ASPCR Detection of the Point Mutation N291S in the LPL Gene

in Homo sapiens DNA with Familial Combined Hyperlipidemia

 

The Fantastic Four

 

Jane Dickson, Meghna Dhir, Luke Friedli, Tim Oja

 

Abstract

 

Familial Combined Hyperlipidemia (FCH) is caused by mutations in the lipoprotein lipase (LPL) gene. The N291S mutation is a particularly common mutation present in FCH patients (Hoffer et al, 1996). This mutation changes the 291st amino acid from asparagine to serine, resulting in protein malfunction while preventing the correct breakdown of lipoproteins. The purpose of our research was to develop a method of detecting this mutation at the molecular level. A PCR diagnostic assay was designed to determine the presence of the N291S mutation in DNA samples extracted from human adherent epithelial cells. Two forward primers (WFP and MFP) and a reverse primer (RP) were developed using the Yaku method to anneal to either wildtype or mutant DNA (Yaku et al, 2008). We hypothesized that the inclusion of an intentional mismatch at the third base pair from the 3’ end of each forward primer would allow for the detection of the N291S mutation using PCR and gel electrophoresis. Following PCR, the amplified products were analyzed by gel electrophoresis to determine the presence of N291S. Our PCR diagnostic assay was unsuccessful in producing a 622bp band using the mutant forward primer with mutant DNA and the wildtype forward primer with wildtype DNA, when paired with a standard designed reverse primer amplifying the target region. As a control, two primers from a similar study published in Arteriosclerosis, Thrombosis, and Vascular Biology unsuccessfully detected the N291S mutation. The members of this research team followed the recommended diet and exercise regimens for a FCH patient to understand the effects of FCH on a college student. We analyzed off-campus food budgeting and on-campus dining accommodations and found that the diet required an average of $20.05 more per week while MSU is making adequate accommodations for students with FCH. Regular blood pressure testing and lipid panels evaluated the effect of these lifestyle changes. We found that our blood pressure and lipid levels decreased to or maintained healthy levels of 120/80 and 200mg/dL, respectively. Our diagnostic assay holds significance because early detection and treatment of FCH leads to the most effective results (Murphy 1974). Our sociological experiment carries important implications because it sought to recognize the challenges faced by FCH patients.  

Figure1:  Non-Specific Binding using GoTaq Green Master Mix from Promega.  The GoTaq Green Master mix cocktail creates an ideal stringency by containing predetermined volumes of Taq polymerase, dNTP’s, a reaction Buffer, and MgCl2.  In order to test our designed primers, we added 1 µl of WFP, 1 µl RP, 2 µl S9 cells, and 21 µl of H2O to 25 µL of the Master mix. The cocktail was run in the PCR machine with a denaturation temperature of 94˚C for an intial cycle of 5 minutes and for 30 seconds for each following cycle.  An annealing temperature of 48˚C was used for 1 minute, the extension was run at 72˚C for 1 minute for each cycle and the final elongation was run for 5 minutes. This cocktail was to determine that our primers could act as a positive control for wildtype DNA.  In lane 2, there was non-specific binding with a stronger band at the desired band length of 622 base pairs.  The possible non-specific binding was most likely due to the low annealing temperature.  However, the greater intensity between the 650 and 500 base pair bands of the 1 Kb Plus Ladder indicates that more favorable results of the desired band at the length of 622 base pairs would have occurred had the temperature been slightly increased.

 

 

Figure 3:  Non-Specific Binding for positive controls of wildtype testing. The first letter for each lane is the type of DNA used, M for mutated DNA and W for wildtype.  The second letter refers to the primers used, P for the control primers and W for the MFP and RP.  The temperatures then indicate the annealing temperature chosen. The cocktails were initially denatured at 94 ºC for 5 minutes and then at 94 ºC for 30 seconds for each of the 30 cycles.  They were then annealed for 1 minute at 30 ºC, 45 ºC, or 50 ºC, and extended for 1 minute at 72 ºC  with a final extension of 5 minutes at 72 ºC.  The highlighted regions emphasize the possible non-specific binding.  The non-specific binding in lane 3 of gel B encouraged the increased temperature for gel A. The region in the smaller box of lane 3 of gel A leads to believe that in the raised annealing temperatures the desired band would be created in the future.  Additionally at the bottom of this lane there are possible primer dimers; however, unsupported due primers not being run alone.  The cocktail in lane 1 of gel A consisted of a negative control because it was the control primers from the Zhang et al, paper for mutated DNA run with wildtype DNA. Future tests are needed to solidify these findings.

