Increasing knowledge of DNA mutations: Designing primers for A455E and F508 gave a band at 800 base

 By: Crystal Fisher, Erich Himebaugh, Theresa Seitz, and Zachary Simons

 

LB 145 Cell and Molecular Biology

Monday 3:00pm-6:00pm

Amanda Gnau, Khaled Hammoud, Mike Busch,

April 27, 2009

 

 

Abstract

 

The purpose of our research was to produce an efficient test to certify the presence of the A455E, a mutation affiliated with pancreatic sufficiency and mild lung disease, and ∆F508, a mutation with more severe lung disease on the CFTR gene sequence in purified S9 cells. Forward (mutant and wild-type) and reverse primers of A455E and ∆F508 were designed to bind precisely 800 base pairs apart to assure dependable bands on the gel electrophoresis. We hypothesize that the mutation should be placed at the 3’ end of the forward mutant primer strand to avoid binding after the mutation causing a false positive. Multiple reaction cocktails were prepared with the DNA template from the S9 purified cell type and corresponding primers of A455E or ∆F508. Polymerase chain reaction (PCR) and gel electrophoresis were performed to determine if the allele-specific primers would bind to the selected DNA target region (A455E or ∆F508), indicating whether the mutation or the wild-type was present on the CFTR.

To better enhance our knowledge of DNA mutations, two Escherichia coli (E. coli) bacterial cell colonies were observed, one was exposed to ultraviolet light (UV) for thirty days and the other was reserved as the control. We hypothesized that the fragments of DNA in the mutant would be longer in length than the control.  Gels were run once a week on both cultures and the DNA fragments present in each were compared.  Fragments represented as bands in the gel electrophoresis were found to be smaller in the control than the mutated DNA supporting our hypothesis.

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Figure 1. Figure is of a gel electrophoresis describing the PCR of S9 cells with specified primers. PCR was run at an annealing temperature of 46° C. Lane 1 is the 1XKB ladder. Lane 2 are the S9 cells with Primer 1 (Forward A455E mutated CFTR) and Primer 5 (Reverse CFTR). Lane 3 are the S9 cells with Primer 2 (Wild-type CFTR) and Primer 5. Lane 4 are the S9 cells with Primer 3 (Forward !F508 mutated CFTR) and Primer 5. Lane 5 are the S9 cells with Primer 4 (Wild-type CFTR) and Primer 5. Bands are present in lanes 4 and 5, which reflect the heterozygous nature of the S9 cells. A band is present in lane 2 at 400 base pairs most likely due to non-specific annealing of our primers.

 

Discussion

Background: Cystic Fibrosis (CF) is a recessive genetic disorder caused by a mutation in the CFTR gene which affects epithelial cell containing organs causing thick mucus build-up (Wright, 2004). Through the examination of the CFTR protein, located on chromosome 7 (Gan et al., 1995), researchers are able to design assays to accurately identify mutations on the gene at different locations. The purpose of this research was to provide more knowledge about mutations and to design an efficient PCR based-test to simultaneously screen for multiple mutations of CF. The two mutations studied were the A455E, which corresponds to a mild form of lung disease (Kerem et al., 1990), and the more severe ∆F508 which causes severe lung disease and pancreatic insufficiency (Chen et al., 2004). They were chosen to investigate if a screening assay could be developed to test for two mutations that cause such different phenotypic effects.

It was hypothesized that using primers with either the A455E or ∆F508 mutations at the 3’ end would be an efficient mechanism to quickly screen for multiple mutations of CF. With the mutation at the end, only primers complementary to a target DNA sequence can bind and cause a band in the gel electrophoresis (Riordan et al., 1989). It was predicted that by placing the mutation at the end of the primer sequence, false positives would not be able to occur. This can happen when the nucleotides of the primer skip over the mutation in the middle and bind everywhere else allowing for the elongation stage of PCR to occur causing a band to appear in the gel (Kwok et al., 1989).

In order to understand mutations more thoroughly which would improve the designing of a PCR based screening assay for CF, the mechanics behind DNA mutations were examined. A colony of E. coli was exposed to ultraviolet light and the lengths of the DNA fragments were observed using gel electrophoresis. UV photons have very short wavelengths which correspond to high amounts of energy greater than the amount needed to break bonds in a DNA strand (Lippke et al., 1981). When bonds between strands break, mutations can occur if those bonds reform differently than the original strand. A form of incorrect re-binding that occurs from UV exposure is pyrimidine dimers which are pairs of thymine and cytosine in a DNA strand. A common form is cyclobutane pyrimidine dimers (Bohr et al., 1985). It was hypothesized that because UV photons contain enough energy to break nucleic bonds, this will cause the DNA fragments to be shorter in length than the original sequences.

Primer Design: Developing correct primers is very important for a multi-assay PCR test because not only must the forward and reverse primers for the A455E mutation correspond to similar annealing temperatures, but they must also work with the annealing temperatures of the primers associated with the ∆F508 (Chen et al., 1998).  Forward primers were designed complementary to the DNA sequences of a CFTR protein representing a wild-type subject as well as DNA containing the A455E or ∆F508 mutations (Kerem et al., 1990).  Strasberg et al performed similar research on the A455E with the mutation at the 2nd nucleotide spot and the purpose of the primers designed in this experiment was to see if placing the mutation at the end of the sequence was an efficient way to screen for CF. The reverse primers were designed to bind to the strand of DNA complementary to the mutation containing strand, 800 base pairs away from the mutant and wild-type primers.  A band was predicted to be present on the gel electrophoresis if both the forward and reverse primers bound to the denatured DNA strand, elongating to form multiple forms of the amplified DNA target sequence (Strasberg et al., 1997).

