AMB4 Flashcards
(26 cards)
Describe single site RE cloning?
identical RE sites flanking insert DNA and vector.
Ligation catalaysed by DNA ligase.
what considersations have to be taken into account in single site RE cloning?
- RE site must be in correct place and no where else.
- Inserts may ligate in opposite orientations
- vector can religate with self - empty vectors
If RE site is not naturally present how can it be added?
site directed mutagenesis
How can you stop religation to prevent empty vectors?
treat vector with alkaline phosphatase, in theory wont ever religate, in practise dont get 100% elimination and can end up with nicked molecule with one strand fused.
- or can also increase insert:vector ratio
why can we ignore the possibility of inserts ligating to each other in single site RE?
there would be no ori so would not be translated.
Describe compatible ends RE cloning?
Makes compatible ends by using two enzymes which create the same sticky ends; therefore being compatible and so can be ligated together. Resulting product will be immune from restriction by original enzymes
What is an example of 2 enzymes that can be used in double enzyme compatible ends cloning?
BamHI - G^GATCC
Bg1II - A^GATCT
Resulting site will be A^GATCC
Describe RE cloning using blunt end cutters?
all termini generated are compatible for ligation = universally compatible ends.
What are the drawbacks of blunt end cutter RE cloning?
- not very efficient - high substrate conc req
- High probability of ligation of empty vectors.
- Inserts will insert in both orientations.
What are 3 examples of blunt end RE cutters?
EcoRV, Hpal, Smal
Describe Golden Gate assembly
Multiple inserts assembled in vector using type11S RE and DNA ligase.
takes adv of type11S RE ability to cut outside of recognition sequence.
Create non palindromic overhangs
What are the advantages of golden gate assembly?
- overhang sequence not dictated by RE, no assembly scar.
- fragment specific sequence of the overhangs allows orderly assembly of multiple fragments
- Restriction site eliminated so digestion and ligation can occur simultaneously. also allows process to be repeated as restriction site not in final plasmid = adds to efficiency.
What are two considerations of golden gate assembly?
PCR amplifcation of inserts means must allow for mutations
inserts and vectors need to be devoid of RE site used.
Describe PCR T/A cloning
under certain condition taq polymerase adds an adenosine to the end.
get adenosine overhang which can ligate to a t overhang on vector.
fast as relies on generation of PCR products that can be used directly.
What does topoiosmerase 1 do, how can this be exploited?
recognises CCCTT sequence and cleaves it
under appropriate conditions reaction generates a c covalent bond between the enzyme and DNA strand via a diphosphoester bridge.
Describe PCR Topo T/A cloning
vector used already contains 3’T linked topoisomerase via a phosphodiester bond at each end of plasmid.
make adenylated insert.
ligation catalysed by topoisomerase itself not by standard ligase
vector wont religate as ends are not compatible.
Describe PCR overlap/extention cloning?
Insert is PCR amplified with chiimeric primers, so final products have overlapping regions with the vector.
vector and insert are then mixed, denatured and annealed.
hybrised insert is then extended by phusion DNAP using vector as template until reached 5’ of insert.
after several PCR cycles the new plasmid with twwo nicks gets accumulated as product.
insert DNA acts like primer and binds to vector, extended by taq
new product transformed into e.coli after Dpnl destroys parent plasmid.
what is a limit of PCR overlap/extention cloning?
number of colonies showing desired gene decreases with insert length.
Describe sequence and ligation independent cloning SILC?
exploits 3’ exonuclease activity of T4 polymerase
treat PCR seq of interest with T4 DNAP under no NTP conditions.
3’ exo activity
design in primer sequences compatibble with vector treated in same way
long overhands created
mix plasmid and vector and they hybridise
nicks are repaired in e.coli once transformed.
How can SILC be simialr to golden gate cloning?
can ligate large number of different inserts in a specific order.
Describe the formation of an entry vector in Gateway cloning
att sequences derived from lambda bacteriophage
- attB1 and attB2 seqa are added to 5’ and 4’ end of gene frag respectively. using gene specific PCR primers and PCR amplification.
- products are mixed with plasmids - donor vectors and BP clonase enzyme mix.
- enzyme mix catalyses recombination and insertion of attB seq contain PCR product inot attP recombination site in vector.
- once casette part of plasmid = entry vector
Which cycles are each clonase used in?
LR = lytic BP = lysogenic
Once the entry vector has been formed in gateway cloning, how can it be used>?
inserts can then be shared with any vectors containing attB/attP sequence by recombiantion, prodcuts are empty entry clone and an expression clone.
can shuffle inserts back and forward between vectors.
In gateway cloning which sites match?
attL1/attL2 and attR1/attR2
attP1/attP2 and attB1/attB2
