Modified inter-simple sequence repeat PCR protocol for use in conjunction with the LI-COR gene ImagIR(2) DNA analyzer

Inter-simple sequence repeat (inter-SSR) PCR was assessed for use in variet y testing of chrysanthemum. This method was modified to allow detailed anal ysis of DNA profiles on a LI-COR Gene ImagIR(2) DNA analyzer system. Protoc ols for unlabeled PCR were unsuccessful in producing labeled products when using infrared (IR) dye-labeled primers. Various modifications to the known protocols were investigated: (i) different ratios of labeled to unlabeled primer; (ii) various annealing temperatures; (iii) the use of an IR genotyp ing kit; (iv) end labeling; and (v) direct incorporation and cycle labeling . Successful amplification using labeled primers only occurred when two con secutive reactions were performed. The first PCR was performed using standa rd protocols for unlabeled reactions. The second PCR used a dilution of the se reaction products as a template and 50% IR-labeled and unlabeled primer. The complete procedure leading to a high-resolution analysis of inter-SSR PCR products on a LI-COR system is reported for the first time. This system allows high-throughput fingerprinting with the potential for applications on a commercial scale.