Development of Software for High-throughput PCR Design

描述（由申请人提供）：本提案的目标是开发新软件，用于基于严格的科学和计算方法设计各种格式的聚合酶链反应（PCR）DNA扩增。DNA软件公司创建了“寡核苷酸建模平台”OMP，以实现各种基于杂交的测定的稳健的自动化高通量设计。新软件将显著提高PCR的可靠性，并将实现高通量PCR应用，从而显著节省成本。将执行三个具体目标：目标1：OMP核心开发。将添加进化算法，以优化多重引物的选择、温度循环方案、[Mg2+]、链浓度和酶的选择。将在OMP中添加用于模拟PCR杂交和聚合酶延伸动力学的新功能。该模块将允许对PCR循环期间发生的反应的时间过程进行准确建模，以便准确预测热循环结束时的产物分布。目标2：为不同格式的PCR开发通用图形用户界面（GUI）和内部功能。GUI将可配置用于许多不同的PCR格式，并且该提案将侧重于完成多重PCR、基因合成、实时PCR和错配引物PCR所需的功能。目的3：用于多重PCR和其他形式的OMP动力学参数化和验证的实验研究。将使用荧光相关光谱和停流动力学对不同条件下双链体形成、靶标解折叠、错配辨别的动力学常数进行系统研究，并将进行几种不同的序列。为了验证我们的软件对PCR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PCR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.