Diagnosis of the G85E – CFTR Mutation of Cystic Fibrosis Through the Use of PCR

 

Written by: Brenton Craggs, revised by: Jillian Braid

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Team Eagle

 

Jillian Braid

Brenton Craggs

Lindsey Foos

 

LB 145

Friday: 11:30a.m. -2:30p.m.

David Malakauskas / Jason Mashni

 

Abstract

Written by: Brenton Craggs, revised by: Lindsey Foos,

and final revision by: Jillian Braid

            G85E-CFTR is a genetic mutation that causes Cystic Fibrosis. Guanine is substituted for adenosine at the 386-nucleotide position in the CFTR gene, thus coding for a glycine instead of a glutamic acid (Decaestecker et al, 2004).  Wild-type CFTR proteins act as chloride channels within epithelial cell membranes (Rowe et al, 2001).  G85E is a missense, class II mutation, where all CFTR proteins in the cell are destroyed (Decaestecker et al, 2004).  Bronchial epithelial cells were used for genomic wild-type DNA purification, utilizing the Generation Capture Column Kit from Qiagen Inc. A protocol for transforming competent e-coli cells was utilized for the G85E plasmid DNA.  Forward and reverse primers were designed to anneal to DNA template in the complementary segments. Both the mutant and wild-type forward primers bind during PCR at the 386th nucleotide of the CFTR gene.  When the PCR reactions cocktails are run through gel electrophoresis, a WT 827 base pair long band can be seen, with a MT band at 680bp.  When the designed MT primers anneal to the mutant G85E-CFTR DNA, and WT primers anneal to the WT DNA sequence at the G85E locus, the presence of the G85E-CFTR mutation can be diagnosed.  For the DIY lab, a survey was created to collect data from a population, including questions about the height of the individuals and their parents.  The data from this supported the hypothesis that nurture (environment) has a significant amount to do with the expression of a gene, rather than just nature (genes).

Discussion

Written by: Brenton Craggs, revised by: Lindsey Foos,

and final revision by: Jillian Braid

            Cystic Fibrosis is a genetic disease caused by a mutation of the CFTR gene, most common in those of European descent. The G85E-CFTR mutation is one of the hundreds of CFTR mutations that cause Cystic Fibrosis. It has a frequency of .2% worldwide for all CFTR mutations, and is most prevalent in the Mediterranean regions (Decaestecker et al, 2004). Symptoms of Cystic Fibrosis include extremely salty sweat, abundant amounts of mucus, sterility, and poor digestive and liver function (Wright et al, 2009).

The purpose for the experiment was to develop a procedure to detect the presence of the G85E CFTR mutation of Cystic Fibrosis, and distinguish between those who do not have the mutation.  This was done using PCR.  Primers were designed to anneal to the segment of DNA on the CFTR gene where the mutation is found. When WT template DNA was used with WT primers, a DNA band 827bp in length could be seen after being run through gel electrophoresis.  Seeing a band with that PCR product, and no band with the control (WT template and MT primers) supports the hypothesis in the way that we were able to confirm that the WT DNA did not have the G85E mutation (Friedman et al, 1997).  When MT template and MT controls underwent PCR and gel electrophoresis, a specific band could not be seen.  However, this and the MT control (MT template with WT primers) both showed nonspecific binding.  If adjustments to the PCR protocol were made, we are optimistic that we could get a MT band.  Without a mutant band at 680bp, our results were inconclusive for the diagnosis of the G85E-CFTR mutation of CF.

            In our DIY we looked at whether external phenotype or genetics have a greater distinguishing factor on the height of individuals.  Our hypothesis is that nature, rather than nurture, is most influential in determining how tall people will grow (Smith, 2007).  When a survey was given to 100 people, we sorted participants into three groups according to height: shorter than both parents, between the height of parents, and taller than both parents.  According to the data acquired, there were many participants from the ÒshortÓ category who were raised on a diet with strict portions.  This number decreased as the height increased. More red meat, and a less strict diet will allow a child to grow taller (Hopkin, 2005).  This supports the hypothesis because the height correlated with the type of diet the child had.  This also supports the research that children with Cystic Fibrosis need a higher fat and higher protein diet, due to the often present pancreatic insufficiency, for normal development to occur (Davis, 2001).  If normal children need higher amounts of fat and meat content for development, then it can be inferred that those growing up with CF would obviously need even higher amounts.  The more better environment that parents of CF patients can provide their children, the more likely theyÕll happily past the average age of 30 years old.

 

 

 

C:\Users\Lindsey\Documents\final gel.bmp

 

 

 

 

Figure 4. Mutant-Type, G85E-CFTR

PCR reaction cocktail was pipetted into the wells of a gel. 10 μl was pippetted into each well for electrophoresis. The DNA was run through the gel and was examined. A band found with wild type DNA template and wild type primers indicates that there is not a presence of the G85E CFTR mutation.  Well 1 has the 1 KB ladder in it.  Well 2 has the wild type DNA in it with the wild type reverse primer and primer 3 in it, and shows a band gap of about 830. Well 3 has the wild-type control in it with the wild-type reverse primer and primer 1 in it, and so it does not have any bands. Well 4 has the mutant DNA in it with primer 1 and the mutant reverse primer in it. Well 5 has the mutant control in it with the primer 3 and the mutant reverse primer in it. The last two wells had non-specific binding, due to a low annealing temperature, and shows multiple faint bands.