An improved and reliable method for microalgae direct PCR

In recent years, methods of microalgal direct PCR have been used frequently, which simplified the DNA isolation procedure and also facilitated identification of unknown natural microalgal isolates. However, our recent attempts to amplify the 18S rRNA gene of Phaeodactylum tricornutum using the protocols reported led to poor repeatability. To explore the reasons for the instability of these methods, one-way analysis of variance was used to analyze the effects of lysis buffer type, cell number, growth stage, and lysis boiling time on the crude DNA content in the lysate. The results showed that the highest content of crude DNA (344 ng μL⁻¹) was obtained with lysis buffer 1% NP-40 (v/v) by using stationary phase cells, which was nearly twice of the content of crude DNA with buffer 0.5% Triton X-100 (207.5 ng μL⁻¹) and three times of those with buffers TE (107.5 ng μL⁻¹) and 1% SDS (108 ng μL⁻¹). It was also found that increase of the number of microalgal cells led to non-linear elevation of the crude DNA content, and the amount of crude DNA from the cells within logarithmic phase was significantly higher than those within stationary phase or decline phase. The effect of lysis boiling time was insignificant when boiling time ranged from 5 to 15 min. The results of PCR amplification with the crude DNAs isolated with different buffers indicated that efficient and repeatable amplification could be realized only when the amount of crude DNA added in 20 μL PCR reaction ranged from 50 to 200 ng, while impacts of lysis buffers themselves on the PCR reactions were negligible. Hence, it was found that the amount of crude DNA in the lysate should be measured and this was a key factor to ensure the positive PCR amplification regardless of which lysis buffer, cell number, growth stage, and lysis boiling time were used. To check the reliability and efficiency of the improved method, a pair of 18S rRNA universal primers for eukaryotic microalgae were designed, which worked very well in the PrimeBlast test and 18S rRNA amplification of microalgal species kept in the laboratory so that the primers could be widely used for microalgal screening combined with improved microalgal direct PCR. Furthermore, transformants of P. tricornutum harboring exogenous gene were screened out successfully via the improved method. Fluorescence microscope observation confirmed the screening results. This improved method can provide reliable technique for the molecular identification of microalgae species and the screen of microalgae transformants.