SNP Analysis DNA Sequencing Joshua Bain Dylan Scordellis

SNP Analysis & DNA Sequencing Joshua Bain & Dylan Scordellis Biology 250, Longwood University Background • The experiment was conducted to analyze SNPs and sequence DNA based off specific genetic traits. • To determine whether a trait was present was to determine if the given trait was heterozygous or homozygous. This was done by looking at the genotype through the SNP sequence provided by Eurofins Genomics (1). • Traits can be either dominant or recessive which helped form the hypothesis for the traits knowing that, for example, blue eyes is recessive so the SNP should show a homozygous blue eyes for someone with a phenotype of blue eyes (2). STEP Figure 1: Steps for Thermocycling. The figure shown represent the steps for thermocycling of the PCR samples. TEMP Initial Denaturation 30 Cycles Final Extension Hold TIME 98°C 5 minute 98°C 55°C 72°C 4°C 5 seconds 20 seconds 1 minute *SNPB uses annealing temperature of 62 o. C, extension for 30 seconds Specific Aim Research Question: The experiment was ran to determine the Hypothesis: If the given trait is present within our physical features, then it sequence of specific traits for the tested individuals. will be a homozygous trait within the SNP sequence. Methods PCR Protocol Swab cheek samples Added primers, water and master mix Gel Electrophoresis Centrifuged samples Gel chamber was prepared Samples & buffer added Purification Analyzing Sequencing data was received from Eurofins Genomics The chromatogram was examined The sequence of the SNPs were analyzed Set to 300 volts for fifteen minutes The products were sent off to Eurofins Genomics for DNA sequencing. Centrifuge samples two times Elution buffer was added Results Individual 1: The sequences for SNPBH as well as the SNP (indicated by the red “A”). Both of the SNPs were identical with the following sequence; TAAATGT. This shows that the trait for blue/brown eyes is homozygous. This is not surprise because individual one had blue eyes present. One thing to notice is if it were heterozygous, then the trait has a possibility of being absent, in this case, the highlighted letter “A” would have been replaced with another nucleotide such as a “G”. The other sequence showed the exact same sequence again. The sequences both were AGTGTTT, but if the waves are checked, there are multiple colors mixed within the nucleotide “G”. This means that this trait is not homozygous, but it is in fact heterozygous for SNP 2. SNPBH (Ind 2) (Blue/Brown Eyes) SNPBH (Ind 1) (Blue/Brown Eyes) 7. 6 ng/µl 21. 2 ng/µl SNPB X 22. 1 ng/µl SNP 2 302 ng/µl X Figure 2: Nanodrop Concentrations: The chart above represents the nanodrop concentrations of each genetic trait per individual. 200 SNP 2 (Ind 1) (Curly Hair) Figure 3: Chromatograms for SNPs. The chromatograms above show the sequences for the SNPs involved in the experiment Conclusions • This study proved that the given traits were expected due to them being present within our physical appearance. The sequences proved that the traits were homozygous and if they were heterozygous, the given nucleotide on the SNP was replaced (1). • The SNPBH sample 2 a was heterozygous even though the subject has a phenotype of blue eyes could be caused by a mutation of pigmentation rather than a mutation of the DNA that would normally be causing blue eyes (3). • The limitations of this study was that we had minimal samples and trials as well as no replications to improve validity. Another experiment that could have been done would be to do the exact same experiment, but with another samples source besides cheek samples and new test subjects (4). 2 b SNPBH 300 2 a Individual 2 1517 1200 1000 900 800 700 600 500/517 400 1 b Individual 1 SNPB (Ind 2) (Bitter Taste) 1 a The blue/brown eyes sequence contained a G (TTAAGTGTC) in the amplicon instead of an A, this indicates the trait is heterozygous. To further back this up the trace, shown in figure 1, at base pair 120 where the amplicon was found has multiple peaks. The multiple peaks, with an otherwise clean sample, indicates that the genotype is heterozygous. The bitter taste trace showed that there were no second peaks throughout the entire trace, shown in figure 2, however, the sequence showed there to be a change in the base pairs signifying a heterozygous trait. The sequence for the bitter taste was GCAGGCACT with the red showing the amplicon. Ladder Individual 2: Figure 4: Gel Samples for the SNP samples & buffer. The figure above represents the gel for the SNP samples. 1 a and 2 a were SNPBH, 1 b was SNP 2, and 2 b was SNPB. References 1. Elston RC. 1984. The Genetic Analysis Of Quantitative Trait Differences Between Two Homozygous Lines. Genetics. 2. Bradley, B. J. , Pedersen, A. and Mundy, N. I. 2009. Brief communication: Blue eyes in lemurs and humans: Same phenotype, different genetic mechanism. Am. J. Phys. Anthropol. , 139: 269 -273. 3. Ian J. Jackson, Homologous Pigmentation Mutations in Human, Mouse and Other Model Organisms. 1997. Human Molecular Genetics, Volume 6, Issue 10, Pages 1613– 1624. 4. Petty RE. 2018. The importance of exact conceptual replications. Behav Brain Sci. 41.