Gene Cloning Flashcards
what are the two purposes of gene cloning?
produce a protein product or produce copies of the gene itself to transform other organisms
why is isolating genes difficult?
very large amount if genetic material and only small amount codes for genes
why is E.Coli commonly used in gene cloning?
has a higher ratio of genes to the length of the genome
how is a recombinant plasmid made?
DNA containing the gene and the plasmid are treated with the same enzyme, mixed together and joined by ligase
what are the two common types of vectors?
E.coli plasmids and bacteriophages
what characteristics does a vector DNA have?
ability to replicate independently, a detectable genetic marker, single sites for one of more restriction enzyme DNA inserted at a specific point
what is the ori site?
where replication begins in a plasmid
how does a bacterial cell protect its own DNA from restriction enzymes?
by adding methyl groups on the restriction sites
where does EcoR1 cleave?
GAATTC between the G and the A
what does Sma1 create?
blunt ends at the restriction site
what do restriction enzymes cut?
the covalent phosphodiester bonds between both strands of DNA
why is E.Coli K-12 a common host of choice?
easily grown and amenable to metabolic studies and highly attenuated
what two important genes does an engineered plasmid have?
ampicillin resistance and lacZ
what does the lacZ gene code for?
beta galactosidase
where is the restriction site in a pUC19 plasmid?
in the lacZ gene, deactivating it
how can cells that have taken up a plasmid be identified?
they form colonies as they will be able to survive ampicillin in the agar
how are colonies that have taken up a transformed plasmid identified?
the transformed plasmid will not have a functioning lacZ gene and so will not hydrolyse the X-Gal sugar in the agar, forming a white colony not blue
