Optimization of PCR parameters for microsatellite markers in barley


Microsatellites are reliable and efficient DNA markers. They have been used for breeding and genetic diversity studies in barley. Although their use is robust compared to other markers, some PCR parameters should be optimized for their successful use. The aim of the present study was to discuss the importance of optimizing certain PCR parameters and to highlight some considerations for specificity and efficiency of PCR reactions. It was found that increasing MgCl2 concentration from 1.5 to 2.5 mM and decreasing the reaction volume from 40 to 25 μl improved the specificity of the reactions. Increasing the Taq DNA polymerase amount from 1 to 2 units and employment of touchdown PCR procedure resulted in amplifications in otherwise unsuccessful reactions. It turned out that primer degradation was a common threat for PCR amplifications, and PCR primers should be checked on gels when an amplification problem is encountered. Some SSR reactions were found to produce artifacts such as stutter bands and heteroduplex DNA formations which could confound the genotyping with these markers. Use of different gel systems to score SSR data was also discussed. It was concluded that each SSR marker should be evaluated as a unique chemical reaction and results of amplifications should be evaluated carefully for a better use of SSR markers in barley research.

Annealing temperature, Genotyping, Polymerase chain reaction, PCR artifact, PCR specificity, Touchdo