Using a Single-Nucleotide Polymorphism to Predict Bitter-

Tasting AbilityCarolina Kit

Timeline

Thursday—Lecture, volunteer aliquotMonday—procedures quiz, BioinformaticsHW: Bioinformatics (use website, not packet)Tuesday—isolate DNA cells, amplify DNA

(PCR) Wednesday—Volunteer pour gelsThursday—digest samples, run gel,

photograph gelTuesday—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

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

read introduction

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

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--SNPWeek before• Label tubes • Pre-set thermo-cyclerBy 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 iceWednesday• 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 cameraBy 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

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

review flow chart

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

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 loading

1. 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

results

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

review results section

Bioinformatics

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

Use website directions as it is most updated

• Complete the worksheet for homework