CN103045554B - 一种制备热启动Taq酶的方法 - Google Patents
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Abstract
本发明公开了一种制备热启动Taq酶的方法，将Taq酶与处理溶液混合，即得热启动Taq酶；所述的处理溶液为肝素溶液、肝素钠溶液或肝素钾溶液。本发明的有益效果是：实现了在PCR反应中，不仅可以在PCR反应的第一步做到热启动，在PCR反应的后续步骤中同样可以实现热启动，从而有效的避免了非特异性序列的扩增的发生，提高了DNA聚合酶的反应特异性、可靠性、均一性和灵敏度，相比现有同类产品，提高了扩增效率，在较低循环数就能从微量、痕量样本中检测、克隆到目的基因，可以很大程度上减少人为误差，适用于常规PCR反应、复杂模板、痕迹模板的扩增和检测。
Description
技术领域
本发明涉及Taq酶的制备方法技术领域，特别是一种制备热启动Taq酶的方法。
背景技术
聚合酶链锁反应，Polymerase chain reaction，简称PCR，是一种分子生物学技术，用于扩增特定的DNA片段，这种方法可在生物体外进行，不必依赖大肠杆菌或酵母菌等生物体。PCR这项技术，被广泛地运用在医学和生物学的实验室，例如用于判断检体中是否会表现某遗传疾病的图谱、传染病的诊断、基因复制，以及亲子鉴定。
PCR用于扩增一小段已知的DNA片断，可能是单个基因，或者仅仅是某个基因的一部分。与活体生物不同的是，PCR只能复制很短的DNA片断，通常不超过10kbp。目前应用的PCR反应需要几个基本组成：DNA模板（template），含有需要扩增的DNA片断；2个引物（primer），决定了需要扩增的起始和终止位置；DNA聚合酶（polymerase），复制需要扩增的区域；脱氧核苷三磷酸（dNTP），用于构造新的互补链；缓冲体系，提供适合聚合酶行使功能的化学环境。
PCR反应在热循环设备中进行，PCR仪可以将反应管加热或冷却到每步反应所需的精确的温度。一般的PCR反应由20到40个循环组成，每个循环包括以下3个步骤：
1.利用高温(93-98℃)使双链DNA分离（熔解）。高温将连接两条DNA链的氢键打断。在第一个循环之前，通常加热长一些时间以确保模板和引物完全分离，仅以单链形式存在。该步骤时间为3分钟。
2.在DNA双链分离后，降低温度使得引物可以结合于单链DNA上（降温或称接合），需30秒。
3.最后，DNA聚合酶由降温时结合上的引物开始沿着DNA链合成互补链（延长）。此阶段的温度依赖于DNA聚合酶。该步骤时间依赖于聚合酶以及需要合成的DNA片断长度。延长速度为1分钟/kb。
PCR反应所必需的DNA聚合酶目前广泛采用嗜热细菌水生栖热菌(Thermus aquaticus，Taq)体内产生的具有热稳定性的DNA聚合酶，因此被称为Taq酶。其作用是通过构建磷酸二酯键将脱氧核苷酸聚合形成脱氧核苷酸链，从而形成双链DNA分子。被广泛用于当前的PCR操作中，Taq酶的缺点是它缺少3'到5'校正外切酶活性，因此在复制DNA时有时会出错，造成DNA序列突变（错误）。为了避免操作过程中产生非特异性序列的扩增，人们对PCR技术进行了改良，发明了热启动聚合酶链式反应（Hot start PCR）。该技术将DNA聚合酶与其他反应物隔绝，或是使用在高热状况下才会活化的聚合酶（热启动Taq酶），该酶在90℃以上才能发挥很好的催化效果，以避免在未达到设定温度前就开始反应。
热启动PCR有以下几种方式：
蜡隔绝法：将除了聚合酶以外的反应物加入PCR管后，加滴熔融的石蜡；待石蜡凝固后再加入聚合酶。放入PCR仪开始反应升温后，石蜡融化浮至最顶层，聚合酶才与其他反应物混合。石蜡可同时作为防止蒸发用。
热启动聚合酶（化学型）：经过甲醛等化学分子修饰的聚合酶，高热下结构改变而启动。
热启动聚合酶（抗体）：带有针对聚合酶的免疫抗体，高热下使抗体分离而启动。
热启动聚合酶（共价键结合）：改良自抗体型，使用小分子的化合物，阻塞聚合酶作用位置，高温下分离。
以上目前普遍使用的方法只能在第1步PCR反应时作到热启动，后续步骤仍难以避免非特异性序列的扩增的发生。
发明内容
