Evaluation of the Forkhead Box P2 gene in Homo sapiens through PCR and Gel Electrophoresis for a 66 Base Pair Product

By: Sydney O’Neill, Jessica Porter, Victor Putz, and Zach Gendron

LB 145 Cell and Molecular Biology

Monday and Wednesday 12:40-2:30

Katy Kesler, and Robert Rotondo

April 20th, 2015

Abstract

Language and the ability to articulate speech are extremely important aspects of the human culture.  The first gene responsible for the the regulation of speech and language was the forkheadbox P2 gene, commonly known as FOXP2 (Enard et. al., 2002).  Mutations in the FOXP2 gene have been associated with a speech - language disorder known as developmental verbal dyspraxia (DVD) (Feuk et al., 2006). The presence of this  gene will be studied with regard to the Homo sapien. The FOXP2 gene will be amplified using a previously published PCR procedure from “Simple and Sensitive Method for Identification of Human DNA by Allele-Specific Polymerase Chain Reaction of FOXP2” (Hiroshige et. al., 2009). The forward primer being used is, 5’-CTCGACTACCTCCTCCAA-3’, HumF and the reverse primer used is, 3’-GTAGTAAGGTATCACTTACC-5’, commonR.  The annealing temperature being used is 58C. The purpose of this study is to amplify and analyze a region of the FOXP2 gene using specific PCR and gel electrophoresis. We hypothesized that allele specific primers will be able to amplify this section of the FOXP2 gene because of previous research and optimal PCR conditions (Hiroshige et al., 2009).  As a result, the hypothesis was accepted and an amplified product of 66 base pairs was obtained.  

Figure 5: Amplification of human FOXP2 gene by PCR and analysis of a 1% agarose gel via gel electrophoresis. A, a target DNA sequence for the FOXP2 gene of 66 base pairs located from 556216 to 556281 was amplified using PCR with the FOXP2-humF and FOXP2-commonR primers (Hiroshige 2009). The PCR cocktail contained 38 µl water, 5µl 10x PCR buffer, 1 µl 10mM DNTPs, 1 µl 100 µM FOXP2-humF, 1µl 100 µM FOXP2-commonR, 1µl template DNA, 1 µl Taq polymerase, 2 µl MgSO4, and a drop of mineral oil. The cocktail was ran in the thermocycler for 25 cycles with an initial denaturing phase at 95°C for 2 minutes then 45s for the rest of the denaturing cycles. Proceeded with an annealing phase of 58°C for 45s, an elongating phase of 72°C for 45 seconds then 5 minutes for a final elongation. The 1% agarose gel was made using TBE (Tris/Borate/EDTA) buffer, water, agarose, and GloGreen was run at 115V for 23 minutes. 2 µl of loading dye and 10 µl of the PCR cocktail was pipetted into wells 2-4 and 4 µl of loading dye and 8 µl of 1kb plus DNA ladder was pipetted into wells 1 and 5. B, the semi-log plot of migration distance vs. molecular size of 1 kb plus DNA ladder was used to analyze the PCR product from the FOXP2 gene. The 1 kb ladder was used and analyzed using the semi log plot shown in which the x-values represent the distance in centimeters in which the bands of the ladder migrated from the well and the y-values represent the size of the molecules in base pairs. The trend line was added in order to obtain the equation that was used to support that the band was actually for the FOXP2 gene DNA.

Discussion

Experiment Summary

Developmental verbal dyspraxia (DVD) is a syndrome that is characterized by the disruption of speech and inability to both convey and understand language (Vernes, 2009). DVD occurs due to a mutation on the FOXP2 gene, located on chromosome seven in humans (Feuk et al, 2006). This mutation results in DVD because the FOXP2 gene encodes a transcription factor, which regulates the expression of a variety of genes responsible for the development of speech and language regions of the brain (MacDermot, 2005). The purpose of this experiment is to amplify and analyze a region of the FOXP2 gene using a specific PCR protocol and gel electrophoresis. We hypothesized that allele specific primers will be able to amplify this section of the FOXP2 gene because of previous research and optimal PCR conditions (Hiroshige et al., 2009).

Original Predictions

We predicted that after running the PCR for the times found and at the annealing temperature of 58°C, we would find and amplify the desired product. A forward primer, FOXP2-humF, and a reverse primer, FOXP2-commonR, were used to amplify a human FOXP2 gene segment (Hiroshige et al., 2009). It was hypothesized that a band of 66 base pairs would be the result of the primers annealing and extending at the correct location of the gene; indicating the presence of the human FOXP2 gene. The forward and reverse primers, 1Rz1F and 1Rz1R respectively, were used to amplify the Rz gene of human Lambda DNA and serve as the positive and negative controls. Successful annealing and extension was hypothesized to produce a 394 base pair band (Taylor et al., 1989), serving as a positive control by confirming ideal PCR conditions. The absence of a band was hypothesized to indicate that, with the lack of a DNA template, there was nothing for primers to bind to; supporting that there is no primer dimer.

Ultimate Findings

Adjustments were made to the PCR cocktail, including the addition of MgSO₄, mineral oil, and DNA concentration, in order to determine the proper conditions for PCR. The addition of MgSO₄ was necessary because it acts as a substrate for Taq polymerase (Cline, 1996), without it no bands were being produced. Mineral oil was also essential in ensuring that the contents of the PCR cocktail did not evaporate during the PCR process (Erlich, 1991). An annealing temperature of 58°C was determined optimal after the calculation of primer melting temperatures and several experimental trials. The results of this experiment, bands at 66 base pairs, directly correlates with the Hiroshige et al. paper. Receiving the same results supports the hypothesis that the allele specific primers used are able to highlight a certain segment of the FOXP2 gene on human DNA.

Future Directions

In the beginning stages of our experiment a problem occurred while running the gel electrophoresis, resulting with what was thought to be bands towards the bottom of 1 kb plus ladder when viewing the gel. We predict that this was caused by a smaller product creating a band with a base pair length of not what was hypothesized. This was revised by running the future gels during gel electrophoresis for a longer time, allowing the irrelevant bands to run off the gel, leaving just our desired product.

During the duration of this experiment a homolog study could not be completed, however if research were to be continued, doing this study would be a great addition to data collected for the FOXP2 gene. The biggest constraint while trying to perform this study was time; the DNA of the Downy Woodpecker needed would have taken too long to ship creating a halt in the experiment. If given six more weeks, the DNA of the Downy Woodpecker would be able to be collected, purified and used in a PCR cocktail to analyze the FOXP2 gene. In correlation with our completed study, allele specific primers would be used to distinguish the FOXP2 gene in the bird, using the same PCR and gel electrophoresis protocol. This would benefit our experiment by supporting the importance of the FOXP2 in the evolution of both humans and other species as a gene necessary for both developing and performing certain communication and verbal skills.