Lecture 13 - Genetic Manipulation II

Question 1

what is PCR based on?

Answer 1

PCR is based on the ability of DNA polymerase to copy a DNA template

Question 2

how is polymerisation initiated in PCR?

Answer 2

polymerisation is initiated at 2 sites by short DNA primers that we design and have chemically synthesised

Question 3

what do primers provide in PCR?

Answer 3

primers provide the 3’ ends for DNA polymerase to use in order to start DNA polymerisation

Question 4

what are the three main steps in the polymerase chain reaction?

Answer 4

(1) denaturation (94C)

(2) cool (annealing - 50C-ish)

(3) extension (70C - for taq polymerase)

then the cycle repeats!

Question 5

what is the key point about the PCR reaction?

Answer 5

the key point about PCR is that it is an exponential reaction - this means that each cycle the number of DNA molecules doubles

Question 6

what do we need to add to a tube in order to carry out a polymerase chain reaction?

Answer 6

we need to add:

(1) template DNA - this can be as little as one copy

(2) multiple copies (billions) of 2 primers - these are designed and chemically synthesised to specifically amplify one part of the template

(3) DNA polymerase

(4) the four dNTPs (bases: A, T, C & G) in sufficient concentration to make DNA we anticipate

Question 7

how long does each cycle last in PCR?

Answer 7

(1) 92C - 1 minute

(2) 50C - 2 minutes

(3) 70C - 2 minutes

Question 8

DNA polymerase used to denature after every step, so to avoid adding it, how have scientists overcame this bore?

Answer 8

they isolated DNA polymerase from thermophilic organisms (originating from hot springs) that work at 70C and are resistant to being destroyed at 92C - thus we only need one DNA polymerase right at the beginning

Question 9

as long as we know the sequence of the DNA we want to amplify we can:

Answer 9

design appropriate primers

Question 10

to aid the cloning of primers, what can we do?

Answer 10

to aid the cloning of primers we can add restriction enzyme cutting sites

Question 11

what is pretty much the minimum required length for a DNA primer?

Answer 11

the minimum length for a DNA primer is 16 base pairs

Question 12

what length are primers usually made to?

Answer 12

often primers are around 20-23 bases long

Question 13

when is PCR the most efficient?

Answer 13

PCR is most efficient if the annealing temperatures are equivalent between the two primers

Question 14

how do the base pairs differ in thermal stability?

Answer 14

C:G base pairs are more thermally stable than A:T base pairs

Question 15

estimating the annealing temperature of a primer:

Answer 15

rough rule-of-thumb is that annealing temperature is

4(C+G) + 2(A+T)

assuming both primers are approximately equivalent

Question 16

how do we isolate our template for PCR?

Answer 16

we identify the gene of interest and isolate the mRNA leaving behind any introns

Question 17

the advantages of cDNA:

Answer 17

• smallest size for the protein encoding sequence (many human genes are huge due to introns)
• if a gene is expressed more in a tissue then there will be proportionally more copies of the mRNA than the DNA

Question 18

how to make cDNA:

Answer 18

(1) dissect tissue and prepare lysine

(2) add polyT bead

(3) the polyA tailed mRNA hybridises to the polyT covalently bound to the magnetic beads

(4) remove extract

(5) add buffer, heat denature removes beads

(6) add polyT primer

(7) add reverse transcriptase (a viral protein) plus dNTPs

now you have a DNA template ready for PCR!

Question 19

significant restriction to cloning:

Answer 19

the requirement for convenient restriction enzymes

Question 20

the advantages of gibson assembly:

Answer 20

• no restriction enzymes needed
• very quick
• multiple molecules can be combines in one reaction
• can be used to introduce site-specific mutations

Question 21

gibson assembly - how does it work?

Answer 21

• need to design the fragments to have 20-30bp at homology at each end
• add “master mix” to get desired outcome

Question 22

master mix components:

Answer 22

• 5’-3’ exonuclease
• DNA polymerase (& dNTPs)
• DNA ligase

Question 23

gibson assembly basic principles:

Answer 23

• at the start only the nuclease has a substrate (finds 5’ ends and chews away one strand, this is a 5’-3’ exonuclease
• once the nuclease has generates three 3’ single strands they can anneal, only then is there a substrate for the polymerase
• the polymerase is more “processive” than the nuclease (i.e: it falls off less often) and works faster thus it ‘wins’
• once the polymerase fills the gap there is a substrate for the ligase. once this has worked, there is no substrate for the nuclease or polymerase

Question 24

what do you need for gibson assembly?

Answer 24

your vector and an insert in which the last 20-30bp of said insert is identical to the vector

Question 25

what do the contents of the “master mix” do?

Answer 25

the master mix inserts your insert into the vector using three different enzymes

Answer 26

(1) the nuclease chews away the vector AND the insert DNA (only one strand) from the 5’-3’ ends and this allows for the two different strands to anneal to each other

(2) then DNA polymerase comes in and fills in the holes to complete the whole DNA strand

(3) once holes are filled in ligase will combine the DNA

now you have a single double-stranded DNA with no nicks or gaps