QuBi/bio203

BIOL 203 Lab 4. Bioinformatics Exercises

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Research in modern molecular genetics increasingly rely on genomic information and computation. The following exercises will expose you to the field of bioinformatics, including the use of online databases and statistical analysis of genetic data.

Introduction

DNA and its organization into genes makes up an organism's genotype. The expression and presentation of those genes in the organism's development, physiology, and physical appearance (physical traits) make up the phenotype of the organism. Phenotypic variations among individuals of a species (e.g., humans) are caused by genotype variations, environmental factors, and interactions between genetic and environmental factors. In other words, phenotypic variations among individuals often have complex, unclear mechanisms and are not necessarily due entirely to genetic differences.

In this lab section, we will explore the concepts of phenotype and genotype by looking at the variations in the TAS2R38 gene, which is responsible for part of the sensation of taste. The taste receptor protein TAS2R38 (taste receptor 2, member 38) has been associated with the ability to taste the bitter compound phenylthiocarbamide (PTC) (Kim et al. 2003). Although most people can taste PTC ("tasters"), a centain percentage of people cannot ("nontasters"). In this experiment, you will test your Taster phenotype as well as determine your Taster genotype and correlate the phenotype with the genotype data. Your results and those of your classmates will be combined to statistically validate if there is such a phenotype-genotype association.

Learning goals and outcomes

• Understand phenotype, genotype, and their association
• Be able to use the NCBI online databases
• Be able to predict genotype frequencies using Hardy-Weinberg equilibrium
• Be able to use the contingency test of genotype-phenotype associations

Part 1. Search for TAS2R38 gene information using NCBI online databases

1. Point your browser to the NCBI Human Genome Resource page
2. Type in the "Find A Gene" search box "TAS2R38" and select "Homo sapiens" from the pull-down menu. Click "Go"
3. Select the first link, which leads to an NCBI Gene Card page. Use the Gene Card to identify the following information on TAS2R38 gene:
   1. NCBI GeneID
   2. Chromosome location
   3. Click on "GenBank" and identify its gene structure, including the length of primary transcript, coding sequences, 5'-UTR and 3'-UTR. Does it have any introns?
   4. Zoom out the Sequence View to find its neighboring genes. Zoom out to read DNA sequences.
4. Click the link to OMIM (under Phenotype) and find phenotypes associated with TAS2R38 gene
   1. What does OMIM stand for?
   2. What are the expected "taster" and "nontaster" frequencies within human populations?
   3. If the ability to taste bitterness is evolutionary advantageous, how are alleles contributing to "nontaster" maintained in population?
   4. Is the correlation between TAS2R38 gene variations and the PTC phenotype variations 100%? If not, what could be the other causes?

Part 2. Predict results of restriction analysis

On a printout of the DNA sequence of TAS2R38 gene (from the GenBank link, see above),

1. Identify 5'-UTR, 3'-UTR, start codon, and stop codon.
2. Identify the regions your PCR primers should bind
3. Identify the base location that contains 785 C/T SNP
4. Copy and paste the expected 302-bp section and locate the Fnu4H1 site using the NEBcutter website
5. What are the expected lengths for the C/C, C/T, and T/T genotypes?

Part 3. Test Hardy-Weinberg Equilibrium

The Hardy-Weinberg Principle predicts the genotype frequencies at a genetic locus in a random-mating population. If two alleles (e.g., C, T) are segregating in a diploid population with respective frequencies of p and q, if the mating is random, and if there is no fitness difference between the two alleles, then it is expected that the genotypes have the following frequencies: C/T (2pq), T/T (q^2), and C/C (p^2).

1. After your PCR/sequencing, collect genotype frequencies in your class as a group
2. Calculate SNP frequencies
3. Predict expected genotype frequencies using H-W Principle
4. Test the goodness-of-fit of observed frequencies with H-W expected frequencies by using Fisher's Exact test
5. Exit Questions
   1. What is your p value? Is it statistically significant?
   2. What are the possible causes of deviation from HWE? Biological? Statistical?

Part 4. Test phenotype-genotype assoication

1. After your PCR/sequencing, collect genotype frequencies in your class as a group
2. Collect both the genotype and phenotype information using the following table
3. Test the goodness-of-fit of observed frequencies with expectations from no-association (Null Hypothesis) by using Fisher's Exact test
4. Exit Questions
   1. What is your p value? Is it statistically significant?
   2. ## What are the possible causes of deviation from HWE? Biological? Statistical?