NDLI: Real-time PCR determination of rRNA gene copy number: absolute and relative quantification assays with Escherichia coli

Content Provider	Springer Nature Link
Author	Lee, Changsoo Lee, Seungyong Shin, Seung Gu Hwang, Seokhwan
Copyright Year	2007
Abstract	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.
Starting Page	371
Ending Page	376
Page Count	6
File Format	PDF
ISSN	01757598
Journal	Applied Microbiology and Biotechnology
Volume Number	78
Issue Number	2
e-ISSN	14320614
Language	English
Publisher	Springer-Verlag
Publisher Date	2007-12-11
Publisher Place	Berlin, Heidelberg
Access Restriction	Subscribed
Subject Keyword	rRNA gene copy number Real-time PCR Absolute quantification Relative quantification Escherichia coli Microbial Genetics and Genomics Microbiology Biotechnology
Content Type	Text
Resource Type	Article
Subject	Medicine Applied Microbiology and Biotechnology Biotechnology

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