Partial 16S rRNA gene sequencing:

This is a useful and widely used technique for identifying bacterial strains at least at the genus level, and often at species level too. The CECT sequences approximately the first 1000 nucleotides of the gene, including the hypervariable regions where most nucleotide differences between species accumulate.

In the event that the result is not sufficiently discriminatory to allow identification at species level, the CECT staff contact the customer to inform them of the results and the possibility of improving the identification by full 16S rRNA gene sequencing or, if necessary, other techniques.

Full 16S rRNA gene sequencing (about 1500 nucleotides):

This technique can improve the result of identification at species level in some bacterial genera. Furthermore, it is essential to describe a new type culture strain of prokaryotic species (Stackebrandt et al., 2002).

Necessary material

Preferably, an active culture of the strain (petri dish or agar slant).

If the customer prefers, we can also work with genomic DNA of the strain. In that case, the genomic DNA must be delivered in cold-storage and be of good quality (A260 / A280 between 1.8 and 2). We require more than 1 microgram of DNA at a concentration of 40-50 ng/µl resuspended in ultrapure water or in TE buffer.

Methodology

The methodology used for partial sequencing is described in Arahal et al. (2008). The DNA is amplified by PCR in a thermocycler using universal primers, 616V: 5´-AGA GTT TGA TYM TGG CTC AG -3´, 699R: 5´-RGG GTT GCG CTC GTT -3´.  PCR products are checked by electrophoresis on agarose gel 1% (w / v) in Tris-Borate-EDTA buffer at 135 V for 25 minutes.

The methodology used for the full sequencing is described in Arahal et al. (2008) and Lucena et al. (2010). The DNA is amplified by PCR using universal primers 616V: 5´- AGA GTT TGA TYM TGG CTC AG -3´ and 699R: 5´- GGG TYK CGC TCG TTR -3´ TTR, to the 5 'end of the gene, and P609D primers: 5’ - YAACGAGCGMRACCC -3’ y P1525R: 5’ - WAGGAGGTRATCCADCC -3’ to the 3 'end. PCR products are checked by electrophoresis on agarose gel 1% (w / v) in Tris-Borate-EDTA buffer at 135 V for 25 minutes.

PCR fragments are sequenced on an ABI 3730xl sequencer (Applied Biosystems) using the same PCR primers at a concentration of 5 nM.

The 16S rRNA gene sequence is analysed on EzTaxon (Kim et al., 2012) and BLAST (Altschul et al., 1997) platforms. The results presented include the overlapping fragment extension, the percentage of similarity and the accession number of the sequence of type strain of the species with the closest identity.

Bibliography

Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 1997. 25, 3389-3402.

Arahal DR, Sánchez E, Macián MC, Garay E. Value of recN sequences for species identification and as a phylogenetic marker within the family “Leuconostocaceae”. Int Microbiol. 2008. 11: 33-39.

Kim OS, Cho YJ, Lee K, Yoon SH, Kim M, Na H, Park SC, Jeon YS, Lee JH, Yi H, Won S, Chun J.  Introducing EzTaxon: a prokaryotic 16S rRNA Gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol. 2012. 62, 716-721.

Lucena T, Pascual J, Garay E, Arahal DR, Macián MC, Pujalte MJ. Haliea mediterranea sp. nov., a new marine gammaproteobacterium. Int. J. Syst. Evol. Microbiol. 2010. 50, 1844-1848.