CN116751840B - 一种石墨相氮化碳纳米片捕获探针的制备及应用 - Google Patents
一种石墨相氮化碳纳米片捕获探针的制备及应用 Download PDFInfo
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Abstract
本发明公开了一种杂交捕获肿瘤基因的石墨相氮化碳纳米片捕获探针制备方法及其应用。所述捕获探针包括石墨相氮化碳纳米片以及固定在所述石墨相氮化碳纳米片上的与目标分子互补的单链核酸探针。本发明通过单链核酸探针与目标DNA碱基互补配对形成稳定的双链DNA，通过固定在石墨相氮化碳上的金粒子，将单链核酸探针和互补配对的靶标DNA固定在石墨相氮化碳纳米片上，从而实现了目标肿瘤基因的富集。本发明与磁珠分离的液相杂交捕获技术相比，减少了基因富集的实验步骤，缩短了富集时间；提高了基因检测灵敏度和准确度，拓展了二维纳米材料在液体活检和靶向重测序等领域的应用。
Description
【技术领域】
本发明属于二维纳米材料应用领域以及目标基因捕获技术领域，特别涉及一种石墨相氮化碳纳米片捕获探针、制备方法、目标基因富集和检测方法及其应用。
【背景技术】
目标基因捕获测序是将感兴趣的基因的目标区域定制成特异性探针，通过杂交分离将目标基因的DNA片段进行富集，而后再利用NGS技术进行测序。选择检测少量的目标区域，可以实现更高的测序深度。目标基因捕获测序技术具备只需消耗少量的成本和时间的优势。在相同成本下，研究者可以研究到更多的样本数量和测到更深的测序深度。作为一个精准、目的性明确的先进技术，它在新一代高通量测序的应用中发挥独特作用。
现有的目标基因捕获技术主要分为固相杂交捕获和液相杂交捕获，主要存在对样本的浓度要求高、操作步骤多、杂交时间长等缺点。而且，由于捕获探针的限制，永远都有一半的目标分子未被捕获到，这使得极低起始量的情况下，检测的灵敏度和准确度大大降低，限制了液体活检、临床穿刺样本等微量样本的检测，或者癌症早期检测的低频突变等的应用。因此，如何实现高效的捕获成为最紧迫的问题。本专利涉及的杂交捕获探针，可以在样本具有较低浓度的目标基因时，依旧能有效的富集和检测目标基因，提高了基因检测的灵敏度和准确度，另外，通过减少实验步骤，缩短检测时间，在合适的条件下实现快速杂交。
【发明内容】
基于二维纳米材料的石墨相氮化碳纳米片捕获探针，可特异性捕获目标肿瘤基因，具有样本量要求低、步骤简单、灵敏度高等优点。
为达到以上目的，本发明所采取的技术方案是提供一种用于杂交捕获肿瘤基因的二维纳米探针的制备方法、基因富集方法。所述的二维纳米探针包括石墨相氮化碳纳米片以及金纳米粒子链接的与目标基因互补的单链核苷酸捕获探针CNNS@Au-ssDNA，所述石墨相氮化碳纳米片为单层片状结构，至少一个维度在100nm范围内。
优选地，所述在石墨相氮化碳纳米片上，通过还原法生成金纳米粒子，所述金纳米粒子直径约为1-10nm。
优选地，所述单链核酸探针通过Au-S键固定在石墨相氮化碳纳米片上的金粒子上。
优选地，所述与目标基因互补的单链核苷酸捕获探针包括肿瘤基因探针库，所述探针库能够与目标的4种突变的肿瘤基因正义链杂交，所述的探针库的核苷酸序列包括了突变位点，如SEQ ID NO.1～SEQ ID NO.9所示。
检测BRAF的探针为SEQ ID NO.1；
检测KRAS的探针为SEQ ID NO.2-3；
检测PIK3CA的探针为SEQ ID NO.4-5；
检测PTEN的探针为SEQ ID NO.6-9；
优选地，所述检测探针的长度为40-100nt，例如可以是40nt、41nt、42nt、43nt、44nt、45nt、46nt、47nt、48nt、49nt、50nt…91nt、92nt、93nt、94nt、95nt、96nt、97nt、98nt、99nt、100nt，优选60nt。
一种用于捕获目标肿瘤突变基因的富集和检测方法，包括以下步骤：提取检测对象的基因组DNA，将基因组DNA片段化，末端修复后加上polyA尾并连接扩增接头，构建样本基因库；将样本基因库和CNNS@Au-ssDNA探针库进行液相杂交后；探针会与目标DNA单链互补配对，固定在金负载的石墨相氮化碳纳米片上；将反应体系进行离心，去除游离的未特异性结合的非靶标DNA单链，洗涤靶向捕获了目标序列的沉淀后，重新悬浮在变性缓冲液中，将杂交的DNA双链打开，离心去除沉淀后得到捕获的目标序列；将捕获的目标序列进行PCR实现目的基因的富集，检测并扩增目标DNA；后续应用NGS技术进行测序分析。
