Results
In this experiment, DNA was extracted from cultured cells, PCR was used to amplify the segment about the R702W mutation, and the resulting products were analyzed using gel electrophoresis. Cultured cells from patients with the R702W mutation, without the mutation, and heterozygous for the mutation will be obtained from Qiagen Inc. The PCR of thegenomic DNA amplified the anticipated products from the R702W locus on the CARD15 gene on chromosome 16. The DNA used was initially unknown before gel electrophoresis. The results contained bands at approximately 1000 base pairs in the three wild-type lanes and no bands at all in the mutant lanes. Thus showing that the DNA is, in fact, homozygous wild-type. Had it been homozygous mutant, only bands at approximately 1000 base pairs would have shown in the mutant wells. Additionally, if it was heterozygous then faint bands would have shown in all wild-type and mutants wells. This occurred because of carefully selected primer design, reactant concentrations, and PCR temperatures and times (Saiki et al, 1998). Thus primers R (annealing at 20,866 base pairs to 20,887 base pairs on the CARD15 gene) and MTF (annealing at 19,866 base pairs to 19,881 pairs on the CARD 15 gene) amplified at 1021 base pair product, and primers R and WTF (annealing at 19,866 base pairs to 19,881 base pairs on the CARD15 gene) amplified a 1021 base pair product (Haenisch, et al. 2009). Also, the forward primers CDF (forward primer designed to anneal to mutant type template) or WTF (forward primer designed to anneal to wild type template) will not anneal when their bases are not completely complimentary to the DNA template bases at the respective annealing loci of the primers, preventing amplification from occurring (Schochetman et al, 1988). The forward primers are known as discriminating primers because they are designed to be completely complimentary to either the mutant type or wild type genotype, but not both (Wittwer et al, 1993). Hence heterozygous DNA will allow for some amplification with both of the discriminating primers. It then follows that amplification of DNA template from the CDF discriminating primer supports the presence of the R702W mutation, which could cause a maladaptive pro-inflammatory response in Paneth cells along with other genetic and environmental factors (Figure 4) (Lala et al, 2003).
We did a preliminary PCR amplification of the 1542 base pair long 16S rDNA locus of Escherichia coli to use as a control throughout our experiment (Haffar et al, 2010). The bands we produced during gel electrophoresis exhibited non-specific binding that we were able to reduce by raising the annealing temperature (Livak et al, 2010). Also, the validity our concentrations of forward primer, reverse primer, dNTPs, buffer, and taq polymerase will supported by these experiments, thus supporting the validity of our Crohn’s experiment.
In our preliminary research and assay design we found that DNA is extracted from samples in four steps: cell lysis, membrane disruption with a detergent, protein removal, and precipitation (Qiagen 2010). We predict DNA yield of purified samples will be in the range of 3-8 μg, because this is the normal theoretical yield of Qiagen’s Generation Capture Column Kit that is being used (Qiagen 2010). This will be determined using spectroscopy; the ratio of absorbance at 260 nm to absorbance at 280 falls within the 1.4-1.9 range when DNA yield is normal (Qiagen 2010). We predict the matrix containing the DNA will be white, again based on the Qiagen assay (Qiagen 2010).
The Jalowiec coping scale is a useful tool in the determination of how patients adapt and deal with diseases (Strickland et al, 2003). Data from filled out surveys gave a picture of what people think they would do if they had Crohn’s (Czaja et al, 2005). Notes were taken during interviews in order to determine what two people who actually have Crohn’s disease do to cope with the disease, and to give a better perspective on the importance of our study. We found that there was a significant difference between different coping styles people said they would use if they had the Crohn’s disease symptoms from the survey. This is supported by statistical analysis of the survey through a chi squared test for independence (Altman et al, 1983). Not at all, hardly at all, neutral, somewhat and very much were assigned values of -2, -1, 0, 1, and 2, respectively. The absolute deviations from zero based on coping style were 1.15 for “face up to the problem, 0.7 for “avoid the problem”, 1.1 for “positive thinking”, 0.95 for “pessimistic thinking”, 0.95 for “release emotions”, 0 for “make yourself feel better”, 0.2 for “find others to rely on”, and 0.2 for “depend on yourself.” Statistical analysis resulted in a T-score of 4.06 and a P-value of 0.0048, rejecting the null hypothesis that there is no significant difference between how people would cope with the genetic disease “given” to them on the survey, because a p-value of less than one rejects the notion that there is no significant difference (Altman et al, 1983).
