Accumulated lipids rather than the rigid cell walls impede the extraction of genetic materials for effective colony PCRs in Chlorella vulgaris

Crystal Jing Ying Tear,Jinchuan Wu,Chanyuen Lim,Hua Zhao

doi:10.1186/1475-2859-12-106

Abstract

BackgroundFailure 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.ResultsHere 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.ConclusionsThe 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.

Highlights

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 the development of an effective colony PCR procedure in C. vulgaris presents a challenge
Our recent efforts on the development of transformation protocols in C. vulgaris require a procedure for rapid identification of transformants by PCR screening
We previously demonstrated that using of Yeast Protein Extraction buffer (Y-PER) with additional boiling step (98°C for 10 mins) led to the effective release of genetic materials from C. vulgaris [8] regardless of growth stage

Summary

Introduction

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 the development of an effective colony PCR procedure in C. vulgaris presents a challenge. In comparison to other microalgae, C. vulgaris has relatively high growth rates, photosynthetic efficiencies and capable of accumulating over 50% lipids (dry cell weight) under nitrogen limited conditions [4,5] Those advantages have led to the great interest in this species for algal oil production and significant efforts have been exercised towards the molecular level understanding and characterization of the lipids pathways and genetic manipulation of Chlorella genes for maximizing its oil contents and optimizing the lipid profiles [4,6]. Diversity studies of microalgae demands a rapid colony PCR protocol for identification of a collection of algal strains by the 18S rRNA analysis [13]

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Results

Conclusion