Mod 10 - Genetic engineering Flashcards
basic principles of molecular cloning
cutting
joining
and propagating recombinant DNA
one way that molecular cloning is useful
reduces complexity of DNA
since u can choose specific sequences and propagate them
to allow large scale production and analysis of purified single sequences
basic steps of molecular cloning
isolate DNA
cut DNA
join to vector (DNA plasmid) = makes recombinant
put recombinant vector into bacteria
and let bacteria grow to amplify recombinant DNA
what is used to cut DNA
restriction endonuclease
cuts palindromic sequence
- this gives sticky ends (5’ overhang bits, see onenote) that can be rejoined
this is temporarily rejoined until ligase comes to covalently join the plasmid and the DNA fragment
what are the three things that can form after an attempt at this molecular cloning technique
desired product = recombinant plasmid
undesired:
unmodified plasmid (very common since the cut ends of the fragment are in close proximity)
or
circularised DNA fragment (usually not a problem cuz it very rarely acc replicates)
how do we prevent the cut ends of the plasmid from just joining back to itself
treat it with phosphatase
cuts of the phosphate ends
how do you make sure that during proliferation of the plasmid, that only the recombinant one is replicated
grown in antibiotic
and the recombinant one holds antibiotic resistance
so all other die
but recombo lives and replicates
two types of DNA libraries
nuclear DNA - (genomic library)
- contains all genes
cDNA library (from mRNA - ie c stands for complementary DNA)
- only expressed genes
- hence why it is cell type specific
- must be converted to DNA to be clone
how to make cDNA library
isolate the mRNA from cytoplasm of a cell
convert it to cDNA via reverse transriptase enzyme
insert it into vector (plasmid) and put it into carrier bacteria
each bacteria can oly take up one recombinant plasmid
so when u grow colonies, each colony represents 1 mRNA sequence
how to make genomic DNA
isolate DNA from nucleus
cut the DNA snip snip via restriction ednonuclease
this is gonna make large fragments (generally 50 kb each)
then put into vector (plasmid)
put into bacteria
and each colony once again, represents a small fragment of the genome
can be identified by DNA hybridisation
see onenote for slide on key differences between cDNA and genomic
:)
Why did we need to use recombo dna in treatment rather than just getting proteins and using those
hard to obtain
Usually from other organisms
So cause immunological problems
And hard to isolate these problems
how do we ensure we have control over the gene’s protein production when making a recombo gene (specificlly for treatments)
ligate the gene downstream from a strong promoter
e.g. lac promoter
something we can easily manipulate
advantages and disadvantages of using bacteria as a host system
adv:
- cheap
- fast
- easy to maintain
-stable
disadv:
- proteins may be insoluble/denatured
- no post tranlational modification like some proteins usually need
advantages and disadvantages of using animal cells as a host system
adv:
- post-translational modification
- soluble, properly folded
disadv:
- expensive
- unstable
