Graham Kenneth J, Holland Michael J
Department of Biochemistry and Molecular Medicine, University of California, School of Medicine, Davis, California 95616, USA.
Methods Enzymol. 2005;395:544-53. doi: 10.1016/S0076-6879(05)95028-3.
We describe PrimerSelect, a program capable of transcriptome-wide design of primer pairs for optimal performance in kinetic reverse-transcriptase polymerase chain reaction (RT-PCR). For the yeast Saccharomyces cerevisiae, PrimerSelect designs primer pairs for 86% of genomic open reading frames (ORFs) using design criteria we previously established to be optimal for kinetic RT-PCR (kRT-PCR)-based transcript quantitation. Primer pairs designed by PrimerSelect for 230 yeast ORFs were evaluated for primer dimer potential, PCR cyclewise yield, and cross-priming. Performance of 95% of these primer pairs is optimal with respect to primer dimer potential and PCR cyclewise yield for quantitating even the rarest yeast transcript. All of the primer pairs produced a single amplicon of the expected size from yeast genomic DNA template. The utility of PrimerSelect for designing primer pairs complementary to ORF sequences defined for multiple isolates of the human bacterial pathogens Helicobacter pylori and Staphylococcus aureus is also demonstrated.
我们介绍了PrimerSelect，这是一款能够在转录组范围内设计引物对的程序，以便在动力学逆转录聚合酶链反应（RT-PCR）中实现最佳性能。对于酿酒酵母，PrimerSelect使用我们先前确定的对基于动力学RT-PCR（kRT-PCR）的转录本定量最为优化的设计标准，为86%的基因组开放阅读框（ORF）设计引物对。对PrimerSelect为230个酵母ORF设计的引物对进行了引物二聚体形成潜力、PCR逐循环产量和交叉引物分析。就引物二聚体形成潜力和PCR逐循环产量而言，这些引物对中有95%的性能对于定量即使是最罕见的酵母转录本也是最佳的。所有引物对都从酵母基因组DNA模板产生了预期大小的单一扩增子。还展示了PrimerSelect在设计与人类细菌病原体幽门螺杆菌和金黄色葡萄球菌多个分离株所定义的ORF序列互补的引物对方面的实用性。
