Synthetic oligonucleotides as quantitative PCR standards for quantifying
microbial genes
Abstract
Real-time quantitative PCR (qPCR) has been widely used to quantify gene
copy numbers in microbial ecology. Despite its simplicity and
straightforwardness, establishing qPCR assays is often impeded by the
tedious process of producing qPCR standards by cloning the target DNA
into plasmids. Here, we designed double-stranded synthetic DNA fragments
from consensus sequences as qPCR standards by aligning microbial gene
sequences. Efficiency of standards from synthetic DNA was compared with
plasmid standards by qPCR assays for different taxonomic and functional
genes involved in C and N cycling, tested with DNA extracted from a
broad range of soils. Results showed that qPCR standard curves using
synthetic DNA performed equally well to those from plasmids for all the
genes tested. Furthermore, gene copy numbers from DNA extracted from
soils obtained by using synthetic standards or plasmid standards were
comparable. Our approach therefore demonstrates that a synthetic DNA
fragment as qPCR standard provides comparable sensitivity and
reliability to a traditional plasmid standard, while being more time-
and cost-efficient.