Pcr Primers To Detect The Assessment Answer

Assignment Task:

Analysis of the NextGen data revealed a suspected somatic mutation in the biopsy sample in which, a thymine (T) has been mutated to an adenine (A) (depicted below in green). Your task is to verify this mutation by designing a PCR assay to specifically detect the adenine mutation in DNA extracted from the cells. 

TASK:

1. Explain in no more than 5 sentences on how your PCR assay will detect the adenine mutation.

2. Carefully design PCR primers to detect the adenine mutation.

3. Calculate Tm and GC content of each primer using the online ‘Tm Calculator’: 

4. Tabulate the DNA sequence of your primers (5`?3`) the Tm’s, the concentration of primers and magnesium chloride along with the expected size of the PCR product. 

5. Highlight region of the DNA that your primers will hybridize to.

6. List the temperature parameters you would use in the PCR to amplify the DNA, along with the number of PCR cycles.

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