A widely used method for calculating the annealing temperature for a primer used

Question

A widely used method for calculating the annealing temperature for a primer used in PCR is 5 degrees below the Tm (ºC), which is computed by this equation 81.5 + 0.41 (%GC) – (675/N), where %GC is the percentage of G and C nucleotides in the oligonucleotide and N is the length of the oligonucleotide. Notice from the formula that both the GC content and the length of the oligonucleotide are variables. Assuming you have the following oligonucleotide as a primer, compute the annealing temperature for PCR. (Note: In reality, this computation provides only a starting point for empirical determination of the most useful annealing temperature.)

Part A: What is the %GC content? 5’-CGCAATAGTCACCGTGCAGTTCCGT-3’ Round your answer to the nearest whole number.

Part B: What is the annealing temperature for this oligonucleotide? 5’-CGCAATAGTCACCGTGCAGTTCCGT-3’ Use your answer from Part A (%GC content) and the length of the oligonucleotide primer (N) to compute the melting temperature (Tm). Remember that you must subtract 5 degrees from the Tm value to calculate the annealing temperature. Round your answer to one decimal place (example: 65.1 or 70.0).

Explanation / Answer

part A: total nucleotide count= 25

GC count=14

% GC= 14/25 X 100= 56%

part B

Tm (ºC) = 81.5 + 0.41 (%GC) – (675/N)

               = 81.5+0.41(56)-(675/25)

               =81.5+22.96-27

                =104.46-27

                 =77.46

                 = (77.46-5)= 72.46

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