PCR and Cloning

Question 0

Why plateau effect in PCR

Answer 0

Thermodynamic phenomenon - large proportion of amplicon increases melting temp

Question 1

Steps in PCR

Answer 1

Initial denaturation (activates fast Taq)
Denaturing (95-98 - formation of single stranded DNA)
Annealing (temp decrease = primer bind, need correct Ta)
Elongation (72 - DNA polymerase extend primers to produce nascent DNA, 30s/bp)
Final extension (72, 5mins)
Hold (4 degrees)

Question 2

Essential components of PCR (8)

Answer 2

Template DNA 
Sequence specific primers (fwd and rev)
dTNPs
Reaction buffer 1X
MgCl2 (enzyme cofactor)
ddH2O
DNA polymerase

Question 3

Wallace rule

Answer 3

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

Question 4

What can influence Tm of primers

Answer 4

Monovalent cations
Divalent cations
Flanking nucleotides
Salt concentration

Question 5

How to create 3’ stability

Answer 5

Avoid 3’ mismatch
3’ base should be a G or a C
(Avoid more than 3 G/Cs at 3’, forms unwanted complexes and could give false priming)

Question 6

Two types of 2dry primer structures

Answer 6

Intraprimer - self-complementarity (hairpin)

Interprimer - primer dimers

Question 7

What to do if PCR doesn’t work

Answer 7

Ta gradient
Mg2+ titration (Mg cofactor for Taq, BUT binds DNA affecting primer template interaction and Taq primertemp interaction) there’re more Mg = less stringent bonding

Question 8

Limitations of PCR

Answer 8

Need sequence and flanking info
High risk of contam
Can only amplify a certain length of DNA (5kb is easy, up to 40kb)

Question 9

Three strategies for cloning PCR products

Answer 9

1. Blunt end PCR cloning - proof reading DNA poly (pfu) generates blunt ends
2. T/A cloning - non-proof reading DNA poly(Taq) have terminal transferase activity (adds adenine on end of product)
3. Restriction site end cloning - NB add bases to 5’ end so RE can bind.

Question 10

Fx of poly histidine tags

Answer 10

Allow protein purification by affinity chromatography

Allow semi-quantitative analysis by immunoblotting

Question 11

Choice of vector depends on

Answer 11

Nature of insert

Downstream applications

Question 12

Expression vectors contain

Answer 12

Promotor
Selection marker
N/C terminal His tags or fusion proteins
Ori

Question 13

Describe reporter gene vectors

Answer 13

Gene of protein easily quantitated (eg. GFP, luciferase) linked to regulatory sequence being tested (eg. Promotor or fusion protein) Changes in transcriptional activity detected by changes in level of reporter gene expression

Question 14

What is special about shuttle vectors

Answer 14

They can replicate in more than one organism (appropriate oris)

Question 15

Define directional cloning

Answer 15

Cutting both vector and insert with two different REs. Ensures insert is inserted in one direction. Efficiency greatly improved.

Question 16

How do you convert a 5’ overhang to a blunt end

Answer 16

Use Klenow and T4 DNA polymerase in presence of dNTPs to fill overhang.

Question 17

How do you convert a 3’ overhang to blunt end

Answer 17

Use exonuclease activity of T4 DNA polymerase in presence of dNTPs

Question 18

What is a linker

Answer 18

A small dsDNA fragment that contains a restriction site. Can convert blunt to sticky.

NB ensure restriction site is not at end of linker

Question 19

What does DNA ligase do

Answer 19

Forms new phosphodiester bond between 5’ P and 3’ OH. Requires energy.

Question 20

What does Alkaline Phosphatase do

Answer 20

Removes 5’ phosphates from vectors preventing self ligation. Reduces non-recombinants

Question 21

Why NB to calculate vector insert ratios

Answer 21

Only two ends to any DNA despite varying size. Therefore one must take into account length when working out number of available ends.

Question 22

How to calculate molar ratios

Answer 22

Look at pink paper

Question 23

What are satellite colonies

Answer 23

Tiny colonies appearing close to true Abx resistant colonies. Due to reduced Abx around true clone.

Question 24

How does blue/white screening work

Answer 24

E. coli plasmid contains lacZ which codes for B-galactosidase. Active B-gal hydrolyses lactose to glucose and galactose. lacZ gene contains multiple cloning site, therefore when insert present = no functional protein. Agar contains x-gal, if x-gal cleaved by B-gal than colony turns blue.