Pfu DNA 聚合酶用于荧光定量PCR的改造研究
Research of Pfu polymerase improvement for Real-time fluorescent quantitative PCR
Abstract
自从1988年Saiki等将耐热性的Taq聚合酶引入PCR反应后，极大地推动了PCR技术的推广，也带动了实时荧光定量PCR（qPCR）技术的兴起，分子生物学发展日新月异。随着技术的推广与应用，人们也逐渐发现了Taq聚合酶自身存在的缺点，其保真性差不适合高保真的PCR，复杂样本扩增能力差需要对样本进行处理以及体系的优化等等。而B族的Pfu聚合酶具有高保真性、高行进性和强耐干扰能力，但是不具有5’-3’外切活性且强3’-5’外切活性会水解游离探针影响荧光增益信号检测而不能用于qPCR。因此本研究希望通过改造Pfu聚合酶，进而用于复杂样本的qPCR检测。 基于Pfu聚合酶的结构和性质，我们采取了两... Since SaiKi et al. used Taq thermostable polymerase in PCR reaction in 1988, this polymerase gave a big boost to the PCR technology development, and promoted the rise of Real-Time Quantitative PCR technology. Molecularbiology developed dramatically. With the popularization and application of PCR technology, people gradually realized the disadvantages of Taq polymerase, its low fidelity is not suit...