Book Review |
Whole Genome Amplification
Edited by S. Hughes and R. Lasken
Scion Publishing Ltd, Bloxham, UK;
2005; ISBN 1-904842-07-0; 193 pp.;
Paperback. With increasingly powerful methods of genomic analysis becoming available in the last few years, the availability of genomic DNA in sufficient quantities can become a limiting factor. This requirement for the unlimited supply of DNA has accelerated the development of reliable methods of amplifying the whole genome.
This book outlines various methodologies that could be adopted to enhance and optimise ‘Whole Genome Amplification’ (WGA). Each chapter contains a concise introduction that leads the reader into the practical approaches of the various subjects covered. The figures and tables are clearly presented and well defined. Furthermore, if greater detail is required, the comprehensive and up-to-date reference list allows further specifics to be obtained. This layout means that the chapters flow well, without hindering understanding with excessive minutiae. Consequently, the book is suitable for both postgraduate and postdoctoral research scientists. It is a timely publication welcomed by both experienced and newcomers to this technology.
The first commonly used WGA methods were the PCR-based technique of using degenerate-oligonucleotide (DOP) and primer-extension pre-amplification (PEP), described in Chapters 2 and 3. DOP-PCR primers have defined sequences at their 5' and 3' ends with a random hexamer sequence between these regions whereas PEP-PCR uses totally degenerate oligonucleotides. The number of potential binding sites is greater in PEP-PCR than DOP-PCR, and hence PEP-PCR has higher amplification efficiency than DOP-PCR. These methods were then subsequently modified in order to reduce the amplification bias as described in Chapters 4–6. The genomic DNA is initially fragmented by using heat, enzymes or by mechanical means. Adaptors are then ligated to the ends of the sheared DNA and PCR performed to produce micrograms of DNA. In many cases where only short degraded DNA are available, such as that isolated from paraffin embedded and formalin fixed tissues, these adaptor-ligation PCR methods of amplification offer a useful tool to use in genotyping and other applications.
Chapter 7 describes a T7 phage RNA polymerase-based linear amplification of genomic DNA. This non-exponential isothermal method of amplification minimises amplification bias and was developed for the use of Chip-chip analysis, where DNA recovered from chromatin immunoprecipitation method is used on DNA microarray analysis.
Another isothermal amplification method is the multiple displacement amplification (MDA). MDA uses phi 29 enzyme and the reaction is carried out at 30°C. It is probably the closest method of DNA replication to that in the living cells. It is capable of replicating approximately 99.8% of the genome with minimum low amplification bias. It is also the only method of amplification capable of producing DNA of high molecular weight, generating fragments of DNA ranging from 2 kb to more than 100 kb in length. Phi 29 enzyme possesses 3'-5'exonuclease proofreading activity, allowing high-fidelity DNA to be produced. However, this method does not work well with degraded DNA. Fragments shorter than 2 kb will not be efficiently amplified and may suffer from reduced locus representation. Thus, this method of amplification may not be suitable to use for forensic purposes or for samples where a high degree of degradation is expected. Chapter 8 gives a comprehensive description of this method.
An application that is often neglected using WGA is within the field of prokaryotic genetics. Once an effective method of cell sorting has been selected, flow-cytometry or serial dilutions, which, incidentally, are covered within Chapter 9, a MDA reaction may be performed. The chapter covers potential downsteam processes that can be utilised in comparative genomic studies, using a single cell as the initial starting point. This opens the door to new opportunities of genomic variation, intra- and interspecies.
Chapter 10 covers the area of genomic amplification tolerant to sample degradation. This is an essential area for retrospective comparative genomic studies of many types of archived material. In brief, formalin-fixed and paraffin-embedded specimens often result in strand breaks, base damage and DNA–protein cross-links within the substrate DNA. An adaptation of the rolling circle amplification (RCA), termed restriction and circularisationaided (RCA–RCA), is approached in this chapter, setting the overall strategies in concise sections. Although one of the smaller chapters, its very much envisaged that this will be an area that will expand in the future.
The 11th and final chapter looks at the area of pre-implantation genetic diagnosis (PGD), using WGA. The approach outlined considers single or multiple cells as a starting material, also the different strategies that can be adopted to achieve amplification. Once again, it is an area for the future; it really highlights the accuracy and versatility of WGA as an application within the field of diagnostic testing.
To conclude, Whole Genome Amplification is an excellent reference guide that would prove to be extremely useful in any molecular biology lab. The examples and recommended protocols are to the point, with supporting ‘trouble shooting’ sections in several of the chapters. Overall, the book is good value, which should be affordable in an academic environment.
Geneservice Ltd, Cambridge, UK
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