Molecular Biology: PCR techniques
Molecular Biology:
PCR Techniques
Author: Prof Estelle Venter
Licensed under a Creative Commons Attribution license.
FAQS
1.
What is PCR?
The PCR is a simple method for producing unlimited copies of a specific DNA sequence in
a test tube which allows a “target” DNA sequence to be selectively amplified several
million-fold in just a few hours. The PCR achieves amplification of a predetermined
fragment of DNA.
2.
Can one do a PCR without knowing the target gene/organism?
To start DNA amplification, the PCR needs specific flanking primers. In order to develop
these primers, the gene sequence of the target gene/organism must therefore be known.
This can be seen as a disadvantage of the PCR.
3.
What is the highest temperature that reverse transcriptase can be used?
This enzyme is not stable and will denature easily. The enzyme will be unstable from 60
°C and higher.
4.
Why do we use the DNA polymerase from Thermus aquaticus in a PCR instead of
normal DNA polymerase from E. coli?
Taq polymerase can withstand high temperature – will not denature at 95 °C.
5.
Why do different PCRs have different annealing temperatures?
Annealing temperature of primers depends on the sequence of the primers. Different
PCRs will have different primer sequences, therefore different annealing temperatures.
6.
How does one determine the correct annealing temperature for primers?
Depends on the length and the sequence of the primers, the formula: Tm= 4(G+C) +
2(A+T) can be used.
7.
Which samples are ideal to be used for PCR?
All diagnostic samples can be used for PCR
8.
Which parameter would you change first if your PCR reaction gave too many
products?
If there are too many products in a PCR reaction then it suggests that primer annealing is
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Molecular Biology: PCR techniques
non-specific. The first parameter to modify would be to increase the annealing temperature
which will increase the specificity of the primers.
9.
What would you do if the PCR reaction gave very little, if any, of the correct
product?
If no product was observed it might suggest that the annealing temperature was too high.
This can be tested by lowering the annealing temperature 3-5 degrees. However, another
possibility is that the primers are not working, in that case, new primers would need to be
designed
10.
Differentiate between a hot start PCR and a touchdown PCR.
Hot start: Protection of Taq polymerase so that PCR only starts after denaturation of DNA
and at the intended specific annealing temperature and not randomly.
Touch down: Initial annealing at a higher temperature as needed for the reaction – after a
few cycles the annealing temperature is decreased – prevent non-specific binding to occur
but has to compete with the already formed correct product - use to make reaction more
sensitive.
11.
What is the plateau effect?
This is the point in a PCR at which running more cycles does not result in a net gain of
specific PCR amplification product.
12.
What formula is used for the calculation of Tm?
Tm = 2(A+T) + 4(G+C)
13.
What are the important criteria to use when designing primers?
a) Length - 18 – 30 nucleotides long
b) Have a target sequence of 100 – 1000 bp (with 5000 bp as practical limit)
c) Primer sequence - Balanced distribution of G/C and A/T rich domains
d) Have 10 – 12 Gs or Cs and a Tm of at least 60 °C
e) Do not form secondary structure
f)
Do not end with AAA –3’, and GGG-3’, etc. (with eukaryotes also avoid the
microsatellite motifs CACA and TGTG)
g) Do not form dimers at the 3’-end. Dimers formed by the last three nucleotides may be
elongated by the Taq polymerase, even if the dimer complex is unstable:
14.
PCR experiments may be disturbed by very small contaminations. Why?
During a PCR minute quantities of DNA are amplified – any foreign DNA with a remote
primer sequence similarity will be amplified.
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Molecular Biology: PCR techniques
15.
Why is the PCR negative control handled after the other samples?
To see if any carry-over DNA occurred during the preparation of the PCR.
16.
Can single-stranded DNA serve as PCR template?
Yes single stranded DNA e.g. cDNA can be used in a PCR.
17.
What is the use of Magnesium in a PCR? Should the concentration regularly be
changed?
Magnesium concentration influences:
Enzyme activity/fidelity, primer annealing, strand dissociation temperatures, product
specificity and the formation of primer-dimer artifacts.
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It is therefore important to determine the ideal Mg concentration for each primer pair for a
PCR. The optimal MgCl2 concentration may vary from approximately 0.5 mM to 5 mM
18.
What is reverse transcribed (RT)-PCR?
When RNA is used as PCR template, it is first converted to cDNA and the polyA in cDNA
can act as part of the template which sequence can act as a primer (- TTT)
19.
What is a nested PCR and when is it used?
It is the re-amplification of a PCR product. Normally the second PCR (nested) uses
primers developed within the first PCR product, the amplicon. It is used to increase the
sensitivity of the PCR but can also be used to distinguish between e.g. serotypes of
organisms, vaccine and wild strains etc.
20.
What is meant by the generic term dNTPs? How do dNTPs influence a PCR?
A generic term referring to the four deoxyribonucleotides: dATP, dCTP, dGTP, dTTP
2+
Imbalanced dNTPs mixtures will reduce polymerase fidelity. dNTPs reduce free Mg , thus
interfering with polymerase activity and decreasing primer annealing. A final concentration
of between 20-200 µM of each results in an optimal balance in yield, specificity and
accuracy.
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