PCR troubleshooting: the essential guide
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| Main Author: | |
|---|---|
| Format: | Book |
| Language: | English |
| Published: |
Wymondham
Caister Acad. Press
2006
|
| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Physical Description: | 80 S. |
| ISBN: | 1904455077 9781904455073 1904455085 9781904455080 |
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| 100 | 1 | |a Altshuler, Michael L. |e Verfasser |4 aut | |
| 245 | 1 | 0 | |a PCR troubleshooting |b the essential guide |c by Michael L. Altshuler |
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Record in the Search Index
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| adam_text | TABLE OF CONTENTS
PREFACE 9
INTRODUCTION 11
APPEARANCES 13
• Unsatisfactory results of PCR 13
CAUSES AND ACTIONS 14
• Do not confront the problem 14
• The nature of PCR and its pathology 15
• Inadequate concentrations of ingredients 18
1. The template 18
2. Inadequate deoxynucleotidetriphosphates 20
3. Inadequate primers 20
4. Inadequate Taq 21
5. Inadequate Mg2+ 21
6. Suboptimal KC1 concentration in the PCR buffer
or the whole of the PCR buffer 21
• Inadequate quality of ingredients 23
l.DNA template 23
Conversion of a DNA solution into a solid body 23
PCR inhibitors 23
Degraded template 23
Verification of the purity of the template
DNA by optical means 24
2. Poor water 24
3. Deoxynucleotidetriphosphates 24
3
4. Poor primers 25
Primers may not be good in practice even if
they are good in design 25
For primer selection use only dedicated
software 25
Difference in Tm balanced by different
primer concentrations 26
Inefficient priming 26
5. Inadequate MgCl2 26
6. Poor buffer 27
7. Poor Taq 27
8. The presence of PCR inhibitors 27
9. Substances that do not inhibit PCR 28
• Inadequate storage of ingredients for the PCR reaction ..29
1. The treacherous refrigerators 29
2. Templates, dNTPs, primers 29
3. Taq polymerase 31
4.MgCl2 31
5. Buffer 31
• The thermocycler 32
1. Conductivity of heat puts a limit
to the mix composition 32
2. The ramp 33
3. Time and temperature 34
4. Dusty, greasy, or fluffy tube wells 36
5. Rapid evaporation 36
6. Suboptimal performance of the thermocycler or its
particular wells 36
7. The tubes are poorly pressed down into the wells
or they have got deformed 37
4
• Faulty target selection 38
• Incomplete DNA denaturation and dispersal 40
1. Template DNA 40
2. PCR fragments 41
3. Hairpins 42
• The Taq enzyme 43
1. Hurdles for Taq polymerase 43
Stable hairpins in the template strand 43
AT rich areas 43
GC rich areas 43
Alternating GC/AT rich regions 43
2. Hot start 44
The improved hot start 44
The role of the reaction volume in the
quasi hot start 45
3. Nonspecific binding of Taq to DNA 46
• Incomplete primer elongation
or premature termination of DNA synthesis 48
1. Under elongation of primers in the late PCR 48
2. Premature termination of the DNA synthesis 50
• Cosolvents or additives or enhancers 51
1. Helix destabilizers 51
2. Helix stabilizers 52
3. Substances that neutralise the PCR inhibitors 53
4. PCR enhancers with poorly understood
mechanism of action 53
• Approaching the limit of the PCR sensitivity 54
5
• Agarose gel electrophoresis 57
1. The band diffuses and disappears 57
2. Short fragments of uneven length migrate
down the gel without any separation 57
3. The band is invisible 57
4. The significance and the insignificance of the salt
concentration in the compared samples 57
5. Bands smear due to their fast movement or the
DNA overload 58
6. Dirty gel support 58
7. Dried well 58
8. DNA sticking in the gel well caused by
inappropriate gel density 58
• Causes for specific nonspecifics
and the false contamination 59
1. Chimeras 59
2. Allele dropout 59
3. Heteroduplexes 60
4. Primer multimers 61
5. Low resolution electrophoresis resulting in
imprecise idea of the correct band position 61
6. Coincidence or the devil 61
• Mineral oil and wax 62
1. Mineral oil 62
2. Wax, paraffin or vaseline 62
• Primer dimers and primer multimers 63
• Short PCR fragments versus long PCR fragments 67
• Avoiding accidents 68
6
CONCLUSIONS 69
• A few words to the novice 69
• A few words to a PCR adept 69
GLOSSARY 71
INDEX 77
7
|
| adam_txt |
TABLE OF CONTENTS
PREFACE 9
INTRODUCTION 11
APPEARANCES 13
• Unsatisfactory results of PCR 13
CAUSES AND ACTIONS 14
• Do not confront the problem 14
• The nature of PCR and its pathology 15
• Inadequate concentrations of ingredients 18
1. The template 18
2. Inadequate deoxynucleotidetriphosphates 20
3. Inadequate primers 20
4. Inadequate Taq 21
5. Inadequate Mg2+ 21
6. Suboptimal KC1 concentration in the PCR buffer
or the whole of the PCR buffer 21
