Methods of molecular identifications and lab. protocols
Preferred primers for sequencing the 5’ end of the translation elongation factor-1α gene (eEF1a1)
Preferred primers for sequencing the 5' end of the translation elongation factor-1α gene (eEF1a1)
Increasingly more informative genes are required to resolve complexes of closely related species in Trichoderma. ITS, which was until recently considered the DNA fragment of choice for identifications at the species level, is now proving inadequate to distinguish closely related species in several Trichoderma clades. Instead, protein coding genes containing large introns have become increasingly valuable to differentiate closely related and recently evolved taxa such as sympatric species.
One gene we recommend is the translation elongation factor gene which has, in Trichoderma, three large introns located in less than a 1000bp span at the 5' end. We have found the protocol that we describe here to be essentially foolproof in sequencing this gene fragment in Trichoderma, but equally effective for essentially all Hypocreales, having used these primers successfully in Metarhizium, Beauveria, Bionectria, Fusarium, etc. Being almost specific to Hypocreales, we have also found these primers useful in examining populations of Hypocreales in soil by cloning and sequencing soil DNA.
Appended to the protocol for tef is an effective protocol developed by Tom Graefenhan for the large intron in RNA polymerase subunit B (RPB2) and reported here with his permission.
>>> tef (eEF1a1)
A 0.9-kb fragment of the 5' end of the translation elongation factor-1α (tef) gene (eEF1a1) containing three major introns can be amplified using the primer pair tef71f (C AAA ATG GGT AAG GAG GAS AAG AC) and tef997R (CA GTA CCG GCR GCR ATR ATS AG) and the following 'touchdown' amplification protocol: 4 min initial denaturation at 94°C, 4 cycles each of 1 min at 94 °C, 90 sec at 70 °C, and 90 sec at 72 °C, followed by 26 cycles with the annealing temperature decreasing by 0.5˚C per cycle from 68˚C to 55˚C, followed by 12 cycles with annealing at 55˚C, and with a final extension period of 7 min at 72°C. In some cases multiple bands may remain after amplification, and the tef1 gene fragment is resolved using the following internal sequencing primers: tef85f (AG GAC AAG ACT CAC ATC AAC G) and tef954r (AGT ACC AGT GAT CAT GTT CTT G).
>>> RPB2 (from Tom Graefenhan)
A 1.2-kb fragment of subunit 2 of the RNA polymerase B gene (RPB2) containing an ITS-like region is amplified using the primer pair RPB2_210up (TGG GGW GAY CAR AAR AAG G) and RPB2_1450low (C ATR ATG ACS GAA TCT TCC TGG T) and the following 'touchdown' amplification protocol: 3 min initial denaturation at 94°C, 5 cycles each of 45 sec at 94 °C, 45 sec at 60 °C, and 2 min at 72 °C, followed by 5 cycles with the annealing temperature decreasing by 1.0˚C per cycle from 58˚C to 54˚C, followed by 30 cycles with annealing at 54˚C, and with a final extension period of 10 min at 72°C. We employ the following internal sequencing primers for RPB2 giving a 0.9kb product: RPB2_210up (above) and RPB2_1150low (GG TTG TGA TCR GGR AAR GGA ATG).
Our standard sequencing protocol is a variation of:
sequencing reactions are prepared using the ABI Prism® BigDye™ Terminator reaction kit (v2.0, Applied Biosystems) in 5 uL volume and 1/8 dilution using ½ BigDye (BioCan Scientific, Mississauga, ON). The cycle sequencing reaction contains the following mix: 1.0 uL BigDye, 1.0 uL ½-BigDye, 0.4 uL of 5 uM primer, 1.6 uL sterile distilled water, 1.0 uL (10-40 ng) PCR template, and employs the following amplification protocol: 40 cycles each of 30 sec denaturation at 96°C, 15 sec annealing at 50°C, and 3 min extension at 60°C. Sequences are obtained using an ABI Prism 3130XL Genetic Analyzer (Applied Biosystems).
Parivash Shoukouhi and John Bissett
Agriculture and Agri-Food Canada
Eastern Cereal and Oilseed Research Centre
Ottawa, Ontario, Canada K1A 0C6
posted by John Bissett on 25 Jun, 2009