Rapid and reliable DNA extraction techniques from trypan-blue-stained mycorrhizal roots: comparison of two methods.
Two improved DNA extraction techniques from trypan-blue-stained root fragments were developed and compared for rapid and reliable analyses. In Method A, 1 cm trypan-blue-stained mycorrhizal root fragments were individually isolated, crushed by bead beating, and purified with Chelex-100 (Bio-Rad). In Method B, DNA extraction was carried out using an UltraClean microbial DNA isolation kit (MoBio Laboratories). DNA was extracted from the mycorrhizal roots of four plant species, quantified by UV absorbance, and PCR-amplified with primers specific to arbuscular mycorrhizal fungi. Although PCR inhibitors might still exist when using Method A, appropriate dilution and employment of nested-PCR overcame this problem. Method B removed PCR inhibitors, but sometimes, depending on the mycorrhizal colonization within the root fragments, it also required nested PCR. In conclusion, both methods enabled us to handle many samples in a short time. Method B provided greater reliability and Method A provided better cost performance. Both techniques can be useful for PCR-based applications to identify species and estimate species composition after measuring mycorrhizal colonization rate with trypan blue staining.
Ishii
Satoshi
S
Department of Agronomy, Iowa State University, 1126 Agronomy Hall, Ames 50011, USA.
Loynachan
Thomas E
TE
D017931:DNA Primers / Q000235:genetics; D004271:DNA, Fungal / Q000032:analysis*; D005658:Fungi / Q000235:genetics; D038821:Mycorrhizae / Q000235:genetics*; D018517:Plant Roots / Q000235:genetics / Q000382:microbiology*; D016133:Polymerase Chain Reaction; D014343:Trypan Blue
D017931:DNA Primers; D004271:DNA, Fungal; D014343:Trypan Blue