Sequencing of protein-coding genes is an increasingly used technique in microbiology laboratories. The analysis of these sequences helps establish phylogenetic relationships and improve identification of species in those strains for which ribosomal DNA analysis does not provide sufficient resolution.
The choice of genes depends on the microbial group to which the isolate belongs. The most commonly used in eukaryotic genes are: ß-Tubulin (TUB2), translation elongation factor (TEF1), actin gene (ACT1) and second largest subunit of RNA polymerase II (RPB2).
Methodology
If users wish to sequence genes, they must provide the citation with the study methodology (sequence of primers, PCR conditions, etc.).
Otherwise, CECT staff will perform a literature search and advise the client on which gene / genes may be useful for the strain under study.
The primers used to amplify different genes are:
- Partial sequencing of the ß-tubulin gene (TUB2): Bt2a / Bt2b (Glass and Donaldson, 1995)
- Partial sequencing of the translation elongation factor (TEF1): with primers tef1fw / tef1rev (O'Donnell et al., 1998), sequencing of a fragment of 0.2 kb (short intron, tef1_int5, between exons 5 and 6 of the gene). With primers EF1728F / EF1-986R (Carbone & Kohn, 1999), sequencing of a 0.3 kb fragment (long intron, tef1_int4, between exons 4 and 5 of the gene).
- Partial Sequencing of the actin gene (ACT1): CA14 / CA5R (Daniel & Meyer, 2003)
- Partial sequencing (with reads in both directions) of the second largest subunit of RNA polymerase II (RPB2): fRPB2-5F (Liu et al., 1999) / fRPB2-414R (Quaedvlieg 2011 et al.).
The DNA is amplified by PCR using previously described and published primers. The PCR products are checked by electrophoresis on agarose gel 1% (w / v) in Tris-Borate-EDTA buffer at 135 V for 25 minutes.
On study completion, the sequence will be sent in FASTA format with a report of the results obtained by comparison with published sequences, including the overlapping fragment extension, identity percentage and the name of the microorganism with the highest degree of sequence identity.
Bibliography
Carbone, I., Kohn, L.M. (1999). A method for designing primer sets for speciation studies in fifilamentous ascomycetes. Mycologia, 91:553–556.
Daniel, H.M. & Meyer, W. (2003). Evaluation of ribosomal RNA and actin gene sequences for the identification of ascomycetous yeasts. International Journal of Food Microbiology, 86:61–78
Glass, N.L., Donaldson, G.C. (1995). Development of primersets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Appl Environ Microb., 61:1323–1330.
Liu, Y.J., Whelen, S. and Hall, B.D. (1999). Phylogenetic relationships among ascomycetes: evidence from an RNA polymerase II subunit. Mol. Biol. Evol. 16(12):1799-1808.
O’Donnell, K., Cigelnik, E. & Nirenberg, H. I. (1998). Molecular systematics and phylogeography of the Gibberella fujikuroi species complex. Mycologia, 90:465–493.
Quaedvlieg, W., Kema, GHJ., Groenewald, JZ., Verkley. GJM., Seifbarghi, S.(2011). Zymoseptoria gen. nov.: new genus to accommodate Septorialike species occurring on graminicolous hosts. Persoonia, 26: 57–69.
