Development of Software for High-throughput PCR Design

描述(由申请人提供)：这项提案的目标是开发新的软件，用于基于严格的科学和计算方法设计各种形式的聚合酶链式反应(PCR)DNA扩增。DNA软件公司已经创建了“寡核苷酸建模平台”OMP，以实现各种基于杂交的分析的强大的自动化高通量设计。新软件将显著提高聚合酶链式反应的可靠性，并将实现高通量聚合酶链式反应应用，从而显著节省成本。将实现三个具体目标：AIM1：OMP核心开发。将增加一种进化算法，以允许对多重反应的引物选择、温度循环方案、[Mg2+]、链浓度和酶的选择进行优化。OMP将增加模拟PCR杂交和聚合酶延伸动力学的新功能。该模块将允许准确地模拟在聚合酶链式反应循环期间发生的反应的时间进程，以便准确地预测在热循环结束时的产物分布。目标2：为不同格式的聚合酶链式反应开发一个通用的图形用户界面和内部功能。图形用户界面将可针对多种不同的聚合酶链式反应格式进行配置，该提案将侧重于完成多重聚合酶链式反应、基因合成、实时聚合酶链式反应和错配引物聚合酶链式反应所需的功能。目的：对多重聚合酶链式反应和其他形式的OMP进行动力学参数化和验证的实验研究。荧光相关光谱和停流动力学将用于系统地研究不同条件下双链形成、靶展开、失配识别的动力学常数，并将执行几种不同的序列。为了验证我们的软件对聚合酶链式反应引物设计的预测，我们将进行一系列精心设计的单靶和多重聚合酶链式反应。

DESCRIPTION (provided by applicant): The goal of this proposal is to develop new software for designing polymerase chain reaction (PCR) DNA amplifications in various formats based upon a rigorous scientific and computational approach. DNA Software Inc. has created the "oligonucleotide modeling platform," OMP, to enable robust automated high-throughput design of a variety of hybridization-based assays. The new software will significantly improve the reliability of PCR and will enable high-throughput PCR applications resulting in significant cost savings. Three specific aims will be performed: Aim1: OMP Core Development. An evolutionary algorithm will be added to allow optimization of the choice of primers for multiplexing, temperature cycling protocol, [Mg2+], strand concentration, and choice of enzyme. New functionality for simulating the kinetics of PCR hybridization and polymerase extension will be added to OMP. This module will allow for the time course of the reactions that occur during PCR cycling to be accurately modeled so that the product distribution at the end of the thermocycling is accurately predicted. Aim2: Develop a generalized graphical user interface (GUI) and internal functionality for PCR in different formats. The GUI will be configurable for many different PCR formats, and this proposal will focus on finishing the functionality required for multiplex PCR, gene synthesis, real-time PCR, and mismatched primer PCR. Aim3: Experimental studies for kinetics parameterization and validation of OMP for multiplex PCR and other formats. Fluorescence correlation spectroscopy and stopped-flow kinetics will be used to perform a systematic study of the kinetic constants for duplex formation, target unfolding, mismatch discrimination under different conditions, and several different sequences will be performed. To validate the predictions of our software for PCR primer design, we will perform a carefully designed series of single target and multiplex PCR reactions.