Can anyone suggest which enzymes I should use for T-RFLP of pmoA gene?
The choice of restriction enzyme will depend on the genes you amplify and subsequently the primers pair you use. For pmoA, MspI (Ho and Frenzel, 2012; Kong et al 2014) or HhaI (Singh et al 2009) have been used previously. But you can’t use those enzymes without doing an in silico digestion of sequence from pmoA. You need to collect sequences from database of pmoA, similar to the samples type you work on but not only, and perform in silico digestion using different enzyme. There is different software, such as MiCA (Shyu et al., 2007), TRiFLe (Junier et al., 2008) or T-RFPred (Fernandez-Guerra et al., 2010). You need to ensure that the enzyme will cleave your sequences. I would advise for pmoA to use multiple enzymes. The selection of the enzyme is extremely important and should be done carefully, have a look on my latest article, although it is not focus on pmoA:
https://www.researchgate.net/publication/280576476_Analysing_the_effect_of_soil_organic_matter_on_bacterial_communities_using_T-RFLP_fingerprinting_different_methods_different_stories?ev=prf_pub
References:
Fernandez-Guerra, A., Buchan, A., Mou, X., Casamayor, E., Gonzalez, J., 2010. T-RFPred: a nucleotide sequence size prediction tool for microbial community description based on terminal-restriction fragment length polymorphism chromatograms. BMC Microbiology 10, 262. doi:10.1186/1471-2180-10-262
Ho, A., Frenzel, P., 2012. Heat stress and methane-oxidizing bacteria: Effects on activity and population dynamics. Soil Biology and Biochemistry 50, 22–25. doi:10.1016/j.soilbio.2012.02.023
Junier, P., Junier, T., Witzel, K.-P., 2008. TRiFLe, a program for in silico terminal restriction fragment length polymorphism analysis with user-defined sequence sets. Applied and Environmental Microbiology 74, 6452–6456. doi:10.1128/AEM.01394-08
Kong, J.-Y., Bai, Y., Su, Y., Yao, Y., He, R., 2014. Effects of trichloroethylene on community structure and activity of methanotrophs in landfill cover soils. Soil Biology and Biochemistry 78, 118–127. doi:10.1016/j.soilbio.2014.07.018
Shyu, C., Soule, T., Bent, S., Foster, J., Forney, L., 2007. MiCA: a web-based tool for the analysis of microbial communities based on terminal-restriction fragment length polymorphisms of 16S and 18S rRNA genes. Microbial Ecology 53, 562–570. doi:10.1007/s00248-006-9106-0
Singh, B.K., Tate, K.R., Ross, D.J., Singh, J., Dando, J., Thomas, N., Millard, P., Murrell, J.C., 2009. Soil methane oxidation and methanotroph responses to afforestation of pastures with Pinus radiata stands. Soil Biology and Biochemistry 41, 2196–2205. doi:10.1016/j.soilbio.2009.08.004
Article Analysing the effect of soil organic matter on bacterial com...
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