Single-tube colony PCR for DNA amplification and transformant screening of oleaginous microalgae
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Recently, several colony PCR methods have been developed to simplify DNA isolation procedure and facilitate PCR-based colony screening efforts in microalgae. A main drawback of current protocols is that cell collection, disruption, and genomic DNA extraction are required preceding the PCR step, making the colony PCR process laborious and costly. In the present study, we have developed a novel procedure that eliminates any steps of DNA extraction and allows the colony screening to be performed in a single PCR tube: algal cells (as low as 5,000) from agar plates or liquid cultures were directly transferred into a PCR tube containing 2× PCR buffer and boiled for 5–10 min depending on different algal strains, followed by addition of other PCR components (dNTPs, primers, and polymerase) and then subjected to conventional PCR reaction. The procedure documented here worked well not only for the model alga Chlamydomonas reinhardtii, but also for the thick-walled oleaginous strains such as Chlorella, Haematococcus, Nannochloropsis, and Scenedesmus with its efficacy independent on amplicon sizes and primer pairs. In addition, screening of Chlorella zofingiensis transformants was achieved using this method. Collectively, our single-tube colony PCR is a much simpler and more cost-effective procedure as compared to those previously reported and has broad applications including gene cloning, strain determination, and high-throughput screening of algae colonies and transformants for biomass and biofuel production.
KeywordsBiofuels Colony PCR High throughput Microalgae Single tube Transformant screening
This work was supported by the Institute of Marine and Environmental Technology, University of Maryland Center for Environmental Science and University of Maryland Baltimore County. We thank Dr. Qiang Hu (Institute of Hydrobiology, Chinese Academy of Sciences) for providing Chlorella sp., Pseudochlorococcum sp., and Scenedesmus sp. strains and Dr. Jian Xu (Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences) for the access to Nannochloropsis genome database.
Conflict of interest
The authors declare no competing interests.
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