Abstract
The ease and effectiveness of colony polymerase chain reaction (PCR) has allowed rapid amplification of DNA fragments and screening of large number of colonies of interest including transformants and mutants with genetic manipulations. Here, we evaluated colony PCR in Chlamydomonas. Individual colonies were treated with 10 mM ethylenediaminetetraacetic acid (EDTA) or Chelex-100 and the resulting clear cell lysate was used for PCR reaction. Either genomic DNA or plasmid DNA incorporated into the genome was equally amplified. We found that the Chelex method is superior to EDTA method in certain cases. This colony PCR technique will bypass the tedious process of isolating genomic DNA for PCR reaction and will make it possible for rapid amplification of genomic DNA fragments as well as rapid large-scale screening of transformants.
References
Harris EH (2001) Chlamydomonas as a model organism. Annu Rev Plant Physiol Plant Mol Biol 52:363–406. doi:10.1146/annurev.arplant.52.1.363
Hofmann MA, Brian DA (1991) Sequencing PCR DNA amplified directly from a bacterial colony. Biotechniques 11:30–31
Kindle KL (1990) High-frequency nuclear transformation of Chlamydomonas reinhardtii. Proc Natl Acad Sci U S A 87:1228–1232. doi:10.1073/pnas.87.3.1228
Pan J (2008) Cilia and ciliopathies: from Chlamydomonas and beyond. Sci China C Life Sci 51:479–486. doi:10.1007/s11427-008-0071-3
Pan J, Snell WJ (2000) Regulated targeting of a protein kinase into an intact flagellum. An aurora/Ipl1p-like protein kinase translocates from the cell body into the flagella during gamete activation in Chlamydomonas. J Biol Chem 275:24106–24114. doi:10.1074/jbc.M002686200
Pan J, Wang Q, Snell WJ (2004) An aurora kinase is essential for flagellar disassembly in Chlamydomonas. Dev Cell 6:445–451. doi:10.1016/S1534-5807(04)00064-4
Sizova I, Fuhrmann M, Hegemann P (2001) A Streptomyces rimosus aphVIII gene coding for a new type phosphotransferase provides stable antibiotic resistance to Chlamydomonas reinhardtii. Gene 277:221–229. doi:10.1016/S0378-1119(01)00616-3
Walsh PS, Metzger DA, Higuchi R (1991) Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. Biotechniques 10:506–513
Ward AC (1992) Rapid analysis of yeast transformants using colony-PCR. Biotechniques 13:350
Zamora I, Feldman JL, Marshall WF (2004) PCR-based assay for mating type and diploidy in Chlamydomonas. Biotechniques 37:534–536
Acknowledgements
We thank all other members of our laboratory for helpful discussion. This work is supported by the National Natural Science Foundation of China (#30671090, #30771084), National Basic Research Program of China (also called 973 program; no. 2007CB914401).
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Muqing Cao and Yu Fu contributed equally to this work.
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Cao, M., Fu, Y., Guo, Y. et al. Chlamydomonas (Chlorophyceae) colony PCR. Protoplasma 235, 107–110 (2009). https://doi.org/10.1007/s00709-009-0036-9
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DOI: https://doi.org/10.1007/s00709-009-0036-9