CN110684605B - 一种核酸祛除剂 - Google Patents
一种核酸祛除剂 Download PDFInfo
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- CN110684605B CN110684605B CN201911003447.3A CN201911003447A CN110684605B CN 110684605 B CN110684605 B CN 110684605B CN 201911003447 A CN201911003447 A CN 201911003447A CN 110684605 B CN110684605 B CN 110684605B
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Abstract
本发明涉及一种核酸祛除剂，属于分子生物学领域，含有以下成分及体积百分比，氧化剂5％‑10％、含氯试剂0.1％‑3％、含酸试剂0.1‑3％、稳定剂0.1％‑2％、表面活性剂1％‑8％和余量的去离子水。本核酸祛除剂无气味，性质温和没有刺激作用，操作简单方便，贮存方便，保存周期长，使用时可以直接将核酸祛除剂喷洒在实验室地面、墙面、桌面、生物安全柜的台面、移液器的表面、各种仪器的外表面、离心管外壁等，也可以直接用核酸祛除剂对仪器、台面、桌面等进行擦拭。使用本试剂数分钟内即可有效降解核酸片段，防止实验室气溶胶污染。
Description
技术领域
本发明属于分子生物学领域，具体地涉及一种核酸祛除剂。
背景技术
PCR技术自1985年问世以来，已经广泛应用于医学、法医学、农学、生物学等所有生命相关的科学，已经成为生命研究领域必备的基础技术之一。特别时基于普通PCR技术开发的荧光PCR技术，其敏感性比普通PCR要高100倍以上，更是受到广大科研人员的青睐，已经成为医学、兽医学领域疫病病原诊断的常用方法。但与此同时，PCR技术独特的原理告诉我们，一个分子经过PCR反应后就会产生数十亿个分子，这种高度敏感性就意味着，在PCR的操作过程中，环境和实验室极其轻微的核酸污染就有可能引起扩增产物的大量扩增，从而导致扩增结果的假阳性。而且一旦实验室污染核酸之后，往往很难清除污染的核酸。实际上，如何避免和防止核酸污染一直是PCR实验室管理的核心和关键问题，即便是一些国家级的实验室也不例外。常见的核酸污染包括：扩增产物的污染、提取核酸的污染、阳性对照的污染、外来核酸的污染等。如何处理这些污染的核酸，科研人员也提出多种处理方法，如核酸酶降解法、紫外线照射法、氯化物氧化法、高温处理法、次氯酸钠浸泡或擦洗法。这些处理方法有的存在严重的气味，影响人们身体健康，有的有的存在腐蚀和破坏仪器的风险。专利(CN 1493582A)提出利用过氧化物在紫外光照、金属离子或它们同时存在下，可以降解生物体或微生物细胞碎液中核酸的方法，但过氧化物不稳定，很短的时间内就降解成水和氧而失去活性；另外，紫外线照射穿透力很弱，仅作用于物体表面，处理效果不佳。因此，寻求一种有效、安全、快速、稳定的核酸去除试剂就成为核酸检测实验室的当务之急。
发明内容
本发明要解决的技术问题在于提供一种核酸祛除剂。本发明研制的核酸祛除剂可以有效解决核酸降解过程中遇到的腐蚀性、刺激性、高效性和稳定性等问题。本发明研制的核酸祛除剂使用方便，贮存周期长，操作简单，在环境中能自然降解，是一种生态型的环保型核酸祛除剂。
本发明是通过以下技术方案实现的：
