I did not specify a particular concentration that would produce optimal efficiency for PCR amplification of the influenza matrix gene because, again, the project was more of a learning experience and is a retrospective look at PCR. However, to restate my hypothesis, I thought that an increase in the MgCl2 concentration would improve the efficiency since a number of the reagents require free magnesium to function.
GENERAL EFFICIENCY (ON THE INITIAL PRODUCT FORMATION)
My hypothesis is true in terms of how increasing magnesium chloride concentration would increase the efficiency of the reaction (in terms of initial product formation efficiency) since we can see that this is the trend in the data chart – an increase in magnesium chloride results in lower Ct values (less time required for product formation). This is probably because reagent kinetics – the magnesium is required by dNTPs, primers, polymerase and also affects annealing temperatures. The increasing magnesium increases the reagent activity and reaction kinetics, which results in a more efficient or quick reaction.
It is uncertain as to whether or not my hypothesis holds true for efficiency in terms of how the products adhere to the theoretical exponential growth since I have no data for support.
OPTIMAL CONCENTRATION FOR EFFICIENT INITIAL PRODUCT FORMATION
The optimal magnesium chloride concentration for efficient initial product formation is debatable. A very general pattern says that the more diluted DNA samples at 3.5mM magnesium chloride is optimal, but the 100 dilution suggests that a concentration greater than 4.0mM may be optimal. We can’t be certain which is true because we are simply dealing with one set of data. In order to be clearer as to what the results say, we would have to collect more data for support.
OPTIMAL CONCENTRATION FOR THEORETICAL GROWTH
In terms of finding the efficiency of how well the PCR product formation adheres to the expected exponential growth, I was unable to carry this out due to time restriction (I couldn’t create a standard growth graph).
Overall, no concentration can be confirmed as the ideal for PCR product efficiency. No confirmations can be made due to lack of supporting evidence and possible errors in the process.
SOURCES OF ERROR
The strange graphs indicate that there is something wrong because the graphs should exhibit similar shapes - therefore we cannot confirm any observations based upon the graphs. The theoretical growth cannot be certain and therefore the Ct values cannot be certain either since the two are related to one another. For more stable data, the graphs must have consistent shape and should not overlap. A possible explanation to the strange graphs could be reagent volume inconsistency.
• Possible sources of error could be rooted to:
• Pipetting Error (reagent volumes, bubble formation – non-uniform in the dilutions and could explain strange graphs).
• The presence of inhibitors may reduce efficiency (inconsistent inhibitor volumes - could account for theoretical product efficiency).
• Primer quality (poor primers cause a poor PCR efficiency)
To avoid having indefinite results, I should have:
• Planned and organized time better
• Begun from the 0mM concentration rather than 2.5mM
• Used a larger range of magnesium chloride concentrations because I may have not reached the optimal value yet
• Increased the magnesium chloride concentrations by smaller increments to be more specific in detecting the optimal concentration
• Collect more sets of results instead of working with one set of results – this will help to validate the data to perhaps show that the information collected was “correct” or “incorrect.” The valid results can then be analyzed rather than the “incorrect” results.
• Use machine/robot measured volumes
• I also thought of further exploring the optimal concentrations in relation to the DNA template length and see if there is any correlation between using different strains of avian influenza (but, again, time restraints prevented this).
The PCR process opened up news ways of studying and analyzing genes and DNA. PCR products can be used for many different purposes;
o Study gene expression
o Gene analyzation
o Mutation detection
o Amplification of complete genes
o Diagnosis of medically important gene deletions or insertions
o Genetic disease diagnosis
o Molecular cloning
o Forensic studies
o Pathogen detection
o Many more
Since researchers and scientists depend heavily upon great quality and reliable results, care must be taken to ensure this. My project has applications in molecular biology where it is sometimes very crucial to have accurate PCR results. Optimization of PCR efficiency is important since it is indirectly related to the accuracy of the PCR products (how close or specific the product compares to the desired sequence).
The efficiency is affected by the magnesium chloride concentration – this concentration in turn affects the performance of the DNA polymerase when replicating DNA. DNA polymerase is the enzyme that forms the new DNA strands – having poor polymerase activity could result in misincorporations and therefore the specificity or accuracy of the sequence is affected.
MgCl2 Efficiency DNA Polymerase Product sequence
Efficiency also affects the accuracy of the products due to the quality of the primers used in the PCR. Poor primer quality could affect the sequence that is to be amplified (primers may anneal to incorrect segment of the DNA, which means inaccurate amplification) – poor primer quality is a major factor in PCR efficiency. Poor efficiency due to poor quality primers is then an indicator of product accuracy.
Primers Efficiency Primer Annealling Product sequence
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