title: General and reliable quantitative measurement of fluorescence resonance energy transfer using three fluorescence channels.
abstract: In this paper, we describe a comprehensive general system adapted for quantitative fluorescence resonance energy transfer (FRET) measurement using signals from three channels of a fluorescence instrument. The general FRET measurement system involves two established methods, as well as two novel approaches. Unlike the previous measurements, which can be taken correctly only when the quantity of the acceptor is greater than or equal to that of the donor, one of our novel methods can overcome this obstacle and take quantitative FRET measurements when the donor is in excess of the acceptor. Hence the general FRET measurement system allowed one to determine the exact distance when the donor and acceptor were present in different quantities, and integrated the methods for quantitative FRET measurements. The uniformity of measured values and utility of each method were validated using molecular standards based on DNA oligonucleotide rulers. We also discussed and validated the use of a novel method for estimating the relative quantities of the donor and acceptor fluorophores when they were not known before an appropriate method of this system can be selected.
authors.affiliation: Medical Systems Biology Research Center, Tsinghua University School of Medicine, Haidian District, Beijing, 100084, China.
authors.forename: Fengbo
authors.initials: F
authors.lastname: Xie
authors.forename: Jiang
authors.initials: J
authors.lastname: Zhu
authors.forename: Cheng
authors.initials: C
authors.lastname: Deng
authors.forename: Guoliang
authors.initials: G
authors.lastname: Huang
authors.forename: Keith
authors.initials: K
authors.lastname: Mitchelson
authors.forename: Jing
authors.initials: J
authors.lastname: Cheng
mesh_terms: D004247:DNA; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry*
chemical_list: D005456:Fluorescent Dyes; D009838:Oligodeoxyribonucleotides; D004247:DNA