Accumulated lipids rather than the rigid cell walls impede the extraction of genetic materials for effective colony PCRs in Chlorella vulgaris.
BACKGROUND
Failure of colony PCRs in green microalga Chlorella vulgaris is typically attributed to the difficulty in disrupting its notoriously rigid cell walls for releasing the genetic materials and therefore the development of an effective colony PCR procedure in C. vulgaris presents a challenge.


RESULTS
Here we identified that colony PCR results were significantly affected by the accumulated lipids rather than the rigid cell walls of C. vulgaris. The higher lipids accumulated in C. vulgaris negatively affects the effective amplification by DNA polymerase. Based on these findings, we established a simple and extremely effective colony PCR procedure in C. vulgaris. By simply pipetting/votexing the pellets of C. vulgaris in 10 ul of either TE (10 mM Tris/1 mM EDTA) or 0.2% SDS buffer at room temperature, followed by the addition of 10 ul of either hexane or Phenol:Chloroform:Isoamyl Alcohol in the same PCR tube for extraction. The resulting aqueous phase was readily PCR-amplified as genomic DNA templates as demonstrated by successful amplification of the nuclear 18S rRNA and the chloroplast rbcL gene. This colony PCR protocol is effective and robust in C. vulgaris and also demonstrates its effectiveness in other Chlorella species.


CONCLUSIONS
The accumulated lipids rather than the rigid cell walls of C. vulgaris significantly impede the extraction of genetic materials and subsequently the effective colony PCRs. The finding has the potential to aid the isolation of high-quality total RNAs and mRNAs for transcriptomic studies in addition to the genomic DNA isolation in Chlorella.
Crystal Jing Ying
CJ
Tear
Chanyuen
C
Lim
Jinchuan
J
Wu
Industrial Biotechnology Division, Institute of Chemical & Engineering Sciences, Agency for Science, Technology and Research (A*STAR), 1 Pesek Road, 627833 Jurong Island, Singapore. zhao_hua@ices.a-star.edu.sg.
Hua
H
Zhao
D002473:Cell Wall / Q000235:genetics / Q000378:metabolism*; D048408:Chlorella vulgaris / Q000235:genetics*; D008055:Lipids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*
D008055:Lipids