TY - JOUR
T1 - Rapid and reliable DNA extraction techniques from trypan-blue-stained mycorrhizal roots
T2 - Comparison of two methods
AU - Ishii, Satoshi
AU - Loynachan, Thomas E.
N1 - Copyright:
Copyright 2008 Elsevier B.V., All rights reserved.
PY - 2004/8
Y1 - 2004/8
N2 - 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.
AB - 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.
KW - Arbuscular mycorrhizal fungi
KW - DNA extraction
KW - Glomales
KW - Trypan blue staining
UR - http://www.scopus.com/inward/record.url?scp=4544312031&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=4544312031&partnerID=8YFLogxK
U2 - 10.1007/s00572-004-0316-3
DO - 10.1007/s00572-004-0316-3
M3 - Article
C2 - 15221577
AN - SCOPUS:4544312031
VL - 14
SP - 271
EP - 275
JO - Mycorrhiza
JF - Mycorrhiza
SN - 0940-6360
IS - 4
ER -