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Single Nucleotide Polymorphisms
Mrs. StewartMedical Interventions
Central Magnet School
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Bell Work
• Why is Taq polymerase used in PCR instead of human polymerase?
• Answer on your own paper
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Objective
• Use laboratory techniques such as DNA extraction, PCR, and restriction analysis to identify single base pair differences in DNA
• Explain how single base pair changes called single nucleotide polymorphisms (SNPs) can be identified through genetic testing and often correlate to specific diseases or traits.
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Single Nucleotide Polymorphisms
• The parts of the human genome that vary by just a single nucleotide
• Abbreviated as SNPs • Pronounced as “snips”
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Effect of SNPs
• Non-coding DNA regions = no effect• Genes that code for proteins = potentially
changing the protein produced
–Different phenotypes–Disease/disorder
EFFECT
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Remember:
• a change in DNA can lead to a change in a protein
• If the protein plays a role in keeping you healthy, serious consequences may occur.
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Using a Single Nucleotide Polymorphism to Predict Bitter
Tasting Ability
Lab Overview
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Gene of Interest
• TAS2R38• On chromosome #7 • Ability to taste “bitter”
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What is your phenotype?
Weak Taster
Non-Taster
Strong Taster
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Step 1: Isolate a sample of your DNA from your cheek cells
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Step 2: Amplifying the Gene of Interest
• Using your DNA sample, you will amplify a 220 base pair region of the PTC gene using PCR.– Specific primers attach to either side of the target sequence
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SNPs for TAS2R38 gene
• In this lab, you will investigate one of the base pair changes or single nucleotide polymorphisms (SNPs) that affects a person’s ability to taste the chemical PTC.
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Genetics Review – Question 1
• The inability to taste PTC is a recessive trait.
• If a capital “T” is is used to designate the dominant allele and a lowercase “t” is used to designate the recessive allele, what is the genotype of a “Nontaster”?
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Answer
• A “Nontaster” carries two recessive alleles and thus has the genotype “tt”.
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Genetics Review – Question 2
• What are the possible genotypes for a “Taster”?
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Answer
• A “Taster” may be homozygous dominant with a genotype of “TT” or heterozygous with a genotype of “Tt”.
• In this lab, you will use the tools of molecular biology to determine your genotype for PTC tasting.
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Step 3: Restriction Analysis
• Restriction enzymes, molecular scissors, recognize specific DNA sequences and cut the nucleotide strands.
• In this part of the experiment, you will use a specific restriction enzyme, HaeIII, to identify a SNP or base pair difference in the amplified segment of the PTC tasting gene.
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HaeIII and TAS2R38 gene
• HaeIII restriction enzyme – 5’ GGCC 5’ ---GG CC--- 3’– 3’ CCGG 3’ ---CC GG--- 5’
• TAS2R38 gene variations
NONTASTER (tt) TASTER (TT)
GGCGGGCACT GGCGGCCACT
CCGCCCGTGA CCGCCGGTGA
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Step 4: Gel Electrophoresis
• Gel Electrophoresis separates DNA fragments based on their molecular weight.
• Once you have digested your DNA sample with the restriction enzymes, run your product on a gel to analyze your results.
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What will gel results show?
Non-taster Taster