Analytical Molecular Biology: Quality and ValidationHelen Parkes, V (Ginny) Saunders In spite of the wide variety and complexity of biological materials, nucleic acids are ubiquitous. DNA is becoming the bioanalyte of choice due to the vast amount of information embedded in its sequence, its robust chemical nature and the range of highly sensitive analytical techniques that have been developed. The results of such analyses can have an important impact on our society both commercially and in terms of the quality of life. Absolute confidence in the data generated is therefore of the utmost importance. This book, produced by LGC as part of the VAM (Valid Analytical Measurement) Programme, introduces the issues of validation and quality to the bioanalytical community, specifically addressing DNA-based analyses. It aims to raise awareness of the factors that can influence the validity of DNA analysis and the production of quality data. Emphasis is placed on VAM principles, as well as additional challenges that are associated with the analysis of real samples, for example, complex food matrices or forensic samples that have been subjected to environmental insult. Information is collated from a variety of sources including literature, discussions and LGC research, and offers constructive advice where possible. |
Contents
Chapter 1 An Introduction to Analytical Molecular Biology | 1 |
Chapter 2 Quality in the Analytical Molecular Biology Laboratory | 9 |
Chapter 3 DNA Extraction | 29 |
Chapter 4 Quantification of Total DNA by Spectroscopy | 47 |
Chapter 5 PCR Factors Affecting Reliability and Validity | 58 |
Chapter 6 Inhibitors and Enhancers of PCR | 81 |
Chapter 7 Random Amplified Polymorphic DNA Analysis | 103 |
Chapter 8 Development of Multiplex PCR | 123 |
Chapter 9 Membrane Hybridisation | 135 |
Chapter 10 Automated Fluorescent DNA Cycle Sequencing | 155 |
Appendix Glossary of Terms | 173 |
| 183 | |
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Common terms and phrases
addition agarose gel amount analysis analytical molecular biology annealing temperature applications assay assessment base pairs binding BioTechniques blot calibration cell components contamination cycle degradation denaturation detection detergents determined dilution DMSO DNA concentration DNA extraction DNA sequencing dNTPs dye primer EDTA effect efficiency enhancers ensure enzyme ethidium bromide exonuclease factors Figure fingerprinting fluorescent forensic gel electrophoresis gene genetic genomic genomic DNA hybridisation rate immobilisation increase inhibition inhibitors labelling laboratory lane lysis magnesium matrix measurements membrane hybridisation method methodology Microbiol molecular biology molecular weight molecule mPCR multiplex PCR non-specific nucleic acid nucleotide obtained optimisation PCR amplification PCR products performance phenol polymerase chain reaction polymorphic potential precipitation primer sets probe procedures protein protocols quantitative RAPD profiles reagents reduce reference materials reproducibility result sample sensitivity single-stranded solution specific standard strand suitable Table Taq DNA polymerase Taq polymerase technique template thermal cycler tion validation µg/ml