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

• ThursdayLecture, volunteer aliquot
• Mondayprocedures quiz, Bioinformatics
• HW Bioinformatics (use website, not packet)
• Tuesdayisolate DNA cells, amplify DNA (PCR)
• WednesdayVolunteer pour gels
• Thursdaydigest samples, run gel, photograph gel
• TuesdayLab 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 Discussionanswer 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 traysuse 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 IIPCR

• 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