Gel electrophoresis

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

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

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

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

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

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

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

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

DNA samples loaded into wells Glycerol also in the loading dye

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

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

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

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)

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.

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.

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.

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.

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

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

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).

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

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.

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:

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

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

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.

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