An extremely simple and effective colony PCR procedure is established for both gram-negative and gram-positive bacteria, yeasts, and microalgae. Among the four lysis buffers examined, Y-PER is observed to be more effective than Tris/EDTA, 0.2 % SDS, and 10 mM EDTA in the extraction of PCR-quality genomic DNA from those microorganisms. Vortexing or pipetting agitation of the cells in Y-PER for 5-10 s was sufficient to release genomic DNA for all the test bacteria and yeasts, and most microalgae. Additional incubation at 98 °C for 5 min for further cell disruption was essential only for Chlorella vulgaris due to its notoriously rigid cell wall.
Inverted repeat and palindromic sequences have the propensity to form non-beta cruciform structures during DNA replication, leading to perturbations within the genome or plasmid replicon. In this study, the tolerance of the Escherichia coli genome to inverted repeat sequences from 25 to 1200 bp was investigated. Genomic inverted repeats were readily created via the homologous insertion of an overlap extension PCR product containing a gene-specific region of the genome together with thyA coding sequence, creating inverted repeat sequences of various lengths flanking the thyA selection marker in the resulting genome. Inverted repeat sequences below 100 bp were stably propagated, while those above and up to 1200 bp were found to be transiently unstable under auxotrophic thymine selection. Excision efficiency improves with increases of the inverted repeat until 600-800 bp, indicating that the genomic stability of inverted repeat sequences is due to secondary structure formation. Its effectiveness of creating precise and scar-free gene deletions was further demonstrated by deleting a number of genes in E. coli. The procedure can be readily adapted for sequence integration and point mutations in E. coli genome. It also has the potential for applications on other bacteria for efficient gene deletions.
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.
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