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.

Question: 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 1

Number of copies obtained after n cycles of PCR = 2^n

Here, n = 33

So, number of copies = 2^33 = 8.589 × 10^9 copies

This is an exponential increase in the number of copies of DNA. This is exclusive to the technique of PCR. PCR is short for polymerase Chain Reaction. It a series of free reactions used for the amplification of a molecule of DNA. The first step is denaturation in which double stranded DNA molecule is denatured to form single stranded DNA molecules at high temperature. The second step is annealing in which primer binds to the three prime end of single stranded DNA molecule. The last step is amplification or elongation in which tag polymerase adds deoxyribonucleotides to the daughter DNA molecule.

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