In Vitro cloning and PCR

Question 1

In Vivo cloning

Answer 1

Amplification of specific sequences of DNA utilizing biological vectors (eg: plasmid cloning)

Question 2

In Vitro cloning

Answer 2

Amplification of specific sequences of DNA without the use of biological vectors.

Question 3

PCR

Answer 3

Polymerase Chain Reaction - repeated, sequence-specific amplification of nucleic acid (usually DNA)

Question 4

Advantages of PCR

Answer 4

1) Quick - may only take a few hours
2) Sequence-specific - ends of the amplified piece of nucleic acid are defined by the design of the primers
3) Relatively cheap compared to other cloning techniques.

Question 5

DNA replication (in plasmids)

Answer 5

• Begins at a specific location: the Origin of Replication’ usually in an area with a large number of A/T pairings.
• Inhibitor proteins attach to the origin of replication and open the strands producing replication forks.
• Helicase binds to the forks and breaks the hydrogen bonds between the nitrogenous base pairs
• single strand binding proteins coat the separate strands to prevent rebinding
• dna polymerase extends the strand from 3’ to 5’
• the leading strand is extended continuously
• the lagging strand is form in parts known as okazaki fragments

Question 6

Three steps of PCR

Answer 6

Denaturation
Annealing
Extension

Question 7

Ingredients for PCR

Answer 7

• Template DNA (target)
• Downstream oligonucleotide primer
• Upstream oligonucleotide primer
• Taq DNA polymerase
• Reaction Buffer (x10 NH4 buffer)
• Cofactor - MgCl2
• Nuclease free water
• Nuclease free light mineral oil (sometimes)
• Deoxyribonucleotide triphosphates (dNTPs)

Question 8

Denaturation

Answer 8

• at around 94’C for approx 60s

The hydrogen bonds are broken leaving 2 single strands of DNA.

Question 9

Annealing

Answer 9

• temperature is reduced to around 50-65’C
• Primers attach to specific sites and remain attached if the bases match exactly.
  A primer sequence made up of G-C would be much stranger due to the 3 hydrogen bonds.

Question 10

Extention - polymerization

Answer 10

Temperature is raised 72’C allowing Taq polymerase to attach to the primers
- the Taq runs the length of the strand using the free nucleotides to copy the strand.
As the reverse primer moves from 5’ to 3’ it can copy the strand in the same time as the forward (no okazaki fragments)
> the cycle repeats 20 - 40 times.

Question 11

Primer design criteria

Answer 11

• must have a primer for each end of the two strands that will define the ends of the molecule synthesised.
• Must have the 3’OH ends pointing towards each other so they flank the sequence required
• Normally sequence specific (unless the are degenerate or universal primers)

Question 12

Primer considerations

Answer 12

Should only bind to the sequence of interest

• Be between 18 and 40 bases long
• high G+C content (increases Tm of the primer::target duplex)
• Avoid A+T at the 3’OH end (can reduce specificity)
• Needs to exactly match the target - DNA polymerase needs a fully matched primer template to begin polymerization.

Question 13

Hot Start PCR protocol

Answer 13

First denaturing 3-4 min before adding enzyme.

Question 14

Calculation of Tm

Answer 14

Tm(°C)=81.5°C+16.6log10{Na+]+0.41(%G+C) –500/n
(where n=number of nucleotides…….)
However if n

Question 15

What is Tm

Answer 15

The temp in °C at which 50% of the DNA duplexes have melted and become single stranded. The ideal annealing temperature of an oligonucleotide is ~ 5°C below Tm.

Question 16

Primer Dimer (PD)

Answer 16

When the forward and reverse primers attach at the 3’ end and are then elongated by DNA polymerase.
These compete with the target sequence for dNTPs.
PDs in ethidium bromide-stained gels are typically seen as a 30-50 base-pair (bp) band or smear of moderate to high intensity and distinguishable from the band of the target sequence, which is typically longer than 50 bp.

Question 17

Primer concentration

Answer 17

Generally in the order of 1μM ….. More is not always better –get lots of non-specific binding if the concentration is too high.

Question 18

MgCl2concentration

Answer 18

Determined by experimentation –0.5mM to 5mM
•High MgCl2= low binding specificity of primers
•Low MgCl2= high binding specificity of primers

Question 19

Which DNA polymerase

Answer 19

Thermo-tolerant DNA polymerase required

• Thermus aquaticus (Taq DNA pol)
• Thermus thermophilus
• Bacillus stearothermophilus

Question 20

DNA needs to be

Answer 20

• fairly clean & reasonably intact
• free from DNA polymerase inhibitors e.g. phenol, EDTA…
• ~125 ng / 25 μl reaction volume if whole genomic (human) DNA (can’t get away with one copy!!)
• at least 100 target copies per reaction volume

Question 21

Avoid contamination

Answer 21

• UV irradiate work area, racks, micropipettes etc.. prior to setting up
• Sterilise all components –UV irradiate reaction mixes just prior to addition of the polymerase and target
• Use filtered tips
• Physical separation of DNA prep areas from PCR areas

Question 22

Optimizing annealing temperature

Answer 22

• Primers have a calculated annealing temperature (Tm)(e.g.54°C)
• Temperature must be confirmed practically
• Temperature steps of 2°C above and below

Question 23

Optimizing Mg2 concentration

Answer 23

• Fidelity of the PCR depends on [Mg2+]
• Vary [Mg2+] in steps of 0.5 mM
• Sometimes a compromise between yield and specificity