Genotypic identification of Forkhead Box P2 found linked allele specific

PCR in Homo sapiens and Xenopus tropicalis

Sydney Kruse, Monica Ward, Samantha Rogers, Emily Liddicoat. Megan Wild

LB 145 Cell and Molecular Biology

Monday and Wednesday 7-9 PM

George Hyde, Zach Kranz, and Melissa Ungkuldee

4/20/2017

www.msu.edu/~krusesyd/

Abstract

The Forkhead Box P2 gene (FOXP2) is linked to speech and language development in the fetus and adult brain of humans (Turner et al., 2013). Mutations of FOXP2 are known to cause the majority of speech disorders including Childhood Apraxia of Speech (CAS) (Morgan et al., 2016). The purpose of this study was to clone the wild-type FOXP2 gene in humans using allele specific PCR and gel electrophoresis, along with the identification of FOXP2 in a previously unstudied homolog, the Tropical Clawed Frog. Human wild type DNA amplified for the FOXP2 gene produced bands at 56 base pairs following PCR and gel electrophoresis (Bruce and Margolis, 2002). Forward and reverse primers were designed by the genomic alignment of several different species in order to ultimately determine if the FOXP2 gene is homologous in the Tropical Clawed Frog. If the FOXP2 gene was present it could imply a similar role in language development in the Tropical Clawed Frog as in humans. Further experiments are needed to manipulate primers to produce bands after gel electrophoresis. It was hypothesized that the use of allele specific primers with calculated annealing temperatures and concentrations of the PCR cocktail manipulated successfully, the FOXP2 gene would appear around 56 base pairs in humans and if present in the Tropical Clawed Frog, 1,234 base pairs (Hiroshige et al., 2009). The published primers were successful in amplifying the target region of human DNA to confirm the presence of the FOXP2 gene. Communication is an important part in animal lives not only including humans, the ability to communicate to future mates is essential for the survival of a species. By studying the genes that are involved in language you can unlock how certain mutations cause problems in other species as well as map how the ability to communicate has evolved throughout evolution. In fact, research has suggested the changes in amino acid coding in humans show the FOXP2 gene to be a target of directional selection during recent human evolution (Hiroshige et al., 2009).

Discussion

The forkhead box P2 (FOXP2) gene, located on chromosome seven, is a gene that is associated with speech and language developments in the brain before and after birth (Murray et al., 2015). The mutant strain of the FOXP2 is responsible for speech disorders, such as childhood apraxia of speech, and can cause decreased oral motor function and quality of speech, specifically difficulties in movement and accurately sequencing speech sounds into syllables, syllables into words, and words into sentences (Morgan et al., 2016). The FOXP2 gene in human DNA, a 56 base pair sequence, was amplified using PCR, primers from a published paper, and gel electrophoresis (Hiroshige et al., 2009). Due to the small size of the amplified region, a 120 ng/µl 50 base pair ladder was obtained from Empirical Bioscience, and a 2% agarose gel was made to accommodate the smaller base pair product. Primers were also designed to find a homologous gene in the Tropical Clawed Frog, using PCR and gel electrophoresis. The presence of a homolog denotes a common ancestor, in this case a common origin for speech and language for humans and the Tropical Clawed Frog (Pearson, 2013). It was hypothesized that allele specific primers and correct annealing temperatures, in combination with PCR and gel electrophoresis, would yield a band at 56 base pairs in length in human DNA (Hiroshige et al., 2009). In addition, it was also hypothesized that PCR and gel electrophoresis, each run with designedF and designedR primers and correct annealing temperatures, would yield a band of 1,234 base pairs in length if the Tropical Clawed frog is homologous to humans (Pearson. 2013).

Original Predictions

Allele specific PCR for the obtained FOXP2 primers amplified the DNA and allowed gel electrophoresis to determine if the given DNA was positive or negative for the FOXP2 gene. There were two different published primers used, FOXP2F and FOXP2R, to evaluate the FOXP2 gene (Hiroshige et al., 2009). A successful annealing of the primers was hypothesized to produce a band of 56 base pairs. In addition, forward and reverse allele specific primers, designedF and designedR, were designed to determine if the Tropical Clawed Frog was an animal homolog. A band around 1,234 base pairs was hypothesized when using the primers for the Tropical Clawed Frog DNA.

