Variety of applications, such as the genotyping of individuals, RWJ 64809 152121-47-6 paternity testing, and monitoring the genetic make-up of plants and animals in agricultural breeding programs. Techniques based on polymerase chain reaction provide a powerful tool for the amplification of minute amounts of initial target sequences. Most PCR protocols involve reactions that amplify a single target. Multiplex PCR is a variation of the conventional technique in which two or more targets are simultaneously amplified in the same reaction. This approach has the potential for greater reliability, flexibility, and cost reduction. As far as we know, nine-target multiplex PCR method has been reported to simultaneously detect eight maize lines as well as the endogenous Zein gene in a single reaction tube, which contains the most targets in reported multiplex-PCR methods. Multiplex PCR is an essential cost-saving technique for large scale scientific, clinical, and commercial applications, such as infectious microorganisms detection, gene expression, whole-genome sequencing, forensic analysis including human identification and paternity testing, the diagnosis of infectious diseases, and pharmacogenomic studies aimed at understanding the connection between individual genetic traits, drug response and disease susceptibility. In recent years, multiplex PCR has emerged as a core enabling technology for high-throughput SNP genotyping. With the rapid development of GM crops, more and more studies have recently described the use of multiplex PCR as a rapid and convenient screening assay for the detection of GMOs. In GM crops such as soybean, maize, and canola, a multiplex PCR system has been developed to detect multiple target sequences using simultaneous amplification profiling. The choice of DNA polymerase is very important for the optimum performance of the PCR. The PhireTM Hot Start DNA polymerase, coupled with a preoptimized primer mix for different multiplex reactions, gave the best results both in terms of reproducibility and robustness. The use of hot start DNA polymerase prevents the formation of misprimed products and reduces primer-dimer formation. As the number of primers increases, the possible sequence dependent interactions between primers of different primer pairs also increase, which results in the formation of primer-dimers. Small differences in amplification efficiencies for the different primer pairs might result in the preferential amplification of some of the PCR products, leaving other PCR products at subdetectable levels.