一种核酸祛除剂，其特征在于含有以下成分及质量百分比，氧化剂5％-10％、含氯试剂0.1％-3％、含酸试剂0.1-3％、稳定剂0.1％-2％、表面活性剂1％-8％余量的去离子水，也即是用去离子水补齐。
进一步，所述的氧化剂包括但不限于过氧化氢、过氧化钠在内的可以释放氧离子的无机或有机氧化物。
进一步，所述的含氯试剂包括但不限于次氯酸钠、次氯酸以及含有次氯酸根的化合物。
进一步，所述的含酸试剂包括但不限于磷酸、乙酸、盐酸、草酸、柠檬酸等有机酸或无机酸。
进一步，所述的稳定剂包括但不限于碳酸盐、硼酸盐或草酸。
进一步，所述的表面活性剂包括但不限于烷基糖苷、异构醇聚氧乙烯醚(TO-10)、聚乙氧基化脂肪醇(AEO-9)在内的非离子表面活性剂、脂肪醇聚氧乙烯醚硫酸钠在内的阴离子表面活性剂。
一种核酸祛除剂，它含有以下成分及质量百分比为过氧化氢5％-10％，次氯酸钠0.1％-3％，草酸0.1-3％，乙酸0.1％-2％，AEO-9 1％-8％，其余用去离子水补齐，最终调整pH值为3.5。
所述氧化氢和次氯酸钠通过氧化作用破坏DNA的二级和一级结构。
所述草酸和乙酸通过氢离子的水解作用破坏DNA的磷酸二酯键和氢键。
所述AEO-9为表面活性剂：通过疏水力非离子表面活性剂与DNA的氢键发生作用降解DNA，且能够增强其他组份的溶解度。
本发明与现有技术相比的有益效果：
本核酸祛除剂的气味温和无刺激，操作简单方便，使用时可以直接将核酸祛除剂喷洒在实验室地面、墙面、桌面、生物安全柜的台面、移液器的表面、各种仪器的外表面、离心管外壁等，也可以直接用核酸祛除剂对仪器、台面、桌面等进行擦拭。本发明的核酸祛除剂不含易燃易爆或有毒性的组份，性质稳定，气味温和没有刺激作用，贮存方便，保存周期长，安全环保。本发明的核酸祛除剂通过与DNA产生的多层面的降解作用，最终将核酸片段完全降解为核苷酸，数分钟内就可以完成核酸的降解作用，能有效避免实验室气溶胶的产生，提供一个良好的实验室操作环境。
附图说明
图1为核酸祛除剂降解DNA核酸的效果图：M表示DNA 2000Marker，1，2：市售核酸祛除剂的厂家A和B，其中泳道1可少量的核酸降解，泳道2未见到明显核酸降解；泳道3为实施例1的核酸祛除剂与核酸体积比为1:1,可见核酸完全降解，泳道4为实施例1的核酸祛除剂与核酸的体积比为1:2，核酸部分降解，泳道5为实施例2的核酸祛除剂，可见核酸完全降解；泳道6-8：未处理的DNA核酸对照组，核酸未见降解。
图2三种核酸降解方式的比较：泳道1：DNA 2000Marker；泳道2：紫外线照射组；泳道3：核酸祛除剂处理组；泳道4：过氧化物处理组。
具体实施方式
结合以下实施例和附图对本发明作进一步说明。但本发明的保护范围不受实施例任何形式上限制。
实施例1
一种核酸祛除剂，含有以下成分及质量百分比为过氧化氢5％，次氯酸钠0.1％，草酸0.1，乙酸0.1％，AEO-9 1％，其余用去离子水补齐，最终调整pH值为3.5。
实施例2
一种核酸祛除剂，含有以下成分及质量百分比为过氧化氢10％，次氯酸钠3％，草酸3％，乙酸2％，AEO-9 8％，其余用去离子水补齐，最终调整pH值为3.5。
实施例3
利用实施例1和实施例2所述核酸祛除剂对基因组进行降解，具体方法如下：
(1)DNA/RNA基因组提取试剂盒提取核酸
①取0.2g猪肝脏样本，加入5倍等重量的生理盐水，研磨均匀后，加入20μL蛋白酶K溶液，混匀，56℃水浴裂解15分钟；
②室温12000转/分钟离心5分钟，取上清200μL加入到一个新的离心管中，再加入200μL缓冲溶液，充分颠倒混匀，70℃放置10分钟；
