Annealing temperature for multiplex PCR was determinedunder empirically chosen primer concentration of0.400 μM. In this study, the annealing temperatures wereinvestigated from 51°C to 62°C and at <57°C annealingtemperature, the unspecific amplifications occurred visibly(Figure 1). As the annealing temperature increased, theunspecific amplification gradually decreased. When theannealing temperature ranged from 57°C to 62°C, allthree primer pairs were efficiently amplified and rare, unspecificamplification products were observed (Figure 2).However, the amplification efficiency decreased obviouslyalong with the increase in annealing temperature.Therefore, the optimum annealing temperature of 57°Cwas selected for multiplex PCR.
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