Derek Wong
Biology 275L-03
Lab 10
Polymerase Chain Reaction (PCR)
Polymerase
Chain Reaction is a technique that allows samples of DNA to be amplified and
multiplied exponentially in a very short period of time. This is done through several process. The first is
through the denaturing of the DNA by using high heat (98C) resulting in two
single-stranded DNA sequences serving as templates for the reaction. The second step is to annealing where primers
bind to single-stranded DNA. The final step is extension of the DNA where
DNA polymerase replicates DNA strand.
The parts of PCR includes the
template, the primers, the dNTPs, the magnesium ions,
and the buffer. The template contains the target sequence to be
amplified. The primers Define the region of DNA that will be amplified. The dNTPs
constitute the growing stand of DNA and the magnesium ion is A
cofactor for the DNA polymerase. The
buffer is used to give the enzyme at work the optimal conditions,
the buffer used here is Taq polymerase.
Another type of PCR used is the RAPD-PCR where unlike
regular PCR, the goal of RAPD-PCR is to amplify
various sized DNA fragments to form a unique banding pattern on an agarose gel.
The purpose of this experiment is to run four different PCR reactions. The first is to amplify a DNA sample from the plasmid DNA isolated previously, the second is to amplify the genomic DNA sample also isolated previously, and for each one a negative control will be ran (hence four in total).
Results:
Figure 1: Agarose Gel Electrophoresis of DNA Samples. This is a depiction of the agarose gel electrophoresis after being ran for 40 minutes at 90volts, and 400 miliamps.
Lane 1: (Our) Vial #4, Lane 2: (Our)Vial #3 (Negative Control),
Lane 3: (Our) Vial #2, Lane 4: (Our) Vial #1 (Negative Control),
Lane 5: DNA Marker,
Other groups: Lane 6: Vial #1, Lane 7: Vial #2, Lane 8: Vial #3, Lane 9: Vial #4.
Table 1: Contents of each Vial
|
Vial # |
Template |
Primers |
|
1 |
dH2O (Negative Control) |
M13F, M13R |
|
2 |
Plasmid DNA |
M13F, M13R |
|
3 |
dH2O (Negative Control) |
ACTF, ACTR |
|
4 |
Genomic DNA |
ACTF, ACTR |
Table 1 shows the contents of each vial including the template and primer used in this experiment.
DNA Marker (top to bottom, bp = base pairs): 100,000 bp; 80,000 bp; 60,000 bp; 50,000 bp; 40,000 bp; 3, 000 bp; 2, 500 bp; 2,000 bp; 1,500 bp; 1, 000 bp; 750 bp; 500 bp; 250bp.
Discussion
In this lab we ran plasmid DNA and genomic DNA through PCR and then ran an electrophoresis gel on them. The results are seen in Figure 1. The plasmid DNA is shown to contain 500 base pairs, and the genomic DNA is shown to contain 1000 base pairs. This means that for the M13F, M13R primers sectioned off and copied only 500 base pairs for the plasmid DNA and the ACTF, ACTR sectioned and copied off only 1000 base pairs.
One of the main problems with PCR is the fact that since so little DNA is needed, there is a high risk of contamination. By running negative controls, we can see if extraneous DNA was placed into the sample. If there are bands in Lane 2 and 4, which are the negative control with dH2O, then it can be determine that the DNA samples ran were contaminated and thus invalid.
Specificity becomes a problem in PCR because if the concentration of the enzyme used for DNA synthesis is above the needed concentration, nonspecific products will result. This will cause a lower concentration in the DNA sample we would want to get from the PCR, since the enzyme (in this case Taq polymerase) will synthesize other fragments of DNA. Fidelity of the DNA yielded may also be a problem. This resultswhen the dNTP concentrations exceed the amount needed and yield the incorrect nucleotide chain. To solve this problem, the concentration of the dNTPs should be lowered. This action, however will not lower the yield.
The best primers are selected from a set of conditions. The first is that their melting temperature should be between 55-72C and should not contain any palindromes. The primer should also not contain any palindromes or complimentary sequences that would result in secondary structures being formed. Another condition is that there should be a high GC content towards the 3’ end to ensure that the primer and the DNA template bind tightly. Primer A has a melting temperature of 68C; Primer B has a melting temperature of 66C; Primer C has a melting temperature of 66C; and Primer D has a melting temperature of 78C. The best primer would be Primer C because it has a melting temperature within the specified range, contains no palindromes, and has a higher GC content towards the GC end.