Enzyme and restriction mapping Flashcards
What type of proteins is genetic engineering use to produce?
- Recombinant proteins
- Insulun
- Interferon
- G-CSF - Transgenic organisms
- Disease models
- Improved agricultural yields
What do nucleases degrade and how?
Degrade nucleic acids by hydrolysing phosphodiester bonds
What does ribonuclease degrade?
Degrades RNA
What does DNase degrade?
Degrades DNA
What does exonuclease degrade?
Degrade from the end of the molecule
What does endonucleases degrade?
Degrade within nucleotide chain
What are the 2 things that restriction endonucleases do?
- Recognise a specific sequence
2. Cut that sequence
Physiology behind restriction
Limit transfer of nucleic acids from infecting phages into bacteria
Where are the many different enzymes of restriction endonucleases from?
Many different enzymes from different bacteria
What can some nucleases produce?
Some nucleases produce an overhang and some produce a blunt end
What are restriction maps?
Map of restriction sites within a molecule
What way is useful for describing plasmids?
Restriction maps are a useful way of describing plasmids
Restriction enzymes in molecular diagnostics
Sickle cell anaemia:
- Single point mutation
- A–>T resulting in Glu–>Val
What can single nucleotide changes do?
Single nucleotide changes can create/destroy restriction enzyme sites
What does DNA ligase do and how?
-Puts 2 fragments together
Does this by repairing nicks in phosphodiesterase backbone
What does DNA polymerase do?
DNA synthesis in 5’ to 3’ direction
What are the uses of DNA polymerase?
- PCR amplification
- Generation of probes
- Blunt ending of DNA overhangs
What do phosphatase’s do?
Hydrolyses a phosphate group off its substrate
What are phosphatase’s used to prevent?
Used to prevent cut plasmids from resealing
What do polynucleotide kinases do?
Adds phosphate to 5’ hydroxyl group of DNA or RNA
What are polynucleotide kinases used for?
- To phosphorylate chemically synthesised DNA so that is can be ligated to another fragment
- To sensitively label DNA so that it can be traced using:
- Radioactively labelled ATP
- Fluorescently labelled ATP
What are probes?
Fragments of ssDNA
What are probes complementary to?
Probes are complementary to the gene of interest
What type of enzyme is a reverse transcriptase?
A RNA dependent DNA polymerase
How are RNA transcriptase collected?
Isolated from RNA-containing retroviruses
What does RNA transcriptase facilitate in the synthesis of?
Synthesises a DNA molecule complementary to a mRNA template using dNTPs
What does reverse transcriptase require?
Requires primers
Priming for Reverse transcription (Random primers)
Cover all of the length of all of the RNA molecule
Priming for reverse transcription (Oligo(dT))
Useful for cloning cDNAs and CDNA libraries, but some might not be full length
