似物理性质的丙烯酸酯衍生物和普通自由基引发剂(偶氮化合物、过氧化物、氧化还原引发剂)(Graememoad,“the chemistry offree radicalpolymerization”,1995)。如上所述,通过含有环氧基的丙烯酸酯单体的重量比可以确定环氧基的含量。
在通过热聚合形成聚合物涂层的情况下,除了含有环氧基的丙烯酸酯单体外,使用含有烷基酯的丙烯酸酯单体。
在通过UV固化形成聚合物涂层中,所述高粘性丙烯酸酯单体可以是二季戊四醇羟基五丙烯酸酯(DPHA)、9-乙二醇二丙烯酸酯(9-EGDA)、季戊四醇三-四丙烯酸酯(PETA)、聚乙二醇400二丙烯酸酯、三丙二醇二丙烯酸酯、三羟甲基丙烷三丙烯酸酯、或二季戊四醇六丙烯酸酯。
即,通过UV固化形成的聚合物涂层包括含有环氧基的丙烯酸酯单体和至少一种上述高粘性丙烯酸酯单体。
在通过UV固化形成聚合物涂层中,含有环氧基的丙烯酸酯单体和高粘性丙烯酸酯单体的重量比可以为0.1∶99.9至100∶0。优选调节用于生物分子固定的含有环氧基的丙烯酸酯单体的含量至10 wt%或更高。
所述自由基聚合可以在90℃或更低进行以防止环氧基的开环反应。
在所述自由基聚合中,通过调节反应持续时间可以调节合成的聚合物的平均分子量。优选自由基聚合进行1~6小时以制备目的涂覆溶液并保证涂覆透明度或防止裂缝。
通过使用过量的如甲醇的醇沉淀终止所述自由基聚合。
自由基聚合后干燥的聚合物可溶于四氢呋喃、二氯甲烷等中,以制备含有0.1~5%聚合物的涂覆溶液。为了防止裂缝形成或提供透明度,优选制备含有1~3%聚合物的涂覆溶液。
待涂覆上述聚合物层的基片可以是普通硅片或玻璃,优选塑料基片,并且更优选透明塑料基片。常规地,加工良好的贵重玻璃主要用作芯片基片。然而,在本发明中,具有易操作性的普通廉价塑料基片用于克服玻璃基片的缺点。通常,术语“透明塑料基片”包括由聚甲基丙烯酸甲酯(PMMA)、聚碳酸酯(PC)、聚降冰片烯、COC(环烯烃共聚物)、氟化聚酰亚胺、聚苯乙烯(PS)、SBC(苯乙烯丁二烯共聚物)、ABS(丙烯腈丁二烯苯乙烯)、SAN(苯乙烯丙烯腈)或聚砜制成的基片。
用于在塑料基片上涂层形成的聚合物涂覆可通过浸渍、喷射、印刷方法等完成。优选在50%或更高的湿度条件下保存后使用涂覆聚合物的塑料基片。如果涂覆聚合物的塑料基片保存在低湿度条件下,可以降低PNA固定效率。
根据本发明,在适当浓度的碱(氢氧化钠、氢氧化钾、碳酸钠、碳酸钾、路易斯酸)存在下,通过如微阵列点样的容易和廉价的方法可以完成生物分子的强固定,即,PNA固定在含有环氧基的塑料基片上。
根据本发明,制备含有碱性催化剂的印刷缓冲液,以将PNA的胺基结合到涂覆在基片上的环氧基。使用喷墨阵列机(inkjet arrayer)注入所述印刷缓冲液,在基片表面上提供容易和廉价的PNA固定。优选地,所述PNA固定在50~60%的湿度条件下在约23℃进行。
本发明提供了使用上述制备的PNA阵列用于特异性PNA/DNA杂交必需的最佳缓冲溶液、反应条件和有效的洗涤方法。
用于PNA/DNA杂交的缓冲溶液可以是含有5X SSC、50mMHEPES、1%SDS和0.1%BSA的缓冲溶液。不需要进行单独的封闭过程以防止非特异性杂交。
PNA/DNA杂交后,可以分别使用含有1X、0.1X、0.01X和0.001XSSC(各5分钟)的四种洗涤缓冲溶液。
下文,将参照以下实施例将更加详细地描述本发明。以下实例用于说明的目的并不意限制本发明的范围。
有益效果
如上述,根据本发明,可以有效和节省成本的方式将探针PNA固定在通用塑料基片上。固定探针PNA的基片的使用能够检测不同的基因变异。此外,预计使用基于PNA优异的物理化学性质的PNA芯片可以克服普通DNA芯片的缺点。
