Primer information
Frequently using primer pairs for multi-loci analysis
Primer set(forward)/(reverse), annealing temperature
PCR condition
For TaKaRa Ex Taq (good at single copy genes and ribosomal genes, and mitochondrial gene)
98℃ 10 sec |
annealing ℃ 30 sec | 35-38 cycle
72℃ 1 min |
For TOYOBO KOD FX Neo (good at ribosomal genes, and mitochondrial gene)
94℃ 2 min
98℃ 10 sec |
annealing ℃ 30 sec | 35-38 cycle
68℃ 1 min |
SSU (nuclear small subunit (18S) ribosomal DNA)
NS1/NS4 42 °C (always using 46 to 48 °C)
NS1: GTAGTCATATGCTTGTCTC
NS4: CTTCCGTCAATTCCTTTAAG
ITS (nuclear ribosomal internal transcribed spacer region)
V9G/LR5, 53 °C
ITS5/ITS4 61.5 °C
V9G: TTACGTCCCTGCCCTTTGTA
LR5: TCCTGAGGGAAACTTCG
ITS5: GGAAGTAAAAGTCGTAACAAGG
ITS4: TCCTCCGCTTATTGATATGC
(Although previously ITS1 was used for standard primer for ITS, I feel something weakly amplified and sometimes amplified non-specific band, thus I dont recommend.)
LSU (nuclear large subunit (28S) ribosomal DNA)
LR0R/LR7 46 °C (always using 48 °C)
LR0R: ACCCGCTGAACTTAAGC
LR7: TACTACCACCAAGATCT
(Although sometimes LR5 is used for reverse primer, the product is too short for analysis, thus I dont recommend.
On the other hand, LR5 is good "sequencing primer", or ITS primer)
RPB1 (DNA-directed RNA polymerase II subunit, rpb1)
RPB1-Af/RPB1-Cr 53 °C
RPB1-Af: GARTGYCCDGGDCAYTTYGG
RPB1-Cr: CCNGCDATNTCRTTRTCCATRTA
RPB2(DNA-directed RNA polymerase II subunit, rpb2)
fRPB2-5F/fRPB2-7cR 58 °C
fRPB2-5F: GAYGAYMGWGATCAYTTYGG
fRPB2-7cR: CCCATRGCTTGYTTRCCCAT
Although some reviewer asked me too high for annealing temperature, but it is good for this region in my experiment (I dont know why).
Strongly recommend DO NOT USE KOD FX NEO, and USE TAQ POLYMERACE due to GC- and AT-rich of each half.
TEF1alpha (Translation elongation factor EF-1 alpha, don't recommend)
PCR pair Tm Cycle
EF1-728F/EF1-986R 58 °C 40 cycle
EF1-728F/TEF1LLErev 55 °C 40 cycle
EF1-983F/EF1-2218R 60 °C 40 cycle
Primer sequence type Reference
EF1-728F CATCGAGAAGTTCGAGAAGG PCR Carbone and Kohn (1999)
EF1-983F GCYCCYGGHCAYCGTGAYTTYAT PCR Rehner and Buckley (2005)
EF1-986R TACTTGAAGGAACCCTTACC PCR Carbone and Kohn (1999)
TEF1LLErev AACTTGCAGGCAATGTGG PCR Jaklitsch et al. (2006)
1567R ACHGTRCCRATACCACCSATCTT Seq Rehner and Buckley (2005)
EF1-2218R ATGACACCRACRGCRACRGTYTG PCR Rehner and Buckley (2005)
Reference
Carbone, I., & Kohn, L. M. (1999). A Method for Designing Primer Sets for Speciation Studies in Filamentous Ascomycetes. Mycologia, 91, 553–556. https://doi.org/10.2307/3761358
Jaklitsch, W. M., Komon, M., Kubicek, C. P., & Druzhinina, I. S. (2006). Hypocrea voglmayriisp. nov. from the Austrian Alps represents a new phylogenetic clade inHypocrea/Trichoderma. Mycologia, 97, 1365-1378. https://doi.org/10.1080/15572536.2006.11832743
Rehner, S. A., & Buckley, E. (2005). A Beauveria phylogeny inferred from nuclear ITS and EF1-alpha sequences: evidence for cryptic diversification and links to Cordyceps teleomorphs. Mycologia, 97, 84–98. https://doi.org/10.3852/mycologia.97.1.84