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.
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). As with the realtime PCR chapter, there is some unnecessary repetition within the chapter. The eighth chapter deals with multiplex PCR and whole-genome PCR. This chapter also covers the mathematics for understanding quantitative PCR.
The quantitative realtime PCR chapter clearly explains the different approaches that are now available, and has a helpful comparison of realtime PCR machines. The post-PCR analysis section of this chapter is quite short and only gives a general introduction to the subject. 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 DNA quantification chapter is particularly helpful – emphasizing the necessity of using an appropriate method for the material under study. The next four chapters cover DNA extraction, DNA quantification, factors affecting PCR reliability and validity, and quantitative realtime PCR. 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 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 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. This book takes the welcome approach of focussing on quality control, quality assurance, optimization, validation and calibration. Given that nucleic acid analyses are now used routinely in forensic and food safety fields, robust methods are highly desirable, if not essential. 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. The analysis of the sequence, diversity and abundance of nucleic acids underpins modern biology.
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