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2.1.3: Test Your Own Genes

Isolate your DNA
1. Amplify Your DNA by PCR
  1. Digest PCR Products with Restriction Enzyme HaeIII
    1. Analyze PCR Products by Gel Electrophoresis
      1. Determine Your PTC Genotype and Phenotype
        Observe the bands on gel & compare digested DNA to uncut control and determine own geneotype
        Obtain PTC paper and test ability to taste the chemical; correlate PTC genotype w/ phenotype
        Gel Electrophoresis results: one band indicates recessive alleles b/c no cut made by HaeIII (my results) meaning unable to taste PTC
        Homozygous Dominant: 2 bands resulting from 1 cut by HaeII on both alleles Heterozygous: 3 bands resulting from one cut on GGCC allele and no cut on GGGC allele
        Goal: compare and correlate our genotype with our phenotype
      2. Place solidified gel in electrophoresis chamber and add TBE buffer
        Load the following into the gel: Lane 1 - DNA Marker - 20ul Lane 2 - Undigested DNA - 10ul Lane 3 - Digested DNA - 15ul
        Run gel at 130V for 30 min.
        TBE Buffer: conducts electricity to allow successful travel of bands
      3. Goal: observe the separation of the bands in order to determine genotype
    2. Label 2 clean 1.5 ml tubes: "undigested" and "digested"
      1. Transfer 15 ul of PCR product into "digested" tube and remainder of the product to "undigested" tube
        Pipet 1 ul of enzyme HaeIII directly into PCR products in "digested" tube
        Transfer digested sample to a .2ml tube
        Incubate tube in thermal cycler for 1 hr
        HaeIII: restriction enzyme that recognizes the GGCC sequence of PTC and cuts between bp to create a blunt end
    3. Goal: to cut the DNA gene at the GGCC seequence and understand difference b/w tasters & non-tasters
  2. Obtain a PCR tube containing a PCR bead
    1. Add 23 ul of PTC primer/loading dye mix to tube
      1. Allow PCR bead to dissolve in the mix
        Use micropipette to transfer 3 ul of the cheek cell DNA into the mix
        Place PCR tube in thermal cycler (programmed for 40 cycles)
        After cycling, store the amplified DNA on ice
        Steps of PCR: 1. Denature at 94 degrees C for 30 sec 2. Anneal at 64 degrees C for 45 sec 3. Extend at 72 degrees C for 45 sec
      2. Primer Loading Dye: adds the primers specific to the TAS2R38 gene, needed to initiate elongation and adds the color needed to see the bands during gel electrophoresis
    2. PCR Bead: contains the taq polymerase, dNTPs , and buffer needed to amplify DNA
  3. Goal: make numerous copies of onyl PTC gene
2. Spit into cup: .9% Saline w/ cheek cells
  1. Take 100 ul and spin/centrifuge - allows separation of cheek cells from saliva
  2. Cells (w/ DNA in nucleus) clump at bottom
    1. Pour off supernatant (spit)
    2. Re-suspend cells: pipet up & down
      1. Withdraw 30 ul of solution and add to tube containing 100 ul of Chelex; heat tube for 10 min.
        Centrifuge again - cell debris sinks to the bottom; supernatant contains DNA
        Transfer 30 ul of supernatant to new 1.5ml tube
        Chelex: protects the DNA from the heat of thermal cycler
        Heat/Thermal Cycler: allows the cells to burst and release the DNA
3. Goal: separate DNA from saliva and other cell parts for amplification
Objective: Determine my genotype for the ability to taste PTC and compare/connect it to my phenotype

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2.1.3: Test Your Own Genes

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	Created by nailahc99 over 8 years ago