Enzyme And Restriction Mapping Flashcards
(29 cards)
What are Nucleases?
These are enzymes that degrade the nucleic acids by hydrolysing phosphodiester bonds
What are the two types of nucleases?
- Ribonuclease (RNAase): Degrade RNA
- Deoxyribonuclease (DNase): Degrade DNA
ExoNuclease: Degrade from the end of molecule
Endoculease: Cleave within the nucleotide chain
What are restriction enzymes?
Restriction: Limit the transfer of nucleic acids from infecting phases into bacteria. May have different enzymes from bacteria
What do restriction enzymes do?
- Recognise a specific sequence
- Cut that sequence
View diagrams
Different restriction enzymes recognise different specific DNA sequences
In Theory, where does a 4 base recognition sequence occur?
Also known as Short Tandem Repeats
Every 256 bases. (4x4x4x4)
In Theory, where does a 6 base recognition sequence occur?
Also known as Short Tandem Repeats
Every 4096 bases (4x4x4x4x4x4)
What is an overhang and what is it produced by?
- Some nucleases produce overhangs
- This is where the DNA sequence is cut between the 5’ and 3’ UTRs leaving a gap between two bases
- This cut then causes another cut of 4 bases on the corresponding sequence causing it to overhang
What are the two types of cuts caused by nucleases?
Overhangs and blunt ends
What are Restriction Enzymes useful for?
- Cloning
- Molecular diagnostics
- Characterisation of plasmids
What type of enzyme could be used to bring two DNA molecules together?
DNA ligaments is used to create a phosphodiester bond between the human DNA and bacterial DNA to create a recombinant DNA molecule
What was restriction enzymes first used to diagnose in Molecular Diagnostics?
Sickle cell Anaemia
How can Restriction enzyme sites be destroyed?
By single nucleotide changes which changes the structure of the site
What are the Restriction maps uses of restriction enzymes?
- Map of restriction sites within a DNA molecule
- Crude way of mapping an unknown molecule
- A Useful way of describing plasmids
What are Plasmid maps?
Essentially tells you where restriction enzymes digests the plasmid
Watch Lecture to get an idea of Mapping
What other enzymes are used when creating recombinant DNA?
- DNA ligase
- DNA polymerase
- Phosphatases
- Polynucleotide kinase
- Reverse transcriptase
What is the function of DNA ligase?
- Repairs nicks in the phosphodiester backbone
- Creates new phosphodiester bonds between the phosphate group and hydroxyl group between the 5’ and 3’
What is the function of DNA polymerase?
- Carry out DNA synthesis in the 5’ to 3’ direction
- To do this, they need a primer
What are the other uses of DNA polymerase?
- PCR amplification
- Generation of probes
- Blunt ending of DNA overhangs
What is the function of Phosphatase?
Hydrolyses the phosphate group off of its substrates
- Cald intestinal alkaline Phosphatase were previously used
- Shrimp Alkaline Phosphatase are now used
Why would you use a Phosphatase?
To prevent cut plasmids from resealing
What is the function of polynucleotide Kinase?
Incorporates a phosphate group from ATP to the substance
- reaction: ATP + Subtrate -> ADP + phosphorylated substrate
- Polynucleotide kinase adds phosphate to 5’ hydroxyl group of DNA or RNA
Why would we use a Polynucleotide kinase?
- To phosphorylase chemically synthesised DNA so that it can be lighted to another form
- To sensitively label DNA so that it can be traced using
- Radioactivity labelled ATP or Fluorescently lebelled ATP