Polymerase Chain Reaction. To design a test to screen for multiple mutations of CF, the presence of the A455E and ∆F508 mutations were tested using polymerase chain reaction. It was predicted that, if either mutation was present on the DNA of the S9 cells, there would be a band present on the gel electrophoresis when the mutant primers were used in the reaction cocktail. This was predicted because the mutant primers were created complimentary to a mutant strand of DNA. Conversely, if the mutations were present, bands would not have shown up on the gel electrophoresis if the wild-type primers were used (Strasberg et al., 1997).

It was also predicted that, if the A455E or ∆F508 mutations were not present on the DNA, there would be a band that showed up on the gel electrophoresis when the wild-type primers were used in the reaction cocktails. This was predicted because the wild-type primers were created complimentary to a wild-type strand of DNA. Conversely, if the mutations were not present, bands would not show up on the gel electrophoresis if the mutant primers were used. 

The results showed that at an annealing temperature of 44°C and a reaction cocktail with 1µl of DNA, a band was present at 400 base pairs in the lane containing the A455E mutant primer. The lanes containing the wild-type A455E and both ∆F508 primers did not show a band. The absence of bands in both lanes containing the ∆F508 primers could indicate that the annealing temperature was not correct for those primers or the concentration of DNA was too little to show up. It is predicted that the absence of bands in both ∆F508 lanes was due to experimental error because if the protocol was correct, a band would appear in one of the lanes because a strand of DNA can either be mutated or wild-type (Kwok et al., 1989).

To make the annealing temperature more compatible with the ∆F508 primers, the temperature was raised to 46°C; the amount of DNA was also raised to 2µl. When gel electrophoresis was performed, ∆F508 showed bands in both the wild-type and mutant lanes. It is predicted that the S9 cells tested are heterozygous for the ∆F508 mutation (Özçelik et al., 1994). In the lane containing the mutant primer, the band was dimmer. CF is a recessive disease meaning that two alleles are needed to suffer from the disease (Campuzano et al.,1996 ). It is predicted that because the wild-type is the dominant allele, the band would show up brighter (Warbuton et al., 1996). The lane containing the A455E mutant primer showed a band at 400 base pairs. The reoccurring bands for mutant A455E at 400 base pairs for both temperatures may have occurred because the primers which were designed to anneal 800 base pairs away were identical to an area on the sequence 400 base pairs away and annealed there. The chances of this happening are very unlikely, but the brightness of the bands indicates that the primer bound to that location multiple times. After the first occurrence of the A455E band at 400, the primers were reanalyzed to confirm that they were designed to bind 800 base pairs away. This showed that the band was not due to incorrect formation of the primers. Even though a band was present in the A455E mutant, it cannot confirm the hypothesis that placing the mutation at the 3’ end of the primer is an efficient screening test because the primers to form a band at 800 base pairs. The bands at the bottom of the ∆F508 lanes are predicted to be primers that did not run off the gel (Rosenmund et al., 1998).

One weakness in the experiment is that two variables were changed in between trials. It cannot be told whether the change of DNA concentration or annealing temperature is the reason for better results in the second trial. If trials were to be run further, only one variable would be changed on between each test.

Exposure of E. coli to Ultraviolet Light: To broaden the knowledge of mechanisms behind DNA mutations, a colony of E. coli was exposed to ultraviolet light for 30 days. A separate colony served as a control and was only exposed to white light. The E. coli that was exposed to the UV light changed to a yellow color while the control was more clear-colored. This observation can be explained by photoelectric induced mutations causing enzyme or protein mis-function which can cause discoloration in cells (Jevon et al., 1998). During the exposure time, new white colored spots arose which were predicted to be new growth of cells since the time of exposure (Hiraga et al., 1989). It was predicted that the high-energy containing photons would break the hydrogen bonds in DNA strands causing shorter fragments than the original sequences. Samples of E. coli cells from the control, yellow colored mutated cells, and white new growth were added to 40 µl of H20 and vortexed. Gels were then run with either 2µl or 5µl of the vortexed cocktail to compare band intensities with different DNA concentrations. The results showed that for each concentration of DNA, a band was present in the control lane while no bands showed in either of the mutant DNA. This data refutes our original hypothesis that the UV would cause the DNA fragments to be shorter. It is predicted that the UV exposed DNA bonds were broken and during reformation, pyrimidine dimers formed from different strands causing sequences longer than the original DNA. The control DNA was short enough to run down the gel, while the newly bonded mutated DNA strands were too long to leave the wells. (Ravanat et al., 2001)

Future Research: If the experiment were able to be run further, there are changes that would be made to the research to obtain more accurate results. Cells that were known to have either the A455E or ∆F508 would be obtained to confirm that any unpredicted results were experimental errors. The reverse primer for the A455E mutation would need to be re-designed because the bands at 300 show that because the primers were designed to bind 800 base pairs away from each other, they were most likely designed incorrectly.  Different annealing temperatures should also be tried. The annealing temperatures in a test to screen for multiple mutations need to be compatible with primers corresponding to both mutations. To obtain more accurate results in examining UV exposed E. coli, each cell type would undergo genomic purification to remove the DNA from the nucleus. This would allow for more accurate results pertaining to DNA fragment length.

If the protocols of these experiments are mastered, more knowledge would be known about the mechanisms behind DNA mutations. Also if an efficient multi-assay screening test was developed, doctors would be able to check for multiple mutations of Cystic Fibrosis at once which would be easy and time efficient.