Forensic Lab Analysis
- Created by: AAntonianannetti
- Created on: 08-05-19 15:13
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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
- Samples must be handled correctly to prevent contamination
- High level of sensitivity can cause issues of contamination
- PCR has the ability to amplify DNA from a template of only a few cells
- 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
- Template DNA - The amount of DNA added to PCR depends on the sensitivity of the reaction.
- 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
- If 1 DNA fragment was present at the beginning of PCR, and 12 cycles were carried out: 1 x 12^2
- 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
- Quality assurance done through standards and guidance
- Prevents contamination
- 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.
- Negatively charged DNA molecules migrate towards the positive end of the field.
- The separation of charged molecules according to size
- 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.
- polymerase chain reaction
- PCR
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