CN106282323B - 基于聚胸腺嘧啶为模板生成铜纳米粒子的高灵敏度dna荧光分析方法 - Google Patents
基于聚胸腺嘧啶为模板生成铜纳米粒子的高灵敏度dna荧光分析方法 Download PDFInfo
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Abstract
本发明公开了基于聚胸腺嘧啶为模板生成铜纳米粒子的高灵敏DNA荧光分析方法。将捕获DNA的3’端固定到磁珠表面,与目标DNA片段杂交后,在末端脱氧核苷酸转移酶的催化作用下,对目标DNA的3’端进行加尾,生成聚胸腺嘧啶,铜离子可以与聚胸腺嘧啶相互作用,抗化学酸钠将二价铜还原成一价铜,一价铜再歧化形成铜纳米粒子附着在目标DNA的聚胸腺嘧啶模板上,铜纳米粒子具有良好的荧光特性,荧光强度与目标DNA的杂交情况相关,通过荧光分析的方法可以实现对DNA片段的高灵敏度、高选择性检测,可应用于分子识别、临床诊断、环境监测及食品安全等领域中DNA样品的检测。
Description
技术领域
本发明属于荧光分析应用领域,具体涉及一种基于聚胸腺嘧啶(PolyT)为模板生成铜纳米粒子(CuNPs)的高灵敏DNA荧光分析方法。
背景技术
高灵敏度高特异性的DNA检测技术在疾病诊断、药物开发、食品安全、环境监测等方面得到了广泛的应用,灵敏度、特异性、检测速率的提高,成本的降低以及更简便的过程控制是目前DNA检测技术发展的趋势。与早期的DNA检测技术如比色法、凝胶电泳、紫外光谱等检测方法相比,采用荧光法的DNA传感器由于其高灵敏度、低成本及便携性等特点受到了越来越多的关注。
文献1(李宏业,范树国、刘玉乐、陈刚、王寰宇,改良聚丙烯酰胺凝胶电泳检测DNA,细胞生物学杂志,1999,21,201-202。)利用改良的聚丙烯酰胺凝胶电泳法,用30%丙烯酰胺,10×TBE,TEMED,10%过硫酸铵配制成10mL的3.5%凝胶,灌胶后采用15V/cm的电压梯度电泳,利用银染法对拟南芥基因组DNA为模板的PCR产物及其克隆于pGEM7Z上的重组质粒限制酶切产物进行检测,DNA片段得到很好的分离,条带显示清晰。目前,这种聚丙烯酰胺凝胶电泳法对于DNA的检测技术已经发展成熟,然而此法需要制胶、聚合酶链式反应,电泳、染色等一系列的过程,消耗时间长,劳动强度大,染料毒性大,银染的DNA片段不能直接回收纯化,且无法用于痕量及突变DNA的检测,因此具有很大的局限性。
文献2(Fernando Patolsky,Yossi Weizmann,Itamar Willner.Redox-ActiveNucleic-Acid Replica for the Amplified Bioelectrocatalytic Detection of ViralDNA.JACS.2002,124,770-772.)将短链DNA探针固定于金电极表面,其与待测的环状病毒DNA部分杂交,在聚合酶的作用下,以长链上未杂交部分的DNA作为母链进行DNA复制,复制原料中的dUTP标记有电活性物质羧酸二茂铁,从而得到具有二茂铁标记的寡聚核苷酸修饰电极,在电极上引入电信号。为了获得放大的检测信号,用葡萄糖氧化酶催化葡萄糖的氧化来加速电子传递。该实验通过测定峰电流的变化,达到特异性的检测病毒DNA的目的。目前,利用二茂铁的电活性来对DNA进行检测的方法已经得到了广泛的应用,然而电化学法背景噪音大,重现性差,且难以用于单核苷酸多态性检测。
发明内容
