Lecture 16 Flashcards
What techniques can follow PCR?
restriction analysis
gel electrophoresis
DNA sequencing
Southern blotting
another PCR (‘nested PCR’)
Look at the following sequence, will A or B be the 6th amino acid?
B
Beuase 1st codon is ATG, which codes for Met. Met is the start codon and is cleaved off. Therefore, the 7th codon along will be the 6th amino acid in the polypeptide.
Restriction sites - so important to understand this in questions about DNA probes (what is a restriction enzyme, what is a restriction site, what happens when they is a mutation to the restriction site? - more about this in group work HAVE A LOOK)
- Sickle cell mutation is a single base substitution
- A>T which destroys an MstII restriction site MstII recognises 5’CTTNAGG
LOOK AT PIC BELOW
- Restriction enzyme = an enzyme that cleaves the DNA fragments at or near the specific recognition site within a molecule e.g. endonuclease is a restriction enzyme
- Restriction site = where a restriction enzyme acts
What happens if the restriction site has been mutated?
Restriction enzyme no longer ‘works’ on the mutated restriction site, DNA fragement is therefore not cut in this place. Therefore, if there is a mutation to the starred restriction site (pic below), and add restriction enzyme and a DNA probe that is complementary to some of the 0.6kb fragrament, get the following fragments:
- 0.4kb
- 1.8kb
- 0.9kb
Where are restriction enzymes found?
Bacteria - only naturally found in bacteria. We use these enzymes commerically in the lab to cut up the DNA at specific sites.
Which pair of primers would you use to PCR?
- As wild type alleles has the ‘working restriction site’, so it would be seperated here creating two shorter fragments
Why can you not use PCR on a blood sample? Comes back ‘negative’
Red blood cells do not have a nucleus, so don’t have DNA!
Why can you not use RNA for PCR?
Taq polymerase does not recognise RNA as template
