The following is the DNA sequence of the wild type allele of Gene Z that you wan
ID: 3480574 • Letter: T
Question
The following is the DNA sequence of the wild type allele of Gene Z that you want to amplify using the polymerase chain reaction (PCR).
5’ – CTCGAGGTGAATATGAAAG-- GENE Z -- CATTTGGCGCGTAATCGATA – 3’
3’ – GAGCTCCACTTATACTTTC-- GENE Z -- GTAAACCGCGCATTAGCTAT– 5’
(A) What are the reaction components that you would need (at minimum) to amplify this DNA sequence.
(B) Which set of parameters below would you use for the PCR reaction in part A?
Set 1: 5’-TACACTTATACTTTC-3’ and 3’-GTAAACCGCGCATTAG-5’
Set 2: 5’-GAGTTACACTTATAC-3’ and 3’-TGGCGAGTAATCGATA-5’
Set 3: 5’-CTCGAGGTGAATAT-3’ and 3’-CCGCGCATTAGCTAT-5’
(C) A PCR reaction is ran in a three-step reaction cycle where each step is performed at a specific temperature: 95° C for step 1, 55° C for step 2, and 70° C for step 3. Briefly explain why the three steps are performed under different temperatures.
Explanation / Answer
a) A PCR reaction requires the following minimum components:
1) Taq DNA polymerase: Taq DNA polymerase is a thermostable polymerase from Thermus aquatics. This polymerase is not inactivated at the denaturation temperature of 940C used in PCR.
2) PCR primers: Forward and reverse primers (18-20 nucleotides) that are specific to the gene have to be added. These primers will anneal to the gene and amplified.
3) dNTPs: A cocktail of for nucleotides need to be added for incorporation into the growing DNA strands.
4) Magnesium ions: Magnesium ions are needed as a cofactor for Taq DNA polymerase.
5) DNA template: The DNA containing the wild type allele of gene Z should be added for this sequence to be amplified.
6. Distilled water: Distilled water is added to dilute the reaction mix to appropriate concentration.
7. PCR buffer: The PCR buffer will have all ions required for optimal activity of Taq DNA polymerase.
b) The PCR primers will be synthesized as follows:
5’ - CTCGAGGTGAATAT-3’ Forward primer
5’ – CTCGAGGTGAATATGAAAG-- GENE Z -- CATTTGGCGCGTAATCGATA – 3’
3’ – GAGCTCCACTTATACTTTC-- GENE Z -- GTAAACCGCGCATTAGCTAT– 5’
Reverse primer 3’-CCGCGCATTAGCTAT– 5
The first and second set will not amplify Gene Z. Only set 3 will amplify the gene Z.
Option Set 3.
c) The first step at 950C is the denaturation step. In this step, the DNA template is denatured by breakage of hydrogen bonds between nucleotides on the two strand. The two strands separate and are available for binding of DNA polymerase. This temperature should be higher than the melting temperature of the gene, where half the bonds between the strands are broken.
In the second step (550C), the forward primer and reverse primer will anneal or bond with the DNA strands. Forward primer will anneal to the 3’ to 5’ strand while the reverse primer will anneal to the 5’ to 3’ strand. Annealing requires a lower temperature than the melting temperature Tm. It also depend on GC content of the gene.
The third step is the extension step. The nucleotides will be added to the annealed primers in the extension step of 700C. Taq DNA polymerase has maximal activity at this temperature.
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