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PCR steps and gel electrophoresis of DNA fragments

Grace Collins English

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  1. Extract the DNA from the sample - look at the introns with the STRs which repeats differ form person to person

  2. Amplification process begins:

    Denaturation- separate the target DNA strand by heating at 95'C for 5 minutes to break the hydrogen bonds.

  3. Annealing- Cool to 55'C, add primers (short DNA strands that provide starting sequence). These normally have flourescent markers attached. This allows DNA polymerase to bind.

  4. Extension- Incubate at 70'C, add nucleotides and DNA polymerase enzyme. During incubation time primers attach to single DNA strand. DNA polymeraes synthesise complementary strands

  5. Repeat for about 25 cycles - exponential so each time the copy of DNA doubles. Make billions of DNA in about 2.4 hours.

  6. Cut up the DNA- using restriciton enzymes yielding thousands of fragments all different sizes

  7. Separate the fragments by length using gel electrophoresis. Dna is negatively charged and gets loaded into wells on an agar gel. The agar gel has an electrodes on either side to make it charged. Negatively charged DNA travels to positively charged terminal. Smaller fragments travel faster.

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Flowchart on Untitled, created by Grace Collins on 23/03/2021.
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