Using a Single Nucleotide Polymorphism (SNP) to Predict

Bitter Tasting AbilityCarolina Kit

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

Prep for LabWeek before• Label tubes • Pre-set thermo-cycler3 students at 8 stations will give their DNABy 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 iceDay 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 cameraDay 3• Set-up incubator at 37 degrees

Background Information

• http://bioinformatics.dnalc.org/ptc/animation/ptc.html

• Important Concepts

-SNPs

-DNA Isolation

-PCR

-Gel Electrophoresis

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.

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

Lab Day 1Part I: isolate DNA

Part II:PCR• Cheek cells

• No Mineral oil for PCR

• Store your PCR samples in the freezer after

Isolation• From cheek cells

• Chelex will be the reagent used

• Boiling step: lyses the cell membrane , releasing DNA and other cell contents

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

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

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

Gel Loading1. Marker2. Partner set 1-U3. Partner set 1-D4. Partner set 2-U5. Partner set 2-D6. EmptyMake sure to recordwhat is in each lane inyour lab notebook

• Resultshttp://bioinformatics.dnalc.org/ptc/animation/ptc.html

Protocol

• http://bioinformatics.dnalc.org/ptc/animation/ptc.html

Bioinformatics

• http://bioinformatics.dnalc.org/ptc/animation/ptc.html

Use website directions as it is most updated

• Complete the worksheet for homework

Sources

• http://www.ornl.gov/sci/techresources/Human_Genome/faq/snps.shtml

• http://www.brooklyn.cuny.edu/bc/ahp/BioInfo/GP/Relationship.html