c) What would you expect to see if DNA was linear? Draw a schematic of the expec

Question

c) What would you expect to see if DNA was linear? Draw a schematic of the expected result on the graph above.

d) Expriments such as that shown in (a) require that the FRET efficiency is calculated precisely. Explain how to calculate the FRET efficiency.

2. (a) The following data describing the FRET cfficiency for a serics of DNA oligomers was obtaincd. Each DNA duplcx has fluorescein attached to the 5' end of one DNA strand and rhodamine attached to the 5' end of the other strand. Note that fluorescein and rhodamine are FRET pairs, with fluorescein as the donor fluorophore and rhodamine as the acceptor fluorophore. Thc closed circles indicate the FRET officiency measurcd and the solid linc is the calculated cfficiency for B form DNA. Explain the shape of the curve. 0.4 0.3 0.2 0.1 6 8 10 12 14 16 18 20 22 bp

Explanation / Answer

At low basepair the double strand DNA helix of both 5' are close together and increase the FRET. If increasing in base pairs leads to increasing the dostance between both 5'prime so decreasing in FRET.

c. If DNA is linear means they are in single strand so there is an opportunity
of both 5' prime ends comes and contact with the flurophores and constant FRET efficiency will absorb.

D. FRET efficiency is then calculated as the following ratio: Fa / (Fd + Fa), where Fa is the acceptor emission and Fd is your donor emission.

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