6.3.5 electrophoresis Flashcards
1
Q
define electrophoresis
A
process used to separate proteins/DNA fragments of different sizes
2
Q
how is electrophoresis able to separate DNA
A
- separates different sized fragments of DNA
- able to separate fragments which differ by 1 base pair
- uses agarose gel plate covered by buffer solution
- electrodes placed in each end of tank = electric current passes through gel when connected to power supply
- DNA has overall negative charge (many phosphate groups) so fragments migrate towards anode
- fragments of DNA have similar surface charge, regardless of size
3
Q
where is electrophoresis widely used
A
gene technology to separate DNA fragments for identification/analysis
4
Q
outline the gel electrophoresis practical (separating DNA)
A
- DNA samples digested with restriction enzymes to cut them (specific recognition sites) into fragments at 30-40 degrees
- tank is set up whilst restriction enzymes cutting DNA
◦ agarose gel made & poured into central
region of tank, whilst combs are in place at
1 end
◦ once gel is set, buffer solution added to
cover gel & end sections
◦ remove comb = leaves wells at 1 end of gel - loading dye added to tubes containing digested DNA
- digested DNA & loading dye added to wells in electrophoresis gel
◦ pipette used & this is held (in buffer
solution) just above 1 of the wells (not
placed right into well as could pierce gel)
◦ loading dye is dense & carries DNA into
well - once wells have been loaded with different DNA samples, electrodes placed & connected to 18V battery
◦ left to run for 6-8 hours
◦ (or) higher voltage power pack can be used
& gel run for shorter time (less than 2
hours) - DNA fragments move though gel at different speeds (smaller travel faster, so travel further)
- at end of period, buffer solution poured away & dye added to gel (adheres to DNA = stains fragments
5
Q
how can electrophoresis separate proteins
A
- same as for separating DNA fragments, but often carried out in presence of charged detergent (eg. sodium dodecyl sulfate) = equalises surface charge of molecules & allows proteins to separate as they move through gel (according to molecular mass)
- ## protein can be separated according to mass & (without SDS) according to surface charge
6
Q
how can electrophoresis be used when separating proteins
A
= analyse types of haemoglobin proteins for diagnosis of conditions, eg:
- sickle cell anaemia (haemoglobin S not regular haemoglobin A)
- aplastic anaemia, thalassaemia & leukaemia (higher amounts of fetal haemoglobin & lower amounts of haemoglobin A)
7
Q
what are DNA probes
A
- short (50-80 nucleotide) single-stranded length of DNA complementary to section of DNA being investigated
- labelled using either radioactive marker (once probe bound to piece of DNA, revealed by exposure to photographic film) or fluorescent marker (emits colour nexposure to UV light)
8
Q
how are DNA probes useful for locating specific DNA sequences
A
- locate certain genes needed for use in genetic engineering
- identify same gene in variety of genomes from different species in genome comparison studies
- identify presence/absence of specific alleles for certain genetic diseases/susceptibility to condition
9
Q
define microarrays
A
scientists can place many DNA probes on fixed surface = DNA microarray
10
Q
describe use of microarrays
A
- applying DNA under investigation to surface can reveal presence of mutated allele which match fixed probes (sample of DNA will bind to complementary fixed probes)
- sample of DNA must be broken into smaller fragments & sometimes amplified using PCR
- can be made with fixed probes in the well (specific for certain sequences found in mutated alleles which cause genetic diseases)
- reference & test DNA samples labelled with fluorescent markers
- test subject & reference marker bind to certain probe
- scan reveals fluorescence of both colours, indicating presence of particular sequence in test DNA complementary