优选地，所述检测对象的基因组DNA来源于人类。
优选地，所述检测对象的DNA提取方式为利用市售的DNA提取试剂盒提取。
优选地，所述基因组DNA片段化的方法包括超声破碎法、核酸内切酶酶切法和转座酶酶切法。
相比现有技术，本发明具有以下优点：
本发明针对常见的肿瘤突变基因进行杂交捕获和富集，提供了一种多位点的肿瘤突变基因检测方法，该方法具有减少实验步骤、缩短检测时间、特异性强、灵敏性高等优点。
【附图说明】
图1为本发明所述的CNNS@Au-ssDNA纳米探针进行杂交捕获肿瘤基因的流程示意图；
图2为本发明实施例1中所需的石墨相氮化碳纳米片CNNS@Au的TEM图；
图3为本发明实施例1中CNNS@Au的粒径分布图；
图4为本发明实施例1中CNNS@Au的XPS谱图；
图5为本发明实施例2中合成的CNNS@Au-PIK3CA ssDNA纳米探针的PCR电泳图；
图6为本发明实施例2中合成的CNNS@Au-PIK3CA ssDNA纳米探针捕获目标肿瘤基因PIK3CA的相对表达量。
【具体实施方式】
下面将结合本发明说明书附图，对本发明实施例作更清楚、完整地描述。本发明所描述的实施例仅是一部分，而不是全部的实施例，在不脱离本发明方法的前提下做出的若干改进和补充都在本发明的保护范围之内。
实施例一
本实施例提供一种CNNS@Au的合成方法。
步骤1：在马弗炉中将三聚氰胺加热至550℃并在该温度下在空气中继续保持4h，使三聚氰胺分子聚合制备块状g-C3N4。
步骤2：将制备的g-C3N4(1g)与20mL H2SO4(98wt％)在50mL烧瓶中混合，并在室温下搅拌8h。然后将混合物缓慢倒入100mL去离子水中并进行超声处理以剥落。而后离心、洗涤、烘干。
步骤3：将获得的粉末(0.3g)放入装有150mL甲醇的烧瓶中，并在65℃加热回流6h。而后离心，80℃干燥，所得产物为石墨相氮化碳纳米片(CNNS)。
步骤4：CNNS@Au通过沉积共还原方法制造,使用CNNS作为载体，HAuCl4作为金源。将10mg CNNS分散在20mL超纯水中，混合物在超声辅助条件下处理30min，然后将104.5μLHAuCl4(24.28mM)水溶液加入上述制备的悬浮液中，搅拌30min。
步骤5：将0.5mL 0.05M柠檬酸钠溶液滴加到悬浮液中。然后，100μL新鲜制备的NaBH4溶液(0.01M)快速加入上述分散液中连续剧烈搅拌(1000rpm)40分钟。随后，将获得的纳米材料用超纯水洗涤并离心回收，在60℃下干燥。所得材料为CNNS@Au。
结果：如图2透射电镜图所示，CNNS呈现超薄片状结构，属于二维纳米材料；并通过DLS进行尺寸和粒径分布，结果如图3所示，CNNS的平均流体力学直径约为120nm；并通过元素分析进行表征，如图4所示，在90eV处出现了Au 4f的峰值，其他相应的元素有对应的峰值，与已报道的相一致，说明已成功负载上了Au纳米粒子。
实施例二
本实施例提供一种CNNS@Au-ssDNA捕获探针的合成方法，所述的探针包括：
与BRAF、KRAS、PIK3CA和PTEN突变的正义链完全互补的ssDNA。
所述的突变类型包括点突变、小片段插入、缺失、融合或拷贝数扩增中任意一种或至少两种的组合。
步骤1：根据探针库合成ss-DNA-SH。
步骤2：所述探针库包括SEQ ID NO.1-9所示的核苷酸序列，其中，检测BRAF的探针为SEQ ID NO.1；
检测KRAS的探针为SEQ ID NO.2-3；
检测PIK3CA的探针为SEQ ID NO.4-5；
检测PTEN的探针为SEQ ID NO.6-9；
