PCR Troubleshooting: The Essential Guide

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PCR Troubleshooting: The Essential Guide | Book

Publisher: Caister Academic Press
Author: Michael L. Altshuler Moscow Research Institute of Medical Ecology
Publication date: March 2006
ISBN-10: 1-904455-07-7
ISBN-13: 978-1-904455-07-3
Price: GB £30 or US $60 (paperback).
Pages: 80

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A unique PCR troubleshooting guide that is an essential companion for anyone who uses the polymerase chain reaction technique. Aimed at a reader with some experience in PCR the book discusses the many and varied problems encountered with PCR together with tips, advice and procedures to obviate rather than overcome the PCR problems. Written in the language of the laboratory with a little humour and a down-to-earth approach, the book is easy to read and understand. If you fail at PCR, consult this book! The advice in these pages is invaluable and is the sort of advice that is not usually found elsewhere.

In the words of the author, remember that there are many other things in life than PCR ... for example, isothermal DNA amplification.

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Reviews:

"... this book is a comprehensive collection of all parameters affecting the quality of a PCR reaction ... a valuable assistant in PCR troubleshooting ..." from ChemBioChem (2006) 7: 1623.

"... a helpful advisor to overcome any kind of PCR problems, it is written by a PCR expert and is useful for every scientist frequently using PCR in the molecular biology laboratory." by Thorsten Eggert Heinrich-Heine Universität Düsseldorf (ChemBioChem)

"... addresses recognized common problems with conventional solutions" from Microbiology Today (2006)

"... a reference point to find more comprehensive information." from Internat. J. Dairy Technol. (2007) 60: 65-66.

"Perhaps the best testimonial is that one of the guys in my lab used it to successfully get a very troublesome PCR to work." from The Biochemist (August 2006).

Key Features:

A unique practical guide
Invaluable tips and advice not usually found elsewhere
Fully cross-referenced
Useful glossary and index
Aimed primarily at experienced PCR users
Low price paperback edition!

Contents:

PREFACE

INTRODUCTION

APPEARANCES

Unsatisfactory results of PCR

CAUSES AND ACTIONS

Do not confront the problem
The nature of PCR and its pathology
Inadequate concentrations of ingredients

1. The template
2. Inadequate deoxynucleotidetriphosphates
3. Inadequate primers
4. Inadequate Taq
5. Inadequate Mg2+
6. Suboptimal KCl concentration in the PCR buffer or the whole of the PCR buffer

Inadequate quality of ingredients

1. 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

2. Poor water
3. Deoxynucleotidetriphosphates
4. 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

5. Inadequate MgCl2
6. Poor buffer
7. Poor Taq
8. The presence of PCR inhibitors
9. Substances that do not inhibit PCR

Inadequate storage of ingredients for the PCR reaction

1. The treacherous refrigerators
2. Templates, dNTPs, primers
3. Taq polymerase
4. MgCl2
5. Buffer

The thermocycler

1. Changes in the PCR mix
2. The ramp
3. Time and temperature
4. Dusty, greasy, or fluffy tube wells
5. Rapid evaporation
6. Suboptimal performance of the thermocycler or its particular wells
7. 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

1. Hurdles for Taq polymerase

Stable hairpins in the template strand
AT-rich areas
GC-rich areas
Alternating GC/AT-rich regions

2. Hot start

The improved hot start
The role of the reaction volume in the quasi hot start

3. Nonspecific binding of Taq to DNA

Incomplete primer elongation or premature termination of DNA synthesis

1. Under-elongation of primers in the late PCR
2. Premature termination of the DNA synthesis

Cosolvents or additives or enhancers

1. Helix-destabilizers
2. Helix-stabilizers
3. Substances that neutralise the PCR inhibitors
4. PCR enhancers with poorly understood mechanism of action

Approaching the limit of the PCR sensitivity

Agarose gel electrophoresis

1. The band diffuses and disappears
2. Short fragments of uneven length migrate
3. The band is invisible
4. The significance and the insignificance of the salt concentration in the compared samples
5. Bands smear due to their fast movement or the DNA overload
6. Dirty gel support
7. Dried well
8. DNA sticking in the gel well caused by inappropriate gel density

Causes for specific nonspecifics and the false contamination

1. Chimeras
2. Allele dropout
3. Heteroduplexes
4. Primer multimers
5. Low resolution electrophoresis resulting in imprecise idea of the correct band position
6. Coincidence or the devil

Mineral oil and wax

1. Mineral oil
2. Wax, paraffin or vaseline

Primer-dimers and primer multimers
Short PCR fragments versus long PCR fragments
Avoiding accidents

CONCLUSIONS

A few words to the novice
A few words to a PCR adept

GLOSSARY

INDEX

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.

How to buy this book

(EAN: 9781904455080, 9781904455073 Subjects: [microbiology] [medical microbiology] [molecular biology] [genomics] [pcr] [molbio])