Enzyme and Restriction Mapping Flashcards
How is insulin, interferon and G-CSF involved with the generation of recombinant proteins?
- Insulin – first protein produced in large amounts
- Interferon – protein involved in fighting viral infections
- G-CSF – factor promotes the formation of bone marrow, radiotherapy patients needs to generate bone marrow. Cancer patients are immunodeficient.
How is genetic engineering involved with the production of transgenic organisms?
- Disease models
* Improved agricultural yields
What do nucleases do?
What does ribonuclease, Deoxyribonuclease, Exonuclease and Endonuclease catalyse?
• Degrade nucleic acids by hydrolysing phosphodiester bonds
• Ribonuclease (RNase): degrade RNA
• Deoxyribonuclease (DNase): degrade DNA
Exonuclease: degrade from end of molecule
Endonuclease: cleave within nucleotide chain
Why are restriction enzymes called restriction enzymes e.g restriction endonucleases?
They are called restriction enzymes because they were discovered in bacteria and there purpose is explained below
Define restriction
Restriction: limit transfer of nucleic acids from infecting phages into bacteria.
What do restriction endonucleases do?
recognise a specific sequence
AND
cut that sequence
What was the first restriction enzyme called and where was it found?
What does it identify and cut?
What does it catalyse?
What does it produce?
- This is the first restriction enzyme found in e.coli -EcoRI
- This enzyme recognises the specific sequence GAATTC
- Once this sequence is found they cleave it, Restriction enzymes catalyse the hydrolysis of phosphodiester bonds. Forming to DNA fragments.
- It will produce an overhand of TTAA and ATT
- It forms a OH and phosphate group on the strands
Different restriction enzymes recognise different specific DNA sequences
Give 3 examples
EcOR I
BamHI
HindIII
How long are recognition sites?
When does a 4 and 6 base recognition sequence occur?
• Recognition sites (aka recognition sequences) are 4-8 base pairs in length, depending on the enzyme, and palindromic.
- A 4 base recognition sequence occurs every 4x4x4x4 = 256 bases
- A 6 base recognition sequence occurs every 4x4x4x4x4x4 = 4096 bases
Some nucleases produce overhangs
Give 2 examples of e.coli and KpnI
On image
Some nucleases give blunt ends
Give an example of Alu 1
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What 3 things are restriction enzymes used for?
- Cloning
- Molecular Diagnostics
- Characterization of plasmids
How can DNA molecules from different sources can be joined together
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How can Restriction Enzymes in Molecular Diagnostics and how can single nucleotide changes create. destroy enzyme restriction sites?
GO OVER THIS
What are restriction maps and why are they useful?
- Map of restriction sites within a molecule
- Crude way of mapping an unknown molecule
- Useful way of describing plasmids
Have a look at a restriction mapping example and analyse it
On pg 6
What does DNA ligase do?
Repairs nicks in phosphodiester backbone
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What does DNA polymerase do?
DNA synthesis in 5’ to 3’ direction
Why do we use DNA polymerase?
- PCR amplification
- Generation of probes.
- Blunt-ending of DNA overhangs.
What does Phosphatase do and why do we use it?
Hydrolyses a phosphate group off its substrate
• Calf intestinal alkaline phosphatase
• Shrimp alkaline phosphatase
Why use a Phosphatase?
To prevent cut plasmids from resealing.
What does Polynucleotide Kinase do?
Kinase: phosphate from ATP to substrate
ATP + SUBSTRATE —–> (Kinase) ADP+ Phosphorlyated-substrate
Polynucleotide kinase adds phosphate to 5’ hydroxyl group of DNA or RNA
Why use a Polynucleotide Kinase?
How can we label DNA?
To phosphorylate chemically synthesized DNA so that it can be ligated to another fragment.
To sensitively label DNA so that it can be traced using:
- radioactively labeled ATP
- fluorescently labeled ATP
What do probes do and how long are they
- Fragment of ssDNA (or RNA)
- 20 – 1000 bases in length
- Complementary to the gene of interest
On image
What does reverse transcriptase do?
Where is it isolated from?
- RNA dependent DNA polymerase
- Isolated from RNA-containing retroviruses
- Synthesizes a DNA molecule complementary to a mRNA template using dNTPs
On image
How can we prime for reverse transcription
Pg 10 of notes