步骤3：将CNNS@Au和ssDNA-SH混合均匀，在37℃下孵育ssDNA偶联CNNS@Au的合成如下：50μL 50mM Tris(2-carboxyethyl)phosphine(TCEP)加入500μL 1μM巯基(SH-)修饰的ssDNA中，混合物在室温下振荡2h以减少二硫键。而后将490μL CNNS@Au与上述混合物混合并振荡24h。将20μL0.1M NaCl以5min的间隔分5次加入CNNS@Au-ssDNA溶液中。所得溶液在15600rpm下离心30min。除去上清液，将沉淀物重新分散在1mL含有0.3MNaCl的10mM PBS(pH 7.4)中。得到CNNS@Au-ssDNA探针。选择PIK3CA作为靶标探针，添加引物通过PCR对溶液进行检测。
结果：如图5所示，CNNS@Au-ssDNA泳道有明显条带，而CNNS@Au泳道，即未加探针的样本没有条带，可说明CNNS@Au-ssDNA已成功制备。
实施例三
本实施例使用实施例2制备的CNNS@Au-ssDNA纳米探针，进行目标肿瘤基因捕获，特异性强。
步骤1：按照说明书的操作，提取检测对象的基因组DNA；
步骤2：使用超声破碎仪将基因组DNA破碎成200bp左右大小的片段；
步骤3：使用T4 DNA聚合酶、T4 PNK和Klenow DNA聚合酶将片段化的DNA进行末端修复后使用Exo(-)Klenow酶添加polyA尾；
步骤4：使用TADNA链接酶在DNA两端加上扩增接头序列；
步骤5：对连接产物进行PCR扩增，构建DNA样本库；
步骤6：设计不同纳米探针，与靶标完全互补PIK3CAE542K G>A(T1)探针、单碱基错配包括PIK3CAE542K G>C(T2)、PIK3CAE542K G>T(T3)和PIK3CAE542K的单核苷酸缺失(T4)的探针及未添加探针的CNNS@Au溶液；
步骤7：将步骤6中CNNS@Au-ssDNA纳米探针和DNA样本库进行杂交，加入甲酰胺，90℃变性5分钟后，65℃杂交12-16h。
步骤8：杂交后，离心收集带目标靶基因的CNNS@Au-ssDNA纳米探针，经过洗涤后，将探针重悬浮于含0.1M氯化钠的PBS溶液中，加入甲酰胺，90℃变性5分钟后，再次离心去除CNNS@Au-ssDNA纳米探针，重复两次后，收集上清，得到捕获的目的基因片段。
步骤9：使用DNA聚合酶对捕获到的目标序列进行扩增。对PCR产物进行质量鉴定。
结果：如图6所示，通过荧光定量对各组结果进行鉴定，只有完全与靶标互补探针组的表达量最高，与其他组相比都有显著差异，其中不含探针的CNNS@Au溶液最低。
以上所述仅为本发明的实施例，并非因此限制本发明的专利范围，凡是利用本发明说明书内容所作的所有等同的变化或变形，都应涵盖在本发明的专利保护范围之内。
Claims (2)
1.一种基于肿瘤突变基因检测的二维纳米捕获探针的制备方法，其特征在于，所述制备方法包括以下步骤：
步骤1：在马弗炉中将三聚氰胺加热至550℃，并在该温度下在空气中继续保持4h，使三聚氰胺分子聚合制备块状g-C3N4，通过超声剥离法以及65℃，6h热回流处理制备出单层石墨相氮化碳纳米片CNNS；
步骤2：通过沉积共还原方法制备CNNS@Au，使用CNNS作为载体，HAuCl4作为金源，柠檬酸钠溶液还原；然后用新鲜制备的NaBH4溶液快速加入还原体系中连续剧烈搅拌40min，获得在石墨相氮化碳纳米片上原位生成的金纳米粒子的复合材料CNNS@Au；
步骤3：设计并合成与BRAF、KRAS、PIK3CA或PTEN正义链互补的SH修饰的单链核酸探针ssDNA-SH；
步骤4：将CNNS@Au和ssDNA-SH混合均匀，在37℃下孵育，ssDNA偶联CNNS@Au得到CNNS@Au-ssDNA探针；
所述ssDNA-SH为与BRAF、KRAS、PIK3CA或PTEN的正义链互补的寡核苷酸片段，具有检测BRAF的探针为SEQ ID NO.1所示的核苷酸序列，具有检测KRAS的探针为SEQ ID NO.2-3所示的核苷酸序列，具有检测PIK3CA的探针为SEQ ID NO.4-5所示的核苷酸序列，具有检测PTEN的探针为SEQ ID NO.6-9所示的核苷酸序列。
2.根据权利要求1所述的一种基于肿瘤突变基因检测的二维纳米捕获探针的制备方法，其特征在于，所述单链核酸探针捕获链ssDNA-SH的3’端修饰有SH键。
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