附图说明
通过结合附图详细描述其示例性实施方式,本发明的上述和其它特征和优点将变得更清楚明了,其中;
图1为说明根据本发明在通用塑料基片上涂覆含有环氧基的聚合物层和将PNA固定到所述聚合物层上的示意顺序图;
图2A和2B分别为根据本发明对于聚合物层中环氧基的含量PNA固定率的评价结果的荧光图像和阵列信息;
图3为图2A的荧光图像的图解定量分析数据;
图4说明PNA阵列的PNA/DNA杂交结果,以确定PNA固定前涂覆聚合物的塑料基片的最佳保存条件;
图5A和5B分别为靶DNA和阵列信息的PNA/DNA杂交结果的荧光图像分析数据;
图6A和6B分别为对于rtL180野生型和rtL180突变体环氧基含量的PNA/DNA杂交结果的图解定量分析数据;
图7A为说明根据本发明用于优化PNA/DNA杂交的连接剂的示意图,和图7B说明不同类型的所述连接剂的化学结构式;
图8A和8B分别为根据本发明用于通过连接剂介导的PNA/DNA杂交的荧光图像和点阵列;
图9A和9B为图8A的荧光图像的图解定量分析数据;以及
图10A和10B为对于探针PNA浓度PNA/DNA杂交检测敏感性的定量分析数据。
具体实施方式
实施例1:通过UV固化制备涂覆含有环氧基的聚合物的基片
以适当的比例(10~90∶80~5∶1~10)混和欲用于掺入环氧基的环氧丙氧基甲基丙烯酸甲酯(GMA)、9-乙二醇二丙烯酸酯(9-EGDA)和光引发剂(Irgacure 184,汽巴-嘉基化学公司)。光引发剂完全溶解后,用旋涂机以500rpm涂覆6秒然后以1,000~4,000rpm涂覆20秒将所得的混合物涂覆在聚甲基丙烯酸甲酯(PMMA)基片上。然后,所得的基片在氮环境中暴露于254nm UV并干燥。上述方法不局限于丙烯酸酯单体和塑料基片,并可以用于多种类型的单体和基片。如此制备的含有环氧基的聚合物具有下式:
-[CH2C(CH3)(C(O)OCH2CHCH2)CH2CH(C(O)O(CH2CH2O)9C(O)CHCH2)]n- 。 用DPHA或PETA代替9-EGDA的UV固化可制备GMA和DPHA或PETA的聚合物。图1为说明根据本发明在通用塑料基片上涂覆含有环氧基的聚合物层并将PNA固定到含有环氧基的聚合物层上的示意顺序图。
实施例2:通过自由基聚合制备涂敷含有环氧基聚合物的基片
以适当的比例(99~10∶1~89∶0.1~0.5∶0.1~0.5)混和欲用于掺入环氧基的环氧丙氧基甲基丙烯酸甲酯(GMA)、甲基丙烯酸甲酯(MMA)、自由基引发剂(2,2,6,6-四甲基-4-哌啶醇,TMPO)和分子量调节剂。在75℃下加热该混合物2小时,然后在90℃下加热0.5~3小时。考虑到该反应混合物的粘性根据MMA的重量比变化极大,在该反应混合物完全固化前终止该反应。当该反应混合物的粘性达到适当水平时,向其加入过量的甲醇,剧烈搅拌并再结晶。将所得的晶体在真空中干燥一天。通过NMR评估如此制得的聚合物的环氧基含量并通过GPC评估其平均分子量。在四氢呋喃中溶解0.1~5wt%的该聚合物。将PMMA基片固定在旋涂机上并通过旋涂机涂覆2ml含有该聚合物的涂覆溶液(参见图1)。 所述的含有环氧基的聚合物具有下式:-[CH2C(CH3)(C(O)OCH2CHCH2)CH2C(CH3)(C(O)OCH3)]n-,并且平均分子量(Mw)为75,000~250,000。用EMA代替MMA以如上述自由基聚合相同的方式可以制备GMA和EMA的聚合物。
