6.3.5: Electrophoresis Flashcards
What is electrophoresis?
- A process used to separate proteins or DNA fragments of different sizes.
- It can separate fragments that differ by only one base pair.
Describe the set up of an electrophoresis kit.
- An agarose gel plate covered by a buffer solution.
- Electrodes placed in each end of the tank so that, when it is connected to a power supply, an electric current can pass through the gel.
Why does DNA have an overall negative charge?
Therefore, where do the DNA pieces migrate to?
- DNA has an overall negative charge due to many phosphate groups.
- The fragments migrate towards the anode (positive electrode).
Does the size of the DNA fragments affect the charges of the fragments?
No, fragments of DNA all have a similar surface charge, regardless of their size.
How is the process different when separating proteins?
- The principle for separating proteins is the same as for separating DNA fragments
- but is often carried out in the presence of a charged detergent such as sodium dodecyl sulfate (SDS).
Why is a charged detergent such as sodium dodecyl sulfate (SDS) used when separating proteins?
To equalise the surface charge on the molecules and allow the proteins to separate as they move through the gel, according to molecular mass.
What happens if a charged detergent such as sodium dodecyl sulfate (SDS) is not used when separating proteins?
The proteins are separated according to their surface charge.
How can the electrophoresis of proteins be used to diagnose (1)sickle cell anaemia (2) aplastic anaemia, thalassaemia and leukaemia?
To analyse the types of haemoglobin proteins:
- In sickle cell anemia, the patient has the haemoglobin S and not the normal haemoglobin A
- Patiens have a higher than normal amounts of fetal haemoglobin (haemoglobin F), and lower amounts of haemoglobin A.
What is a DNA probe?
A short (50-80 nucleotides) single-stranded length of DNA that is complementary to a section of the DNA being investigated.
What are the two ways that you could label a DNA probe?
- A radioactive marker
- A fluorescent marker
How can a radioactive marker be used to label a DNA probe?
-Usually with ^32P in one of the phosphate groups in the probe strand. Once the probe has annealed (bound), by complementary base pairing, to the piece of DNA, it can be revealed by exposure to photographic film.
How can a fluorescent marker be used to label a DNA probe?
What other uses are there for fluorescent markers?
- By using a fluorescent marker that emits a colour on exposure to UV light.
- Fluorescent markers may also be used in automated DNA sequencing.
Probes are useful in locating specific DNA sequences. Give examples…
- To locate a specific gene needed for use in genetic engineering.
- To identify the same gene in a variety of different genomes from different species when conducting genome comparison studies.
- To identify the presence or absence of a specific allele for a particular genetic disease or an allele that gives susceptibility to a particular condition.
What is a DNA microarray?
-A fixed surface that scientists can place a number of probes on.
What will applying DNA to the surface of a DNA microarray reveal?
- Applying the DNA under investigation to surface can reveal the presence of mutated alleles that match the fixed probes.
- The DNA will anneal to any complimentary fixed probes
