L1

Question 1

What was the recombinant DNA revolution based on?

Answer 1

DNA cloning

Question 2

What is DNA cloning

Answer 2

the ability to propagate large amounts of same individual DNA fragments

Question 3

how was DNA cloning made possible

Answer 3

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

Question 4

how is DNA cloned

Answer 4

amplified in PCR or cloning individual fragments using E.coli

Question 5

What did Arber show

Answer 5

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

Question 6

What did Hamilton Smith do

Answer 6

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

Question 7

What are the 1st hypothesis of Hamilton Smith

Answer 7

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

Question 8

What is the 2nd hypothesis of Hamilton Smith

Answer 8

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

Question 9

what is the 3rd hypothesis of Hamilton Smith

Answer 9

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

Question 10

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

Answer 10

true

Question 11

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

Answer 11

true

Question 12

What did Arber and co discover about the restriction modification systems

Answer 12

• 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

Question 13

Give a summary of the restriction modification systems

Answer 13

• 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

Question 14

true or false, RE have symmetric recognition sites

Answer 14

true

Question 15

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

Answer 15

true

Question 16

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

Answer 16

1bp-1kb; ssdna

Question 17

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

Answer 17

0.5-20kb; plasmid cloning

Question 18

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

Answer 18

50kb-10Mb

Question 19

What is the recognition site of RE: Sau3A

Answer 19

GATC

Question 20

what is the expected frequency of Sau3A

Answer 20

1/256 bp

Question 21

What kind of cutter is Sau3A

Answer 21

4 cutter

Question 22

What is the recognition site of EcoRI

Answer 22

GAATTC

Question 23

What is the expected frequency of EcoRI

Answer 23

1/4 kb

Question 24

what kind of cutter is EcoRI

Answer 24

6 cutter

Question 25

what is the expected frequency of NotI

Answer 25

1/65 kb

Question 26

what is the recognition site of NotI

Answer 26

G C GG CC G C

Question 27

what kind of cutter is NotI

Answer 27

8 cutter

Question 28

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

Answer 28

true

Question 29

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

Answer 29

true

Question 30

What questions do you ask when constructing a restriction map ?

Answer 30

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

Question 31

what is T4 DNA ligase

Answer 31

it can join ends of DNA together

Question 32

where did T4 DNA ligase come from

Answer 32

- enzyme purified from cells infected with from bacteriophage T4

Question 33

how does T4 DNA ligase join the ends of DNA together

Answer 33

- 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

Question 34

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

Answer 34

true

Question 35

true or false, we can clone using sticky ends

Answer 35

true

Question 36

what are sticky ends

Answer 36

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.

Question 37

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)

Answer 37

true ...

Question 38

how is directional cloning achieved

Answer 38

by using pairs of enzymes with different cohesive termini

Question 39

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

Answer 39

true

Question 40

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

Answer 40

true

Question 41

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

Answer 41

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

Question 42

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

Answer 42

1. ) chemical treatments: Ca2+, Rb2+, heat shock | 2. ) electroporation: Apply square pulse of current- "pores" in wall membrane

Question 43

what are the most common plasmid vectors

Answer 43

based on ColE1 (class of ColE1 is pMB1)

Question 44

what are the current plasmid vectors which are based on ColE1

Answer 44

pMB1 pBR322 pUC

Question 45

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

Answer 45

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

Question 46

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

Answer 46

true

Question 47

true or false, pBR322 has Amp Tet

Answer 47

true

Question 48

true or false, pUC has Amp lac alpha

Answer 48

true

Question 49

what is a multiple cloning site (MCS) aka polylinker

Answer 49

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

Question 50

how are MCS made

Answer 50

- 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

Question 51

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

Answer 51

true

Question 52

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

Answer 52

true

Question 53

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

Answer 53

true