在使用前将涂覆聚合物的基片在50%或更高的湿度条件下保存4小时。如果涂覆聚合物的基片在低湿度条件下保存,可以降低环氧基和胺基之间的反应效率,导致信号降低。图4说明PNA阵列的PNA/DNA杂交结果以确定PNA固定前涂覆聚合物的塑料基片的最佳保存条件。将该涂覆含有环氧基的聚合物的塑料基片暴露于多种湿度条件(10、20~30、>50%)。在图4中,PM/MM比表示在DNA/PNA杂交中完全匹配(PM)与不匹配(MM)的平均荧光信号比值,并且单个碱基差异导致PM和MM之间的信号差异。较高的PM/MM比表示探针PNA对靶DNA更优异的特异性。参考图4,当所述涂覆聚合物的基片在30%或更高、优选50%或更高的湿度条件下保存时,信号敏感性(PM/MM比)更优异。
实施例3:PNA-Cv3固定
在本实施例中,用实施例1和2制备的涂覆聚合物的塑料基片评估了对于GMA含量的PNA固定率。为此,在上述基片上固定Cy3-标记的PNArtL-180w(PNAGENE.Inc)。根据以下实施例4进行印刷溶液制备和点样。通过调节GMA含量,所述塑料基片具有20%、30%、40%和50%的不同的环氧含量。通过所述Cy3-标记的PNA rtL-180w的荧光强度可以直接确定PNArtL-180W的固定率。在本实验中,试验了不同的PNA浓度,即,500、400、300、200和100 nM。图2A和2B中分别说明了固定在含有不同含量的环氧基的基片上的PNA的荧光图像和阵列信息。图3为图2A的荧光图像的图解定量分析数据。在图3中,不含PNA-cy3的点样组合物的荧光强度(S/B比)用作本底信号。参考图3,在20%和30%的环氧含量,PNA固定率是最大的。甚至在40%和50%的环氧含量,PNA固定无问题发生。在本实施例中所用的PNArtL-180w具有以下序列:
rtL180w:N末端(5')-GTTTCTCC*TGGCT-C末端(3')-Cy3(SEQID NO:2)
实施例4:印刷溶液制备和PNA阵列制造
在50μM蒸馏水中溶解PNA寡核苷酸(13-mer,PANAGENE,Inc.)。作为所述PNA寡核苷酸,使用PNAA、rtL-180w和rtL-180m。PNA A用作PNA/DNA杂交的阳性对照。所述rtL-180w是HBV(乙型肝炎病毒)RNA聚合酶(GeneIn有限公司)的特异序列,并且所述rtL-180m是导致拉米夫定(lamivudine)抗性的HBV RNA聚合酶的变化序列。所述rtL-180w和rtL-180m之间的差异仅仅为一个碱基(C-A)。所述rtL-180w和rtL-180m用作用于鉴定SNP(单核苷酸多态性)的检测序列。0.1N NaOH溶液用于辅助PNA固定。对于各PNAA、rtL-180w和rtL-180m,以1∶1~0.5的比例混和通过在蒸馏水中溶解PNA寡核苷酸制得的溶液和所述NaOH溶液,并在96-孔板中上样。用喷墨阵列机(Cartesian)将制备的样品点样在PMMA基片上,然后在50%或更高的湿度和23~24℃的温度下保存16小时,以诱导环氧基和胺基之间的充分反应。在点样期间,增加湿度以均匀地维持点大小。
在本实施例中所用的探针PNA寡核苷酸(13-mer)具有以下序列:
探针A(人工序列):N末端(5')-TTCCACCAGATGG-C末端(3')(SEQ ID NO:1)
探针W(rtL-180w):N末端(5')-GTTTCTCC*TGGCT-C末端(3')(SEQ ID NO:2)
探针M(rtL-180m):N末端(5')-GTTTCTCA*TGGCT-C末端(3')(SEQ ID NO:3)。
实施例5:芯片上的反应和SNP检测
