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Essentials of nucleic acid analysis: a robust approach.
JT Keer, L. Birch eds. 2008.
Cambridge, UK: RSC Publishing. £79.95 (hardback). 248 pp.
The analysis of the sequence, diversity and abundance of nucleic acids underpins modern biology. There are many books available that introduce the uninitiated to the delights of nucleic acid analysis, mostly in the form of recipe collections, and such books succeed or fail by the attention to detail given to optimization, standardization and trouble-shooting protocols. Given that nucleic acid analyses are now used routinely in forensic and food safety fields, robust methods are highly desirable, if not essential.
This book takes the welcome approach of focussing on quality control, quality assurance, optimization, validation and calibration. The first chapter sets the context for the rest of the book, introducing ‘Valid Analytical Measurement’ principles, which will be familiar to those working in ISO17025 and similar environments. The second chapter discusses quality in the molecular laboratory and provides pointers to the relevant ISO and other standards, and stresses the need for sample and metrological traceability. The third chapter introduces the general principles of method validation and the concepts of accuracy, precision, limit of detection, limit of quantification, measurement uncertainty, etc., and then gives a worked example of the validation of a realtime PCR procedure.
The next four chapters cover DNA extraction, DNA quantification, factors affecting PCR reliability and validity, and quantitative realtime PCR. The DNA quantification chapter is particularly helpful – emphasizing the necessity of using an appropriate method for the material under study. The PCR chapter has clear guidelines for dealing with inhibitory materials present in DNA from animal samples, but, alas, it is poor on plant DNA. The post-PCR analysis section of this chapter is quite short and only gives a general introduction to the subject. The quantitative realtime PCR chapter clearly explains the different approaches that are now available, and has a helpful comparison of realtime PCR machines. This chapter also covers the mathematics for understanding quantitative PCR.
The eighth chapter deals with multiplex PCR and whole-genome PCR. As with the realtime PCR chapter, there is some unnecessary repetition within the chapter. It does, however, mention untemplated nucleotide addition, but does not give practical advice on minimizing the difficulties that it can cause (beyond saying that it is easier to optimize the addition of the extra nucleotide).
The following chapter, on RNA, includes more data from real experiments than is typical of the other chapters, and uses these experiments as a way of conveying tips and tricks. The authors deal with extraction, quantification, quality assessment and qPCR in an engaging way. As in the PCR chapter, the emphasis is on RNA of animal origin and therefore those with green fingers should look elsewhere for guidance on extracting plant RNA, although much of the chapter is of general interest.
The tenth and final chapter deals with microarrays, and is almost entirely based on the use of arrays in the analysis of gene expression. There is a helpful discussion of the basic types of array and the applications of arrays. A good part of the chapter deals with obtaining reliable, consistent data from microarray experiments. Much of the inconsistency in microarray experiments is due to the complexity of the procedure, and the chapter considers each step in turn with recommendations for minimizing the inconsistency. The authors also include pointers to international and national initiatives that have evolved in order to foster the adoption of uniform methods and standardized techniques.
In a book such as this, one would expect a chapter on robust methods for DNA sequencing, but neither sequencing nor resequencing is covered. One of the shortcomings of this kind of book is that, due to the time scales involved, the science and technology have moved on since the ink dried on the authors' submissions. This book, for instance, does not cover next-generation sequencing (Solexa, 454, Solid, etc.) and nor does it cover the more modern forms of genotyping (e.g. the Illumina Golden Gate system) that have revolutionized nucleic acid analysis in the last two years. It would seem to be self-evident that quality management principles should be applied to techniques that generate such enormous volumes of raw data.
Whilst the book is primarily directed at laboratories within the professional analytical sector, it would be of immense benefit to many undergraduates and recent graduates. The book is well produced, with clear diagrams and illustrations of key points. In summary, it is a book that is recommended reading for anyone involved in the analysis of nucleic acids, particularly those using PCR-based techniques.