 

 

 

 

Discussion

Background

Familial Combined Hyperlipidemia is a hereditary disorder characterized by high levels of LDL, cholesterol, and, triglycerides in affected individuals (Brunzell et al, 1983).  These symptoms are the result of abnormalities in lipoprotein metabolism, which is caused by a defective form of the enzyme lipoprotein lipase (LPL) (Reymer et al, 1995).  The N291S mutation is one of many that can occur in the LPL gene, resulting in a single base pair substitution from adenine to guanine (Zhang et al, 1995).  This mutation is common to a high frequency of FCH patients (Reymer et al, 1995).  In our study, we attempted to design a PCR diagnostic test that can conclusively confirm whether or not an individual carries the N291S mutation. As a second goal, our experiment investigated the impact that FCH has on a college student. We focused on how a college student could accommodate the necessary diet on and off campus. In order to do this, the researchers followed a diet and exercised regularly much like an FCH patient would be required (Cabezas et al, 1993).

Original Predications

We hypothesized that our designed primers would differentiate between wildtype and mutant DNA during the PCR, which in turn would result in specific band patterns following gel electrophoresis­­­.  In the case of wild-type DNA, the WFP was predicted to produce a 622bp band when paired with the RP, while the MFP was predicted to show no band after gel electrophoresis. The band would be 622 bps long because the 5’ ends of the forward primers are 622 bp’s away from the 5’ end of the RP.  This was because the single intentional mismatch on the third base pair from the 3’ end, incorporated based on the Yaku method, on the WFP should not affect annealing and subsequent extension of the primer, provided that our concentrations and annealing temperatures were adequate (Yaku et al, 2008).  In comparison, the MFP included both the intentional mismatch on the same base pair and the mutation on the last base pair or the 3’ end, therefore preventing extension of the primer because there would be three base pairs on the 3’ end of the primer, which do not anneal due to the two mismatches.  In the case of mutant DNA, the MFP was predicted to produce a 622bp band, while the WFP was predicted to show no band. Both MFP and WFP contain the intentional mismatch; however, WFP contains an additional mismatch at the site of the mutation that will prevent extension of the primer (Yaku et al, 2008). We hypothesized that MSU provides the necessary resources and accommodations that would allow a student with FCH to have a college experience similar to that of an unaffected student.  We predicted that the health variables being measured, which included blood pressure, cholesterol level, triglycerides, HDL, and LDL levels, would show a steady decrease to healthier levels for two reasons (Wong et al, 2000).  The first comes from our knowledge that a wide variety of foods are served on campus and that the cafeterias would provide healthier options to fit within our diet (Shannon et al, 1997).  The second reason involves the availability exercise facilities on campus.

Genomic Purification

After completing genomic purification, a purity ratio of 2.176 was found through a spectrophotometer, showing more DNA then protein was extracted. 

Results and Ultimate Findings

After a total of six different PCR diagnostic trials, PCR detection of the N291S mutation was unsuccessful. Additional trials are required in order to detect the mutation in both the wildtype and mutant DNA (Roux, 2009).

Successful detection of the Rz gene of bacteriophage lambda was utilized to determine the cocktail ingredients for the wildtype DNA cocktail that included the wildtype DNA forward and reverse primers. The other cocktail was adapted from a previously successful PCR detection of the N291S mutation (Zhang et al, 1995). It was found that the paper cocktail used more reaction buffer than the lambda cocktail. After running both cocktails simultaneously for all the trials, it was apparent that non-specific binding occurred in the lanes that contained the lambda cocktail. This can be attributed to the fact that excess buffer can affect the optimal annealing temperatures (Roux, 2009).

During trial 1, we ran both cocktails at the annealing temperatures 48°C and 51°C, which showed that our targeted region was not amplified. Thus, we decided to drop the temperatures to 35°C and 40°C and slowly increase the temperatures as necessary. Temperatures were then increased to 30°C and 45°C. In the lanes that were ran at 45°C, there appeared to be non-specific binding. Non-specific binding occurs when the primers anneal to multiple points on the DNA (Ausubel, 1987). A method to reduce non-specific binding is to raise the annealing temperature (Roux, 2009). Our lasts gels were then run at 46°C and 48°C and continued to show slight non-specific binding. A very faint band appeared around the predicted band length of 622 base pairs for the paper cocktail using wildtype DNA and wildtype primers, however another trial is needed to confirm the presence of the band. However, the lane also showed possible primer dimer, which could be attributed to incorrect primer design, causing the primers to anneal to each other (Cha et al, 1993).

