So, where were we?. The risk of getting skin cancer, particularly melanoma, can be reduced by taking precautions in the sun. In this workshop we will focus more on the association between disease and genes.
(a locus, plural loci, is the specific location of a particular DNA sequence)
there is much less evolutionary pressure
because of less UV radiation at higher latitudes.
9% in GWAS had the higher risk variant
91% have the lower risk variant
Odds Ratio 1.67 for malignant melanoma
(Odds ratio is the odds of getting the disease for someone who has the harmful variant compared to the odds of getting the disease for someone who doesn’t have the harmful variant.)
the skin pigment.
27% in GWAS had the higher risk variant
73% had the lower risk variant
Odds Ratio 1.29 for malignant melanoma
50% have this higher risk variant
50% have this lower risk variant
Odds Ratio 0.85 for malignant melanoma
Restriction Length Polymorphism analysis and PCR.
2 metres per cell!
3 billion letters long (i.e. ACTG and so on).
Like a piece of string.
Specific enzymes can cut the DNA at specific nucleotide sequences. e.g. GTTAAC is cut by one sort of enzyme and TTAA cut by another.
RFLP is defined as a variation in the number of restriction sites, or nucleotide sequences, in a specific DNA region of one individual compared with another.
RFLP analysis – a specific region of DNA (usually within or near the gene of interest)is amplified using Polymerase Chain Reaction (PCR).
PCR makes millions of copies of DNA – enough to work with and test.
A PCR machine (thermalcycler) automates the process. It effectively ‘photocopies’ DNA.
Uses a gel made of seaweed (agarose). It is porous thus allowing DNA strands to ‘wiggle’ through.
The DNA fragments have been pre-prepared.
Enzymes have been added that cut the DNA at a sequence associated with the FH mutation.
DNA has an overall negativecharge due to its phosphate backbone.
When a current is run through the gel the DNA fragments will move from the negative toward the positive electrode.
SmallDNA fragments are able to move further than larger ones.
Brian’s sample (E) will be loaded in Lane 5
Anna’s (B) is loaded in lane 2 and Colin (D) lane 4
Gels are run in an electrophoresis tank for approximately 30 minutes at 150 volts.
Check for bubbles at the electrodes.
After, gels are placed in a DNA stain solution for 5 mins.
They are then de-stained in distilled water for 20 mins. or more until the DNA banding can be clearly seen in the gel.
Down the left-hand side the number of sequence repeats in locus K are shown.
Can you find the repeat sequence of DNA which is shared by all those with the disease *?
1 2 3
1 2 3 4 5 6
(Anna) (Colin) (Brian)
1 = DNA standard markers
3 & 6 = controls for FH patient
What are the parents? (use the sheet)