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Retrieval of glycoside hydrolase family 9 cellulase genes from environmental DNA by metagenomic gene specific multi-primer PCR.

Authors
  • Xiong, Xiaolong1
  • Yin, Xiaopu
  • Pei, Xiaolin
  • Jin, Peng
  • Zhang, Ao
  • Li, Yan
  • Gong, Weibo
  • Wang, Qiuyan
  • 1 Center for Biomedicine and Health, College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, 310015, China. , (China)
Type
Published Article
Journal
Biotechnology Letters
Publisher
Springer-Verlag
Publication Date
May 01, 2012
Volume
34
Issue
5
Pages
875–882
Identifiers
DOI: 10.1007/s10529-012-0855-1
PMID: 22261869
Source
Medline
License
Unknown

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.

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