Primer Design for Multiplex Polymerase Chain Reaction and Multiplex Isothermal Recombinase Polymerase Amplification

• Main Authors: Chou, Yu-Pao, 周育葆
• Other Authors: Huang, Hsien-Da
• Format: Others
• Language: en_US
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/65p49j

碩士 === 國立交通大學 === 生物資訊及系統生物研究所 === 106 === At present, most of the deoxyribonucleic acid (DNA) amplification techniques such as polymerase chain reaction (PCR). PCR relies on the thermal cycle machine, through denaturation, annealing, extension, the process requires precise control the temperature. Recombinase polymerase amplification (RPA) technology is developed by TwistDx in 2006. First, it makes the primer sequence and protein form a complex, the complex will find the location of the homologous sequence on the DNA template and open double-stranded DNA helix structure. Next, amplification was performed by recombinase polymerase. The temperature of the whole process is maintained at about 37 to 42°C, which will allow the DNA amplification technology get a new breakthrough. DNA amplification technology at constant temperature will no longer need to rely on the thermal cycle machine, enhances this DNA amplification technology’s portability and convenience. However, the most important part of the technology is how to design primers to make the RPA correctly amplify the target gene or sequence. In addition, design primers for multiplex PCR or RPA, it needs to avoid the two primers because the sequence with excessive similarity leads to form primer dimers so that reduce the amplification efficiency. So far, it is still not found that someone provides a primer design for multiplex RPA platform. In this study, we collect RPA primers from literature, and statistics out RPA primer features and integrate the recommendations of primer design from literature. Next, according to as above, use a series of bioinformatics methods like we use Primer3 to generate candidate primer groups, and then we use Bowtie to confirm the specificity of each primer pairs. Finally, the genetic algorithm was used to find out optimized primer group that the temperature between the two primers will not be too high to form primer dimers. In this study, we respectively designed primer sets for multiplex PCR and multiplex RPA to provide future experimental verification, such as gel electrophoresis, next-generation sequencing or Nanopore MinION sequencing platform. In summary, this study develops a web platform and a standalone tool allows users to design multiplex PCR or RPA primer sets that meet their own experimental needs.