title: Soil clone library analyses to evaluate specificity and selectivity of PCR primers targeting fungal 18S rDNA for denaturing-gradient gel electrophoresis (DGGE).
abstract: We evaluated the fungal specificity and detection bias of four fungal 18S rRNA gene (18S rDNA) primer sets for denaturing-gradient gel electrophoresis (DGGE). We constructed and compared clone libraries amplified from upland and paddy field soils with each primer set (1, NS1/GCFung; 2, FF390/FR1-GC; 3, NS1/FR1-GC; and 4, NS1/EF3 for the first PCR and NS1/FR1-GC for the second PCR). Primer set 4 (for nested PCR) showed the highest specificity for fungi but biased specific sequences. Sets 1, 2, and 3 (for single PCR) amplified non-fungal eukaryotic sequences (from 7 to 16% for upland soil and from 20 to 31% for paddy field soil) and produced libraries with similar distributions of fungal 18S rDNA sequences at both the phylum and the class level. Set 2 tended to amplify more diverse fungal sequences, maintaining higher specificity for fungi. In addition, clone analyses revealed differences among primer sets in the frequency of chimeras. In upland field soil, the libraries amplified with primer sets 3 and 4, which targeted long fragments, contained many chimeric 18S rDNA sequences (18% and 48%, respectively), while the libraries obtained with sets 1 and 2, which targeted short fragments, contained fewer chimeras (5% and 10%, respectively).
authors.affiliation: National Institute for Agro-Environmental Sciences, 3–1–3 Kannondai, Tsukuba, Ibaraki, 305–8604, Japan. yuko422@affrc.go.jp
authors.forename: Yuko
authors.initials: Y
authors.lastname: Takada Hoshino
authors.forename: Sho
authors.initials: S
authors.lastname: Morimoto
mesh_terms: D017931:DNA Primers / Q000235:genetics*; D004271:DNA, Fungal / Q000235:genetics; D058645:Denaturing Gradient Gel Electrophoresis; D005658:Fungi / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D015723:Gene Library; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D012337:RNA, Ribosomal, 18S / Q000235:genetics*; D012988:Soil Microbiology; D013045:Species Specificity
chemical_list: D017931:DNA Primers; D004271:DNA, Fungal; D012337:RNA, Ribosomal, 18S