Question

A research associate working for Intragene Therapeutics prepares an investigative PCR reaction to screen human tissue samples for specific mutations. He uses 55 ng of genomic DNA from one tissue sample in a single PCR reaction, to amplify a 1,246 bp fragment. The PCR protocol requires a final concentration for two primers of 1.5 uM each, and a final concentration of 180 uM for dNTPs, in a total PCR reaction volume of 65 uL. During the PCR process, the fragment is amplified for 33 cycles. He quantifies the amount of PCR products to be 74 ng/uL.

3. How many copies of the fragment are produced in his PCR reaction? Assume that the target sequence is present in only one copy in the genome, as in a haploid human genome. Answer to the nearest whole number. Please show all work.

CORRECT ANSWER IS 16,667 COPIES OF TARGET SEQUENCE.

Answer 1

Sorry, but that is not the correct answer, even when starting with one single copy of the DNA fragment, much more copies are obtained by 33 cycles. I guess that number (16,667) is the number of initial human genomes, that is before PCR. Let us calculate that number here and we will calculate the asnwer with your number and ours.

- First ours

Okay, so we have to calculate first how many initial copies of the target sequence we have. We start with 55ng fo genomic DNA, and human genome (the whole diploid genome) weights 3.59x10-12g, and a whole human genome contains 2 copies for this fragment (because it is diploid). How many ng does the human genome weights?

Now, we have 55ng, that is how many human genomes?

55ng/0.00359ng = 15,320.33 human genomes

How many copies of the fragment are located there considering the human genome is diploid?

(15,320.33)(2) = 30,640.66 fragment copies

Now, this number is going to get duplicated in every cycle. The equation then is:

(30,640.66)(233) = 2.63x1014 fragment copies after PCR

- Now let's try with your initial number of human genomes

We have 16,667 genomes, we are diploid so that is 33,334 initial fragment copies.

(33,334)(233) = 2.86x1014 fragment copies after PCR

The numbers are pretty similar actually, that variation may come from different sources of human genome mass.