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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Acknowledgments
This work was supported by the National Natural Science Foundation of China (30900253, 20906016 and 21006018), the Technology Research and Development Program of Hangzhou (20101131N03) and the Normal Science Project of Zhejiang Province (2009C31086).
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Xiong, X., Yin, X., Pei, X. et al. Retrieval of glycoside hydrolase family 9 cellulase genes from environmental DNA by metagenomic gene specific multi-primer PCR. Biotechnol Lett 34, 875–882 (2012). https://doi.org/10.1007/s10529-012-0855-1
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DOI: https://doi.org/10.1007/s10529-012-0855-1