Gene Tech - In Vivo Flashcards
What is in vivo cloning?
(In the body)
Transferring the fragments of DNA to a host cell using a vector
What do promoter regions do?
Tell RNA polymerase where to start.
What do terminator genes do?
Tell RNA polymerase where to stop
What does DNA ligase do?
Bind two bases that have lined up with sticky ends together
Why are restriction enzymes important?
Can cut DNA so that all fragments will have the same complementary ends.
What happens in the Isolation stage of in vivo cloning?
- DNA fragments are produced using restriction endonuclease.
- Promoter regions are added so that RNA polymerase and transcription factors can attach and begin transcription.
- Terminator regions are added to detach RNA polymerase.
What happens during the Insertion stage of in vivo cloning?
- DNA fragment is inserted into a vector
- This is used to transport the DNA into the host cell.
- Plasmids are commonly used - these are circular lengths of DNA, found in bacteria, and contain genes for antibiotic resistance.
- The same restriction endonucleases are used at one of these ABR genes to break the plasmid loop.
- DNA fragments mix with the opened up plasmid and become incorporated and the join is made permanent using DNA ligase.
Why is the same restriction endonuclease enzyme used to break the plasmid loop?
Ensures that the sticky ends of the opened-up plasmid are complementary to the sticky ends of the DNA fragment.
What happens during the Transformation stage of in vivo cloning?
- DNA fragment is mixed in a medium containing calcium ions and a change in temperature is brought which make the bacterial membrane permeable.
- This allows the plasmids to pass through the cell-surface membrane into the cytoplasm.
What happens during the identification stage of in vivo cloning?
- Marker genes are used are used to identify whether a gene has been taken up by bacterial cells.
There a three types: - Antibiotic resistant genes
- May make an easy to see fluorescent protein
- May produce an enzyme
How are antibiotic resistant marker genes used?
- These bacteria can be grown on a culture containing an antibiotic.
How are fluorescent marker genes used in in vivo cloning?
Bacterial cells that have not taken up the gene will not fluoresce.
How do enzyme markers in in vivo cloning work?
- This gene produces lactase which turns particular substrates blue.
- So when these bacterial cells are grown on a medium with these particular substrates, they will turn blue.
Advantages of in vivo cloning
- Gene therapy, useful when we want to introduce a gene into a living organism
- Almost no risk of contamination
- DNA copied is very accurate
- Precisely cuts out specific genes
- Can be used to produce large quantities of gene products, commercial and medical use
What are the stages of in vivo cloning?
Isolation
Insertion
Transformation
Identification