本发明的目的是为了检测痕量的DNA片段,包括对完全互补、单碱基错配、三碱基错配和对照序列的鉴别,提供一种高灵敏度高特异性DNA荧光分析方法。
实现本发明目的的技术解决方案为:一种基于聚胸腺嘧啶为模板生成铜纳米粒子的高灵敏DNA荧光分析方法,包括如下步骤:
步骤一、将链霉亲和素磁珠(SA-MB)悬浮在PBS缓冲液中,加入3’端修饰生物素的捕获DNA(cDNA),反应结束后进行磁分离并用PBS缓冲液清洗多次;
步骤二、将固定了捕获DNA的磁珠复悬在Tris-HCl缓冲液中,加入目标DNA(tDNA)片段,杂交结束后进行磁分离并用Tris-HCl缓冲液清洗多次;
步骤三、杂交后的磁珠复悬在1×TdT缓冲液中,加入TdT、脱氧胸腺三磷酸(dTTP),催化结束后加热以终止反应;
步骤四、再将步骤三中的反应混合物转移到含NaCl的3-(N-吗啉基)丙磺酸(MOPS)缓冲液中,加入抗坏血酸钠及硫酸铜溶液后,进行荧光分析。
步骤一中,所述的链霉亲和素磁珠的浓度为0.5~1.5mg/mL,cDNA的浓度为0.5~1.5μM,cDNA的序列为5’-AAC CAT ACA ACC TAC TAC CTC A-Biotin-3’,反应条件为室温振荡0.5~1.5h。
步骤二中,所述的tDNA浓度为0.1nM~10nM,选用序列为5’-TGA GGT AGT AGG TTGTAT GGT T-3’的完全互补的DNA片段,序列为5’-TGA GGT AGT AGG TTG TGT GGT T-3’的单碱基错配的DNA片段,序列为5’-TGA GGT ATT AGA TTG TGT GGT T-3’的三碱基错配的DNA片段或序列为5’-ACT TAC CTT TGC TCA TTG ACG A-3’的对照DNA片段;反应条件为37℃下振荡0.5~1.5h。
步骤三中,所述的TdT的浓度为0.5~1.5U/μL,dTTP的浓度为2~3mM,反应条件为37℃下振荡5~7h,反应终止条件为60~80℃下加热5~15min。
步骤四中,所述的抗坏血酸钠溶液浓度为4~6mM,硫酸铜溶液浓度为1nM~0.1M,荧光激发波长为320~360nm。
与现有技术相比,本发明的显著优点是:
1、采用荧光法进行分析具有很高的灵敏度,且背景噪声小、成本低、方法简便、分析时间短。
2、通过TdT在tDNA末端原位聚合胸腺嘧啶,荧光信号得到了显著放大。
3、以PolyT为模板,加入还原剂及二价铜离子后能够立即生成CuNPs,极大程度上简化了分析过程。
4、此方法具有很高的灵敏度,且对单核苷酸多态性具有很好的鉴别能力。
附图说明
图1是本发明基于PolyT为模板生成CuNPs的高灵敏DNA荧光分析示意图。
图2是本发明实施例2中的的透射电镜图(其中,a为对照序列分析结果,b为互补序列分析结果)。
图3是本发明实施例3中的铜离子浓度优化图(其中,a为荧光光谱的比较,b为峰值的比较)。
图4是本发明实施例4中的CuNPs荧光稳定特性图(其中,a为荧光光谱随时间的变化,b为峰值随时间的变化)
图5是本发明实施例5中的对不同浓度tDNA片段的检测特性图(其中,a为荧光光谱的比较,b为峰值的比较)。
图6是本发明实施例6中的对不同序列DNA片段的荧光响应对比图(其中,a为荧光光谱的比较,b为峰值的比较)。
具体实施方式
下面通过实施例对本发明作进一步的说明,其目的是为更好理解本发明的内容,但所举的实施例并不限制本发明的保护范围:
实施例1:基于PolyT为模板生成CuNPs的紫外和荧光光谱表征。
