Real-time PCR determination of rRNA gene copy number: absolute and relative quantification assays with Escherichia coli.
Real-time polymerase chain reaction (PCR)-based methodology for the determination of rRNA gene (rrn) copy number was introduced and demonstrated. Both absolute and relative quantifications were tested with Escherichia coli. The separate detection of rRNA gene and chromosomal DNA was achieved using two primer sets, specific for 16S rRNA gene and for D-1-deoxyxylulose 5-phosphate synthase gene (dxs), respectively. As dxs is a single-copy gene of E. coli chromosomal DNA, the rrn copy number can be determined as the copy ratio of rrn to dxs. This methodology was successfully applied to determine the rrn copy number in E. coli cells. The results from absolute and relative quantifications were identical and highly reproducible with coefficient of variation (CV) values of 1.8-4.6%. The estimated rrn copy numbers also corresponded to the previously reported value in E. coli (i.e., 7), indicating that the results were reliable. The methodology introduced in this study is faster and cost-effective without safety problems compared to the traditionally used Southern blot analysis. The fundamentals in our methodology would be applicable to any microorganism, as long as having the sequence information of the rRNA gene and another chromosomal gene with a known copy number.
Lee
Changsoo
C
Microbial Ecology Laboratory, Department of Microbiology and Environmental Change Institute (ECI), National University of Ireland, Galway, Republic of Ireland.
Lee
Seungyong
S
Shin
Seung Gu
SG
Hwang
Seokhwan
S
D004926:Escherichia coli / Q000235:genetics; D018628:Gene Dosage; D020459:Genes, rRNA; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D014166:Transferases / Q000235:genetics
D014166:Transferases; C109461:deoxyxylulose-5-phosphate synthase