title: Retrieval of glycoside hydrolase family 9 cellulase genes from environmental DNA by metagenomic gene specific multi-primer PCR.
abstract: A new method, termed metagenomic gene specific multi-primer PCR (MGSM-PCR), is presented that uses multiple gene specific primers derived from an isolated gene from a constructed metagenomic library rather than degenerate primers designed based on a known enzyme family. The utility of MGSM-PCR was shown by applying it to search for homologues of the glycoside hydrolase family 9 cellulase in metagenomic DNA. The success of the multiplex PCR was verified by visualizing products on an agarose gel following gel electrophoresis. A total of 127 homologous genes were amplified with combinatorial multi-primer reactions from 34 soil DNA samples. Multiple alignments revealed extensive sequence diversity among these captured sequences with sequence identity varying from 26 to 99.7%. These results indicated that significantly diverse homologous genes were indeed readily accessible when using multiple metagenomic gene specific primers.
authors.affiliation: Center for Biomedicine and Health, College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, 310015, China.
authors.forename: Xiaolong
authors.initials: X
authors.lastname: Xiong
authors.forename: Xiaopu
authors.initials: X
authors.lastname: Yin
authors.forename: Xiaolin
authors.initials: X
authors.lastname: Pei
authors.forename: Peng
authors.initials: P
authors.lastname: Jin
authors.forename: Ao
authors.initials: A
authors.lastname: Zhang
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
authors.forename: Weibo
authors.initials: W
authors.lastname: Gong
authors.forename: Qiuyan
authors.initials: Q
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D002480:Cellulase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D017931:DNA Primers / Q000235:genetics; D014644:Genetic Variation; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D054892:Metagenome; D008969:Molecular Sequence Data; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D012987:Soil / Q000737:chemistry*
chemical_list: D017931:DNA Primers; D012987:Soil; D004247:DNA; D006026:Glycoside Hydrolases; D002480:Cellulase