After several failed attempts to amplify the targeted region, it was found that the wildtype and mutant forward designed primers did not have a 40-60% G/C content, or G/C clamp. G/C bonds are very important in primers due to three hydrogen bonds between the nitrogenous bases (Roux, 2009). The three hydrogen bonds provide strength and promote efficiency during the annealing phase in PCR (Ausubel, 1987). From some recalculation, it was also apparent that the melting temperatures for each the forward and reverse primers for both mutant and wildtype were calculated incorrectly (Roux, 2009). The temperatures were all a few degrees lower than they should have been. This can be a possible reason for the non-specific binding that is visible gel trials 4, 5, and 6 that unsuccessfully amplified the targeted region.  To strengthen our experiment, we performed a negative control (Cha et al, 1993). We tested the wildtype DNA with the CRP and CFP. It was predicted that no bands would appear on the gel. However, after running the gel, it was found that some faint bands did appear in multiple spots on the gel. This could be attributed to a flaw in primer design, causing primers to anneal to random places on the DNA (Roux, 2009). In terms of our sociological experiment, our hypothesis was supported and we found that a college student at MSU would be able to make sufficient accommodations to follow a healthy lifestyle. No significant decrease was shown for blood pressure. Our lipid profile test, showed a decrease in overall cholesterol for each member, but varied for the LDL, HDL, and triglyceride levels. Variation can be attributed to the fact that this diet only lasted four weeks, and more stable results are usually seen over a longer period (Cabezas et al, 1993). It was also found an average of 45% of the foods were consumable on campus.  For someone living off campus, it was found that more money was spent and fewer items were brought at each grocery trip, due to the fact that healthier options are more expensive (Drewnowski et al, 2005).

Future Directions

With an unsuccessful PCR assay, many parts of the experiment could be improved. The first three trials of gels showed no bands at all. After much research, it was found that the annealing temperatures were calculated incorrectly. To fix this problem, we dropped the annealing temperatures to 30°C and then raised it to try to find the optimal annealing temperature. After raising the temperatures, we found non-specific binding appeared in many of the lanes (Figure 3). In order to reduce non-specific binding, the temperature should be raised by 2°C each time and the duration of the annealing and extension steps should be shortened (Cha el at, 1993). Also, the initial denaturation time could be lengthened to increase the likelihood that the template DNA is fully denatured (Pal et al, 1991). The denaturation time that we used was five minutes; this could be increased to seven.  Non-specific binding can also be reduced by varying the amounts of Mg2+ added to each cocktail (Roux, 2009). Lower Mg2+ concentration can reduce non-specific binding because Taq polymerase is an enzyme that depends on magnesium in order to provide thermo-stability (Cha el at, 1993). Possible primer dimer was also visible (Figure 3). To refute or support this, both the primers should be run together  (Roux, 2009). If given more time, we would redesign the primers so that each would have an increased G/C content to strengthen bonding. We would use Primer-BLAST, and Oligo Design for assistance. After some research, it was found that “Hot Start” PCR could improve our results by reducing the chances of primer dimer and non-specific binding (Cha el at, 1993). Hot Start PCR is a technique that raises the temperature above the annealing temperature of the reaction components before the taq is added, thus increasing specificity (Roux, 2009). Another method to reduce non-specific binding is called nested PCR, which uses two sets of primers in two successive PCR trials (Roux, 2009). The product of the first pair of primers is then used in the second PCR, with has a set of primers that are different from the first reactions. Therefore, this increases the specificity of the amplified DNA, and reducing non-specific binding (Pal et al, 1991). Mutant DNA would be obtained from Dr. Paul Reymeror from the University of Illinois would allow for further testing to amplify the N291S mutation with respective primers.

In order to improve our sociological assay, a long-term diet could be adapted for three months and a deeper analysis of all the different meal times could be taken, which includes looking at both breakfast and lunch.

 

Still-frame video: http://www.youtube.com/watch?v=fzS9oeqNZvw.