Caister Academic Press

Causes and Actions

from: PCR Troubleshooting: The Essential Guide (Author: Michael L. Altshuler). Caister Academic Press, U.K. (2006)

Abstract

  • Do not confront the problem
  • The nature of PCR and its pathology
  • Inadequate concentrations of ingredients
    • The template
    • Inadequate deoxynucleotidetriphosphates
    • Inadequate primers
    • Inadequate Taq
    • Inadequate Mg2+
    • Suboptimal KCl concentration in the PCR buffer or the whole of the PCR buffer
  • Inadequate quality of ingredients
    • DNA template
      • Conversion of a DNA solution into a solid body
      • PCR inhibitors
      • Degraded template
      • Verification of the purity of the template DNA by optical means
    • Poor water
    • Deoxynucleotidetriphosphates
    • Poor primers
      • Primers may not be good in practice even if they are good in design
      • For primer selection use only dedicated software
      • Difference in Tm balanced by different primer concentrations
      • Inefficient priming
    • Inadequate MgCl2
    • Poor buffer
    • Poor Taq
    • The presence of PCR inhibitors
    • Substances that do not inhibit PCR
  • Inadequate storage of ingredients for the PCR reaction
    • The treacherous refrigerators
    • Templates, dNTPs, primers
    • Taq polymerase
    • MgCl2
    • Buffer
  • The thermocycler
    • Changes in the PCR mix
    • The ramp
    • Time and temperature
    • Dusty, greasy, or fluffy tube wells
    • Rapid evaporation
    • Suboptimal performance of the thermocycler or its particular wells
    • The tubes are poorly pressed down into the wells or they have got deformed
  • Faulty target selection
  • Incomplete DNA denaturation and dispersal
  1. Template DNA
  2. PCR fragments
  3. Hairpins
  • The Taq enzyme
    • Hurdles for Taq polymerase
      • Stable hairpins in the template strand
      • AT-rich areas
      • GC-rich areas
      • Alternating GC/AT-rich regions
    • Hot start
      • The improved hot start
      • The role of the reaction volume in the quasi hot start
    • Nonspecific binding of Taq to DNA
      • Incomplete primer elongation or premature termination of DNA synthesis
        • Under-elongation of primers in the late PCR
        • Premature termination of the DNA synthesis
  • Cosolvents or additives or enhancers
    • Helix-destabilizers
    • Helix-stabilizers
    • Substances that neutralise the PCR inhibitors
    • PCR enhancers with poorly understood mechanism of action
  • Approaching the limit of the PCR sensitivity
  • Agarose gel electrophoresis
    • The band diffuses and disappears
    • Short fragments of uneven length migrate
    • The band is invisible
    • The significance and the insignificance of the salt concentration in the compared samples
    • Bands smear due to their fast movement or the DNA overload
    • Dirty gel support
    • Dried well
    • DNA sticking in the gel well caused by inappropriate gel density
  • Causes for specific nonspecifics and the false contamination
    • Chimeras
    • Allele dropout
    • Heteroduplexes
    • Primer multimers
    • Low resolution electrophoresis resulting in imprecise idea of the correct band position
    • Coincidence or the devil
  • Mineral oil and wax
    • Mineral oil
    • Wax, paraffin or vaseline
  • Primer-dimers and primer multimers
  • Short PCR fragments versus long PCR fragments
  • Avoiding accidents
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