Enzyme and Restriction Mapping Flashcards
List some examples of recombinant proteins and how transgenic organisms can be utilised?
Recombinant proteins:
- insulin
- interferon
- G-CSF (granulocyte-colony stimulating factor).
The capacity we’ve got to manipulate DNA has resulted in the production of transgenic organisms. These have been used as:
- 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. Restriction
They do two things:
- Recognise specific DNA sequences (different enzymes recognise different specific DNA sequences)
- Cut that sequence (catalyse the hydrolysis of phosphodiester bonds)
What are some features of recognition sites?
Recognition sites are 4-8 base pairs in length, depending on the enzyme, and are palindromic (reads the same backward as forward).
While some nucleases produce an overhang, some nucleases produce a blunt end.
In theory:
- a 4 base recognition sequence occurs every 4x4x4x4 = 256 bases
- a 6 base recognition sequence occurs every 4x4x4x4x4x4 = 4096 bases
(it is ‘4x’ because there is a probability of 1/4 for each nucleotide base being in that position).
What are some uses of restriction enzymes?
They are crucial for:
- Cloning (e.g. joining DNA molecules from different sources like Humans and Bacterial).
- Molecular Diagnostics.
- Characterisation of plasmids.
- Restriction Maps
What can a single nucleotide change do a restriction enzyme site?
Single nucleotide changes can create/destroy restriction enzyme sites. (E.g. - Ddel site is lost in sickle cell anaemia).
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.
What is the function of DNA ligase?
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?
DNA synthesis in 5’ to 3’ direction.
- PCR amplification
- Generation of probes
- Blunt-ending of DNA overhangs
What is a phosphatase and why is it used?
It is an enzyme that hydrolyses a phosphate group off its substrate. It is used to prevent cut plasmids from resealing.
Sources of phosphatase:
- Calf intestinal alkaline phosphatase.
- Shrimp alkaline phosphatase.
What is the function of polynucleotide kinase?
Polynucleotide kinase adds a phosphate group to the fragment of DNA (from ATP to substrate). Its function is the opposite of phosphatase. It adds phosphate to 5’ hydroxyl group of DNA or RNA.
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).
- Normally around 20-1000 bases in length.
- It is complementary to the gene of interest.
What is the function of the reverse transcriptase enzyme?
It is a RNA-dependant DNA polymerase. It is isolated from RNA-containing retroviruses (E.g. - HIV).
It synthesises a DNA molecule complementary to an mRNA template using dNTPs.
The Reverse Transcriptase requires primers. How does Priming for Reverse transcription occur in Random primers and Oligo(dT) primers in PCR?
Reverse transcriptase requires primers.
- Random primers: cDNAs up to 700bp but will cover all of the length of all of the RNA molecules.
- Oligo(dT) primers: Useful for cloning cDNAs and CDNA libraries, but some might not be full length
There are also gene specific primers.
