1. Using a Single Nucleotide Polymorphism (SNP) to Predict Bitter Tasting Ability Carolina Kit

2. Timeline Day 1: Lecture Day 2: Isolate DNA, then amplify using PCR methods Day 3: Pour gels (lab service) Day 4: Digest cells (lab service—1 from each group—start at lunch), run and photograph gel

3. Prep for Lab Week before Label tubes Pre-set thermo-cycler 3 students at 8 stations will give their DNA By Day 1 10 mL of.9% NaCl solution (.9g NaCl/100ml water) in cups (24) 100 uL 10% chelex into 1.5mL tube (24) 22.5uL of PTC primer/loading dye (24) 20uL pBR322/BstNI marker (8) paper cups TBE 20x dilute to 1x to use (150mL TBE with 2850mL dwater) Ice buckets with ice Day 2 Pour 2% gels, add ethidium bromide (200ng/mL final or 1uL of 10mg/mL stock in gel prepared from 50mL), 10 well comb, TBE buffer (10 grams agarose add up to 500mL TBE buffer) Prepare UV trans. and camera Day 3 Set-up incubator at 37 degrees

4. Background Information http://bioinformatics.dnalc.org/ptc/animatio n/ptc.htmlhttp://bioinformatics.dnalc.org/ptc/animatio n/ptc.html Important Concepts -SNPs -DNA Isolation -PCR -Gel Electrophoresis

5. SNPs Single Nucleotide Polymorphisms http://www.youtube.com/watch?v=tJjXpiWKMyA For a variation to be considered a SNP, it must occur in at least 1% of the population. SNPs, which make up about 90% of all human genetic variation, occur every 100 to 300 bases along the 3-billion-base human genome.

6. Relationship between Phenotype and Genotype Relationship: The genotype ("internally coded, inheritable information") codes for the phenotype (outward, physical manifestation) 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. Lab Day 1 Part I: isolate DNA Part II:PCR Cheek cells No Mineral oil for PCR Store your PCR samples in the freezer after

8. Isolation From cheek cells Chelex will be the reagent used Boiling step: lyses the cell membrane, releasing DNA and other cell contents

9. PCR- Polymerase Chain Reaction Sections of DNA can be recognized and reproduced Based on cell respiration (meiosis) Three steps: 1) Denature (seperation of two strands of DNA) at 90 degrees 2) Anneal (binding of DNA primer to the seperated strands) at 72 degrees 3) Elongate (elongation of the strands using the DNA primer with heat stable DNA polymerase) at 50 degrees PCR tutorials: http://learn.genetics.utah.edu/content/labs/pcr/ http://www.dnalc.org/resources/animations/pcr.html

10. 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

11. Gel Electrophoresis A process that uses electricity to separate charged molecules, such as DNA fragments, RNA, and proteins, on a gel slab Larger sections of DNA is the slower it moves through gel DNA is negative so it runs to the positive The buffer in the gel is conductive and allows the electricity to flow through the gel and stimulate the DNA

12. 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 Resultshttp://bioinformatics.dnalc.org/ptc/animation/ptc.htmlhttp://bioinformatics.dnalc.org/ptc/animation/ptc.html

14. Protocol http://bioinformatics.dnalc.org/ptc/animatio n/ptc.htmlhttp://bioinformatics.dnalc.org/ptc/animatio n/ptc.html

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

16. Sources http://www.ornl.gov/sci/techresources/Hum an_Genome/faq/snps.shtmlhttp://www.ornl.gov/sci/techresources/Hum an_Genome/faq/snps.shtml http://www.brooklyn.cuny.edu/bc/ahp/BioIn fo/GP/Relationship.htmlhttp://www.brooklyn.cuny.edu/bc/ahp/BioIn fo/GP/Relationship.html