title: Real-time PCR measurement by fluorescence anisotropy.
abstract: We have developed an instrument for monitoring real-time PCR using fluorescence anisotropy, enabling an assay chemistry in which the fluorescence from a labeled primer elucidates amplification. The instrument holds the sample temperature constant to within +/-0.03 degrees C during measurement in the extension phase of each PCR cycle and achieves 0.116 mP FA resolution. Primer conjugation with Alexa-Fluor 488, when compared with other fluorophores, is shown to provide the greatest FA range between primer and product. Comparable reproducibility and linearity of the crossing point for a range of target copy numbers is observed between the FA-based assay run in our instrument and the SYBR green assay run in commercial instrumentation. Reproducibility is also consistent with Poisson-distributed experimental error in aliquoting starting copies, a theoretical limit to instrument/assay performance.
authors.affiliation: BioInstrumentation Laboratory, Department of Mechanical Engineering, and Division of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. bcrane@mit.edu
authors.forename: Bryan L
authors.initials: BL
authors.lastname: Crane
authors.forename: N Catherine
authors.initials: NC
authors.lastname: Hogan
authors.forename: Hiroko
authors.initials: H
authors.lastname: Sudo
authors.forename: William G
authors.initials: WG
authors.lastname: Thilly
authors.forename: Ian W
authors.initials: IW
authors.lastname: Hunter
mesh_terms: D005454:Fluorescence Polarization / Q000379:methods*; D016678:Genome / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000295:instrumentation* / Q000379:methods*; D013696:Temperature; D013997:Time Factors