Real-time PCR measurement by fluorescence anisotropy.
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.
Crane
Bryan L
BL
BioInstrumentation Laboratory, Department of Mechanical Engineering, and Division of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. bcrane@mit.edu
Hogan
N Catherine
NC
Sudo
Hiroko
H
Thilly
William G
WG
Hunter
Ian W
IW
D005454:Fluorescence Polarization / Q000379:methods*; D016678:Genome / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000295:instrumentation* / Q000379:methods*; D013696:Temperature; D013997:Time Factors