Fluorescence is a widely used quantitative method of detection. For example, in
ID: 166983 • Letter: F
Question
Fluorescence is a widely used quantitative method of detection. For example, in flow cytometry, two different dyes can be used and separately interrogated to label DNA in terms of the total amount of C-G vs A-T content in individual chromosomes. This is shown schematically below. Explain concisely how we can tell the two dyes apart. (10 points, + bonus points if you correctly draw, use, and explain energy level diagrams for both) Explain concisely how we can tell how much of each type of base pair (C-G and A-T) and dye is present. If a piece of a chromosome is missing, what happens to the two signals?Explanation / Answer
Q.No a
Each fluorescent dye has different emission and excitation range. The DNA composition of specific chromosomes in suspension is measured using DNA specific dyes Hoechst 33258 (HO) and Chromomycin A3 (CA3)
Q.No b
The base pair complementarity between the nucleotide probe which can be labelled and the target sequence are visualized by chromosome preparation and allowing physical mapping. Two DNA fluorescent dyes HO and CA3 with non-overlapping emission spectra and different base pair affinity such as GC and AT rich DNA are used in combination. HO exhibits a high quenched fluorescence signal on chromosomes.
Q.No c
Long exposure times are required to detect specific two signals. Amplification can be done either in linear or logarithmic mode and it depends on the dynamic range of signal.
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