Abstract:

The aims were to optimize the protocol of genomic DNA extraction with sodium hydroxide hydrolysis method in spinach, and provide the basis for marker-assisted selection of large-quantity breeding materials. The young leaves of spinach were used as materials, according to the PCR amplification results, the effects of NaOH concentrations, water bath temperatures and methods, and Tween 20 concentrations on the extracted quality of DNA from young spinach leaves were studied. The result showed that the effect of PCR amplification with DNA extracted by boiling water bath for 1 min was much better than those extracted by ambient room temperature treatment. Leaf samples could also be directly incubated at 99°C for 4 minutes in a thermal cycler instead of the boiling water bath, omitting the grinding and centrifuge steps. At this moment, the best extraction solution for a perfect PCR amplification result was 0.4 mol/L NaOH+ 0.5% Tween 20. The higher value A₂₆₀/A₂₃₀ of the extracted DNA was, the better result PCR amplification had. So A₂₆₀/A₂₃₀ was the best criterion for evaluating DNA quality. This study optimized the protocol of genomic DNA extraction with sodium hydroxide hydrolysis method, obtained the ideal PCR amplification.

Key words: spinach, DNA extraction, sodium hydroxide hydrolysis method, DNA quality assessment, marker-assisted selection