Gene Cloning and Manipulation

Question 1

basic pcr reaction

Answer 1

94C: denature DNA
lower T ~ 55C: primer annealing
72C: DNA synthesis

Question 2

primer design (4 ways)

Answer 2

• cloned DNA, sequence known (want to amplify a smaller section of)
• genomic data - genome projects/ ESTs/ search with BLAST
• use amino acid sequence to work back
• design primers for the conserved features of a protein family

Question 3

other key features of primers

Answer 3

20-30 bp
avoid 3’ mismatches (extension is from here)
G/ C at 3’ good (3 H bonds)
similar annealing temp for both primers
no internal complementarity/ complementarity between primers (primer dimers would form)

Question 4

polymerase error

Answer 4

Taq doesn’t have 3’ to 5’ exo so a significant error rate, 1/10,000. so use a different enzyme which does have 3’-5’ exo. eg Phusion.
problems are then cost and yield.

Question 5

PCR: product size difficulties

Answer 5

amplification efficiency decreases for larger products as get more truncated products, which cannot be used for more PCR.

• use longer extension times
• shorten denaturation time to reduce product depurination (cleavage at purine residues)
• use glycerol/ DMSO to lower the strand sep and denaturation temps, so reducing the effects of high temps.

Question 6

overcoming non-specific priming in PCR (5 ways)

Answer 6

• manipulate temps
• manupulate cation concs (Mg2+ = cofactor for polymerase but also affects DNA duplex stability)
• touchdown pcr
• hot start pcr
• nested pcr

Question 7

touchdown PCR

Answer 7

start with annealing temp > predicted and decrease for subsequent rounds (stepwise) so 1st rxns have most stringent annealing conditions

Question 8

hot start PCR

Answer 8

• mispriming pay occur if priming and DNA synthesis have started before the denaturation temp is reached
• add polymerase later/ use a polymerase which only becomes active after the denaturation temp is first reached

Question 9

nested PCR

Answer 9

2 rounds of pcr, 2nd set uses products of the first, with new primers which anneal within the correct product - very unlikely to amplify incorrect product here.

Question 10

good features of cloning vectors

Answer 10

MCS
antibiotic resistance gene
origin of replication
lacZ’ allows blue-white selection

Question 11

3 potential products from mixing and ligating the PCR product and linearised vector

Answer 11

insert or vector can self ligate

vector with insert = desired product

Question 12

key features of the e coli host (4)

Answer 12

• high efficiency of transformation
• doesn’t have restriction enzymes which would degrade DNA
• recombination deficient
• debilitation

Question 13

blue-white selection

Answer 13

want white colonies, blue colonies do not have the insert in the MCS so are able to produce functional B Gal (form blue pigment from X Gal)