Question

'High Resolution Melt ' - technique Q1.What are the key scientific principles which form the basis of this technique: Q2.What are the necessary controls, standards, and data processing. (Describe and state the purpose of these.) 1. Controls: 2. Standards: 3. Data processing (e.g. cleaning/trimming, normalisation, etc): Q3.Interpretation: What are the possible outcomes (list 3 outcomes) for this technique and how each of these outcomes should be interpreted with the 'controls/standards' stated above? A B Q4.For each of the possible outcomes described in the interpretation section, explain which follow up analyses are needed (where applicable). Use the letter (A, B, C) assigned in the “Interpretation” section to refer to each of these. Q5.What are the limitations for this technique?

Answer 1

-High Resolution Melt (HRM) is a tool used in molecular biology to study about nucleic acid, more specifically dsDNA. The job of the technique is to scan for any possible polymorphisms and mutations present in the dsDNA. The different DNA samples have their respective different dissociation characteristic, which is taken into consideration over here and melting curves are plotted accordingly.

-The main requirement of the technique is the presence of PCR which works in order to amplify the targeted region of the DNA. This region is referred to as the amplicon. Other than the amplicon there are controls which solve the purpose to plotting the melting curve graph.
-Once the amplicon is ready, our next step is to incorporate the intercalating dye into the system such as LC green. If the dye comes in exposure to a dsDNA, it will bind and cause fluorescence. Hence, during melting a sudden drop in the fluorescence is observed as the dyes no more find dsDNA to bind to.
-Limitations associated with HRM are obviously it's complexity as gel based technique are more user friendly and simple and HRM is one of the first non gel based molecular biology techn. Another disadvantage is it's cost efficiency.