Week 4 (genetic Engineerig And Cloing) Flashcards
To work with a gene, it must be:
- pure
- homogeneous
- available in sufficient quantity
What is gene cloning?
Cloning is making many identical copies (of the same gene)- The amplification of a particular DNA sequence.
The term also describes the isolation of a particular stretch of
DNA from the rest of the cells genome
1 bacterial colony=
1 clone
What are the properties of a good vector?
- Be independent of the host chromosome
- Be copied by the cell
- Be maintained throughout the population of cells (every cell contains copies of the vector)
- Allow selection for cells containing the vector (as opposed to those that do not)
- It is beneficial if there is a secondary selection system which allows identification of cells containing recombinant vector
What criteria must the vector meet to have beneficial properties?
(1+2) Vectors must have an origin of replication
(3) Vectors carry a defined copy number
(3+4) Vectors carry a selectable marker e.g. antibiotic resistance
(5) vectors carry an additional selectable marker, which is inactivated by the insertion of a foreign gene
How is the vector cut?
- DNA can be cut at specific sequences by enzymes called restriction endonuclease to create linear DNA molecules
- The vectors are engineered to have just one recognition site for each group of restriction enzymes
- These recognition sites are grouped close together at one location in the plasmid (this will allow for secondary selection later on)
- Creation of single stranded sticky ends with a 5’ overhang (GATC)
- DNA ligase repeals phosphodiester bonds
- Sequences of the sticky ends of the gene complement the sequence of the plasmids sticky ends
- Transformation: puts plasmid DNA inside a cell using ice cold CaCl2 which is then heat shocked(this process is very inefficient- <1 in 10,000)
- Vector replication in which multiple copies of the same vector are made inside the cell according to the copy number encoded on the plasmid
- Selection/ cell multiplication so only cells contains the plasmid can grows
Give an example of a restriction site
BamHI
Explain secondary selection in the process of cloning?
- The restriction sites are located within a non-essential gene that produces an observable phenotype e.g betagalactosidase, which makes a blue colour
- if the plasmid re-ligands to itself in the absence of the gene, the beta galaxtosidase gene is reformed and the colony will be blue
- if the gene is inserted into the restriction site, the betagalactosidase gene (restriction site) will be disrupted and the cell will be white
Where is the restriction site cleaved by endonucleases found?
In the betagalactosidase (blue) gene not the antibiotic resistance gene
What is self ligation?
Reformation of the original plasmid without incorporating foreign DNA
What are some uses of gene cloning?
Expression- the gene product may be therapeutic, or commercially useful
Investigation- gene sequence
Manipulation- mutation of certain bases to examine how the encoded protein works
How is the bacterium’s own DNA protected from cleavage?
By methylation
Why are restriction nucleases so useful?
The enzyme will always cut a particular DNA molecule at the same sites therefore for a given sample of DNA, a particular restriction nuclease will reliably generate the same set of DNA fragments
What are plasmid vectors?
Small, circular molecules of double stranded DNA derived from plasmids that occur naturally in bacteria cells
How can the clone DNA fragment be readily recovered?
By cutting it out of the plasmid DNA with the same restriction endonuclease that was used to insert it and then separating it from the plasmid by gel electrophoresis
