Using Single Nucleotide Polymorphism (SNP) to Predict...

Using Single Nucleotide

Polymorphism (SNP)

to Predict

Bitter Tasting Ability

Part II: !

Digestion and Analysis of an Amplified Region

of the Bitter Taste Receptor TAS2R38 Gene

In The Last Lab:

•! You sampled some of your

own DNA and amplified a

short region of chromosome 7.

•! This region codes for part of

TAS2R38 gene, bitter taste

receptor gene.

In Today’s Lab:

•! You will digest some of your amplified PCR

product with the restriction enzyme, Hae III

•! And use agarose gel electrophoresis to

determine your genotype

•! You will also taste a sample of PTC paper and

determine your phenotype

•! You will compare the results of your genotype

with those of your phenotype – record on

board

•! Finally, you will use class data to determine if

the class is in Hardy-Weinberg equilibrium

Digestion of TAS2R38 With

Restriction Enzyme, Hae III

Agarose Gel Electrophoresis

Digesting PCR Sample with HAE III

Label your original PCR tube with a “U” for undigested; leave this sample on ice while the other sample is incubated with HaeIII. !

Get your PCR tube from your instructor or TA (need your code)

•! Transfer 16 µl (0.016 ml) of your PCR product (amplified DNA) to a

flat top PCR tube

•! Label this tube “D” for “digested” and add your code.

•! This DNA sample will be digested with the restriction enzyme, HaeIII

•! Ask your instructor or TA to add 1µl (0.001 ml) of the restriction

enzyme, HaeIII, to the “D” tube

•! Place this tube in the thermal cycler set for 37oC

and incubate for at least 30 minutes.

http://highered.mcgraw-hill.com/olc/dl/120078/bio37.swf

http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#

(choose restriction endonucleases)

Agarose Gel Electrophoresis of

Amplified PCR Products

•!You will share an agarose gel (2%) with 2 other people

•!Load both the undigested and digested samples in separate wells of the gel

•!Undigested - 10 µl

•!Digested - 16 µl

•! Also load the Marker, a 100bp ladder, in both end lanes of the gel

•! http://www.sumanasinc.com/webcontent/animations/content/gelelectrophoresis.html

•! http://www.wellesley.edu/Biology/Concepts/Html/gel.html

Agarose Gel

Run the gel for 30-40 minutes at 130 Volts; remove gel and stain it with ethidium bromide

Staining DNA - Ethidium Bromide

•!Bring your gel to your TA who

will transfer it to the plastic

container and add ethidium

bromide stain to the DNA bands

•!Ethidium bromide intercalates

between the base pairs of the

DNA ladder

•!Stain the gel ~20 minutes

•!Destain ~ 10-15 minutes

•!View gel using UV light to make

the bands fluoresce

•!Analyze photo; attach it to your

worksheet

Determining Your Genotype

http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#

Choose RFLP-restriction fragment length polymorphism

Determining your PTC Phenotype

•! Taste the Control paper first (no PTC)

•! Taste the PTC paper

•! Place strip in your mouth for a few seconds

•! Remove it and wait; it takes a few seconds

to taste PTC

•! Record both your genotype and phenotype

on board

•! You will need these results to complete the

post-lab assignment

Class Results Genotype Phenotype

strong taster

Phenotype

weak taster Phenotype

non-taster

TT

Tt

tt

* Record the class results on your worksheet.

Worksheet

•! Do sample calculations

•! Calculate expected allele and genotype

frequencies using the sample data

given:

•! T/T: 15

•! T/t: 50

•! t/t: 35

•! Do Chi square calculation to determine

if the sample population is in Hardy-

Weinberg equilibrium http://www.pbs.org/wgbh/nova/sciencenow/0302/01.html (12minutes)

Personal DNA Testing

Next Lab

DUE: C-fern Lab Report

Pre-lab on RPI-LMS:

Genetics & Medicine Simulation Lab:

Sickle Cell Alleles

Quiz: Using SNPs to Predict Bitter Tasting Ability

•! How well does TAS2R38 genotype predict PTC-tasting

phenotype?

•! What does this tell you about classical dominant/recessive

inheritance?

The presence of a T allele generally predicts tasting, although

heterozygotes are more likely to be weak tasters. Even in a relatively simple genetic system such as PTC tasting, one allele rarely has complete

dominance over another. This experiment examined only one of several mutations in the TAS2R38 gene that influence bitter tasting ability.

Variability in taste perception is likely affected by processing in the brain,

which involves numerous other genes. (E.G., There are about 25-30 genes that code for bitter taste receptors. Also the genes for three of the cranial

nerves involved in taste perception.) Thus, perception of bitter tasting substances like PTC is complex and does not exactly follow the simple

Mendelian inheritance pattern.

The forward primer incorporates part of the HaeIII recognition site, GGCC. How is this different from the sequence of the human TAS2R38 gene?

Mismatch between

forward primer & alleles

Formation of HaeIII Recongition Site

in Amplified PCR Product

http://www.pbs.org/wgbh/nova/body/tongue-taste.html

Making sense of SNPs and

the Albuterol Story

http://www.genome.gov/Pages/Education/DNADay/

TeachingTools/MakingSNPsMakeSense.html