L2 PCR

Question 1

replisome

Answer 1

proteincomplex that handles replication in bacteria

Question 2

DNA gyrase

Answer 2

releases tension in DNA to prevent twists and knots on DNA strands

Question 3

DNA helicase

Answer 3

breaks apart dsDNA

Question 4

DNA polymerase iii

Answer 4

performs replication on leading strand
downstream

Question 5

DNA polymerase i

Answer 5

performs replication on lagging strand
upstream

Question 6

dNTP
ddNTP

Answer 6

• dNTP = dioxy NTP = sugar based, they have one extra OH-group

-ddNTP= they have no extra OH-group

Question 7

nucelase, exonuclease

Answer 7

• nuclease = breaks down nts
• exonuclease = degrades nts, for ex. after there’s been a mistake
  -> DNAP has exonuclease acitivity

Question 8

DNA ligase

Answer 8

connects fragments

Question 9

PCR

Answer 9

= used to amplify DNA

Question 10

primer

Answer 10

= synthesized DNA
- around 20 nts
-> too long: will form hairpins
-> too short: not specific enough, can bind anywhere and not just where you want it to

Question 11

PCR general process

Answer 11

heat up DNA => dsDNA seperates
cool down DNA and hope primer anneals before complementary sequence

Question 12

how does primer win “competition”?

Answer 12

• 2 things competing:
  1. primer annealing to template strand
  2. complementary sequence annealing to template strand
• primer wins because there’s a looot of primer which makes it favourable

Question 13

adding overhangs

Answer 13

= a way to use PCR to add a sequence to another sequence

1st cycle: overhang (~20 nts) added to 5’-end of primer, primer anneals to template strand and gets replicated

2nd cycle: overhang has been added to DNA and new overhang can be added with new primer etc

Question 14

overlap extension PCR

Answer 14

= used to add sequence to middle of target

step 1: add 4 primers, 2 to each strand, the two in the middle have complementary overhangs
=> 2 PCR reactions happen at once

step 2: the leading strand on one of the two dsDNA formed align with the lagging strand of the other (where DNAP can go 5’->3’) and DNAP performs replication so a new dsDNA is formed with the overhangs in the middle
-> the other strands can’t form new dsDNA since DNAP can’t go 3’->5’

Question 15

problem with PCR (mutations)

Answer 15

• Taq polymerase used => good! can handle 90 degrees Celcius
  -> but, has no proofreading
• in lab: 15-20 cycles of PCR performed => many mutations which we can’t get rid of

Question 16

solution to problem with PCR

Answer 16

= filtrate DNA!
- put all DNA into plasmids via ligation reaction
- transform plasmids into E. coli that takes up one plasmid each where it gets replicated
- some E. coli will then hold plasmids with no mutations
=> find them via DNA sequencing