Rapid,High Quality DNA Isolation from Cypress (Cupressus sempervirens L.) Needles and Optimization of the RAPD Marker Technique

A protocol for extracting high quality DNA from cypress (Cupressus sempervirens L.), a gymnosperm of economic and ecological importance to the Mediterranean, is presented using the commercially supplied DNeasy^TM Plant Mini Kit (QIAGEN GmbH, Germany). Additional steps were introduced in the QIAGEN protocol to significantly enhance DNA quantity and quality. For one sample, the procedure can be completed in less than one hour, and more than 10 samples can be processed in a day. DNA yield and purity were monitored by gel electrophoresis and by determining absorbance at UV (A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀). Both ratios were between 1.7 and 2.0, indicating that the presence of contaminating metabolites was minimal. The average DNA yield obtained from 100 mg starting material was around 22 g, which compares very favorably with numbers indicated by the manufacturer. Additionally, restriction and PCR analyses of the extracted DNA showed its compatibility with downstream applications. Using this DNA, the parameters for the randomly amplified polymorphic DNA (RAPD) protocol were optimized based on the use of (1) an AmpliTaq^® DNA Polymerase, Stoffel fragment (Perkin-Elmer), and (2) a high initial denaturation temperature (97.5 °C). Reproducible amplification products were achieved in all PCR reactions. Seven to eight bands per primer were obtained with most individuals. This represents a number at the high end of published results with other plant species.