Download 1.PtI.SNPs and TAS2R38 Bitter Taste Receptor Gene.v3

Using Single Nucleotide
Polymorphism (SNP)
to Predict
Bitter Tasting Ability
Part I: Isolation of DNA and Amplification of
Target Sequence, a Short Region of the Bitter
Taste Receptor TAS2R38 gene
The PCR Song
There was a time when to amplify DNA,
You had to grow tons and tons of tiny cells.
Then along came a guy named Dr. Kary Mullis,
Said you can amplify in vitro just as well.
Just mix your template with a buffer and some primers,
Nucleotides and polymerases, too.
Denaturing, annealing, and extending.
Well it’s amazing what heating and cooling and heating will do.
PCR, when you need to detect mutations.
PCR, when you need to recombine.
PCR, when you need to find out who the daddy is.
PCR, when you need to solve a crime. (repeat chorus)
Scientists for better PCR
http://www.youtube.com/watch?v=x5yPkxCLads
What Are Single Nucleotide
Polymorphisms (SNPs)?
•! Polymorphism - refers to the presence of more than one
allele of a gene in a population
–! The frequency of this allele is greater than 1% of the
population
–! It is stable.
–! The above distinguish it from a mutation.
•! A SNP is a specific type of allele
–! caused by a small genetic change within a DNA
sequence
–! result of replacement of one single nucleotide
with any one of the other three nucleotides = SNP
Of a person’s DNA sequences, 99.9% will be identical to that of another person.
Of the 0.1% difference, over 80% will be single nucleotide polymorphisms (SNPs).
Why are SNPs Important?
•! Most SNPs occur outside protein-coding regions and thus
are phenotypically silent—the equivalent of mile markers
on the side of the highway
•! Others affect protein sequences.
•! Both types of SNPs can serve as landmarks in the search
for genes associated with disease, drug response and
complex phenotypes
•! E.g., Asthma drug, Albuterol
http://www.genome.gov/Pages/Education/DNADay/TeachingTools/MakingSNPsMakeSense.html
Testing for SNPs in the
TAS2R38 Gene
•! Amplify a short region of Chromosome 7
•! Determine if you are a taster or non-taster for
a bitter-tasting substances
•! homozygous or heterozygous (taster)
or
•! homozygous (non-taster)
•! phenylthiocarbamide (PTC),
bitter tasting chemical
TAS2R38 Gene
Digestion of TAS2R38 With
Restriction Enzyme, Hae III
Agarose Gel Electrophoresis
Today:
Amplify 221bp sequence of TAS2R38 Gene
Harvesting Cheek Cells
•! Source of DNA - cheek cells
•! Use saline mouth wash
•! Swish vigorously between cheeks and gums
for at least 30 seconds
•! Return mouth wash to original tube
Harvesting Cheek Cells
•!Transfer 1ml (1000µl) of mouth wash to microfuge tube
* Invert mouthwash tube to resuspend cells before
making transfer
•!Centrifuge to pellet cells
•!Check the size of your pellet
•! Show your pellet to your TA or instructor
•! You need at least a match-head size pellet to get good
results
Show your TA
your pellet!
Harvesting Your DNA
•! Pour supernatant into cup
•! Don’t lose your pellet!
•! Resuspend pellet until no clumps of cells
remain
•! Transfer 30µl of resuspended cells to Chelex
tube, and close and lock the lid of the tube
•! Boil contents for 10 minutes, then cool on ice
Harvesting Your DNA
•!
•!
•!
•!
Vortex briefly, then centrifuge for 2 minutes
This step pellets the Chelex and cellular debris.
DNA is in supernatant
Transfer 30µl of supernatant to clean microfuge tube
labeled DNA - avoid pipetting any Chelex beads as
these will interfere with the PCR reaction
Amplifying TAS2R38 Region of Your DNA
1.! Get PCR tube with Ready-to-go bead from your TA
2.! Label it with your CODE on both sides of the tube
Ready-to-go bead contains everything needed for amplification of DNA
(except Primer and DNA):
•!
•!
•!
•!
Taq polymerase
dNTPs
Mg2+
Buffer
2. Have your TA/instructor add PTC Primer Mix
- 22.5µl of PTC Primer Mix will be added to your tube
- let the bead dissolve
3. Add 2.5µl of your DNA. Spin contents briefly. Place your tube in PCR
rack in ice bucket near thermal cycler.
You are ready for PCR!
Amplifying the Targeted Sequence
of the TAS2R38 Gene
http://www.dnalc.org/resources/animations/index.html!
Amplifying the Targeted
Sequence of the TAS2R38 Gene
•!Thermal cycler is used for Polymerase Chain
Reaction - PCR
•!Programmed to change temperatures
•!Amplifies small selected region of DNA
!http://www.sumanasinc.com/webcontent/animations/content/pcr.html
Polymerase Chain Reaction
"Beginning with a single molecule of the genetic material
DNA, the PCR can generate 100 billion similar molecules
in an afternoon. The reaction is easy to execute. It
requires no more than a test tube, a few simple reagents
and a source of heat. The DNA sample that one wishes to
copy can be pure, or it can be a minute part of an
extremely complex mixture of biological materials. The
DNA may come from a hospital tissue specimen, from a
single human hair, from a drop of dried blood at the
scene of a crime, from the tissues of a mummified brain
or from a 40,000-year-old wooly mammoth frozen in a
glacier."
Kary Mullis
Amplifying the Targeted Sequence
of the TAS2R38 Gene
http://www.pbs.org/wgbh/nova/sciencenow/0302/01.html (12minutes)
NextWeek:
Second part of Using SNPs
•! Pre-lab (Pt. II)
•! Quiz on C-fern life cycle