Recombinant DNA and Cloning Vectors Flashcards
What are non-primate lentiviruses used for?
β vectors used to integrate DNA in mammalian cells
What are baculoviruses used for?
β vectors used in combination with recombinant expression in insect cells
What are artificial chromosomes used for?
β introducing large segments of DNA
β Used because large pieces of DNA are unstable and unlikely to be incorporated into plasmids
What are plasmids?
β Discrete circular dsDNA molecules found in many but not all bacteria
β Are a means by which genetic information is maintained in bacteria
β genetic elements (replicons) that exist and are replicated independently of the bacterial chromosomes
What can plasmids be exchanged between?
β bacteria within a restricted host range
What are vectors?
β A piece of DNA that is circular and foreign DNA can be inserted within this
How are vectors used?
β The plasmid is cut so the ends of the plasmid are complementary with the PCR product
β piece of DNA can be ligated
What are the 6 important features of plasmid vectors?
1) they can be linearised at one or more sites in non-essential stretches of DNA
2) can have DNA inserted into them
3) can be re-circularised without loss of the ability to replicate
4) are often modified to replicate at high multiplicity within a host cell
5) Contain selectable markers
6) relatively small in size
What are the steps to use a bacterial plasmid as a vector?
β Linearise it at a particular restriction site
β generate a PCR product of the gene you want which is then restricted
β Include within the primer sequence of the gene a restriction enzyme site
β plasmid is restricted to allow insertion of a DNA product
β gene is then ligated
How do you select the plasmids that have taken up the gene?
β The plasmid can be put into e.coli
β It is then plated onto agar containing antibiotic that corresponds to the antibiotic resistance gene that has been inserted
β only the plasmids that contain the gene will grow and form colonies
β The colony can then be cultured and isolated
β confirm insertion by restriction mapping a clone
Give three reasons why plasmids are used as recombinant tools?
β Plasmids can express a recombinant gene in a living organism of choice
β you can add or modify control elements
β alter properties of the gene product
What are 5 recombinant proteins in clinical use?
β Human insulin β Interferons β Erythropoietin β Factor XIII β Tissue plasminogen activator
What is the effect of adding control elements to a plasmid?
β Make genes inducible or express the gene to high levels
What are the requirements for cloning a defective gene to be expressed in large amounts in bacteria?
β Ability to replicate in bacteria β Maintained at a high copy number β modified origin of replication β selectable (has an antibiotic marker) β Ampicillin resistance gene β Easy to manipulate - cut and rejoin β Multiple cloning site (MCS)
What control elements are needed for expression in bacteria?
β Shine dalgarno sequence (ribosomal binding site for prokaryotes)
β Bacterial promoter
β Transcriptional terminator