PCR Flashcards
What is PCR and what is it used for ?
PCR = Polymerase Chain Reaction
Widely used technique to amplify DNA in vitro.
When did the first publications concerning PCR occur ?
Which scientists won a nobel prize working on PCR and with whom ?
“First” publications: 1985, but there are earlier suggestions (Khorana group, Kleppe 1971)
Nobel Prize in Chemistry 1993: Kary Mullis for PCR, shared with Michael Smith for site directed mutagenesis
What are the 3 formal steps of PCR ?
1) Strand separation of double stranded DNA (
denaturation, melting)
2) Annealing of primers
3) Extension of primers (DNA synthesis by DNA polymerase)
How many PCR cycles are required to obtain our first desired dsDNA fragments ?
3 cycles.
What are the most common uses of PCR ?
What are these based on ?
- “Sequence-specific” detection (and amplification) of DNA
- Generation of DNA for specific applications (e.g. sequencing)
- Engineering of DNA (mutagenisis and much more) - Quantitation of DNA
all the above are based on an exponential amplification of DNA (both strand are being amplified).
Total product count after n cycles: 2^n (including starting sequence and intermediate products)
What are PCR-like processes ?
These are processes when only one strand is amplified, due to priming to only one strand total product count after n cycles: n (excluding original strand, if present) therefore also called linear amplification e.g. used for sequencing reactions.
At what temperatures do strand separation (denaturation), primer annealing and extension occur ?
Denaturation = 94-96 degrees
Primer Annealing = 65
Extension = 72
What were the DNA polymerases known in 1985 ?
What are their properties ?
DNA polymerases known in 1985 included DNA polymerase I (or Pol I) originally purified from
E.coli, but ubiquitous in prokaryotes (1956), involved in DNA repair (and DNA replication) has three main enzymatic activities :
5’->3’ DNA-dependent DNA polymerase, primer dependent, template dependent
3’->5’ exonuclease [relevant for proofreading]
5’->3’ exonuclease [“nick translation”]
Why is the 5’->3’ exonuclease activity often unwanted in molecular biology reactions ?
Because the main function of the 5’ to 3’ exonuclease activity is to remove ribonucleotide primers that are used in DNA replication.
What is the Klenow fragment, or Klenow polymerase ?
An in-vitro proteolytic treatment of Pol I can remove the domain necessary for 5’->3’exonuclease activity, yielding the Klenow fragment (or Klenow polymerase) –> enzyme used in 1985 publication.
What is the [porblem of using the Kelow polymerase for PCR ?
How was this problem overcome ?
Problem : Klenow works best at about 37 degrees –> denaturation requires high temperatures –> Klenow will be deactivated => requires addition of new enzyme at every cycle
Solution : find an enzyme that is heat-stable in PCR 1986/1987 : Taq polymerase isolated from thermophilic bacterium Thermus aquaticus, stability about 30min at 95 degrees, or 9 min at 97.5 degrees
What is the problem with using tag polymerase for PCR ?
Taq has no 3’->5’ exonuclease activity = no proofreading
What is DNA denaturation dependant on ?
- Salt ion concentrations: higher ionic strength => more stable base pairing
- Base pair composition: G/C more stable than A/T => initially local melting at AT rich
- pH: basic conditions favor stand separation
Why is full separation required ?
Otherwise, there is a possibility of very fast snap-back renaturation.
What are the main contraints linked to PCR ?
One buffer system for all the PCR steps :
- PCR enzyme has to work well in this buffer
- PCR enzyme should be stable at denaturing conditions
Further technical constraints :
- temperature dependency of buffer pH
- evaporation and condensation in reaction vessel (heated lid, mineral oil cover layer)
- denaturation at 94-96 degrees, appropriate buffer system, salt concentrations
What is the most critical/complex step in PCR ?
Primer annealing process.
How long would a primer have to be to amplify the entire human/mouse genome (assuming random genome)?
A 16mer, because 4^16 ~ 4,300,000,000 > 3Gbases complexity.
What should the melting temperature be ?
It should be such that at the melting temperature, 50% of the sequence has annealed.
What is the Wallace rule ?
Wallace rule = a rule for calculating the melting temperature Tm of short oligonucleatides : Td = 2oC(A+T) + 4oC(G+C)
Rule works for 14 - approx 30mers, with one sequence filter bound, at 0.9M NaCl
What is the Bloton and McCarthy rule ?
Bolton and McCarthy (modified, e.g. Maniatis):
Tm = 81.5oC + 16.6 (log10[M+]) + 0.41(%[G+C]) - 675/n), works ~ 14-70mers, [M+] <0.4M
Give an example of more sophisticated method that can be used to calculate the Tm of an oligonucleotide ?
Nearest-neighbor thermodynamics (software packages).
How long should the annealing period usually be ?
Usually short e.g. ~10-30s
Why is “hot start” PCR used ?
To minimize unspecific priming + the formation of primer dimers at their 3’ ends, followed by extension.
What does DNA Pol require for its activity ?
- an oligonucleotide-primed template
- free dNTPs (dATP, dTTP, dGTP, dCTP)
- divalent cations, usually Mg2+, plus monovalent ions
What is the temperature of the extension phase ?
e.g. 68-72oC; usually close to optimal temperature of polymerase (i.e. 75-80oC for Taq)
How long should the extension stage last ?
Long enough to synthesize full length of PCR product (e.g. 1min/kb for Taq)
What are the potential consequences if the annealing temperature is much lower than the extension temperature ?
What does this impose ?
Primers may separate from target at extension temperature.
Thus, some extension should ready occur at annealing temperature.
How could primers be designed for an optimal annealing-extension transition ?
We can design primers so that annealing temp is not much lower than extension temp or design the same temperature for annealing and extension.
What is the error rate of Klenow ? - of Taq ?
Klenow : 1/50,000
Taq : 1/2,000 (or worse)
What do DNA Pol error rates depend on ?
Reaction conditions :
- [dNTP]
- [Mg2+]
What is Pfu ?
Pfu = thermostable DNA Pol found in the hyperthermophilic archaeon Pyrococcus furiosus, has 3’–>5’ exonuclease (proofreading) activity, but is slower that Taq
All DNA Pol’s seems to have both advantages and flaws.
What can be envisaged to overcome this problem ?
Add in a mixture of polymerases.
Why is blunt end cloning not efficient ?
No sticky ends.
What overhangs are produced by Taq ?
How can these be used ?
3’A overhangs –> can be efficiently exploited for TA cloning
What overhangs are produced by Pfu ?
NONE (I think).
What are the two main ways of generating a restriction fragment from a PCR product ?
- Cut at site within PCR product –> works only if suitable sites are present
- Add sites to primers (these sites should be absent in target sequence) –> digestion of the PCR product will generate a DNA with EcoRI and MluI overhangs
What are the three steps to indtroduce a mutation via PCR ?
- Generate two separate PCR products, two external primers, two internal primers with mutated sequence
- Combine the two PCR product and run new PCR with the two external primers
- Final product
If we start with one copy of our DNA fragment, what is the yield after n cycles ?
2^n
If we start with 1ng of target DNA, calculate 40 cycles.
1,100 kg of DNA
If we start with 1pg of target DNA, calculate 25 cycles.
What about w/ a cycle efficiency of 1.8 ? - 1.5 ?
Efficiency of 2 : 33.5 ug
- —————- 1.8 : 2.4ug
- —————- 1.5 : 25.5 ng
What is the risk of a large number of cycles ?
justify your answer.
Increased error :
a) each final product has undergone a synthesis n times
b) reaction conditions usually conditions deteriorate with increased cycle count
