PCR DNA fingerprinting Gel electrophoresis DNA Profiling PCR DNA fingerprinting Gel electrophoresis

Gel Electrophoresis https://www.youtube.com/watch?v=ZxWXCT9wVoI

Electrophoresis A technique used to separate and identify chemicals. Uses electrical voltage to separate Useful for large biological molecules (e.g. DNA) DNA becomes electrically charged in a solution with the correct pH.

Method 1) Sample placed in a well in a gel 3) Electrodes placed at each end of tank 4) Electric voltage applied and runs between electrodes. Particles with a charge move towards the opposite electrode. 2) Buffer solution to keep pH constant.

Method Negative particles move towards the positive electrode -  + Positive particles move towards the negative electrode +  -

Speed of movement The speed at which it travels depends on: How big the charge is (e.g. -1, -2, -3) How large the particle is (smaller moves further)

DNA Electrophoresis DNA fragments are dissolved in a solution that gives them a NEGATIVE charge. The fragments move towards the POSITIVE electrode. The speed depends on their size After 24 hours the current is switched off. The bands of dyed DNA show how far each fragment moved in the time.

DNA Profiling Everyone (except identical twins) has unique DNA DNA profiling looks at these differences The DNA being tested is broken into fragments by enzymes and then analysed by electrophoresis Because everyone is different, everyone produces a different pattern of bands.

A B C D E F G

Uses of DNA profiling Forensic Investigations – identifying murderers, rapists, criminals, bodies

Uses of DNA profiling Checking parentage – in people and animals

Uses of DNA profiling Wildlife protection – detecting illegally traded animals

Uses of DNA profiling Testing food – for genetically modified ingredients.

What is Polymerase Chain Reaction? It makes possible: the detection of pathogenic virus or bacteria DNA fingerprinting (identifying individuals) other applications involving DNA manipulation Makes possible the detection of pathogenic virus or bacteria, DNA fingerprinting (identifying individuals) and others involving DNA maniupaltion

Polymerase Chain Reaction

What is Polymerase chain reaction? (PCR) PCR is a technique that is used to amplify one sample of DNA thousands of times over to create a large enough DNA sample for extensive analysis. i.e. each time a PCR cycle is performed the total amount of DNA is doubled. It is In Vitro amplification of DNA.

What are some uses of PCR? PCR can be used for many applications Paternity Tests – The child’s tandem repeats are compared to the mother’s and the father’s. Detecting mutations- Comparing one persons DNA to the DNA of the person with the mutation

The use of PCR in forensics To create enough DNA from a small sample to create a DNA profile.

What is needed? 1. The DNA fragment to be copied 2. DNA polymerase – for PCR it is taken from bacteria that live in hot springs so it is tolerant to heat 3. Primers – short pieces of DNA that bind to the start of each end of fragment – also help to prevent both ends from joining back up 4. Nucleotides 5. Thermocycler – a computer controlled machine that varies temperatures precisely for set time periods

Grad student doing PCR without a thermal cycler

Let’s visualize it first https://www.youtube.com/watch?v=eEcy9k_KsDI

The process of PCR Step 1: Denature the DNA “Denature” means to separate the DNA strands into 2 separate strands This involves heating the DNA sample up to 96 degrees!

The process of PCR Step 2: Anneal the DNA - “annealing” means to add In this step 2 primers are added to the 2 separated DNA strands. ( 1 on each strand) The temperature is “cooled” to 65 degrees, this help the primers bind to the DNA. The primer is an attachment that signals to a polymerase where to start synthesising (making) new DNA

The process of PCR Step 3: “Extension” of DNA- New DNA Created 2 Polymerase molecules attach to the 2 Primers on the 2 DNA strands and move along the strand. As they move along they create new “complementary” DNA. Temperature goes up to 72 degrees

The process of PCR Done! By heating the DNA to separate it into 2 strands (Denaturation) Cooling the DNA to add 1 primer to each of the 2 DNA strands (Annealing) And by Adding 1 polymerase to each strand to synthesise DNA from where the primer attached (Extension) You just doubled the mount of DNA present.

Animation http://www.dnalc.org/resources/animations/pcr.html

Making more DNA Every time you do a Cycle of PCR you double the DNA. You can do this process as many times as needed to create millions of strands of DNA. Cycle Number DNA copies None 2 1st cycle 4 2nd cycle 8 3rd cycle 16

PCR simulation http://learn.genetics.utah.edu/content/labs/pcr/

Why is DNA profiling not conclusive Contamination of DNA can happen at any of the several stages involved in DNA profiling Only a small portion of the DNA is analysed It is possible to have two identical profiles from unrelated individuals Identical twins will have the same profile