Enzyme and restriction mapping Flashcards
List some examples of recombinant proteins
- insulin
- interferon
- G-CSF
The capacity we have to manipulate DNA has resulted in the production of transgenic organisms, how have these been used?
- disease models
- to improve agricultural yields
What are nucleases??
Enzymes that degrade nucleic acids by hydrolysing phosphodiester bonds
What are the two main types of nucleases?
1) Ribonucleases (RNase), which degrade RNA
2) Deoxyribonucleases (DNAse), which degrade DNA
- exonucleases degrade from the end of the molecule
- endonucleases cleave within the nucleotide chain
Describe restriction endonucleases
Their main function is restriction - they limit the transfer of nucleic acids from infecting phages into bacteria
What do restriction endonucleases do?
Recognise a specific sequence and cut it
What are some features of recognition sites?
They are 4-8 base pairs in length, depending on the enzyme, and are palindromic
Whilst some nucleases produce an overhang, some produce a blunt end
What does palindromic sequence men?
A DNA locus whose 5’-to-3’ sequence is identical on each DNA strand. The sequence is the same when one strand is read left to right and the other strand is read right to left. Recognition sites of many restriction enzymes are palindromic.
Describe restriction maps
It is a map of restriction sites within a molecule that requires the use of restriction enzymes - a crude way of mapping an unknown molecule, and a useful way of describing plasmids
Can DNA restriction be reversed?
DNA molecules from different sources can be joined together, and if their overhangs are compatible we can put them together with DNA ligase (whose function is to create phosphodiester bonds between the fragments)
What are some uses of DNA polymerase?
- PCR amplification
- generation of probes
- blunt-ending of DNA overhangs
What is a phosphatase enzyme?
An enzyme that hydrolyses a phosphate group off its substrate, we use them to prevent cut plasmids from resealing
What is a polynucleotide kinase?
An enzyme which adds a phosphate group to the fragment of DNA. It has the opposite function to phosphatase
Why would we use a polynucleotide kinase?
- to phosphorylate chemically synthesised DNA so it can be ligated to another fragment
- to sensitively label DNA so it can be traced using radioactively/fluorescently labelled ATP
Define a probe
A fragment of ssDNA/RNA which is 20-1000 bases in length
it is complementary to the gene of interest