Through the interviews we also found that crohn’s disease does affects ones life in many ways, but mainly through severe abdominal pain, frequent hospital visits, expensive medicine, and having to plan your life around knowing where the bathroom is. Contrary to the survey, the most important coping mechanism used by the interviewee’s seemed to be friends and family, or in Jalowiec terms “support from others (Strickland et al, 2003).” Detection of several genetic mutations related to Crohn’s disease is possible through PCR (Laghi et al, 2005) (Leong et al 2003) (Lesage et al 2002). The interviewee’s indicated that there could be potential discrimination from insurance companies or employers if a complete diagnosis existed, but they also agreed that the benefits of being able to mentally prepare for the disease would far outweigh those risks.
We did a preliminary PCR amplification of the 1542 base pair long 16S rDNA locus of Escherichia coli to use as a control throughout our experiment (Haffar et al, 2010). The bands we produced during gel electrophoresis exhibited non-specific binding that we were able to reduce by raising the annealing temperature (Livak et al, 2010). Also, the validity our concentrations of forward primer, reverse primer, dNTPs, buffer, and taq polymerase will supported by these experiments, thus supporting the validity of our Crohn’s experiment.
In our preliminary research and assay design we found that DNA is extracted from samples in four steps: cell lysis, membrane disruption with a detergent, protein removal, and precipitation (Qiagen 2010). We predict DNA yield of purified samples will be in the range of 3-8 μg, because this is the normal theoretical yield of Qiagen’s Generation Capture Column Kit that is being used (Qiagen 2010). This will be determined using spectroscopy; the ratio of absorbance at 260 nm to absorbance at 280 falls within the 1.4-1.9 range when DNA yield is normal (Qiagen 2010). We predict the matrix containing the DNA will be white, again based on the Qiagen assay (Qiagen 2010).
The Jalowiec coping scale is a useful tool in the determination of how patients adapt and deal with diseases (Strickland et al, 2003). Data from filled out surveys gave a picture of what people think they would do if they had Crohn’s (Czaja et al, 2005). Notes were taken during interviews in order to determine what two people who actually have Crohn’s disease do to cope with the disease, and to give a better perspective on the importance of our study. We found that there was a significant difference between different coping styles people said they would use if they had the Crohn’s disease symptoms from the survey. This is supported by statistical analysis of the survey through a chi squared test for independence (Altman et al, 1983). Not at all, hardly at all, neutral, somewhat and very much were assigned values of -2, -1, 0, 1, and 2, respectively. The absolute deviations from zero based on coping style were 1.15 for “face up to the problem, 0.7 for “avoid the problem”, 1.1 for “positive thinking”, 0.95 for “pessimistic thinking”, 0.95 for “release emotions”, 0 for “make yourself feel better”, 0.2 for “find others to rely on”, and 0.2 for “depend on yourself.” Statistical analysis resulted in a T-score of 4.06 and a P-value of 0.0048, rejecting the null hypothesis that there is no significant difference between how people would cope with the genetic disease “given” to them on the survey, because a p-value of less than one rejects the notion that there is no significant difference (Altman et al, 1983).
Through the interviews we also found that crohn’s disease does affects ones life in many ways, but mainly through severe abdominal pain, frequent hospital visits, expensive medicine, and having to plan your life around knowing where the bathroom is. Contrary to the survey, the most important coping mechanism used by the interviewee’s seemed to be friends and family, or in Jalowiec terms “support from others (Strickland et al, 2003).” Detection of several genetic mutations related to Crohn’s disease is possible through PCR (Laghi et al, 2005) (Leong et al 2003) (Lesage et al 2002). The interviewee’s indicated that there could be potential discrimination from insurance companies or employers if a complete diagnosis existed, but they also agreed that the benefits of being able to mentally prepare for the disease would far outweigh those risks.
