Enzyme and Restriction Mapping Flashcards
List some examples of recombinant proteins.
- insulin
- interferon
- G-CSF
The capacity we’ve got to manipulate DNA has resulted in the production of transgenic organisms.
List how those have been used.
- disease models
- improved agricultural yields
What are nucleases? What are the two types of nucleases?
They are enzymes that degrade nucleic acids by hydrolysing phosphodiester bonds.
There are two types:
RIBONUCLEASE (RNAse): degrade RNA
DEOXYRIBONUCLEASE (DNAse): degrade DNA
- Exonuclease: degrade from end of molecule
- Endonuclease: 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.
There are many different enzymes from different bacteria.
They do two things:
- recognise a specific sequence
- cut that sequence
What are some features of recognition sites?
Recognition sites are 4-8 base pairs in length, depending on the enzyme, and are palindromic.
While some nucleases produce an overhang, some nucleases produce a blunt end.
Describe restriction maps.
It is a map of restriction sites within a molecule.
It is 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.
If their overhangs are compatible, we would 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
Another enzyme used is phosphatase.
What is a phosphatase?
It is an enzyme that hydrolyses a phosphate group off its substrate.
Why would we use a phosphatase?
To prevent cut plasmids from resealing.
Another enzyme used is polynucleotide kinase.
What is the function of this enzyme?
It adds a phosphate group to the fragment of DNA. Its function is the opposite of phosphatase.
Why would we need to use a polynucleotide kinase?
- to phosphorylate chemically synthesised DNA so that it can be ligated to another fragment
- to sensitively label DNA so that it can be traced using:
- radioactively labelled ATP
- fluorescently labelled ATP
Define a probe.
It is a fragment of ssDNA (or RNA).
It is normally around 20-1000 bases in length.
It is complementary to the gene of interest.
Yet another enzyme we use is reverse transcriptase.
What is its function?
It is an RNA-dependant DNA polymerase. It is isolated from RNA-containing retroviruses.
It synthesises a DNA molecule complementary to an mRNA template using dNTPs.
