Part A Calculate the annealing temperature of the two PCR pr

Part A) Calculate the annealing temperature of the two PCR primers that will anneal to this template.

5’-ACTTCGACTGGCG(5000bp)GCTTACGTAGCTTAGGCT-3’ 3’-TGAAGCTGACCGC(5000bp)CGAATGCATCGAATCCGA-5

Part B) Assuming the region of DNA you wish to amplify is 5kb in size, calculate the time that one PCR cycle will take. Assume the Taq polymerase adds 3000 bp per minute. Assume that the melting step takes 30s, the annealing step takes 30s, and the PCR thermal cycler takes 30s to ramp from one temperature to the next.

How long will ONE PCR cycle take?
How long will 35 PCR cycles take?
If you started with 200 target sequences, how many copies of it would you have after 35 cycles?

Solution

Answer part A) There are various ways of calculating the annealing temperature of primers for a PCR reaction. and an optimum annealing temperature can be known only by running a temperature gradient PCR.

though, we will be using a thumb-rule formula for calculating annealing temperature;

Tm = 64.9 + 0.41 (%GC) - (500/length of primer)

in the given case, we have two primers 5\'-ACTTCGACTGGCG-3\' as forward primer and 3\'-CGAATGCATCGAATCCGA-5\' as reverse primer.

Tm for primer one will be;

Tm = 64.9 + 0.41 (62) - (500/13)

= 64.9 + 25.4 - 38.5

= 51.8*C

Tm for primer 2 will be;

Tm = 64.9 + 0.41 (50) - (500/17)

= 64.9 + 20.5 - 27.8

= 57.6 *C

and the annealing temperatures are Tm-5*C, so the annealing temperature for primer 1 is 46.8*C and for primer 2 is 52.6*C.

Answer part B) We must assume the conditions given in the question (though actual PCR requires initial denaturation and final extension as well).

in the assumption, one PCR cycle involves melting of 30sec, annealing of 30sec, extension time for 5kb and ramping time 30sec.

Taq polymerase adds 3kb per minute which means 1kb in 20sec and 5kb in 100sec. it can be rewritten as 5kb extension in 1min40sec.

a PCR cycle starts when the PCR machine achieves the melting temperature.

So, one PCR cycle will take 30sec for melting + 30sec for ramping + 30 sec for annealing + 30 sec for ramping + 1min40sec for extension. = 3min 40sec for one cycle.

similarly, 35 cycles will require 35 x 3min40sec = 128min20sec.

If we started with 200 target sequences then after 35 cycles, we will end up having 200 x 2³⁵ copies of initial sequence.