A linear dichroism technique for quantitative PCR applications Research shows that an intrinsic signal from DNA has the potential to be an interference-free, specific, and label-free probe for real-time quantitative polymerase chain reaction.evelopment and progression. David J. Halsall, Timothy R. Dafforn, Rachel Marrington, Eugene Halligan, and Alison Rodger The high sensitivity and wide dynamic range of real-time polymerase chain reaction (RT-PCR) have made it the technique of choice for sensitive, specific nucleic acid quantitation, especially in the field of molecular diagnostics. This article explores the novel application of flow-aligned linear dichroism (faLD) as a detection method for RT-PCR. Two intrinsic properties of the DNA helix are exploited by faLD: its ability to absorb ultraviolet (UV) light and its asymmetric structure. Consequently, unlike most current methods, faLD does not require costly exogenous labels or time-consuming post-PCR processing. Common interferences, such as those caused by artifactual PCR primer self-concatamerization (so-called primer-dimers), are effectively negated. This is because, though they are optically active, they lack the asymmetry of the intended PCR product. Another advantage of the method is its ability to detect long PCR products. Also, it offers the possibility of detecting mutations concurrently with nucleic acid quantitation during the PCR process. Flow-aligned linear dichroism has potential as a method for detecting exclusively polymeric DNA. Molecules absorb light because the electric field of the radiation pushes their electrons in a particular direction at a particular wavelength. When all the molecules in a sample are oriented, the electrons in the sample all are characterized by the same preferred net displacement direction. Shining polarized lightthat is, light whose electric field moves in only one directionenables the absorbance to be varied. Linear dichroism (LD) is the measure of the difference in absorbance between two perpendicular polarizations of light. This can be deconvoluted to yield information about the
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B. Daya Reddy
With 34 Illustrations
Department of Mathematics
University of Iowa
Iowa City, IA 52242
B. Daya Reddy
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