Introduction to PCR and Animal Genotyping

pcr-genotyping-intro-v7

In a follow up to our overview on DNA methods, we wanted to discuss PCR (polymerase chain reaction) which is one of the most sensitive and versatile techniques in molecular biology. PCR is a technique which selectively amplifies any targeted DNA from a complex mixture based on a set of framing primers. These primers are ~20 base oligonucleotides which we can (1) design based on a sequenced genome and (2) make/order based on solid-phase chemical synthesis. PCR has many applications (see partial list below) but is to test for a particular gene/mutation (i.e. “genotype”) in an animal (see figure above).

  • purify bulk DNA for:
    • sequencing
    • structural analysis
    • engineering
  • measure DNA/RNA concentration for:
    • gene expression level (mRNA)
    • gene copy #
    • viral/bacterial load
  • modify DNA (w/ primers) for:
    • site-directed mutagenesis
    • protein expression
    • reporter cloning

If you want to test a mouse for a gene/mutation you can use any piece of tissue because the DNA is the same in every cell of the mouse’s body. When the mice are alive and breeding, however, you should only use the tip of the mouse’s tail to minimize its discomfort/pain. With this sample in hand you then:

  1. Extract the DNA from this tail-tip using a commercially available extraction kit
  2. PCR amplify (~30 cycles) the target gene/mutation using primers, DNA polymerase (usually in a kit) and 1-2 μL of your DNA extract
  3. Run an Agarose Gel (containing ethidium bromide to make the DNA fluorescent) on the entire PCR amplification sample
    • One band typically means your mouse is homozygous (with two mutant genes OR two wild-type genes)
    • Two bands typically means your mouse is heterozygous (with one mutant AND one wild-type gene)

pcr-genotyping-intro-v7

The automated thermocycler programs that are used for PCR amplification cycle are generically illustrated above and are made up of three steps, repeated iteratively:

  1. Denaturing the DNA (to allow primer access the targeted DNA)
  2. Annealing (to allow primers to bind to the targeted DNA)
  3. Extension (to allow the DNA polymerase to synthesize new strands of targeted DNA

 

REFERENCES:

  1. Beaucage, S.L.; Iyer, R.P. Advanced in the Synthesis of Oligonucleotides by the Phosphoramidite Approach. Tetrahedron, 1991, 48, 2223-2311.
  2. Saiki, R.K.; Gelfand, D.H.; Stoffel, S.; Scharf, S.J.; Higuchi, R.; Horn, G.T.; Mullis, K.B.; Erlich, H.A. Primer-Directed Enzymatic Amplification of DNA with a Thermostable Polymerase. Science, 1988, 239, 487-491.
  3. Cheng, S. Fockler, C.; Barnes, W.M., Higuchi, R. Effective amplification of long targets from cloned inserts and human genomic DNA. PNAS, 1994, 91, 5695-5699.
  4. Alberts, B. Molecular Biology of the Cell 5th Ed. Garland Science 2008
  5. Murphy, K. Janeway’s Immunobiology 8th Ed. Garland Science 2012

 

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