Use of artificial DNA with multiple probe sites as reference DNA templates for quantitative real-time PCR to examine methanogen communities

Abstract

Isolation of reference DNA templates for quantitative real-time PCR assays is an expensive, labor-intensive and time-consuming process if they are not readily available. Two artificial DNA templates with multiple probe sites were designed for quantifying methanogens and their 10 subgroups, based on the methyl coenzyme M reductase gene (mcrA). Their standards were comparable to each other. PCR amplification efficiencies (cycle vs. cumulative fluorescence) of the artificial DNAs were also comparable to those of the observed methanogen groups from anaerobic digesters. The artificial templates can be alternatives to the actual references.

Keywords:

• Quantitative PCR
• artificial DNA
• methanogens
• mcrA.

Acknowledgment

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (MEST) (NRL program, R0A-2008-000-20044-0), and RP-Grant 2012 of Ewha Womans University.