A new approach to retrieve full lengths of functional genes from soil by PCR-DGGE and metagenome walking.
Metagenomes are a vast genetic resource, and various approaches have been developed to explore them. Here, we present a new approach to retrieve full lengths of functional genes from soil DNA using PCR-denaturing gradient gel electrophoresis (DGGE) followed by metagenome walking. Partial fragments of benzoate 1,2-dioxygenase alpha subunit gene (benA) were detected from a 3-chlorobenzoate (3CB)-dosed soil by PCR-DGGE, and one DGGE band induced by 3CB was used as a target fragment for metagenome walking. The walking retrieved the flanking regions of the target fragment from the soil DNA, resulting in recovery of the full length of benA and also downstream gene (benB). The same strategy retrieved another gene, tfdC, and a complete tfdC and two downstream genes were obtained from the same soil. PCR-DGGE allows screening for target genes based on their potential for degrading contaminants in the environment. This feature provides an advantage over other existing metagenomic approaches.
Morimoto
Sho
S
National Institute for Agro-Environmental Sciences, 3-1-3 Kannondai, Tsukuba, Ibaraki, 305-8604, Japan. shomo@niaes.affrc.go.jp
Fujii
Takeshi
T
D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D016678:Genome; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012987:Soil / Q000032:analysis*; D012988:Soil Microbiology
D012987:Soil; D004247:DNA