采用本发明所述的基于PolyT为模板生成CuNPs的高灵敏DNA荧光分析,其过程示意图见图1。将1mg/mL SA-MB悬浮在20μL的PBS缓冲液中,加入1μM的3’端修饰生物素的cDNA(序列为5’-AAC CAT ACA ACC TAC TAC CTC A-Biotin-3’),室温振荡反应1h,反应结束后进行磁分离并用PBS缓冲液清洗多次,再将固定了cDNA的磁珠复悬在20μL的Tris-HCl缓冲液中,加入10nM互补DNA片段(序列为5’-TGA GGT AGT AGG TTG TAT GGT T-3’),37℃下振荡反应1h,杂交结束后进行磁分离并用Tris-HCl缓冲液清洗多次,杂交后的磁珠复悬在20μL的1×TdT缓冲液中,加入1U/μL的TdT、2.5mM的dTTP,37℃下振荡反应6h,催化结束后在70℃下加热10min以终止反应,将反应混合物转移到300μL含NaCl的MOPS缓冲液中,加入5mM抗坏血酸钠及1mM硫酸铜溶液后,进行紫外和荧光分析,荧光激光波长为340nm。结果表明:基于PolyT为模板生成CuNPs具有良好的荧光特性,该荧光分析方法具有可行性及高效性。
实施例2:基于PolyT为模板生成CuNPs的透射电镜表征。
采用本发明所述的基于PolyT为模板生成CuNPs的的高灵敏DNA荧光分析,操作步骤如同上述实施例1。其中,选用10nM序列为5’-TGA GGT AGT AGG TTG TAT GGT T-3’的互补DNA片段作为目标分析物,10nM序列为5’-ACT TAC CTT TGC TCA TTG ACG A-3’的DNA片段作为对照,其透射电镜图如附图2所示,其中,a是对照组,b是实验组,由图可见,CuNPs仅在tDNA与cDNA互补配对后才会生成。
实施例3:铜离子浓度优化。
采用本发明所述的基于PolyT为模板生成CuNPs的的高灵敏DNA荧光分析,操作步骤如同上述实施例1。其中,选用10nM序列为5’-TGA GGT AGT AGG TTG TAT GGT T-3’的互补DNA片段作为目标分析物,改变铜离子浓度依次为1nM、10nM、100nM、1μM、10μM、100μM、1mM、10mM、100mM,分析不同的铜离子浓度对本发明DNA荧光分析结果的影响,其分析结果如附图3所示,其中,a为荧光光谱的比较,b为峰值的比较,由图可知,铜离子浓度为1nM、10nM、100nM、1μM、10μM、100μM时,随着浓度的增加,荧光强度显著增强,而在浓度高于1mM的情况下,随着浓度的增加,荧光强度显著降低,因此在实际检测过程中,选用100μM作为适宜的铜离子浓度。
实施例4:CuNPs荧光稳定性实验。
采用本发明所述的基于PolyT为模板生成CuNPs的的高灵敏DNA荧光分析,操作步骤如同上述实施例1。其中,选用10nM序列为5’-TGA GGT AGT AGG TTG TAT GGT T-3’的互补DNA片段作为目标分析物,铜离子浓度为100μM,检测产生的CuNPs的荧光随时间的变化,其结果如附图4所示,其中,a为荧光光谱随时间的变化,b为峰值随时间的变化,由图可知,在静置1h以后,CuNPs仅下降不到35%的荧光,具有较好的荧光稳定性,因此适用于荧光分析应用。
实施例5:基于PolyT为模板生成CuNPs的的高灵敏DNA荧光分析对不用浓度DNA片段的荧光响应特性。
