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

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

2. 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)

3. 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

4. Background Information • http://bioinformatics.dnalc.org/ptc/animation/ptc.html read introduction

5. Single nucleotide polymorphism DNA Science textbook: page 296-297 • 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

6. 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.

7. 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

8. 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

9. Protocol • http://bioinformatics.dnalc.org/ptc/animation/ptc.html review flow chart

10. Lab Day 1Part 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

11. Lab Day 2Part 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

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

13. results • http://bioinformatics.dnalc.org/ptc/animation/ptc.html review results section

14. Bioinformatics • http://bioinformatics.dnalc.org/ptc/animation/ptc.html Use website directions as it is most updated • Complete the worksheet for homework