Journal of Applied Phycology

, Volume 26, Issue 4, pp 1719–1726 | Cite as

Single-tube colony PCR for DNA amplification and transformant screening of oleaginous microalgae

  • Jin Liu
  • Henri Gerken
  • Yantao Li


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.


Biofuels 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.


  1. Blanc G, Duncan G, Agarkova I, Borodovsky M, Gurnon J, Kuo A, Lindquist E, Lucas S, Pangilinan J, Polle J, Salamov A, Terry A, Yamada T, Dunigan DD, Grigoriev IV, Claverie JM, Van Etten JL (2010) The Chlorella variabilis NC64A genome reveals adaptation to photosymbiosis, coevolution with viruses, and cryptic sex. Plant Cell 22:2943–2955PubMedCentralPubMedCrossRefGoogle Scholar
  2. Cao M, Fu Y, Guo Y, Pan J (2009) Chlamydomonas (Chlorophyceae) colony PCR. Protoplasma 235:107–110PubMedCrossRefGoogle Scholar
  3. Chisti Y (2007) Biodiesel from microalgae. Biotechnol Adv 25:294–306PubMedCrossRefGoogle Scholar
  4. Giorno F, Mazzei R, Giorno L (2013) Purification of triacylglycerols for biodiesel production from Nannochloropsis microalgae by membrane technology. Bioresour Technol 140:172–178PubMedCrossRefGoogle Scholar
  5. Gonzalez-Ballester D, Pootakham W, Mus F, Yang W, Catalanotti C, Magneschi L, de Montaigu A, Higuera J, Prior M, Galvan A, Fernandez E, Grossman A (2011) Reverse genetics in Chlamydomonas: a platform for isolating insertional mutants. Plant Methods 7:24PubMedCentralPubMedGoogle Scholar
  6. Hu Q, Sommerfeld M, Jarvis E, Ghirardi M, Posewitz M, Seibert M, Darzins A (2008) Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances. Plant J 54:621–639PubMedCrossRefGoogle Scholar
  7. Kilian O, Benemann CSE, Niyogi KK, Vick B (2011) High-efficiency homologous recombination in the oil-producing alga Nannochloropsis sp. Proc Natl Acad Sci U S A 108:21265–21269PubMedCentralPubMedCrossRefGoogle Scholar
  8. La Russa M, Bogen C, Uhmeyer A, Doebbe A, Filippone E, Kruse O, Mussgnug JH (2012) Functional analysis of three type-2 DGAT homologue genes for triacylglycerol production in the green microalga Chlamydomonas reinhardtii. J Biotechnol 162:13–20PubMedCrossRefGoogle Scholar
  9. Li Y, Han D, Sommerfeld M, Hu Q (2011) Photosynthetic carbon partitioning and lipid production in the oleaginous microalga Pseudochlorococcum sp (Chlorophyceae) under nitrogen-limited conditions. Bioresour Technol 102:123–129PubMedCrossRefGoogle Scholar
  10. Li X, Moellering ER, Liu B, Johnny C, Fedewa M, Sears BB, Kuo M-H, Benning C (2012) A galactoglycerolipid lipase is required for triacylglycerol accumulation and survival following nitrogen deprivation in Chlamydomonas reinhardtii. Plant Cell 24:4670–4686PubMedCentralPubMedCrossRefGoogle Scholar
  11. Litaker RW, Vandersea MW, Kibler SR, Reece KS, Stokes NA, Steidinger KA, Millie DF, Bendis BJ, Pigg RJ, Tester PA (2003) Identification of Pfiesteria piscicida (Dinophyceae) and Pfiesteria-like organisms using internal transcribed spacer-specific PCR assays. J Phycol 39:754–761CrossRefGoogle Scholar
  12. Liu J, Zhong Y, Sun Z, Huang J, Sandmann G, Chen F (2010) One amino acid substitution in phytoene desaturase makes Chlorella zofingiensis resistant to norflurazon and enhances the biosynthesis of astaxanthin. Planta 232:61–67PubMedCrossRefGoogle Scholar
  13. Liu J, Huang J, Jiang Y, Chen F (2012) Molasses-based growth and production of oil and astaxanthin by Chlorella zofingiensis. Bioresour Technol 107:393–398PubMedCrossRefGoogle Scholar
  14. Lucas-Salas LM, Castrillo M, Martínez D (2013) Effects of dilution rate and water reuse on biomass and lipid production of Scenedesmus obliquus in a two-stage novel photobioreactor. Bioresour Technol 143:344–352PubMedCrossRefGoogle Scholar
