L1 Flashcards
What was the recombinant DNA revolution based on?
DNA cloning
What is DNA cloning
the ability to propagate large amounts of same individual DNA fragments
how was DNA cloning made possible
made possible by the discovery of restriction enzymes, ligase, development of plasmid vectors and transformation
how is DNA cloned
amplified in PCR or cloning individual fragments using E.coli
What did Arber show
- that phage DNA from different hosts had different methylation patterns
- also showed that incoming DNA undergoes cleavage if it is from a different strain
What did Hamilton Smith do
he isolated an enzyme from e.coli that cut DNA at specific nucleotide sequences
What are the 1st hypothesis of Hamilton Smith
that the restriction in phage titre is the result of a “restriction enzyme” that cuts DNA at specific recognition sequences
What is the 2nd hypothesis of Hamilton Smith
that different strains of E.coli have restriction enzymes and methylation enzymes that recognize different sequences
what is the 3rd hypothesis of Hamilton Smith
that host DNA is protected from cleavage by its own restriction enzymes because it methylates the same sequences
true or false, did restriction modification systems evolve as a defense against phage
true
true or false, Arber, Nathans and Smith won the 1978 Nobel Prize in Medicine
true
What did Arber and co discover about the restriction modification systems
- enzyme activities were discovered from the ‘restriction’ of virus titre during infection between bacterial strains
- the recognition sites for DNA cleavage and methylation were sequence specific and identical within strains
- Both REs and methylating enzymes were purified and characterised biochemically
Give a summary of the restriction modification systems
- R-M systems evolved as bacterial defence system against bacteriophages
- Host MEs methylate cellular DNA (including phage) at specific sequences
- Host REs cleave DNA at specific sequences
- The recognition sequences of the REs and MEs are the same for any strain of E.coli
- Methylated DNA is protected from cleavage by its own RE
- During replication, hemi-methylated DNA is recognized and the new strand is methylated
- Viral infection of a new strain leads to a “race” between host restriction systems and host modification systems to recognise their sites
- only in very rare cases does methylation win and protect the incoming phage DNA so that it can replicate
true or false, RE have symmetric recognition sites
true
true or false, gel electrophoresis is an essential tool for cloning and different gel systems resolve different sized fragements
true
fragment size range for gel: acrylamide, type of DNA it sequences
1bp-1kb; ssdna
fragment size range for gel: agarose, type of DNA it sequences
0.5-20kb; plasmid cloning
fragment size range for gel: PFGE, pulsed gel field electrophoresis
50kb-10Mb
What is the recognition site of RE: Sau3A
GATC
what is the expected frequency of Sau3A
1/256 bp
What kind of cutter is Sau3A
4 cutter
What is the recognition site of EcoRI
GAATTC
What is the expected frequency of EcoRI
1/4 kb
what kind of cutter is EcoRI
6 cutter
what is the expected frequency of NotI
1/65 kb
what is the recognition site of NotI
G C GG CC G C
what kind of cutter is NotI
8 cutter
true or false, the frequency of cleavage is changed if GC content of organism is altered
true
true or false, mammalian genomes are depleted for CG dinucleotide pairs (1/5 of expected)
true
What questions do you ask when constructing a restriction map ?
for multiple RE digests:
- linear or circular?
- estimate total size
- estimate # cuts per RE
- use single cutter as anchor point
- identify differences in digests +/- anchor RE
- draw possible map and check
what is T4 DNA ligase
it can join ends of DNA together
where did T4 DNA ligase come from
- enzyme purified from cells infected with from bacteriophage T4
how does T4 DNA ligase join the ends of DNA together
- reconnects phospho-diester bonds between nucleotides
- only requirement is a free 3’-OH and a free 5’-PO4
- blunt or annealed sticky ends are substrates
- need (ribose) ATP as energy source
- Mg2+ as cofactor
true or false, any EcoRI end can base pair with any other EcoRI end
true
true or false, we can clone using sticky ends
true
what are sticky ends
a single-stranded end of DNA or RNA having a nucleotide base sequence complementary to that of another strand, enabling the two strands to be connected by base pairing: produced in the laboratory with the use of restriction enzymes for genetic engineering purposes.
true or false, for plasmids and inserts cut with the same enzyme, any restriction fragment can be cloned in any orientation (for typical RE like EcoRI)
true …
how is directional cloning achieved
by using pairs of enzymes with different cohesive termini
true or false, ligase can also join blunt termini, gives even more flexibility since all termini can be made blunt by DNA pol I
true
true or false, the ligase activity of topoisomerase 1 is fast and efficient
true
what kind of annealing enzyme do the new cloning variations use now
topoisomerase 1
site specific recombinases
long sticky ends: 20-40 bp overhanging ends anneal stably without ligase –> ligation in vivo
What are the two methods used to introduce plasmid DNA into bacteria (DNA transformation)
- ) chemical treatments: Ca2+, Rb2+, heat shock
2. ) electroporation: Apply square pulse of current- “pores” in wall membrane
what are the most common plasmid vectors
based on ColE1 (class of ColE1 is pMB1)
what are the current plasmid vectors which are based on ColE1
pMB1
pBR322
pUC
how many copy number do each of the 3 mentioned plasmid vectors (pMB1, pUC, pBR322) make
pMB1 : 15 copies/cell
pBR322 : 30 copies/cell
pUC: 200 copues/cell
true or false, addition of lac alpha gives simple, blue/white screen for inserts; removal of unwanted RE sites
true
true or false, pBR322 has Amp Tet
true
true or false, pUC has Amp lac alpha
true
what is a multiple cloning site (MCS) aka polylinker
a series of recognition sites for restriction enzymes placed back to back
how are MCS made
- made in vitro using oligo synthesizer
- allows many RE fragments to be cloned in a single vector
- MCS is inserted in frame at the beginning of the lac coding region without disrupting it
true or false, insertional inactivation is used to identify clones with inserts
true
true or false, when a blue colony is observed means that there is no DNA insert present because enzyme cleaved X gal
true
true or false, if white colony is observed means that there is no cleavage of X gal, and DNA insert is present
true
