Real-time PCR-based method for the estimation of genome sizes.
The fast and reliable estimation of the genome sizes of various species would allow for a systematic analysis of many organisms and could reveal insights into evolutionary processes. Many methods for the estimation of genome sizes have already been described. The classical methods are based on the determination of the phosphate content in the DNA backbone of total DNA isolated from a defined number of cells or on reassociation kinetics of high molecular weight genomic DNA (c(0)t assay). More recent techniques employ DNA-specific fluorescent dyes in flow cytometry analysis, image analysis or absorption cytometry after Feulgen staining. The method presented here is based on the absolute quantification of genetic elements in a known amount (mass) of genomic DNA by real-time quantitative PCR. The method was evaluated on three different eukaryotic species, Saccharomyces cerevisiae (12.1 Mb), Xiphophorus maculatus (550 Mb) and Homo sapiens sapiens (2.9 Gb), and found to be fast, highly accurate and reliable.
Institut für Biochemie, FB 08, Justus-Liebig-Universität Giessen, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany.
Jochen
J
Wilhelm
Alfred
A
Pingoud
Meinhard
M
Hahn
D000818:Animals; D003532:Cyprinodontiformes / Q000235:genetics; D004247:DNA / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D016678:Genome; D006238:Haploidy; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012269:Ribosomal Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D016159:Tumor Suppressor Protein p53 / Q000235:genetics
D004271:DNA, Fungal; D012269:Ribosomal Proteins; D016159:Tumor Suppressor Protein p53; C044152:ribosomal protein S3; D004247:DNA