Forensic Lab Analysis

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  • Lab Analysis
    • PCR
      • polymerase chain reaction
        • Developed in 1985 by Kary Mullis
        • First used in 1988 to analyse the skeletal remains of a 3 year old girl
        • Components
          • Template DNA - The amount of DNA added to PCR depends on the sensitivity of the reaction.
            • PCR has the ability to amplify DNA from a template of only a few cells
              • High level of sensitivity can cause issues of contamination
                • Samples must be handled correctly to prevent contamination
                  • PPE
          • PCR Primers - (at least two) Short synthetic DNA fragments that bind to the specific areas of DNA priming the amplification reaction.
            • Define the region of the genome that will be amplified
          • DNA Polymerase - The enzyme that puts together the building blocks
            • Joins the nucleotides onto the new DNA strand
          • dNTP's - The building blocks used to make the DNA copies
            • A primer
        • PCR Calculation of how many DNA fragments will be produced after X PCR cycles.
          • If 1 DNA fragment was present at the beginning of PCR, and 12 cycles were carried out: 1 x 12^2
            • During every cell cycle, DNA is doubled
      • Electrophoresis
        • PCR is needed to allow electrophoresis to later occur
        • The movement of charged particles in a fluid or a gel under the influence of an electric field.
          • The separation of charged molecules according to size
            • Smaller molecules are able to navigate out of the gel faster than larger molecules.
            • The gel will have multiple lanes where the sample can run
              • When the wells are loaded, they are staggered so if a sample were to leak into the adjacent lanes, it wouldn't match up to a measurable allele.
                • Prevents contamination
                  • Quality assurance done through standards and guidance
                    • Forensic Science Regulator
                    • Scientific Advisory Group DNA Analysis and Methods
                    • Consequences of poor quality assurance
          • Negatively charged DNA molecules migrate towards the positive end of the field.
      • Stages of PCR
        • 1) Denaturation - (90 degrees) DNA samples are heated to separate into two strands. Hydrogen bonds are broken, leaving a single stranded 'template' DNA
        • 2) Annealing - (50-65 degrees) Hydrogen bonds are able to form between the primers and template DNA.
          • Primers attach to complementary strands
        • 3) Extension (72 degrees) New strands of DNA are synthesised with taq polymerase.
          • Nucleotides that are complementary to the template are added to the nascent DNA strand at the rate of 40-60 per second.
          • Taq polymerase is a thermostable  DNA polymerase enzyme
            • From Thermus acquaticus bacteria
            • Ability to withstand the protein-denaturing conditions required for PCR
        • 4) Denaturation New strands of DNA separate and the process continues.

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