Reference no: EM133516320 , Length: word count:1000

Molecular Theory, Data & Application in the Animal Sciences

Question 1. Describe a) how PCR works with reference to its ingredients and b) using 2 examples, outline why we might wish to use this technique.

Question 2. Describe a) how agarose gel electrophoresis works and b) with reference to one example, outline why we might wish to use this technique.

Question 3. You run a set of qPCR reactions to estimate mtDNA copy number from 3 genomic DNA samples derived from the following tissues: white fat, liver and cardiac muscle.

You have designed primers pairs to selectively amplify mitochondrial and nuclear DNAin separate reaction tubes. The reported cycle thresholds are as follows:

Tissue	nDNA Ct	mtDNA Ct
White fat	28.2	24.1
Liver	27.5	20.8
Cardiac muscle	26.3	17.4

Estimate the mtDNA copy numbers for each tissue explaining your working.Note that your species has a diploid genome.
What does the result suggest about the metabolism of the 3 tissues and why?

Question 4. Chromatography is a laboratory technique for the separation of a mixture of compounds and metabolites into its components. Liquid chromatography uses the liquid mobile phase where gas chromatography uses the gaseous mobile phase. In a liquid chromatography platform, explain what happens to the metabolites in a plasma sample during the sample preparation steps mentioned below:
- Step 1: Plasma mixed with of ice-cold acetonitrile and kept at 4°C for 20 min.
- Step 2: The mixture of plasma and acetonitrile centrifuged for 10 minutes and supernatant is harvested.
- Step 3: The supernatant is evaporated using a vacuum centrifuge until complete drying.
- Step 4: Metabolites are resuspended in 200 μL of water/ACN/formic acid.
- Step 5: The supernatant from step 4 is centrifuged again and injected into the chromatography column.