1. Gel electrophoresis

2. Definition
Separation of DNA fragments according to size, based on movement through a gel medium when an electric field is applied.

3. DNA negatively charged
Organic molecules such as DNA are charged. DNA is negatively charged because the phosphates (red circles) that form the sugar-phosphate backbone of a DNA molecule have a negative charge

4. DNA cut up using restriction endonucleases
Demonstrate on the board using diagrams
Students to complete exercise of cutting up a plasmid using restriction enzymes
Give them W/S and instruct to cut the plasmid using different enzymes and then look at the restriction fragments
Ask – Why do they cut in different places?
Reinforce that this can be applied to any DNA – example human DNA
Called restriction digestion
Different DNA will produce different fragment sizes
Can be PCR’d DNA or DNA produced by clones or ‘natural state’ DNA

5. A bit like electronic chromatography
The fragments are seperated according to size in process called gel electrophoresis

6. Make up the gel which the DNA will be put into
Square tray
2-3 cm of agarose gel which is left to set, can also be made of starch or polyacrylamide, special comb put in so that there are small wells left in the gel

8. A comb is put in the gel to create holes which we call wells

10. Dye added to the DNA
Makes the sample visible when it is put into the agarose wells

11. Buffer solution added to the tank
This ensures that the electric current goes through the whole tank and that maintains that ions can move in the solution

12. DNA samples loaded into wells
Glycerol also in the loading dye

13. Electrical current applied to the chamber
Safety cover is put over the top and the current is switched on
The dye will migrate through the gel toward the positive electrode, as will the DNA
Depending on how much voltage is applied and how warm the gel is and size and shape of molecules will depend on how fast the mols move through the gel
Smaller fragments will move easier so they will be closer to the positive electrode
Once the dye has moved through the gel to the buffer, the electrical current is switched off and gel is removed from the tray

14. DNA is stained using ethidium bromide
Gel is stained using ethidium bromide which binds to DNA it shows up as bands in UV light
Draw attention to the fact that small mols are at the bottom of the gel and large ones stay nearest to the wells

15. How to read a Gel! Gels are read in base pairs.
Wells
Gels are read in base pairs.
Gels are read with guides called ladders.
….the size of the DNA band will be predetermined..the actual size of your gel bands will determine whether this particular DNA is present

16. Stages of DNA Profiling
Cells are broken down
to release DNA
If only a small amount of DNA is available it can be amplified using the polymerase chain reaction (PCR)

17. Stages of DNA Profiling
Step 2:
The DNA is cut into fragments using restriction enzymes.
Each restriction enzyme cuts DNA at a specific base sequence.

18. Stages of DNA Profiling
Fragments are separated on the basis of size using a process called gel electrophoresis.
DNA fragments are injected into wells and an electric current is applied along the gel.

19. Stages of DNA Profiling
DNA is negatively charged so it is attracted to the positive end of the gel.
The shorter DNA fragments move faster than the longer fragments.
DNA is separated on basis of size.

20. Stages of DNA Profiling
A radioactive material is added which combines with the DNA fragments to produce a fluorescent image.
A photographic copy of the DNA bands is obtained.

21. Stages of DNA Profiling
The pattern of fragment distribution is then analysed.

22. Uses of DNA Profiling
DNA profiling is used to solve crimes and medical problems

23. Crime
Forensic science is the use of scientific knowledge in legal situations.
The DNA profile of each individual is highly specific.
The chances of two people having exactly the same DNA profile is 30,000 million to 1 (except for identical twins).

24. Biological materials used for DNA profiling
Blood
Hair
Saliva
Body tissue cells

25. DNA Profiling can solve crimes
The pattern of the DNA profile is then compared with those of the victim and the suspect.
If the profile matches the suspect it provides strong evidence that the suspect was present at the crime scene (it does not prove they committed the crime).
If the profile doesn’t match the suspect then that suspect may be eliminated from the enquiry.

26. Example A violent murder occurred.
The forensics team retrieved a blood sample from the crime scene.
They prepared DNA profiles of the blood sample, the victim and a suspect as follows:

27. Was the suspect at the crime scene?
Suspects Profile
Blood sample from crime scene
Victims profile

28. Solving Medical Problems
DNA profiles can be used to determine whether a particular person is the parent of a child.
A childs paternity (father) and maternity(mother) can be determined.
This information can be used in
Paternity suits
Inheritance cases
Immigration cases

29. Example: A Paternity Test
By comparing the DNA profile of a
mother and her child it is possible to
identify DNA fragments in the child
which are absent from the mother and
must therefore have been inherited
from the biological father.

30. Is this man the father of the child?
Mother
Child
Man