Using a Single-Nucleotide Polymorphism to Predict Bitter- Tasting Ability Carolina Kit

Timeline Thursday—Lecture, volunteer aliquot Monday—procedures quiz, Bioinformatics HW: Bioinformatics (use website, not packet) Tuesday—isolate DNA cells, amplify DNA (PCR) Wednesday—Volunteer pour gels Thursday—digest samples, run gel, photograph gel Tuesday—Lab write-up due (after break)

Write-up Annotate handout Data draw a gel and mark each banding site, staple picture to lab that you turn in to me Results and Discussion—answer all parts Bioinformatics worksheet

Background Information n/ptc.htmlhttp://bioinformatics.dnalc.org/ptc/animatio n/ptc.html read introduction

Single nucleotide polymorphism DNA Science textbook: page Point mutation Most mutations are rare in a population, so to be helpful, SNPs must have a population frequency of 1% A region of linkage is called haploblock because it is inherited without recombination like haploid in mDNA A set of SNPs, markers, within the haploblock are inherited as a haplotype. Different populations inherit different SNPs with the haploblock This info. is great for linkage studies The hope is to make a map, find disease genes in populations of unrelated people

Genotype and Phenotypes The TAS2R38 polymorphism was specifically selected to demonstrate the relationship between genotype and PTC- tasting phenotype, because it has no known relationship to disease states or sex determination. TAS2R38 alleles are inherited in a Mendelian fashion and can give indications about family relationships.

Prep. For lab--SNP Week before Label tubes Pre-set thermo-cycler By Tuesday 10 mL of.9% NaCl solution (.9g NaCl/100ml water) in 15 mL plastic tube (15) 100 uL 10% chelex into 1.5mL tube (15) 22.5uL of PTC primer/loading dye (30) 10uL of Restriction enzyme HaeIII (15) 20uL pBR322/BstNI marker (8) paper cups TBE 20x dilute to 1x to use (150mL TBE with 2850mL dwater) Tuesday Ice buckets with ice Wednesday Pour 2% gels, add ethidium bromide (200ng/mL final or 1uL of 10mg/mL stock in gel prepared from 50mL), 6 well comb, TBE buffer (10 grams agarose add up to 500mL TBE buffer) Prepare UV trans. and camera By Thursday Set-up water bath 37 degrees

Preparing gels ___ grams agarose Add up to ___mL buffer Melt in microwave, let cool Set up trays—use 6 well comb Add 1uL ethidium bromide/50uL of solution Pour about 30-50mL into each tray

Protocol n/ptc.htmlhttp://bioinformatics.dnalc.org/ptc/animatio n/ptc.html review flow chart

Lab Day 1 Part I: isolate DNA Part II:PCR We are doing cheek cells Work with a partner in your group (15 sets in the class) we will use the heat block at set 9 No Mineral oil for PCR I will store your PCR samples in the freezer after PCR

Lab Day 2 Part III: Digest Part IV: electrophoresis Make sure to label with a “D” and “U” At step 5, use the water bath instead of thermo-cycler Skip step 9, we already added ethidium bromide Test your bitter taste

Gel loading 1.Marker 2.Partner set 1-U 3.Partner set 1-D 4.Partner set 2-U 5.Partner set 2-D 6.Empty Make sure to record what is in each lane in your lab notebook

results n/ptc.htmlhttp://bioinformatics.dnalc.org/ptc/animatio n/ptc.html review results section

Bioinformatics n/ptc.htmlhttp://bioinformatics.dnalc.org/ptc/animatio n/ptc.html Use website directions as it is most updated Complete the worksheet for homework