The DNA sample under high temperature such as 940C, this is the optimum temperature for the hydrogen bonds to break is called denaturing. The DNA double strand will be separated to single DNA strands. Therefore the bases are free to pair with their complementary bases present in the nucleotide solution.
The binding or annealing where the base pairing occurs, according to a very strict rules, where Adenine (A) pairs with Thymine (T) and Cytosine (C) pairs with Guanine (G) and mRNA the Adenine pairs with another base called Uracil (U) under a certain temperature lower then the temperature used to break the hydrogen bonds. At each cycle of the DNA the amplification expands.
As the reaction continues the raise of the temperature up to the 720C and the DNA polymerase adds nucleotides at the end of each primer and new strands are formed using the same base-pairing rule.
As this process called cycle, occurs the number of DNA copies will increases, or elongate.
After each cycle of the DNA replication the light at 488 nm is passed through the Polymerase Chain Reaction tube and some energy is absorbed and light at 522 nm is emitted.
Ct is the point when we start to see the light emitted. The detection of fluorescence is proportional to the DNA present in the sample. Lower the DNA to detect the fluorescence higher the threshold value.
As mentioned above, there are many variants of this gene, we tested only the CYP2D6 *4 variant. The presence of this gene will mean that the mRNA it is not processed properly and the mature mRNA will not be available for the synthesis of the protein CYP2D6.
... and the ones that won't