• Inadequate quality of ingredients 23
l.DNA template 23
Conversion of a DNA solution into a solid body 23
PCR inhibitors 23
Degraded template 23
Verification of the purity of the template
DNA by optical means 24
2. Poor water 24
3. Deoxynucleotidetriphosphates 24
3
4. Poor primers 25
Primers may not be good in practice even if
they are good in design 25
For primer selection use only dedicated
software 25
Difference in Tm balanced by different
primer concentrations 26
Inefficient priming 26
5. Inadequate MgCl2 26
6. Poor buffer 27
7. Poor Taq 27
8. The presence of PCR inhibitors 27
9. Substances that do not inhibit PCR 28
• Inadequate storage of ingredients for the PCR reaction .29
1. The treacherous refrigerators 29
2. Templates, dNTPs, primers 29
3. Taq polymerase 31
4.MgCl2 31
5. Buffer 31
• The thermocycler 32
1. Conductivity of heat puts a limit
to the mix composition 32
2. The ramp 33
3. Time and temperature 34
4. Dusty, greasy, or fluffy tube wells 36
5. Rapid evaporation 36
6. Suboptimal performance of the thermocycler or its
particular wells 36
7. The tubes are poorly pressed down into the wells
or they have got deformed 37
4
• Faulty target selection 38
• Incomplete DNA denaturation and dispersal 40
1. Template DNA 40
2. PCR fragments 41
3. Hairpins 42
• The Taq enzyme 43
1. Hurdles for Taq polymerase 43
Stable hairpins in the template strand 43
AT rich areas 43
GC rich areas 43
Alternating GC/AT rich regions 43
2. Hot start 44
The improved hot start 44
The role of the reaction volume in the
quasi hot start 45
3. Nonspecific binding of Taq to DNA 46
• Incomplete primer elongation
or premature termination of DNA synthesis 48
1. Under elongation of primers in the late PCR 48
2. Premature termination of the DNA synthesis 50
• Cosolvents or additives or enhancers 51
1. Helix destabilizers 51
2. Helix stabilizers 52
3. Substances that neutralise the PCR inhibitors 53
4. PCR enhancers with poorly understood
mechanism of action 53
• Approaching the limit of the PCR sensitivity 54
5
• Agarose gel electrophoresis 57
1. The band diffuses and disappears 57
2. Short fragments of uneven length migrate
down the gel without any separation 57
3. The band is invisible 57
4. The significance and the insignificance of the salt
concentration in the compared samples 57
5. Bands smear due to their fast movement or the
DNA overload 58
6. Dirty gel support 58
7. Dried well 58
8. DNA sticking in the gel well caused by
inappropriate gel density 58
• Causes for specific nonspecifics
and the false contamination 59
1. Chimeras 59
2. Allele dropout 59
3. Heteroduplexes 60
4. Primer multimers 61
5. Low resolution electrophoresis resulting in
imprecise idea of the correct band position 61
6. Coincidence or the devil 61
• Mineral oil and wax 62
1. Mineral oil 62
2. Wax, paraffin or vaseline 62
• Primer dimers and primer multimers 63
• Short PCR fragments versus long PCR fragments 67
• Avoiding accidents 68
6
CONCLUSIONS 69
• A few words to the novice 69
• A few words to a PCR adept 69
GLOSSARY 71
INDEX 77
7 |
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| language | English |
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| spelling | Altshuler, Michael L. Verfasser aut PCR troubleshooting the essential guide by Michael L. Altshuler Wymondham Caister Acad. Press 2006 80 S. txt rdacontent n rdamedia nc rdacarrier DNA polymerases Polymerase chain reaction Polymerase-Kettenreaktion (DE-588)4256726-9 gnd rswk-swf Polymerase-Kettenreaktion (DE-588)4256726-9 s DE-604 HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=014842317&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Altshuler, Michael L. PCR troubleshooting the essential guide DNA polymerases Polymerase chain reaction Polymerase-Kettenreaktion (DE-588)4256726-9 gnd |
| subject_GND | (DE-588)4256726-9 |
| title | PCR troubleshooting the essential guide |
| title_auth | PCR troubleshooting the essential guide |
| title_exact_search | PCR troubleshooting the essential guide |
| title_exact_search_txtP | PCR troubleshooting the essential guide |
| title_full | PCR troubleshooting the essential guide by Michael L. Altshuler |
| title_fullStr | PCR troubleshooting the essential guide by Michael L. Altshuler |
| title_full_unstemmed | PCR troubleshooting the essential guide by Michael L. Altshuler |
| title_short | PCR troubleshooting |
| title_sort | pcr troubleshooting the essential guide |
| title_sub | the essential guide |
| topic | DNA polymerases Polymerase chain reaction Polymerase-Kettenreaktion (DE-588)4256726-9 gnd |
| topic_facet | DNA polymerases Polymerase chain reaction Polymerase-Kettenreaktion |
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