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PLAN
To express in very general terms, the focus of my project involves a reaction called PCR (polymerase chain reaction – further explained in context, section 3). PCR is a reaction that reproduces vast quantities of DNA. I will specifically be using reverse-transcription real-time PCR (real-time PCR involves sophisticated equipment to detect product , reverse transcription is a step that converts RNA to DNA) to produce copies of a segment of the influenza virus (the matrix gene).
PCR, like all reactions, involves reactants/reagents; the intention of my project is to find the optimal concentration of one of the reagents (magnesium chloride) - that would produce products with efficiency. How I will examine the efficiency is in terms of:
1. The initial amount of product formed in a given time (somewhat like speed - I am looking at the concentration that requires least number of cycles/time for product formation to occur) – This involves analyzing Ct values (the cycle number at which product forms).
2. How well PCR product formation adheres to the theoretical/expected exponential growth – This involves analyzing standard product-formation graphs.
The optimal conditions (concentrations or volumes) vary from reaction to reaction depending upon many factors, but I will be specifically finding the optimal concentration of magnesium chloride for PCR efficiency. Four concentrations were use: 2.5, 3.0, 3.5, 4.0 mM.
The procedure of collecting data begins with making serial dilutions – all PCRs require serial dilutions of the DNA that is to be duplicated and this allows for visualization of product (serial dilutions are controls and they are changes in the DNA concentration). The second step in the process is to extract the DNA that is to be amplified – PCR requires a template of the DNA in order to reproduce it (sometimes the template comes from RNA instead and in such a case, an extra step called reverse transcription is required to convert RNA to DNA – such as the influenza virus). After the DNA is extracted, the volumes of component parts for PCR are calculated, measured and mixed together in a mastermix. After aliquoting, the real-time reaction can be set up and data can be collected via the computer connected to the SmartCycler (device in which the real-time PCR takes place). The data that is collected indicates the amount of product formed.
The first analyzation for efficiency requires looking at the minimal number of cycles (essentially, the time) it takes for initial product formation; for this we look at Ct values. The Ct. values (cycle/time at which the product is first visualized) are scrutinized to see where the value no longer changes or where it increases after a steady decline. This particular Ct. value indicates that any more increase in magnesium chloride will have no further effect upon efficiency.
The second analyzation for product efficiency involves a standard fluorescent graph of DNA amplification and comparing how close the results are in relation to the standard. The most efficient concentration will result in values that deviate the least from the values of the standard graph. Another method is to create a standards graph of the logarithm of the concentration versus the Ct value – the best magnesium chloride concentration would produce a graph with a slope closest to -3.33 (-3.2 to -3.4 are acceptable) – again, the expected values can be used to compare.
The results generally showed that an increase in magnesium chloride concentration decreases the Ct. values – this means that high concentrations increase the efficiency – but to a certain extent. However, a specific magnesium chloride concentration could not be designated as the optimal for product efficiency due to insufficient proof/evidence.
In terms of the efficient initial product formation: Very tentatively, it can be vaguely said that the more diluted DNA samples (less concentrated samples) of 3.5mM magnesium chloride concentration is the ideal concentration based on general observation of Ct. values.
In terms of the product adherence to theoretical growth: A comparison to a standard fluorescent graph could not be done due to time constraints, so it is unknown as to which concentration is efficient in terms of theoretical growth. In addition, the log[concentration] method could not be done.
Due to the fact that only one set of data was used to analyze and draw conclusions, no optimal magnesium chloride concentration for product-formation efficiency can be confirmed; data must be further collected.
My project may seem somewhat pointless since a national laboratory would obviously have optimized reaction reagents to use – most of the time spent in the laboratory was for observation and therefore more of a learning experience so the project is a retrospective look at PCR.
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