In Vitro cloning and PCR Flashcards
In Vivo cloning
Amplification of specific sequences of DNA utilizing biological vectors (eg: plasmid cloning)
In Vitro cloning
Amplification of specific sequences of DNA without the use of biological vectors.
PCR
Polymerase Chain Reaction - repeated, sequence-specific amplification of nucleic acid (usually DNA)
Advantages of PCR
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.
DNA replication (in plasmids)
- 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
Three steps of PCR
Denaturation
Annealing
Extension
Ingredients for PCR
- 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)
Denaturation
- at around 94’C for approx 60s
The hydrogen bonds are broken leaving 2 single strands of DNA.
Annealing
- 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.
Extention - polymerization
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.
Primer design criteria
- 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)
Primer considerations
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.
Hot Start PCR protocol
First denaturing 3-4 min before adding enzyme.
Calculation of Tm
Tm(°C)=81.5°C+16.6log10{Na+]+0.41(%G+C) –500/n
(where n=number of nucleotides…….)
However if n
What is Tm
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.
Primer Dimer (PD)
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.
Primer concentration
Generally in the order of 1μM ….. More is not always better –get lots of non-specific binding if the concentration is too high.
MgCl2concentration
Determined by experimentation –0.5mM to 5mM
•High MgCl2= low binding specificity of primers
•Low MgCl2= high binding specificity of primers
Which DNA polymerase
Thermo-tolerant DNA polymerase required
- Thermus aquaticus (Taq DNA pol)
- Thermus thermophilus
- Bacillus stearothermophilus
DNA needs to be
- 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
Avoid contamination
- 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
Optimizing annealing temperature
- Primers have a calculated annealing temperature (Tm)(e.g.54°C)
- Temperature must be confirmed practically
- Temperature steps of 2°C above and below
Optimizing Mg2 concentration
- Fidelity of the PCR depends on [Mg2+]
- Vary [Mg2+] in steps of 0.5 mM
- Sometimes a compromise between yield and specificity