采用本发明所述的基于PolyT为模板生成CuNPs的的高灵敏DNA荧光分析,操作步骤如同上述实施例1。其中,选用序列为5’-TGA GGT AGT AGG TTG TAT GGT T-3’的互补DNA片段作为目标分析物,浓度分别为0.1nM、0.2nM、0.4nM、0.6nM、0.8nM、1nM、2nM、4nM、6nM、8nM、10nM,分析不同浓度tDNA片段的荧光响应特性,分析结果如附图5所示,由图可知,在该检测浓度范围内,荧光强度随着tDNA浓度的增加而增加,具有良好的线性关系,检测极限为98.2pM,荧光响应特性较好且灵敏度较高。
实施例6:基于PolyT为模板生成CuNPs的的高灵敏DNA荧光分析对不用序列DNA片段的荧光响应特性。
采用本发明所述的基于PolyT为模板生成CuNPs的的高灵敏DNA荧光分析,操作步骤如同上述实施例1。其中,以完全互补(5’-TGA GGT AGT AGG TTG TAT GGT T-3’)、单碱基错配(5’-TGA GGT AGT AGG TTG TGT GGT T-3’)、三碱基错配(5’-TGA GGT ATT AGA TTGTGT GGT T-3’)、对照(5’-ACT TAC CTT TGC TCA TTG ACG A-3’)的DNA片段作为目标分析物,对不同序列DNA片段进行荧光分析,分析结果如附图6所示,由图可知,本发明的DNA荧光分析方法对于不同序列的DNA片段具有不同的荧光响应,因而可以有效区分单核苷酸多态性,具有高特异性和选择性。
Claims (4)
1.一种基于聚胸腺嘧啶为模板生成铜纳米粒子的高灵敏DNA荧光分析方法,其特征在于,该方法为非诊断用的分析方法,包括如下步骤:
步骤一、将链霉亲和素磁珠悬浮在PBS缓冲液中,加入3’端修饰生物素的捕获DNA,反应结束后进行磁分离并用PBS缓冲液反复清洗,其中,所述的链霉亲和素磁珠的浓度为0.5~1.5mg/mL,3’端修饰生物素的捕获DNA的浓度为0.5~1.5μM;
步骤二、将固定了捕获DNA的磁珠复悬在Tris-HCl缓冲液中,加入3’端与捕获DNA的5’端形成平末端的目标DNA片段,杂交结束后进行磁分离并用Tris-HCl缓冲液反复清洗,其中,所述的目标DNA浓度为0.1nM~10nM;
步骤三、杂交后的磁珠复悬在1×TdT缓冲液中,加入TdT、脱氧胸腺三磷酸,催化反应在目标DNA的3’端原位聚合胸腺嘧啶后加热以终止反应;
步骤四、再将步骤三中的反应混合物转移到含NaCl的3-(N-吗啉基)丙磺酸缓冲液中,加入抗坏血酸钠及硫酸铜溶液后,进行荧光分析;
其中,
目标DNA的序列为5’-TGA GGT AGT AGG TTG TAT GGT T-3’,
捕获DNA的序列为5’-AAC CAT ACA ACC TAC TAC CTC A-Biotin-3’。
2.如权利要求1所述的基于聚胸腺嘧啶为模板生成铜纳米粒子的高灵敏DNA荧光分析方法,其特征在于,步骤一中,反应条件为室温振荡0.5~1.5h。
3.如权利要求1所述的基于聚胸腺嘧啶为模板生成铜纳米粒子的高灵敏DNA荧光分析方法,其特征在于,步骤三中,所述的TdT的浓度为0.5~1.5U/μL,脱氧胸腺三磷酸的浓度为2~3mM,反应条件为37℃下振荡5~7h,反应终止条件为60~80℃下加热5~15min。
4.如权利要求1所述的基于聚胸腺嘧啶为模板生成铜纳米粒子的高灵敏DNA荧光分析方法,其特征在于,步骤四中,所述的抗坏血酸钠溶液浓度为4~6mM,硫酸铜溶液浓度为1nM~0.1M,荧光激发波长为320~360nm。
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