6.3 – Manipulating genomes DNA Profiling 6.3 – Manipulating genomes

Specification Reference

Learning Objective Success Criteria Understand the principles and uses of electrophoresis for separating nucleic acid fragments or proteins. Describe the procedure involved in separating nucleic acids and proteins using electrophoresis. Describe where using DNA probes is useful and explain how they work. Explore the uses of microarrays.

Electrophoresis gel showing radioactively labelled fragments of DNA. What is Electrophoresis? Electrophoresis is used to separate DNA fragments based on their size. What are the enzymes used to cut DNA into smaller fragments called? Electrophoresis is accurate enough to separate fragments of DNA that differ by only one base in length. Restriction Enzymes Electrophoresis gel showing radioactively labelled fragments of DNA.

The Principle Which fragments would get there first? The technique relies on the movement of DNA through a gel matrix. Larger fragments are hindered and tend to move more slowly than small fragments. But what causes the fragments to move at all? DNA is negatively charged due to the many phosphate groups in each nucleotide In an electric field, DNA fragments will move towards the anode (+ve electrode). Which fragments would get there first?

Why is the gel not run indefinitely? The DNA fragments are placed in ‘wells’ at one end of a gel plate. The gel is made of agarose or polyacrilamide, which is similar to agar. Remember that separation occurs because longer strands get caught up in the gel, whereas shorter strands move more quickly through it. Cathode (-) Anode (+) Wells Why is the gel not run indefinitely?

The Apparatus

The Procedure The DNA samples are first treated with restriction enzymes to fragment them. They are placed into wells at the cathode end of the gel. The gel is immersed in electrolyte/buffer solution. A current is passed through the solution for a fixed time. The fragments of DNA move towards the anode due to the electrical gradient. Shorter lengths of DNA move faster than longer lengths and so move further in the fixed time. The position of the fragments can be shown using a dye, or fluorescent/radioactive labelling.

The Results If the fragments have been radioactively labelled, placing photographic film over the gel will produce something like this: Further analysis may be required on the fragments. They can be lifted from the gel by Southern Blotting. A nylon sheet is simply placed on the gel and pressed firmly, transferring the fragments.

Task Read the section titles ‘Separating Proteins’ on page 226. What are the similarities and differences in the procedure used to separate proteins, compared to that of separating DNA? The procedure can be used to diagnose certain conditions. What are they?

DNA Probes

DNA Probes If one particular fragment that contains a particular gene is being searched for, DNA probes can be used to locate it. DNA probes are simple, short and single-stranded sections of DNA. They will bind (anneal) to complementary sections of other DNA strands. Being labelled in some way, they make this ‘other DNA’ easily identifiable. Labelling with radioactivity Labelling with fluorescence

Microarrays To save time when screening for particular genes, scientists can place a variety of different probes onto a fixed surface known as a microarray. The DNA under investigation is applied to the microarray. If any genes of interest are present, they will anneal to their complementary probe.

Plenary Answer the following questions: What type of bonding is responsible for annealing? Why is electrophoresis described as ‘similar to chromatography’? Why must an electric field be applied over a gel for a fixed period of time? Why is an electrolyte/buffer mix required to immerse the gel? Suggest why it is important that DNA probes are relatively short molecules.

Learning Objective Success Criteria Understand the principles and uses of electrophoresis for separating nucleic acid fragments or proteins. Describe the procedure involved in separating nucleic acids and proteins using electrophoresis. Describe where using DNA probes is useful and explain how they work. Explore the uses of microarrays.