Consider the following 45 base-pair (bp) DNA sequence:
1 10 20 30 40
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5’-CGCACCTGTGTTGATCACCTAGCCGATCCACGGTGGATCCAAGGC-3’
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3’-GCGTGGACACAACTAGTGGATCGGCTAGGTGCCACCTAGGTTCCG-5’
Tip: If you want to see a review of PCR, we recommend this amazing animation and virtual lab made by the University of Utah.
You also have a collection of short DNA primers:
Primer 1: 5’-CGTGGA-3’
Primer 2: 5’-TGTGTT-3’
Primer 3: 5’-ATCCAA-3’
Primer 4: 5’-CCTTGG-3’
You put together several reaction mixtures, run them in the thermocycler under conditions that should produce a product if the reaction mixture is correct.
All mixtures contain:
The 45 bp DNA fragment
DNA polymerase that can function at high temperature
The necessary buffer conditions
For each mixture, indicate whether or not the PCR reaction would produce a product. If a product would be produced, give the length of that product in base-pairs (bp); if no product would be produced, give zero “0” for the length.
Reaction Added to Mixture Length of Resulting Product
dATP, dGTP, dCTP, dTTP
Primer 2
Primer 4
To determine whether or not PCR reaction would produce a product, we need to check if the primers are complementary to the DNA template and if the necessary nucleotides (dATP, dGTP, dCTP, dTTP) are present.
Let's analyze each reaction mixture:
- Reaction mixture 1:
- Added to Mixture: dATP, dGTP, dCTP, dTTP, Primer 2, Primer 4
- Length of Resulting Product: To determine the length of the resulting product, we need to identify if both Primer 2 and Primer 4 are complementary to any region within the 45 bp DNA fragment.
- Checking Primer 2 (5’-TGTGTT-3’): The sequence TGTGTT is not found in the given DNA fragment. Therefore, Primer 2 will not bind to the DNA template, and no product will be produced. The length of the resulting product is 0 bp.
- Checking Primer 4 (5’-CCTTGG-3’): The sequence CCTTGG is found at position 32-37 in the given DNA fragment. Since Primer 4 is complementary to a region within the DNA fragment, it will bind to the DNA template, and a product will be produced. The length of the resulting product will be the distance between the binding sites of Primer 4, which is 37-32 = 5 bp.
Therefore, the length of the resulting product in reaction mixture 1 is 5 bp.
In summary:
Reaction 1: dATP, dGTP, dCTP, dTTP, Primer 2, Primer 4
Resulting product length: 5 bp