本发明的目的在于克服现有技术的缺点，提供一种不仅可以在PCR反应的第一步做到热启动，在PCR反应的后续步骤中同样可以实现热启动的热启动Taq酶的制备方法。
本发明的目的通过以下技术方案来实现：一种制备热启动Taq酶的方法，将Taq酶与处理溶液混合，即得热启动Taq酶；
所述的处理溶液为肝素溶液、肝素钠溶液或肝素钾溶液。
所述的Taq酶与处理溶液混合液中肝素、肝素钠或肝素钾的浓度为0.0001U/ml以上。
将肝素、肝素钠或肝素钾溶于去离子水至溶液终浓度为0.0001U/ml以上，将Taq酶加入该溶液中；将反应产物采用透析、过柱或超滤方法纯化，即得热启动Taq酶。
在PCR反应液中加入肝素、肝素钠或肝素钾，使肝素、肝素钠或肝素钾的终浓度为0.0001U/ml以上。
本发明具有以下优点：本发明实现了在PCR反应中，不仅可以在PCR反应的第一步做到热启动，在PCR反应的后续步骤中同样可以实现热启动，从而有效的避免了非特异性序列的扩增的发生，提高了DNA聚合酶的反应特异性、可靠性、均一性和灵敏度，相比现有同类产品PCR反应可减少5～10个循环即可获得特异性扩增条带，提高了扩增效率，在较低循环数就能从微量、痕量样本中检测、克隆到目的基因，可以很大程度上减少人为误差，适用于常规PCR反应、复杂模板、痕迹模板的扩增和检测。
附图说明
图1为本发明实施例3的扩增结果图。
具体实施方式
下面结合实施例对本发明做进一步的描述，本发明的保护范围不局限于以下所述：
实施例1：
一种制备热启动Taq酶的方法，将Taq酶与处理溶液混合，即得热启动Taq酶。
所述的处理溶液为肝素溶液、肝素钠溶液或肝素钾溶液，所述的Taq酶与处理溶液混合液中肝素、肝素钠或肝素钾的浓度为0.0001U/ml以上。
其具体操作方法为：
将肝素、肝素钠或肝素钾溶于去离子水至溶液终浓度为0.0001U/ml以上，将Taq酶加入该溶液中。将反应产物采用透析、过柱或超滤方法纯化，即得热启动Taq酶。
实施例2：
一种制备热启动Taq酶的方法，将Taq酶与处理溶液混合，即得热启动Taq酶。
所述的处理溶液为肝素溶液、肝素钠溶液或肝素钾溶液，所述的Taq酶与处理溶液混合液中肝素、肝素钠或肝素钾的浓度为0.0001U/ml以上。
其具体操作方法为：
在PCR反应液中加入肝素、肝素钠或肝素钾，使肝素、肝素钠或肝素钾的终浓度为0.0001U/ml以上，PCR反应液中的Taq酶与肝素、肝素钠或肝素钾溶液混合后，即得热启动Taq酶。
实施例3：
将本发明的热启动Taq酶应用于PCR，以Foregene PCR Easy为例，以模板2.5ul（2.5ng），2×PCR Easy Mix25ul，10uM Primer11ul，10uM Primer21ul，补ddH2O至50ul，按如下反应条件反应：94℃3min热变性，94℃变性30sec，55℃退火30sec，72℃延伸1min，共25～40个循环，最后72℃保5min。同时以未处理过的DNA聚合酶（5U/ul）做平行对照。结束PCR反应后，取反应产物5ul，琼脂凝胶电泳检测，结果见图1，从图1可知，采用热启动耐高温DNA聚合酶扩增强度明显增加。图中，1、2、3：普通Taq酶扩增，1a、2a、3a：热启动Taq酶扩增，M：100bp-1KB Marker，1-1a：ZmIVR(225bp)，2-2a：BnPEP(248bp)，3-3a：D-lgl。
Claims (2)
1.一种制备热启动Taq酶的方法，其特征在于：将Taq酶与处理溶液混合，即得热启动Taq酶，其中，所述的处理溶液为肝素溶液、肝素钠溶液或肝素钾溶液，混合的具体操作方法是：将肝素、肝素钠或肝素钾溶于去离子水至溶液终浓度为0.0001U/ml，将Taq酶加入该溶液中；将反应产物采用透析、过柱或超滤方法纯化，即得热启动Taq酶。
2.根据权利要求1所述的一种制备热启动Taq酶的方法，其特征在于：混合的具体操作方法是：在PCR反应液中加入肝素、肝素钠或肝素钾，使肝素、肝素钠或肝素钾的终浓度为0.0001U/ml，PCR反应液中的Taq酶与肝素、肝素钠或肝素钾溶液混合后，即得热启动Taq酶。
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