③加入200μL无水乙醇，充分震荡混匀15秒；
④将上述溶液加入到一个吸附柱中，12000转/分钟离心30秒，倒掉废液，将吸附柱放回收集管中；
⑤加入500μL洗涤液到吸附柱中，12000转/分钟离心30秒，倒掉废液，将吸附柱放回收集管中；
⑥加入600μL漂洗液到吸附柱中，12000转/分钟离心30秒，倒掉废液，将吸附柱放回收集管中；
⑦复步骤⑥；
⑧将吸附柱放回收集管中，空离吸附柱12000转/分钟离心2分钟，倒掉废液，将吸附柱插入一新的离心管内，室温静置2分钟以彻底挥发酒精；
⑨向吸附柱膜中央滴加50μLTE，室温静置2分钟，12000转/分钟离心2分钟，收集洗脱液即提取的核酸。
(2)核酸祛除剂降解试验
①取上述(1)中提取的核酸20μL，分别加入20μL实施例1和实施例2所述的核酸祛除剂；设置一个实验组加入10μL实施例1的核酸祛除剂。
②颠倒混匀后室温放置3分钟；
③加入4μL 10×Loading Buffer终止反应；
④通过1.5％琼脂糖核酸凝胶电泳分析核酸降解的效率。
(3)试验结果分析
凝胶电泳结果可以明显看出，核酸与等量的核酸祛除剂作用3分钟后，大分子的DNA基因组被降解为小片段，如图1中的3和5泳道。
实施例4
一种核酸祛除剂，其特征在于含有以下成分及质量百分比，氧化剂5％-10％、含氯试剂0.1％-3％、含酸试剂0.1-3％、稳定剂0.1％-2％、表面活性剂1％-8％、去离子水补齐。
所述氧化剂和含氯试剂通过氧化作用破坏DNA的二级和一级结构，氧化剂包括但不限于过氧化氢、过氧化钠在内的可以释放氧离子的无机或有机氧化物；含氯试剂包括但不限于次氯酸钠、次氯酸以及含有次氯酸根的化合物。
所述含酸试剂通过氢离子的水解作用破坏DNA的磷酸二酯键和氢键；含酸试剂包括但不限于磷酸、乙酸、盐酸、草酸、柠檬酸等有机酸或无机酸。
所述稳定剂能够保持过氧化物等易降解物质的稳定性，可以使核酸祛除剂在室温下保存一年以上，其降解作用没有发生明显改变。稳定剂包括但不限于碳酸盐、硼酸盐或草酸。
所述表面活性剂：通过疏水力非离子表面活性剂与DNA的氢键发生作用降解DNA，且能够增强其他组份的溶解度。表面活性剂包括但不限于烷基糖苷、异构醇聚氧乙烯醚(TO-10)、聚乙氧基化脂肪醇(AEO-9)等非离子表面活性剂、脂肪醇聚氧乙烯醚硫酸钠等阴离子表面活性剂。
核酸与等量的所述的核酸祛除剂作用3分钟后，大分子的DNA基因组被降解为小片段。
实施例5：不同试剂的降解作用比较
利用本实施例1的核酸祛除剂，分别与过氧化氢、紫外线照射等多种方法进行试验比对，观察这些处理方式降解核酸的效果。其中，待比较的核酸为提取的质粒pET32a，一组取50mg，用核酸祛除剂按照正常浓度使用，作用3分钟，一组取50mg，用紫外线照射30分钟，距离0.5m，波长是254nm，一组取50mg，加入过氧化氢至终浓度为0.01M，作用5分钟。实验结果见图2。从中可以明显看出，紫外线处理组有很少一部分核酸发生了降解，残留的核酸很明亮；过氧化物处理组比紫外线处理组降解的核酸稍多一些，但仍有大量核酸没有降解，残留的核酸仍比较清晰；而加入核酸祛除剂组的核酸基本降解完毕，在电泳的泳道中只留下模糊的核酸痕迹。
Claims (1)
1.一种核酸祛除剂，它含有以下成分及质量百分比为：过氧化氢5%-10%，次氯酸钠0.1%-3%，草酸0.1-3%，乙酸0.1%-2%，AEO-9 1%-8%和余量的去离子水， pH值为3.5。
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