Molecular Biology: PCR techniques

Molecular Biology:

PCR Techniques

Author: Prof Estelle Venter

Licensed under aCreative 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 tolerate?
    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 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:

  1. 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.
  2. 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.
  3. Can single-stranded DNA serve as PCR template?
    Yes single stranded DNA e.g. cDNA can be used in a PCR.
  4. 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.
    It is therefore important to determine the ideal Mg2+ concentration for each primer pair for a PCR. The optimal MgCl2 concentration may vary from approximately 0.5 mM to 5 mM
  5. 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)
  6. 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.
  7. 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
    Imbalanced dNTPs mixtures will reduce polymerase fidelity. dNTPs reduce free Mg2+, thus interfering with polymerase activity and decreasing primer annealing. A final concentration of between 20-200 M of each results in an optimal balance in yield, specificity and accuracy.

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