Enzymes and restriction mapping Flashcards
What are restriction enzymes?
Restriction enzymes are considered as molecular scissors, as you can use them to cut DNA in specific locations. They are mostly produced by bacteria. Restriction enzymes work like a defence system for bacteria. They ensure that virus/fudge (bacterial fudge- enemy which kills and invades bacteria by putting their genome inside) doesn’t survive & cleave the DNA of it thus protecting the bacteria.
What are some applications of restriction enzymes?
Genetic engineering. Generation of recombinant proteins:
- Insulin; protein crucial for diabetic patients
- Interferon; involved in antiviral defences
- G-CSF (Granulocyte colony stimulating factor)- promotes formation of bone marrow (in cancer patients)
What are Nucleases?
Proteins that degrade nucleic acid by hydrolysing phosphodiester bonds.
What are the two main types of nucleases?
Ribonuclease (RNase) - degrade RNA
Deoxyribonuclease (DNase) - degrade DNA
-Exonuclease- Degrade from end of molecule
-Endonuclease- Cleave within nucleotide chain
What does ‘restriction’ mean in terms of restriction enzymes?
Limit transfer of nucleic acids from infecting phages into bacteria.
What two things do restriction endonucleases do?
They are restriction enzymes. They:
- Recognise a specific sequence. Each restriction enzyme will only recognise one type of specific sequence (will not cleave just any sequence) and upon binding to it;
- Cut/cleave that sequence into fragments. Does this by catalysing the hydrolysis of phosphodiester bonds..
What are the different specific DNA sequences (recognition sites) of the restriction enzymes EcoR I, BamHI and HINDIII?
EcoR I: 5’..GAATTC..3’
3’..CTTAAG..5’
BamHI: GGATCC, CCTAGG
HindIII: AAGCTT, TTCGAA.
Restriction sites (Aka recognition sequences) are 4-8 bp in length and (Depending on enzyme) are palindromic.How many times does a 4 base and 6 base recognition sequence occur?
4 base occurs every 4x4x4x4 = 256 bases
6 base occurs every 4x4x4x4x4x4x= 4096 bases
Do all nucleases produce overhangs?
No. Some produce overhangs (e.g EcoRI and KpnI), however some produce a blunt end. (e.g ALu I). Sticky ends ligate better than blunt ends.
What are restriction enzymes crucial for?
Cloning, molecular diagnostics, characterisation of plasmids.
What is the effect of base changes on restriction enzyme sites?
Single nucleotide changes can create/destroy restriction enzyme sites. E.g. Ddel site (5’CTNAG3’) Is lost in sickle cell anaemia due to single base change of GAG to GTG.
What are restriction maps?
Map of restriction sites within a molecule. They are a good way of mapping an unknown molecule and a useful way of describing plasmids.
What does DNA ligase do?
Repairs nicks in phosphodiester backbone and creates a phosphodiester bond for that backbone.
Hence can covalently seal two diff DNA molecules.
Why is using DNA polymerase good?
It can create blunt ends which is useful for incompatible DNA ends. DNA pol creates blunt ends of DNA overhangs so you can seal both molecules as blunt ends, as it used for DNA synthesis in 5’ to 3’ direction. Thus, two DNA molecules can be ligated.
What does phosphatase do? Why use a phosphatase?
Hydrolyses a phosphate group off its substrate at 5’ end (removes it)
Use it to prevent cut plasmids from resealing – if we dephosphorylate/remove the phosphate from digested plasmid, it will not be able to re-circularise (as it doesn’t have phosphate group)
