iFRET: an improved fluorescence system for DNA-melting analysis.
Fluorescence resonance energy transfer (FRET) is a powerful tool for detecting spatial relationships between macromolecules, one use of which is the tracking of DNA hybridization status. The process involves measuring changes in fluorescence as FRET donor and acceptor moieties are brought closer together or moved farther apart as a result of DNA hybridization/denaturation. In the present study, we introduce a new version of FRET, which we term induced FRET (iFRET), that is ideally suited for melting curve analysis. The innovation entails using a double-strand, DNA-specific intercalating dye (e.g., SYBR Green I) as the FRET donor, with a conventional FRET acceptor affixed to one of the DNA molecules. The SNP genotyping technique dynamic allele specific hybridization (DASH) was used as a platform to compare iFRET to two alternative fluorescence strategies, namely, the use of the intercalating dye alone and the use of a standard FRET pair (fluorescein as donor, 6-rhodamine as acceptor). The iFRET configuration combines the advantages of intercalating dyes, such as high signal strengths and low cost, with maintaining the specificity and multiplex potential afforded by traditional FRET detection systems. Consequently, iFRET represents a fresh and attractive schema for monitoring interactions between DNA molecules.
Howell
W Mathias
WM
Center for Genomics and Bioinformatics, Karolinska Institute, S-171 77, Stockholm, Sweden. Mathias.Howell@cgb.ki.se
Jobs
Magnus
M
Brookes
Anthony J
AJ
D000483:Alleles; D020029:Base Pairing / Q000187:drug effects / Q000235:genetics; D052160:Benzothiazoles; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015342:DNA Probes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003959:Diamines; D004735:Energy Transfer; D019793:Fluorescein / Q000737:chemistry; D005454:Fluorescence Polarization / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000494:pharmacology; D007364:Intercalating Agents / Q000494:pharmacology; D008957:Models, Genetic; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D009691:Nucleic Acid Denaturation / Q000187:drug effects; D009693:Nucleic Acid Hybridization / Q000187:drug effects; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods; D011804:Quinolines; D012235:Rhodamines / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013698:Templates, Genetic
D052160:Benzothiazoles; D015342:DNA Probes; D003959:Diamines; D005456:Fluorescent Dyes; D007364:Intercalating Agents; D009930:Organic Chemicals; D011804:Quinolines; D012235:Rhodamines; C026188:rhodamine 6G; C098022:SYBR Green I; D004247:DNA; D019793:Fluorescein