L10 - lab tools for DNA manipulation Flashcards
(22 cards)
what are the 5 basic steps in gene cloning
- contruction of recombinant DNA molecule/plasmid
- Transport into the host cell
- multiplication of plasmid/DNA molceule in bacteria
- division/proliferation of bacterium host cell
- produces clones with identical copies of the recombinant DNA
= bacteria is essentially used as a factory to replicate the DNA
is the contruction of the recombinant DNA plasmid in vivo or in vitro
in vitro
= outside of the cell in a test tube
CRISPR is in vivo on the other hand as it happens inside the cells
what are the functions of these key enzymes:
1. exonuclease
2. restriction endonuclease
3. phosphatase
4. ligase
5. DNA polymerase
exonuclease. = shortening
restriction endonuclease = cutting
phosphatase = modifying
ligase = joining
DNA polymerase = lengthening
compare the effets of DNase 1 and a restriction endonuclease
DNase cuts both aingle and doub;e-stranded DNA at non-specific locations with a PREFERENCE of C and T bases
Restriction endonuclease cuts double stranded DNA at specific locations (restriction site)
= one produces lots of random cuts and one can only can at certain/specific locations
what are the components of the restriction/m0dification defence system obf bacyteria against phages
Endponuclease = cleaves and cuts foreign DNA inserted
methyltransferase = methylates host DNA preventing endonucleases acting on itself
which TYPE of restriction endonucleases do we mostly use for gene editing and how do they work
Type 2 REs
cuts DNA at defined positions close to or within their recognition sequnces
= simple to use
what determines the name given to a Restriction endonuclease
which microorganism the enzyme was discovered in
classed into different TYPES according to enzytmatic behavior
what type of sequnces do type 2P REs recognise
palindromic sequences 4-8bps in length
= same sequnce in one direcetion as it is on the other strand in the opposite direction
what comformation do type 2 REs form to bind and cleave double stranded DNA
homodimer
2 identical proteins bind togther prior to binding to DNA
bind in opposite directions to allow the palindromic sequnce on both strands to be recognised and cleaved
what occors when the homodimer type 2P restriction endonuclease binds to DNA
bends the DNA 50° , widens minor groove and compresses major groove
= causes unstacking of the bases
unstacking is when the base pairs come apart and the ‘stacked coin’ formation of the base apir on top of one another come apart to reveal areas of DNA for editing
what does widening of the minor groove when a T2P RE binds allow/cause
brings phosphodiester bonds/linkages ckoser to the active site of each monomer enzyme
what is the only T2 RE to cause major bending when it binds to DNA
EcoRV
what type of ends (sticky or blunt) are created from EcoRV
blunt ends
= both monomers cut at the same point on each strand
what is an ‘ambigious’ sequence
simiular but not identical sequence
what is ‘Star’ activity of some restriction enzymes
cut similiar/ambigious sequnces but not identiocal to their recogntion sequence
how large are type 2S restriction endonucleases
400-600 amino acids in length
= slightly larger than Type 2p
what do type 2S RE recognise and compare this to Type 2P
asymetric DNA sequences
= do not read the same forwards and backwards
= each strand is a different sequnence
this is compared to the palindromix sequnces recognised by T2P
how do Type 2S RE bind and cleave
bind as a monomer and form ‘transient homodimers’
have seperate recognition and cleavage sites unlike T2P
the 2 monomers can bind to sequnces far apart from one another
briefly dimerise causing a loop to form of the inbetween DNA and allowing cleavage (as a homodimer) to occor
what is a key point about Type 2S REs that seperates them from T2P
seperate recogntion and cleavage domnains
linked togther by a short polypeptiude connector
= causes ‘shifted cleavage’ to one side of the recognised sequence
what do the sticky ends produced by Type 2s redsult from - 3 points
the sequnce of the overhang depnds on whatever DNA bases are downstream from where the cleavage occurs
Different Type IIS enzymes cut different distances away from the recognition site depending on the ‘reach’ of the enzyme
Because of the helical shape and flexibility of DNA, the exact cut site might shift by a base or two.
summarise differences between Type 2P and 2S enzymes
2P:
recongition and clevagae domain are the same = cuts within the target sequnce
blunt ends
palindromic sequnces
binds as a homodimer pre-formed
2P:
recognition and clevage domains are seperate held togther by ‘connector’= cuts outside the target sequnce
sticky ends depnding on enzymes ‘reach’
binds as monomers do DNA and forms ‘transient homodimers’
causes DNA looping
asymmetrical sequences