用蒸馏水洗涤实施例4中制备的固定PNA的PMMA基片5分钟以除去残留的NaOH,然后无单独封闭杂交。使用含有5X SSC(含有氯化钠和柠檬酸钠的pH 7.0的缓冲液)、50mMHEPES、1%SDS和0.1%BSA的杂交缓冲液。用名为花青3(Cy3)的荧光染料标记的DNA(1pM~100nM)作为靶标。杂交溶液中的靶DNA在94℃变性5分钟,然后在42℃孵育60分钟。通常,相对于DNA,PNA具有高一度的熔解温度(Tm)(每一个碱基)。为了除去非特异性PNA/DNA杂交,用1XSSC缓冲液、0.1X SSC缓冲液、0.01X SSC缓冲液和0.001X SSC缓冲液进行洗涤(各5分钟)。然后,将PNA阵列完全除去水分,并用荧光检测激光扫描仪(Axon Instrument有限公司)在焦点位置65和PMT 400的条件下测定DNA/PNA杂交引起的荧光信号。图5A和5B中分别说明使用不同靶DNA和阵列信息的PNA/DNA杂交结果的荧光图像。与人工PNA序列互补的DNA寡核苷酸(阳性对照)、用于鉴定普通HBV感染的DNA寡核苷酸和用于鉴定抗拉米夫定HBV感染的DNA寡核苷酸分别用作图5A中(A)、(B)和(C)中的靶DNA。参考图5A和5B,探针PNA与对应的靶DNA特异性杂交。图6A和6B分别说明对于rtL-180w和rtL-180m的环氧基含量PNA/DNA杂交结果的图解定量分析数据。从图6A和6B,可以看出在20%和30%的环氧含量发生最大水平的PNA/DNA杂交。
在本实施例中所用的靶DNA寡核苷酸(13-mer)具有以下序列:
靶A:5'Cy3-CCATCTGGTGGAA-3'(SEQ ID NO:4)
靶W:5'Cy3-AGCCAG*GAGAAA-3'(SEQ ID NO:5)
靶M:5'Cy3-AGCCAT*GAGAAA-3'(SEQ ID NO:6)。
实施例6:通过PNA连接剂的PNA/DNA杂交
为了优化PNA/DNA杂交,将起间隔区作用的C5-8含有胺基的酸连接到PNA的胺端基。图7A为说明根据本发明用于优化PNA/DNA杂交的连接剂的示意图,和图7B说明不同类型连接剂的化学结构式。在图7B中,O-连接剂为2-(2-氨基乙氧基)乙氧基乙酸,M-连接剂为2-氨基乙氧基乙酸,和C-连接剂为6-氨基己酸。所述含有胺基的酸通过肽键与PNA连接,并且如上述实施例,含有胺基的酸的胺基与含有环氧基的聚合物层的环氧基反应,以将PNA固定到所述聚合物层上。与PNA连接的连接剂防止固定的PNA在表面上聚集,并使PNA分子对靶DNA分子有代表性。结果,起间隔区作用的连接剂改善了探针PNA对靶DNA的可接近性。在本实施例中,使用环氧含量为20%和30%的涂覆含有环氧基的聚合物的基片提供PNA固定和PNA/DNA杂交的最大效率。除了使用连接剂外,以与实施例4和5相同的方式进行PNA固定和PNA/DNA杂交。图8A和8B分别说明用于通过不同类型的连接剂介导的PNA/DNA杂交的评价结果的荧光图像信息和阵列信息。图8B的点阵列中表示连接剂和PNA的类型。在图8B中,H2O+NaOH为阴性对照,rtL-W(M)为没有连接剂的普通野生型(突变体)PNA,O-W(M)为连接O-连接剂的rtLW(M)PNA,M-W(M)为连接M-连接剂的rtLW(M)PNA,和C6-W为连接C6-连接剂的rtLW PNA。PNA-cy3用作位置标记。图9A和9B为图8A的荧光图像的图解定量分析数据。在图9A和9B中,rtLW(M)为无连接剂的普通野生型(突变体)PNA,rtLW(M)-3为连接O-连接剂的rtLW(M)PNA,rtLW(M)-4为连接M-连接剂的rtLW(M)PNA,和rtLW(M)-5为连接C6-连接剂的rtLW(M)PNA。从图9A和9B,可以看出,相对于无连接剂的探针PNA,连接连接剂的探针PNA显示对靶DNA更优异的特异性。
在本实施例中所用的连接连接剂的探针PNA的序列如下:
