DNA cloning Flashcards
what is DNA cloning?
method to amplify a specific piece of DNA using a bacterial cell
what is a plasmid?
small circular piece of DNA that is extra chromosomal
predominantly found in bacteria
provide antibiotic resistance & increase virulence
what are cloning vectors?
a DNA molecule, usually a plasmid, that is able to:
incorporate a foreign DNA fragment
be introduced into a cell & replicate
what are the 3 features of cloning vectors that make it easier to insert DNA?
origin of replication
antibiotic resistant gene
cloning sites
what is the purpose of have an origin of replication in a plasmid?
enables recognition by DNA replication enzymes that enable a plasmid to reproduce itself
allows amplification of plasmid
what is a multiple cloning site?
a DNA region that contains many restriction enzyme recognition sites next to each other
what are multiple cloning sites also known as?
polylinker sites
DNA cloning and restriction process
cut the cloning vector with a restriction endonuclease
cut DNA of interest with same restriction endonuclease
mix the cut cloning vector & DNA of interest together
ligate fragments together using DNA ligase
insert ligated DNA into bacteria
grow host cells under selective conditions on agar plate containing antibiotic
extract amplified recombinant plasmid DNA & analyse
how do corresponding sticky ends align
through hydrogen bonding
why dont bacteria destroy their own DNA with their restriction enzymes?
they methylate their DNA which masks the restriction site from being recognised by the restriction enzymes
where do restriction enzymes usually cut?
at palindromic sequence
DNA ligation process
incubate cut fragments of vector & insert DNA with DNA ligase
DNA ligase creates covalent phosphodiester bonds to join different DNA molecules
ATP needed
transformation process
E.coli made competent to take up DNA by treatment with magnesium chloride
cells are kept cold to prevent pores closing
mix E.coli cells with DNA & heat shock to 42 degrees celsius for plasmid DNA uptake from medium
plasmid replication then occurs
what are the possible products of transformation?
plasmid and insert = ampicillin resistant
plasmid without insert = ampicillin resistant
no plasmid = no ampicillin resistance = no colonies
what are the different screening techniques to find the recombinant plasmids containing the desired DNA?
blue/white screening colony PCR restriction mapping functional screening southern blot analysis DNA sequencing
