MG (Electrophoresis) Flashcards
define gel electrophoresis
procedure that uses an electrical current to separate out DNA fragments, RNA fragments or proteins depending on their size
Outline the steps in gel electrophoresis including those leading to it and after it
- Extraction of DNA from sample.
- Fragmentation of DNA using restriction endonuclease enzymes
- Separation using electrophoresis (small fragments travel furthest).
- These bands are colourless but are revealed using a radioactive or fluorescent probes.
How does gel electrophoresis work?
- Phosphates in the backbone of DNA, are -vely charged
- DNA fragments are placed in wells at the top of an agarose gel.
- An electric current is applied over it.
- Agar is actually a ‘mesh’, which resists the movement of the DNA fragments through it.
- The DNA moves towards the +ve electrode, but at different rates.
- Small fragments get there quicker.
What is the ladder used for?
A tool we can use to compare base length sequences to that of known sequence lengths. Can be used to give estimates of the size.
Steps in gel electrophoresis in detail…
- DNA samples are first digested with restriction enzymes to cut them at specific recognition sites into fragments. This is carried out at 35-40C and may take up to an hour.
- tank is set up. Agarose gel poured into the central region of the tank whilst combs are placed at one end.
- Once the gel is set, buffer solution is added so that the gel is covered and the end sections of the tank contain buffer solution. Now the comb can be carefully removed, leaving wells at one end. (Ensure that the wells are at the end closest to the negative electrode)
- Take the fragmented DNA samples, using a micropipette add the same volume of loading dye to each (loading dye helps the samples to sink to the bottom of the wells and makes them easier to see)
- Next add a set volume of a DNA sample to the first well (make sure the micropipette is in the buffer solution and just above the opening of the well) don’t stick the tip too far into the well or you might pierce the bottom
- Repeat ensuring a new pipette tip is used for each sample, and a note is made of what sample is added to each well
- Place lid onto the gel box and connect the leads from the gel box to the power supply. The electrical current is then passed through the gel.
- DNA fragments are negatively charged so move through the gel to the anode (positive electrode), let the gel run until the dye is about 2cm from the end.
- Wearing gloves remove the gel, cover the surface of the gel in a staining solution, then rinse the gel. The bands of different sizes will now be visible on the gel.
What can using gel electrophoresis on proteins be used for?
Used to analyse the types of haemoglobin proteins for diagnosis of conditions such as;
- Sickle cell anaemia, where a patient has haemoglobin S and not the normal haemoglobin A
- Aplastic anaemia (no new blood cells produced), thalassaemia (abnormal amounts of haemoglobin produced) and leukaemia, where the patients have higher than normal amounts of fetal haemoglobin (haemoglobin F), and lower than normal amounts of haemoglobin A.
How are proteins prepared to be separated by gel electrophoresis?
- Proteins are denatured to unfold, to expose charges.
- a charged detergent such as sodium dodecyl sulfate (SDS), equalises the surface charge on the molecules and allows the proteins to separate as they move through the gel, according to their molecular mass and then, without SDS, according to their surface charge.
- Electrophoresis can be carried out on RNA fragments following the same basic method as DNA fragments.
What’s a DNA probe
- a short single-stranded length of DNA that is complementary to a section of the DNA being investigated
- labelled with radioactive marker (P-32) in one of the phosphate groups in the probe strand. Once the probe has annealed, by complementary base pairing, to the piece of DNA, it can be revealed by exposure to photographic film.
- or labelled with a fluorescent marker that emits a colour on exposure to UV light.
Probes are useful in locating specific DNA sequences, for example…
- To locate a specific gene needed for use in genetic engineering
- To identify the same gene in different species when conducting genome comparison studies
- To identify the presence or absence of a specific allele for a genetic disease
what are DNA microarrays
different probes placed on a fixed surface
How can DNA microarrays be made?
- made with fixed probes, specific for certain sequences found in mutated alleles that cause genetic diseases, in the well
before making microarrays DNA must be…
broken into smaller fragments, and it may also be amplified using PCR.
why are reference and test DNA samples labelled with fluorescent markers?
Where a test subject and a reference marker both bind to a particular probe, the scan reveals fluorescence of both colours, indicating the presence of the particular sequence in the test DNA.
