Part 1. Preface

"A broad road full of wonderful prospects is about to open up before you," my boss said hinting at my impending firing, after taking a look at an empty gel. "A clear example of complete professional degeneracy," he greeted me the next time. He is a fine man, I like him very much. His greetings, congratulations and wishes of good fortune outside his laboratory have goaded me into an idea to compile a PCR troubleshooting guide of a novel type accompanied by a brief consideration of ways to obviate rather than overcome the PCR problems. If you fail at PCR, consult this book. Then try to figure out the most probable cause of your failure. I wouldn't let this guide slip into a full-scale manual, so inevitably it has had to be oriented to a reader with some experience in PCR. If you are a PCR greenhorn, keep several manuals close at hand while reading it. Although there is a considerable overlap with other troubleshooting guides, some of the most obvious advice, the kind that you generally remember and is invariably mentioned in other textbooks and guides, has been deliberately omitted from my book (detailed account of the hot start, the trick of touchdown, commonly accepted rules for primer selection, the significance of contamination, etc.). The advice that you will find in these pages is the sort of advice that is not usually found elsewhere and that is often the most useful.

Part 2. Introduction

Part 3. Appearances

Unsatisfactory results of PCR

Part 4. Causes and Actions

• 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

Part 5. Conclusions

Part 6. Glossary

Part 7. Index