Vectors lecture Flashcards
(13 cards)
What is a vector?
A molecule that is used to transport cloned sequences between biological hosts and the tube.
Common properties of vectors
Ability to promote autonomous replication
Contain a gene marker (usually dominant) for selection
Unique restriction sites to facilitate cloning of insert DNA
minimum amount of non-essential DNA to optimise cloning
Describe plasmids
Bacterial cells contain small circular DNA.
Plasmid vectors are used to clone DNA ranging in size from several base pairs to several thousands of base pairs.
What do plasmid vectors contain
A gene that permits selection, a selectable marker
Selective gene ampr which encodes the enzyme b-lactamase which encodes the enzyme b-lactamase which inactivates ampicillin
Lac-Z
Disadvantages of plasmids
Fragments must be small
Standard methods of transformation are inefficient
Where are genes introduced into cloning vectors?
At one of the restriction sites present in the polylinker.
Bacteriophage lambda: features, lambda viral genome
head, tail, tail fibres
DNA with a 12 base ssDNA sticky ends at both ends which are complementary in sequence and can hybridise to each other.
How does bacteriophage lambda insert its DNA?
Lambda tail fibres absorb to a cell surface receptor. The tail contracts and the DNA is injected.
The DNA circularises at the cos site and the lambda begins its life cycle in the E.coli host.
Cosmid vectors
Cosmids are plasmids with one or two Lambda Cos sites. Clone large inserts
Presence of Cos site permits in vitro packaging of cosmid DNA into lambda particles
Advantages of cosmid vectors
Strong selection for cloning or large inserts
Infection process rather than transformation for entry of chimeric DNA into E.coli host
Maintain cosmids as phage particles in solution
Retroviral vectors
Used to introduce new or altered genes into the genomes of human and animal cells.
Retroviruses are RNA viruses.
The viral RNA is converted into DNA by the viral reverse transcriptase and is efficiently integrated into the host genome.
Any foreign or mutated host gene introduced into retroviral genome will be integrated into the host chromosome and can reside there indefinitely.
Retroviral vectors are widely used to study oncogenes and other human genes.
What is binding of primers affected by?
MgCl2 concentration
Temperature (increased specificity at higher temperature)
Yeast artificial chromosomes
Linear DNA vectors that resemble a normal yeast chromosome. Contain telomeres that stabilise chromosome ends and centromeres that ensure chromosome partitioning between two daughter cells and a selective marker gene
