Question

Why does Reference strand-mediated conformation analysis (RSCA) needs a fluorophore on its probe? Is it because without the fluorophore probe, the signal (Etbr) would be too weak? Does more mismatches in the heteroduplex causes the ethylBr not bind well and give weak signal and may go undetected in the gel? And how do you order those heteroduplexes in the gel, more mismatches in the heterdoplux->less stable, so less migration and on top of the gel. Less mismatches, more stable heterduplex -> better migration on the bottom of the gel? http://imgur.com/0VP5bjq

Answer 1

Answer on Question #54048 – Biology – Biochemistry

Question:

Why does Reference strand-mediated conformation analysis (RSCA) needs a fluorophore on its probe? Is it because without the fluorophore probe, the signal (Etbr) would be too weak? Does more mismatches in the heteroduplex causes the ethylBr not bind well and give weak signal and may go undetected in the gel? And how do you order those heteroduplexes in the gel, more mismatches in the heteroplux->less stable, so less migration and on top of the gel. Less mismatches, more stable heterduplex -> better migration on the bottom of the gel?

Answer:

Reference strand-mediated conformation analysis (RSCA) is used to determine the difference in the heteroduplexes separation in polyacrylamide gel. An automatic laser scanning system is used for this analysis: heteroduplexes labelled with fluorophore are detected. Fluorophore is used to exclude from the analysis heteroduplexes formed of two strands of different samples as shown in the figure below. Ethidium bromide binds to all DNA molecules. Fluorophore tagged strand gives opportunity: (1) to determine the product of hybridization and (2) to do it automatically.

More mismatched heteroduplexes are less stable and have decreased rate of migration in gel. That is why these molecules are on top of the gel.

The figure below describes the principle of this method and obtained results.

Fig. 1. Diagrammatic representation of RSCA. The conformation of each HLA allelic fragment is controlled by combining the locus specific PCR product with an FLR DNA molecule. As the sense strand of the FLR contains a fluorescent label (Cy5), only duplexes containing this sense strand

will be detected using a laser detection system such as an automated DNA sequencer. For heterozygous loci, two variable fluorescent heteroduplex signals will be observed in addition to the fluorescent homoduplex signal. [Adapted from (16)]

Tissue Antigens 1998: 82: 57-66

J.R. Arguello et al., 1998

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