6717954
Description
Mind Map by fionnghuala hammon, updated more than 1 year ago
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Created by fionnghuala hammon
about 9 years ago
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IN VIVO CLONING
- What is in vivo cloning
- isolation and the amplification of an individual gene sequence
- by insertion of that sequence into an organism where it can be replicated
- involves the construction of novel DNA molecules by joining DNA from different sources
- product is recombinant DNA
- product is recombinant DNA
- involves the construction of novel DNA molecules by joining DNA from different sources
- by insertion of that sequence into an organism where it can be replicated
- isolation and the amplification of an individual gene sequence
- why clone DNA
- A particular gene can be isolated and its nucleotide sequence determined
- Control sequences of DNA can be identified and analysed
- Protein, Enzymes, RNA functions can be investigated
- Mutations can be identified e.g. gene defects
- Organisms can be engineered for specific purpose
- Organisms can be engineered for specific purpose
- Mutations can be identified e.g. gene defects
- Protein, Enzymes, RNA functions can be investigated
- Control sequences of DNA can be identified and analysed
- A particular gene can be isolated and its nucleotide sequence determined
- how is DNA cloned
- Cell based DNA cloning
- Cell free DNA cloning e.g. PCR
- Cell based DNA cloning
- restriction enzymes
- role of restriction enzymes
- only found in prokaryotes
- endonucleases and ubiquitous
- endonucleases and ubiquitous
- over 100 enzymes have bee nisolated from bacteria
- nomenclature e.g. Eco RI and
- nomenclature e.g. Eco RI and
- why do bacteria produce these enzymes
- Protection from foreign DNA--- phages (bacterial viruses)
- Protection from foreign DNA--- phages (bacterial viruses)
- only found in prokaryotes
- role of restriction enzymes
- restriction endonucleases
- host enzymes that prevent the invasion of foreign DNA such as viral DNA by cutting them up
- Restriction
- Restriction
- these enzymes cut away at the foreign DNA rather than chewing them away from the ends
- Endo nucleuses
- Endo nucleuses
- these enzymes recognise a specific DNA sequence (4-12bp) which is twofold symmetry and cut both DNA strands
- host enzymes that prevent the invasion of foreign DNA such as viral DNA by cutting them up
- Vector features
- must contain a replicon that enables it to replicate host cells
- Several Marker genes
- Unique cleavage sites
- for expression must contain control elements such as promoters terminators ribosome binding sites
- for expression must contain control elements such as promoters terminators ribosome binding sites
- must contain a replicon that enables it to replicate host cells
- vectors
- must have an origin of replication
- allow the vector as well as the DNA to amplify the host cell
- antibiotic resistant genes
- allow host cell to grow on selective media
- can selectively amplify this specific vector in the host cell
- multiple cloning sites
- allow insertion of foreign DNA
- must have an origin of replication
- cloning a gene in a bacteria plasmid
- isolation of vector and gene source DNA
- insertion of DNA in vector
- introduction of cloning vector into cells
- cloning of cells and foreign genes
- identification of cell clonescarrying the gene of interest
- identification of cell clonescarrying the gene of interest
- cloning of cells and foreign genes
- introduction of cloning vector into cells
- insertion of DNA in vector
- isolation of vector and gene source DNA
- DNA can be inserted into the cell by...
- Transformation
- Electroporation
- Protoplast fusion
- Microinjection
- Gene gun
- Transformation
- disadvantages to using bacterial host cells
- May not be able to use information in a eukaryotic gene
- due to prokaryotes and eukaryotes use different enzymes and regulatory mechanisms during transcription and translation
- due to prokaryotes and eukaryotes use different enzymes and regulatory mechanisms during transcription and translation
- cant make the post-translational modifications required to produce some eukaryotic proteins
- May not be able to use information in a eukaryotic gene
- SLIDES 27-33 ARE NOT INCLUDES IN THIS CONEPT MAP
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