Consider the following 45 base-pair (bp) DNA sequence:
1 10 20 30 40
| . | . | . | . | .
5’-CGCACCTGTGTTGATCACCTAGCCGATCCACGGTGGATCCAAGGC-3’
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3’-GCGTGGACACAACTAGTGGATCGGCTAGGTGCCACCTAGGTTCCG-5’
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
1
dATP, dGTP, dCTP
Primer 2
Primer 4
unanswered
2
dATP, dGTP, dCTP, dTTP
Primer 2
unanswered
3
dATP, dGTP, dCTP, dTTP
Primer 2
Primer 4
unanswered
4
dATP, dGTP, dCTP, dTTP
Primer 1
Primer 3
unanswered
5
dATP, dGTP, dCTP, dTTP
Primer 2
Primer 3
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To determine whether a PCR reaction would produce a product, we need to check if the primers can bind to the target DNA sequence. The primers will bind to their complementary sequences in the DNA template, and the polymerase will then synthesize DNA in the region between the primers.
Let's analyze each reaction mixture:
1. Reaction 1:
- Primers: Primer 2 and Primer 4
- The primer Primer 2 (5'-TGTGTT-3') has a perfect match (complementary) in the DNA template, so it can bind.
- The primer Primer 4 (5'-CCTTGG-3') also has a perfect match in the DNA template and can bind.
- The resulting product will be the region between these two primers, which is 12 base-pairs (bp) long.
2. Reaction 2:
- Primers: Primer 2
- The primer Primer 2 (5'-TGTGTT-3') can bind to the DNA template because it has a perfect match.
- However, since there is no second primer to bind to the template, no product will be produced (length = 0).
3. Reaction 3:
- Primers: Primer 2 and Primer 4
- The primer Primer 2 (5'-TGTGTT-3') has a perfect match in the DNA template, so it can bind.
- The primer Primer 4 (5'-CCTTGG-3') also has a perfect match in the DNA template and can bind.
- The resulting product will be the region between these two primers, which is 12 base-pairs (bp) long.
4. Reaction 4:
- Primers: Primer 1 and Primer 3
- The primer Primer 1 (5'-CGTGGA-3') has a perfect match in the DNA template, so it can bind.
- The primer Primer 3 (5'-ATCCAA-3') does not have a perfect match in the DNA template, so it cannot bind.
- Since there is no second primer binding to the template, no product will be produced (length = 0).
5. Reaction 5:
- Primers: Primer 2 and Primer 3
- The primer Primer 2 (5'-TGTGTT-3') has a perfect match in the DNA template, so it can bind.
- The primer Primer 3 (5'-ATCCAA-3') does not have a perfect match in the DNA template, so it cannot bind.
- Since there is no second primer binding to the template, no product will be produced (length = 0).
So, the lengths of the resulting products in base-pairs (bp) are as follows:
1. Reaction 1: 12 bp
2. Reaction 2: 0 bp
3. Reaction 3: 12 bp
4. Reaction 4: 0 bp
5. Reaction 5: 0 bp