Some Applications of Linear Dichroism Spectroscopy - Science Assignment Help

Assignment Task:

Task:

This exercise is to replace Experiment 1b where a kinetics experiment (in that case chemiluminescence of luminol) was designed to measure the concentration of an oxidizing agent (in that case H2O2). This exercise focuses on the technique of linear dichroism data for DNA and uses the signal to explore Some applications of linear dichroism spectroscopy

Linear dichroism is the difference in absorbance of light polarised parallel to an orientation direction and light polarised parallel to it. Molecules only absorb light when the electric field of light can push along the transition polarisation direction. A symmetric aromatic molecule such as anthracene will only absorb light if the electric field pushes the electrons in the molecular plane and along the long axis of the molecule (254 nm) or along the short axis of the molecule (280 nm). Less symmetric molecules such as DNA bases will absorb light if the electrons are pushed in number of different directions, but all in the place of the aromatic group. If a solution of polymeric DNA is flowed past the detection window of the spectrometer with horizontal flow, the DNA molecules preferentially align along the flow direction. At 260 nm the absorbance of the DNA is due to transitions in the plane of the base pairs. In this exercise all the experiments are done in a Couette flow cell which endlessly flows the solution past the detection windo in a horizontal direction stretching out the DNA so the helic axis is along the flow direction and the bases are perpendicular. You may find this reference is helpful. “Validation of new microvolume Couette flow linear dichroism cells”, Rachel Marrington, Timothy R. Dafforn, David J. Halsall, James I. MacDonald, Matthew Hicks and Alison Rodger. The Analyst, 2005, 1608?1616. DOI: 10.1039/b506149k

1. DNA linear dichroism

a. Sketch how the DNA molecules are oriented with respect to the light beam, the flow direction and the horizontal polarisation direction.

b. At 260 nm indicate the polarisation of transitions relative to the horizontal flow direction.

c. What sign will the Linear dichroism be?

2. Polymerize chain reactions The Polymerize Chain Reaction (PCR) is the name given to the reaction used to produce samples of DNA from a very small number of DNA molecules by repeatedly separating the strands of the DNA then using each strand as a template to make a new DNA molecule. Thus the number of DNA molecules doubles each cycle. The progress of the reaction can be measured by a number of techniques of which linear dichroism is ideal as it only gives a signal for the product DNAs.

a. Consider a solution of 100 µg/mL double stranded DNA. DNA is made up of covalently linked pairs of aromatic nucleic acid bases called a base-pair which lie perpendicular to the helix axis. This DNA has 1500 base pairs per molecule.

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