Results and Ultimate Findings

After running multiple different PCR cocktails, the temperatures for the annealing stage of the lambda cocktail that yielded the best results were 50.5°C and 57.4°C with a concentration of 1 µl of lambda DNA and 5 µl of each primer. In the lambda experiment, the RZ gene was amplified and this served as a positive control within the overall experiment. The RZ gene was amplified using PCR and then read using gel electrophoresis, where bands were shown at 396 base pairs (Toba, 2011). The lambda test, as a positive control, ensured that PCR and gel electrophoresis were able to produce results. There was a secondary positive control in the E.coli experiment. The concentrations for this experiment were the same as the lambda experiment, with different primers and DNA. The annealing temperatures for E.coli was run on a gradient from 44.0°C to 46.6°C. Nuclease free water served as a negative control, where the PCR cocktail was held constant and nuclease free water was substituted for the lambda. There were no bands expected, in order to ensure that no contamination occurred during PCR and gel electrophoresis.

The human DNA had to purified in order to be used in the PCR cocktail. For DNA purified with QIAGEN’s Blood and Cell Culture DNA Mini Kit from Qiagen Inc., the yielded genomic DNA had to be run through a spectroscope (Alameda, CA). The yield of genomic DNA depends on the number of cells and the capacity of the genomic tip used. The concentration of DNA can be determined using the absorbance at 260 nm, which should be between 0.1 and 1.0 to be accurate. The purity can also be determined by calculating the ratio of absorbance at 260 nm and 280 nm. A pure DNA solution should have a ratio between 1.7 and 1.9. DNA absorbs light at 260 nm and contaminants absorb at 280 nm. The more contaminants there are, such as proteins, the lower the ratio. However, DNA isn’t the only thing that absorbs DNA at 260 nm. If there is too much absorbance at 260 nm the ratio will be too high to be pure DNA (Mitra. 2004). The sample of human DNA that was purified with QIAGEN’s Blood and Cell Culture DNA Mini Kit from Qiagen Inc. (Alameda, CA) was calculated to be 1.9865. The ratio was too high and most likely had contaminants in addition to DNA. This was most likely why this sample of DNA did not yield any bands.

PCR was performed on human DNA to amplify the FOXP2 gene. By performing multiple experiments, the optimal annealing temperature was found to be 55℃. The human results at 56 base pairs confirmed the hypothesis that the correct annealing temperatures and primers would yield the correct band length. The Tropical Clawed Frog DNA went through similar rigorous testing, however, there were no bands produced for the multiple different annealing temperature trials. The Tropical Clawed Frog DNA yielded insufficient results and did not support our hypothesis. The frog trials generated a primer dimer as well as a band appearing in the well, which denotes a region that was too large when amplified. This is most likely due to primers that did not anneal to the desired part of the frog genome.

Figures

Figure 1. Amplification of human genome using PCR and gel electrophoresis. (A) A target region of human genome was amplified using FOXP2F and FOXP2R primers to make a product 56 base pairs long. A master mix for PCR was made using 152 µl nuclease free water, 28 µl of 10x PCR buffer, 2 µl of Taq Polymerase, 4 µl of dNTPs, and 4 µl of each primer. This master mix was pipetted into three, 49 µl reaction samples, and 1 µl of DNA was added to each sample. The samples were ran for 30 seconds at 94°C for the denaturing phase, 55°C for 1 minute for the annealing phase, and for 15 seconds at 65°C for the elongation phase for a total of 35 cycles. The initial step ran for 9 minutes at 95 °C and the final step at 60°C for 30 minutes. A 2% agarose gel was made with 49 ml of 1X TBE (Tris/Borate/EDTA) buffer, 1.0 g of 100% agarose powder, and 5 µl of 10000x SYBR Safe. This gel ran for 15 minutes at 110 V. A 1kb plus DNA ladder with a concentration of 1 µg/µl was used in well one, with an 8 µl: 1 µl: 1 µl nucleus free water to DNA to loading dye ratio. Well two was loaded with PCR product annealed at 55°C, well three at 55°C, well four at 55°C, well five at 55°C, well six was loaded with a positive control ran at 50°C, and well seven was loaded with a 1kb plus DNA ladder with a concentration of 1 µg/µl, with an 8 µl: 1 µl: 1 µl nucleus free water to DNA to loading dye ratio. The gel was placed under an ultraviolet light to observe the bands created in the PCR product. (B) Migration distance vs molecular size of 1 kb Plus DNA ladder was plotted and analyzed using a semi-log plot. The x-values are the distance in centimeters that the bands in the ladder traveled away from the wells and the y-values are the base pair length of the corresponding bands. The trend line was created to form an equation for analysis of the base pair length of the amplified human genome. The distance traveled by the PCR product from the well (3.95 cm) was inputted for the x-value of the equation. The y-value derived from this calculation is the base pair length of the product (56 bp) for the lane that produced a slight band (lane 3 and 4) around 3.95 cm in distance. Indicating that most likely the primers did not anneal to the genome, but instead themselves. The R2 value is the fit of the trend line, where a value of 1 would represent a perfect fit.