W(rtL180w)-连接剂:5'Cy3-AGCCAG*GAGAAA-3'-连接剂
M(rtL180m)-连接剂:5'Cy3-AGCCAT*GAGAAA-3'-连接剂。
实施例7:用于PNA/DNA杂交的PNA阵列的敏感性评价
以低于公知的PNA的浓度的10uM或5uM PNA点样实施例1和2中制备的涂覆聚合物的基片。根据实施例4和5中描述的方法评价PNA/DNA杂交中靶DNA的敏感性。点样5uM PNA(低浓度)的杂交强度特征显示与25uM PNA相似的水平。这可能是因为,由于比DNA更高的Tm值,PNA显示比DNA高的结合亲和力,并且由于无荷电率,所以无排斥力。图10A和10B说明对于探针PNA的浓度PNA/DNA杂交的检测敏感性的定量分析数据。
Claims (10)
1.一种PNA(肽核酸)芯片,其中,含有目的DNA序列的探针PNA固定在用含有环氧基的聚合物涂层的塑料基片上,
其中,所述含有环氧基的聚合物为由下面式1表示的含有环氧基的丙烯酸酯单体和高粘性丙烯酸酯单体的共聚物:
-[CR3R3-CR1R3-X]n-, (1)
其中,R1是C3-12含有环氧基的酯,R3是氢或C1-16烷基,和X是高粘性丙烯酸酯单体,n为10~2,000,其中所述高粘性丙烯酸酯单体选自由二季戊四醇羟基五丙烯酸酯(DPHA)、9-乙二醇二丙烯酸酯(9-EGDA)、季戊四醇三-四丙烯酸酯(PETA)、聚乙二醇400二丙烯酸酯、三丙二醇二丙烯酸酯、三羟甲基丙烷三丙烯酸酯和二季戊四醇六丙烯酸酯组成的组中;
或者,所述含有环氧基的聚合物是由下面式2表示的含有环氧基的丙烯酸酯单体和能够粘着于所述塑料基片上的粘性丙烯酸酯衍生物的共聚物:
-[CR3R3-CR1R3-CR3R3-CR2R3]n-, (2)
其中,R1是C3-12含有环氧基的酯,R2是C2-16烷基酯,和R3是氢或C1-16烷基,n为10~2,000。
2.权利要求1所述的PNA芯片,其中,所述塑料基片为由选自由聚甲基丙烯酸甲酯(PMMA)、聚碳酸酯(PC)、聚降冰片烯、COC(环烯烃共聚物)、氟化聚酰亚胺、聚苯乙烯(PS)、SBC(苯乙烯丁二烯共聚物)、ABS(丙烯腈丁二烯苯乙烯)、SAN(苯乙烯丙烯腈)和聚砜组成的组中的材料制成的透明塑料基片。
3.权利要求1所述的PNA芯片,其中,所述粘性丙烯酸酯衍生物选自由甲基丙烯酸甲酯(MMA)、丙烯酸乙酯、甲基丙烯酸乙酯(EMA)、丙烯酸正丙酯、甲基丙烯酸正丙酯、丙烯酸异丙酯和甲基丙烯酸异丙酯组成的组中。
4.权利要求1所述的PNA芯片,其中,所述含有环氧基的聚合物中环氧基的含量为20~30wt%的范围。
5.权利要求1所述的PNA芯片,其中,含有胺基和存在或不存在醚的C5-8羧酸连接剂与所述探针PNA的胺端基连接。
6.一种制造根据权利要求1所述的PNA芯片的方法,该方法包括:
以10~90∶80~5∶1~10的比例混和含有环氧基的丙烯酸酯单体、高粘性丙烯酸酯单体和光引发剂;
在塑料基片上涂覆所述混合物;
通过紫外光固化所述混合物;以及
在塑料基片上点样探针PNA印刷溶液。
7.一种制造根据权利要求1所述的PNA芯片的方法,该方法包括:
以10~99∶1~89∶0.1~0.5的比例混和含有环氧基的丙烯酸酯单体、能够粘着于所述塑料基片上的粘性丙烯酸酯衍生物和自由基引发剂;
自由基聚合所述混合物;
将通过在溶剂中溶解所得的聚合物制得的含有聚合物的溶液涂覆到塑料基片上;以及
在塑料基片上点样探针PNA印刷溶液。
8.权利要求6或7所述的方法,其中,所述探针PNA印刷溶液含有0.01~1.0M的碱以辅助PNA固定。
9.权利要求7所述的方法,还包括在点样前将含有聚合物的溶液涂层的塑料基片在30%或更高湿度条件下保存4小时或4小时以上。
