IMED1002 Study Guide - Final Guide: In Situ Hybridization, Southern Blot, Dna Ligase
MANIPULATING DNA II
Define recombinant DNA
DNA from 2 sources that are isolated, then inserted into a bacterial cell for its own machinery to
replicate genetic material in it.
To make recombinant DNA in vivo (within bacterial cells), a DNA fragment can be inserted into a
vector which can be used to carry and amplify the gene of interest. This insertion requires the use of
REs (restriction enzymes). Type II REs cut small DNA at specific sequences that are usually 4 to 6 base
pairs long and that are palindrome.
REs are endouleases ad therefore ut y digestig DNA at iteral phosphodiester bonds. This
leaves behind the 3 OH and 5” P groups meaning that fragments with ____________ ends can
anneal. The final step makes use of the enzyme DNA ligase which covalently links these two groups
therefore re-establishing the _____________ ______.
Describe how REs are named and the difference between blunt and sticky ends
Derive from name of bacteria, where:
First letter = genus
Second letter = species
Additional letters = strains
Number = numerical order of discovery
Blunt ends = when DNA cut by RE is straight across ad a e joied together eve if its ot
complimentary base paired
Sticky ends = when DNA cut by RE creates overhangs, which reanneals by complimentary base
Define:
Cloning – In vivo: tracking the replication machinery of bacterium into amplifying recombinant DNA
containing the gene, creating lots of copies of DNA of interest
Selectable marker – it is AB resistant and provides a way to selet the ateria thats otaied i
plasmid
MCS – Region with several RE recognition sequences that provide cloning sites for insertion of DNA
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Document Summary
Dna from 2 sources that are isolated, then inserted into a bacterial cell for its own machinery to replicate genetic material in it. To make recombinant dna in vivo (within bacterial cells), a dna fragment can be inserted into a vector which can be used to carry and amplify the gene of interest. Type ii res cut small dna at specific sequences that are usually 4 to 6 base pairs long and that are palindrome. Res are endo(cid:374)u(cid:272)leases a(cid:374)d therefore (cid:858)(cid:272)ut(cid:859) (cid:271)y digesti(cid:374)g dna at i(cid:374)ter(cid:374)al phosphodiester bonds. This leaves behind the 3(cid:859) oh and 5 p groups meaning that fragments with ____________ ends can anneal. The final step makes use of the enzyme dna ligase which covalently links these two groups therefore re-establishing the _____________ ______. Describe how res are named and the difference between blunt and sticky ends.