  15. Moniz MBJ, Kaczmarska I (2010) Barcoding of diatoms: nuclear encoded ITS revisited. Protist 161:7–34PubMedCrossRefGoogle Scholar
  16. Packeiser H, Lim C, Balagurunathan B, Wu J, Zhao H (2013) An extremely simple and effective colony PCR procedure for bacteria, yeasts, and microalgae. Appl Biochem Biotechnol 169:695–700PubMedCrossRefGoogle Scholar
  17. Parmar A, Singh NK, Pandey A, Gnansounou E, Madamwar D (2011) Cyanobacteria and microalgae: a positive prospect for biofuels. Bioresour Technol 102:10163–10172PubMedCrossRefGoogle Scholar
  18. Radakovits R, Jinkerson RE, Fuerstenberg SI, Tae H, Settlage RE, Boore JL, Posewitz MC (2012) Draft genome sequence and genetic transformation of the oleaginous alga Nannochloropis gaditana. Nat Commun 3:686PubMedCentralPubMedCrossRefGoogle Scholar
  19. Radha S, Fathima A, Iyappan S, Ramya M (2013) Direct colony PCR for rapid identification of varied microalgae from freshwater environment. J Appl Phycol 25:609–613CrossRefGoogle Scholar
  20. Saade A, Bowler C (2009) Molecular tools for discovering the secrets of diatoms. Bioscience 59:757–765CrossRefGoogle Scholar
  21. Sluiman HJ, Guihal C, Mudimu O (2008) Assessing phylogenetic affinities and species delimitations in Klebsormidiales (Streptophyta): nuclear-encoded rDNA phylogenies and its secondary structure models in Klebsormidium, Hormidiella, and Entransia. J Phycol 44:183–195CrossRefGoogle Scholar
  22. Stoeglehner G, Narodoslawsky M (2009) How sustainable are biofuels? Answers and further questions arising from an ecological footprint perspective. Bioresour Technol 100:3825–3830PubMedCrossRefGoogle Scholar
  23. Vieler A, Wu G, Tsai C-H, Bullard B, Cornish AJ, Harvey C, Reca I-B, Thornburg C, Achawanantakun R, Buehl CJ, Campbell MS, Cavalier D, Childs KL, Clark TJ, Deshpande R, Erickson E, Armenia Ferguson A, Handee W, Kong Q, Li X, Liu B, Lundback S, Peng C, Roston RL, Sanjaya Simpson JP, TerBush A, Warakanont J, Zäuner S, Farre EM, Hegg EL, Jiang N, Kuo M-H, Lu Y, Niyogi KK, Ohlrogge J, Osteryoung KW, Shachar-Hill Y, Sears BB, Sun Y, Takahashi H, Yandell M, Shiu S-H, Benning C (2012) Genome, functional gene annotation, and nuclear transformation of the heterokont oleaginous alga Nannochloropsis oceanica CCMP1779. PLoS Genet 8:e1003064PubMedCentralPubMedCrossRefGoogle Scholar
  24. Wan M, Rosenberg JN, Faruq J, Betenbaugh MJ, Xia J (2011) An improved colony PCR procedure for genetic screening of Chlorella and related microalgae. Biotechnol Lett 33:1615–1619PubMedCrossRefGoogle Scholar
  25. Wang SB, Hu Q, Sommerfeld M, Chen F (2004) Cell wall proteomics of the green alga Haematococcus pluvialis (Chlorophyceae). Proteomics 4:692–708PubMedCrossRefGoogle Scholar
  26. Wijffels RH, Barbosa MJ (2010) An outlook on microalgal biofuels. Science 329:796–799PubMedCrossRefGoogle Scholar
  27. Yoon K, Han D, Li Y, Sommerfeld M, Hu Q (2012) Phospholipid:diacylglycerol acyltransferase is a multifunctional enzyme involved in membrane lipid turnover and degradation while synthesizing triacylglycerol in the unicellular green microalga Chlamydomonas reinhardtii. Plant Cell 24:3708–3724PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  1. 1.Institute of Marine and Environmental TechnologyUniversity of Maryland Center for Environmental ScienceBaltimoreUSA
  2. 2.Institute of Marine and Environmental TechnologyUniversity of Maryland Baltimore CountyBaltimoreUSA
  3. 3.Department of Applied Sciences and MathematicsArizona State University Polytechnic CampusMesaUSA

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