10.一种检测SNP(单核苷酸多态性)的方法,该方法包括:
将含有靶DNA的反应样品应用于权利要求1至5任一项的PNA芯片;
探针PNA与靶DNA进行杂交;
洗涤所述PNA芯片以除去非特异性反应产物;以及
基于PNA/DNA杂交检测荧光信号。
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KR1020040047677A KR100766752B1 (ko) | 2004-06-24 | 2004-06-24 | 에폭시기를 갖는 중합체가 코팅된 플라스틱 기판을 이용한pna 칩 |
KR10-2004-0047677 | 2004-06-24 | ||
KR1020040047677 | 2004-06-24 | ||
PCT/KR2005/001920 WO2006001627A1 (en) | 2004-06-24 | 2005-06-21 | Pna chip using plastic substrate coated with epoxy group-containing polymer, method of manufacturing the pna chip, and method of detecting single nucleotide polymorphism using the pna chip |
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KR100653975B1 (ko) * | 2004-11-30 | 2006-12-05 | 한국과학기술원 | 집코드 방식을 이용한 pna 칩 및 이의 제작방법 |
KR100725579B1 (ko) * | 2005-02-16 | 2007-06-08 | 주식회사 엘지생명과학 | 에폭시기를 갖는 중합체가 코팅된 플라스틱 기판을 이용한마이코박테리아 유전자형 검출용 pna 칩 및 그를 이용한마이코박테리아 유전자형 검출 방법 |
US9051667B2 (en) * | 2007-10-26 | 2015-06-09 | Wisconsin Alumni Research Foundation | Substrate independent copolymers for biofunctionalization |
KR100943421B1 (ko) * | 2007-12-24 | 2010-02-19 | 연세대학교 산학협력단 | 에폭시기와 불포화이중결합을 갖는 광중합성 단량체 및이를 함유한 광중합 조성물 |
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WO2012120129A1 (en) | 2011-03-10 | 2012-09-13 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods and pharmaceutical composition for the treatment of infectious diseases |
US10006909B2 (en) | 2012-09-28 | 2018-06-26 | Vibrant Holdings, Llc | Methods, systems, and arrays for biomolecular analysis |
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