DNA Profiling Flashcards
What are the main 5 steps of DNA profiling?
- Extracting the DNA
- Digesting the sample
- Separating the DNA fragments
- Hybridisation
- Seeing the evidence
How do you extract the DNA in the first stage of DNA profiling?
PCR is used to replicate the sample found.
The small sample found is heated to 95 degrees, this separates the strands and breaks the H bonds between the base pairs
The heat is lowered to 55 degrees and the strands are separated and primers attach to the ends
The temperature is raised to 77 degrees and free floating nucleotides are added
The process repeats again
What enzyme is used to separate the strands of DNA in PCR?
Taq polymerase
How do you carry out the process of digesting the sample in the second stage of DNA profiling?
Strands of DNA cut into small fragments using special enzymes called restriction endonucleases
Different enzymes cut DNA at a specific nucleotide sequence
All restriction endonucleases make two cuts at defined introns
How does separating the DNA in the third stage of DNA profiling occur??
This is achieved through electrophoresis
The sample from the previous stage is placed in a well at the cathode end of a gel medium. In the gel is a matrix and a buffer solution to control the pH of the sample
The smaller fragments of the DNA move quicker through gel to the anode end of the gel
The gel is then placed in an alkali solution which denatures the DNA into separate strands
Place nylon membrane into gel and the fragments are transferred on to it! (Southern blotting)
Why does DNA travel to the anode in electrophoresis??
The anode is positive, and DNA is slightly negatively charged due to the phosphate group in the nucleotide
So the negative DNA is attracted to the positive anode
How is hybridisation carried out in the 4th stage of DNA profiling
Radioactive or fluorescent DNA probes are now added in excess to the DNA fragments, these complimentary probes attach to the strands under certain conditions of pH and temp
Excess probes are washed off
What happens in the fifth stage of DNA profiling (seeing the evidence) ?
If radioactive labels are added to the DNA, X-ray images are taken of the paper/membrane
If florescent labels are added to the DNA, the paper/membrane is placed under UV light
Fragments give a pattern of bars - the DNA profile - which is unique to every individual except identical siblings
What is DNA profiling used for??
Criminal investigations
Paternity tests
Identifying individuals at risk of developing particular diseases
