A PCR-Based Assay That Detects CFTR N1303K in Genomic and cDNA Optimally with the Addition of 3mM MgCl2
by: Gillian Cann, Hannah Kamil, Jeremy Levine, and Brittany VanRaaphorst
Abstract: Written by: Hannah Kamil, revised by Jeremy Levine and Brittany VanRaaphorst
A fast and accurate test for the presence of the Cystic Fibrosis mutation
N1303K mutation would be very beneficial because researchers could locate
the existence of the mutation in patients with cystic fibrosis. The
purpose of this study was to develop a PCR based assay to detect the
cystic fibrosis mutation N1303K. In addition, we tested the addition of
MgCl2 to PCR in concentrations ranging from 1.5mM to 10.5mM, to determine
the optimal concentration. We hypothesized that a concentration of 3mM
would produce the most vibrant and specific bands visualized by gel
electrophoresis based on previous findings. After transforming E. coli
will plasmids containing the CFTR N1303K gene, the plasmids were isolated
and replicated using PCR. This assay required a standard PCR cocktail with
Taq polymerase and four primers, created and obtained by diagramming the
CFTR genome. Reverse primer 1 and the cDNA forward were hypothesized to
anneal at 50ºC and produce a band 504 base pairs long. Reverse primer 2
and the genomic forward primer were hypothesized to bind to the wild type
DNA at 50ºC and produce a band 526 base pairs long. Performing PCR and gel
electrophoresis on both wild type and mutated DNA produced bands of
hypothesized lengths, thus creating a simple test for this particular CFTR
mutation (Henegariu et al, 1997). The effects of MgCl2 on PCR were
compared by gel electrophoresis and supported our hypothesized optimal
concentration of 3mM using both wild type DNA and N1303K positive plasmid
DNA. Thus we conclude that the sensitivities of PCR-based assays used to
study CFTR mutations such as N1303K could benefit from the addition of
MgCl2 to their original design.
Figures: Written by: Gillian Cann, revised by Brittany VanRaaphorst and Hannah Kamil
Correct gel electrophoresis of our DIY. Both genomic wild-type CF DNA and plasmid N1303K mutated DNA were used. 4ul of MgCl2 was added in varying concentrations to each reaction cocktail. The wells, and the bands that came from them are numbered one to eight, from left to right, restarting the count for each new gel, with well #1 being the ladder in each gel. The gels themselves are numbered from left to right as well, ranging from one to three. In gel one, the reaction cocktails that were inserted into wells two, five, and seven contained wild-type CF DNA with the appropriate primers; wells three, six, and eight contained plasmid N1303K mutated DNA with the appropriate primers. Wells two and three were the control; five and six contained an additional MgCl2 concentration of 1.5mM; seven and eight contained an additional MgCl2 concentration of 3.0mM. The progression continues in the same manner for gels two and three, alternating between wild type and mutant DNA, with the MgCl2 concentrations as labeled.
Discussion:Written by: Jeremy Levine, revised by Hannah Kamil and Gillian Cann
There are many types of mutations that cause the same, or very similar, effect. N1303K is one of many mutations that cause cystic fibrosis. Since it is like most other mutations in how it changes the DNA, the same methods used to detect the presence of other mutations can be used to detect N1303K’s presence. By creating a primer that attaches specifically to the change in DNA made by the N1303K mutation, it became possible to use polymerase chain reaction (PCR) to amplify that specific section of DNA, making it easy to detect if the mutation was present within the DNA sample.
The first group of primers which were originally designed to detect the N1303K mutation did not successfully bind and amplify the chosen segment of DNA for either wild-type or mutant. All of the old primers were created with cDNA, but the wild-type cystic fibrosis (CF) DNA was genomic. In addition, the primer which coded for the N1303K mutation used with mutated DNA was mistakenly designed to run in the same direction as the other primer in the reaction cocktail, and so no segment of DNA was amplified. Four new primers were designed to solve these problems. Upon testing the newly designed primers, it was found that they did bind to and amplify the mutated DNA, allowing for simpler detection of the presence of this mutation (N1303K) in DNA samples.
