Fluorescent fragment size analysis

With respect to detection for CE, laser-induced fluorescence (LIF) detection is an attractive approach as a result of the sensitivity attainable despite the short path length. For this reason, it is well suited to microchip electrophoretic analysis of a variety of analytes including DNA fragment sizing and sequencing.The excitation source is usually a continuous-wave argon ion laser, which is focused in a capillary or on a microchannel in a microchip. 

The present analysis relies on - and extends - the comprehensive theoretical study of Refs. [23,24] on the multi-state interactions in the manifold of the X — E states of Bz+. Like this recent work, it utilizes an ab initio quantum-dynamical approach. In Refs. [23,24] we have, in addition, identified strong coupling effects between the B — C and B — D electronic states, caused by additional conical intersections between their potential energy surfaces. A whole sequence of stepwise femtosecond internal conversion processes results [24]. Such sequential internal conversion processes are of general importance as is evidenced indirectly by the fluorescence and fragmentation dynamics of organic closed-shell molecules and radical cations [49,50]. It is therefore to be expected that the present approach and results may be of relevance for many other medium-sized molecular systems. 

Figure 7.3 UV versus LIF detection of the CE separation of a 53 base pair RT-PCR product from the RNA of the polio virus vaccine, Sabin 3. An Hae Ill-digested d>X174 DNA marker was coinjected with the PCR product for size determination—note the 72 bp fragment. The same Sabin 3 concentration was used for each analysis, whereas the marker total DNA concentration varied from 200 mg/mL for UV analysis, to 20 mg/mL for LIF analysis. Note the unambiguous pattern observed with LIF for the Sabin 3 fragment compared to UV detection of the same fragment. Full scale UV detection, 0.005 absorbance unit (AU) LIF detection, 10 relative fluorescence units (RFU). [Reproduced with permission from Schwartz et al., J Capillary Electrophor 1 36 (1994). Copyright ISC Technical Publications, Inc.]...

After amplification, tlie products can be detected by various methods. Simple gel electrophoresis with ethidium bromide staining may suffice. When greater accuracy is required, one of the primers can be fluorescently labeled so that after PCR the fragments are accurately sized on a DNA sequencing device. Alternatively, some form of hybridization assay can be used to verify or analyze the amplified product. Automated methods are always attractive and closed-tube methods are particularly advantageous in the clinical laboratory. Adding a fluorescent dye or probe before amplification allows thermocyclers equipped with optical detection to analyze the reaction as it progresses (real-time PCR) or after the reaction is complete (endpoint measurement) without need to process the sample for a separate analysis step. 

Gel electrophoresis is a method for analyzing DNA. Electrophoresis separates DNA or protein by size or electrical charge. The DNA runs towards the positive charge as it separates the DNA fragments by size. The gel is treated with a DNA-binding dye that fluoresces under ultraviolet light. A picture of the gel can be taken and used for analysis. 

Figure 2 A schematic view of multiplex PCR analysis of STRs. In a test tube, some STRs from the sample s DNA (A) are amplified by PCR (B) using fluorescent-labeled primers. After the addition of an internal standard (red-labeled fragments) (C) the DNA fragments obtained at the end of the PCR are separated by capillary electrophoresis according to their size (D) and detected at the end of the capillary. Each peak (E) is then electronically labeled with the name of the corresponding allele. The profiles in blue, green, and black present the alleles detected for the STRs VWA, D21S11, andTHOI, respectively. The red profile displays two peaks from the internal standard.

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