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2014 Genetics Topic 3: RG > L1 > Flashcards

L1 Flashcards

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1
Q

What was the recombinant DNA revolution based on?

A

DNA cloning

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2
Q

What is DNA cloning

A

the ability to propagate large amounts of same individual DNA fragments

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3
Q

how was DNA cloning made possible

A

made possible by the discovery of restriction enzymes, ligase, development of plasmid vectors and transformation

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4
Q

how is DNA cloned

A

amplified in PCR or cloning individual fragments using E.coli

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5
Q

What did Arber show

A
  • that phage DNA from different hosts had different methylation patterns
  • also showed that incoming DNA undergoes cleavage if it is from a different strain
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6
Q

What did Hamilton Smith do

A

he isolated an enzyme from e.coli that cut DNA at specific nucleotide sequences

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7
Q

What are the 1st hypothesis of Hamilton Smith

A

that the restriction in phage titre is the result of a “restriction enzyme” that cuts DNA at specific recognition sequences

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8
Q

What is the 2nd hypothesis of Hamilton Smith

A

that different strains of E.coli have restriction enzymes and methylation enzymes that recognize different sequences

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9
Q

what is the 3rd hypothesis of Hamilton Smith

A

that host DNA is protected from cleavage by its own restriction enzymes because it methylates the same sequences

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10
Q

true or false, did restriction modification systems evolve as a defense against phage

A

true

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11
Q

true or false, Arber, Nathans and Smith won the 1978 Nobel Prize in Medicine

A

true

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12
Q

What did Arber and co discover about the restriction modification systems

A
  • 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
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13
Q

Give a summary of the restriction modification systems

A
  • 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
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14
Q

true or false, RE have symmetric recognition sites

A

true

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15
Q

true or false, gel electrophoresis is an essential tool for cloning and different gel systems resolve different sized fragements

A

true

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16
Q

fragment size range for gel: acrylamide, type of DNA it sequences

A

1bp-1kb; ssdna

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17
Q

fragment size range for gel: agarose, type of DNA it sequences

A

0.5-20kb; plasmid cloning

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18
Q

fragment size range for gel: PFGE, pulsed gel field electrophoresis

A

50kb-10Mb

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19
Q

What is the recognition site of RE: Sau3A

A

GATC

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20
Q

what is the expected frequency of Sau3A

A

1/256 bp

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21
Q

What kind of cutter is Sau3A

A

4 cutter

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22
Q

What is the recognition site of EcoRI

A

GAATTC

23
Q

What is the expected frequency of EcoRI

A

1/4 kb

24
Q

what kind of cutter is EcoRI

A

6 cutter

25
Q

what is the expected frequency of NotI

A

1/65 kb

26
Q

what is the recognition site of NotI

A

G C GG CC G C

27
Q

what kind of cutter is NotI

A

8 cutter

28
Q

true or false, the frequency of cleavage is changed if GC content of organism is altered

A

true

29
Q

true or false, mammalian genomes are depleted for CG dinucleotide pairs (1/5 of expected)

A

true

30
Q

What questions do you ask when constructing a restriction map ?

A

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
31
Q

what is T4 DNA ligase

A

it can join ends of DNA together

32
Q

where did T4 DNA ligase come from

A
  • enzyme purified from cells infected with from bacteriophage T4
33
Q

how does T4 DNA ligase join the ends of DNA together

A
  • 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
34
Q

true or false, any EcoRI end can base pair with any other EcoRI end

A

true

35
Q

true or false, we can clone using sticky ends

A

true

36
Q

what are sticky ends

A

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.

37
Q

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)

A

true …

38
Q

how is directional cloning achieved

A

by using pairs of enzymes with different cohesive termini

39
Q

true or false, ligase can also join blunt termini, gives even more flexibility since all termini can be made blunt by DNA pol I

A

true

40
Q

true or false, the ligase activity of topoisomerase 1 is fast and efficient

A

true

41
Q

what kind of annealing enzyme do the new cloning variations use now

A

topoisomerase 1
site specific recombinases
long sticky ends: 20-40 bp overhanging ends anneal stably without ligase –> ligation in vivo

42
Q

What are the two methods used to introduce plasmid DNA into bacteria (DNA transformation)

A
  1. ) chemical treatments: Ca2+, Rb2+, heat shock

2. ) electroporation: Apply square pulse of current- “pores” in wall membrane

43
Q

what are the most common plasmid vectors

A

based on ColE1 (class of ColE1 is pMB1)

44
Q

what are the current plasmid vectors which are based on ColE1

A

pMB1
pBR322
pUC

45
Q

how many copy number do each of the 3 mentioned plasmid vectors (pMB1, pUC, pBR322) make

A

pMB1 : 15 copies/cell
pBR322 : 30 copies/cell
pUC: 200 copues/cell

46
Q

true or false, addition of lac alpha gives simple, blue/white screen for inserts; removal of unwanted RE sites

A

true

47
Q

true or false, pBR322 has Amp Tet

A

true

48
Q

true or false, pUC has Amp lac alpha

A

true

49
Q

what is a multiple cloning site (MCS) aka polylinker

A

a series of recognition sites for restriction enzymes placed back to back

50
Q

how are MCS made

A
  • 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
51
Q

true or false, insertional inactivation is used to identify clones with inserts

A

true

52
Q

true or false, when a blue colony is observed means that there is no DNA insert present because enzyme cleaved X gal

A

true

53
Q

true or false, if white colony is observed means that there is no cleavage of X gal, and DNA insert is present

A

true

2014 Genetics Topic 3: RG (10 decks)

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