Once a subject has been diagnosed with Cystic Fibrosis, it is important to discover which mutation or mutations are causing the disease. The specific mutation must be known, because different mutations can lead to slightly different symptoms. These symptoms are similar enough that they all fall under the name Cystic Fibrosis, but may differ enough to require slightly different treatments. The N1303K mutation, as an example, is a “‘severe’ mutation with respect to the pancreas,” but it is not linked to “the severity of lung disease” that is common in Cystic Fibrosis (Osborne, 1992). Knowing this, it might become possible to plan variations in the treatment which would not have been done if the precise mutation causing the Cystic Fibrosis had not been known.
Before being put into general use, however, further tests need to be done on the primer specific to the mutation. Testing our primers showed that the primers designed for the mutated DNA binds to DNA containing the N1303K mutation, and primers designed for wild-type CF DNA binds to wild-type DNA. This test was necessary, because before the primer that has been developed can be used as a part of a test for the N1303K cystic fibrosis mutation, it must be ascertained the mutated primer will not viably attach to wild type DNA. This was easily tested and accomplished by running a second agarose gel alongside the other one; with wild type DNA in place of the N1303K mutated DNA. This gel should show no bands because the mutation-specific primer did not attach to the wild-type DNA. This wild-type test acts as another type of control, as it possesses the same arrangement of primers and other possible variables that had been used in the initial experiment, with only the DNA differing. This type of test has been used in similar situations to protect against the chance of a false positive occurring (Shi, 2007).
There are several techniques to determine the length in base pairs of DNA. This is crucial when using PCR because the primers used are a specific length in base pairs, and are designed to amplify a specific segment of DNA. Extracting and purifying the DNA and then testing the concentration and absorbency using a spectrometer is one of these techniques. The extracted DNA will then be used as a DNA template when performing PCR.
When performing polymerase chain reaction, the Taq Polymerase
requires free magnesium, beyond the magnesium that binds the DNA and
dNTPs. The concentration of
magnesium also influences how well the primer anneals to the DNA template
(Khosrachinia, 2007). This presents an interesting
question: Would altering the magnesium concentration in a PCR cocktail
provide better band results on the agarose gel? When and if the dNTP concentration
increases, the efficiency of polymerase chain reaction is hindered without
an increase in magnesium concentration as well. The goal of our DIY was to find
the optimum magnesium concentration, while keeping the dNTP concentration
constant, in which polymerase chain reaction will work most
effectively. This was
determined by how the bands appear on the gel during electrophoresis.
In a previous study, it was shown that the most effective concentration for magnesium (in the form of MgCl2) is around 3.0 mM when the dNTP concentration is around 200 uM each (Henegariu et al, 1997). By increasing the magnesium concentrations up to a certain point, the bands should become more precise, eliminating any smudges on the gel. Magnesium enhances Taq polymerase and enabled it to replicate DNA more effectively, but only to a certain concentration. After that point however, the bands become less focused and dim. Too little magnesium will decrease the amount of products because dNTPs will not bind, and the primer will not attach to the DNA template efficiently. Too much magnesium will result in several unspecific products, which will show up blurry on the gel because magnesium inhibits Taq polymerase in high concentrations. By altering the magnesium concentration in our PCR reaction cocktails, we found the optimum concentration, that which produced the brightest and most specific results on the agarose gel for the amplification of both the N1303K mutation and wild-type DNA, to be 3.0mM. This is close to the optimum concentration found in the previous study (Henegariu et al, 1997).
In the study discussed above it was shown that, in addition to magnesium concentrations, annealing temperatures and the concentration of dNTPs also have an affect on PCR and the results viewed after gel electrophoresis (Khosrachinia, 2007). For instance, by combining the addition of 2mM MgCl2 and 200 uM of each dNTP the best PCR conditions were obtained for products visualized by gel electrophoresis. The study concluded that with increasing dNTP concentrations, higher concentrations of MgCl2 were required to achieve optimal results. When considering varying annealing temperatures with the addition of MgCl2, the annealing temperature must also be increased which leads to less unspecific binding (Henegariu et al, 1997). After determining the concentration of MgCl2 that presented the brightest and most easily viewed results for observing amplified DNA segments containing the N1303K mutation for cystic fibrosis, we would like to experiment with both annealing temperatures and dNTP concentrations to produce the best band possible. This will enable future PCR-based assays regarding a detection of the N1303K mutation to be more effectively visualized by gel electrophoresis.
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Note: Due to technical constraints, it was not possible to insert or utilize subscripts while building this webpage. Therefore, all mentions of magnesium chloride's chemical formula are displayed as MgCl2, and should be read as MgCl subscript(2).