EAGER: BISH: Isothermal solid-phase PCR using electrostatic control of DNA hybridization

这项拟议的研究调查了迄今为止未知的静电DNA杂交的基本特征。也就是说，产量、动力学速率、变性长DNA的能力和支持足够数量的杂交循环，以建立有效的PCR扩增条件。具体目的是：1.构建用于电极拴系引物杂交和延伸的荧光检测仪，建立消失波荧光实时检测仪，用于测定固相PCR变性、退火期和延伸期的动力学和产率。对静电等温聚合酶链式反应进行了原理验证和优化。将使用寡核苷酸模板在实时PCR模式下演示线性和指数扩增。将确定有效的静电脉冲周期以及最佳表面和溶液条件。将开发扩增方案。

0939048VainrubThe proposed research investigates the hitherto unknown fundamental characteristics of electrostatic DNA hybridization. Namely; the yield, kinetic rates, ability to denature long DNA and to support a sufficient number of hybridization cycles, in order to establish conditions for efficient PCR amplification. The specific aims are to: 1. Construct a fluorescent detector for hybridization and extension of electrode tethered primers.An evanescent wave fluorescent real-time detector will be built to measure the kinetics and yieldof denaturation, annealing, and extension stages in solid-phase PCR.2. Prove-in-principle and optimize electrostatic isothermal PCR. Linear and exponentialamplification will be demonstrated in real-time PCR mode using oligonucleotide templates.Efficient electrostatic pulse cycles and optimal surface and solution conditions will be defined.Amplification protocols will be developed.