Abstract
We describe a two-step PCR strategy using tagged highly degenerate primer (THDP-PCR) targeting copper-containing membrane-bound monooxygenases (CuMMO) genes for community analysis of methane- or ammonia-oxidizing bacteria. This strategy consists of a primary CuMMO gene-specific PCR followed by a secondary PCR with a tag as a single primer. This strategy remarkably increases the divergence of CuMMO gene amplicons while maintaining PCR efficiency without obvious amplification bias. This THDP-PCR strategy can be extended to other functional gene-based community analysis with design of new highly degenerate primer covering target functional gene sequences.
Keywords: Tagged highly degenerate primer, Two-step PCR, CuMMO
Background
Gene types in CuMMO superfamily are divergent and existent primer sets can only cover some CuMMO types (Tuomivirta et al., 2009). To cover the divergent types of genes in the CuMMO superfamily, highly degenerate primers are inevitable, but previous strategies using highly degenerate primers have limitations when applied to environmental samples, like low PCR efficiency or non-specific amplification (Ledeker and De Long, 2013). We recently used a two-step PCR strategy with tagged highly degenerate primers, designated THDP-PCR, to amplify a wide range of genes in the CuMMO family with satisfactory PCR efficiency and no obvious amplification bias (Wang et al., 2017).
Materials and Reagents
Equipment
Software
Procedure
Note: Conventional (one step) PCR efficiency is not satisfactory for highly degenerate primers with degeneracy higher than 48 based on our experiment.
Data analysis
Acknowledgments
This protocol was adapted from our previous studies (Wang et al., 2017). This work was supported by the National Natural Science Foundation of China (NSFC) [grant numbers 31470222, 31170114].
References
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