- Email: [email protected]
The impossible takes a little longer
BOOKS
The impossible takes a little longer In situ PCR Techniques by Omar Bagasra and John Hansen Wiley-Liss, 1997. £32.50 pbk (142 pages) ISBN 0 471 15946 8 The technique of in situ PCR is a relatively recent invention and is not routine in many laboratories, unlike the individual techniques of PCR amplification and in situ hybridization. This is partly because the combination of the techniques appears to achieve the impossible. The principle is to perform a PCR amplification of target DNA or an RT–PCR of target RNA in situ on either tissue sections or on cell spreads on modified microscope slides. The amplification products are then visualized by in situ hybridization to the newly synthesized DNA. The sceptics among us say that this cannot possibly work reliably because the amplification products will diffuse away throughout the reaction solution and lead to unlocalized signals after in situ hybridization. Bagasra and Hansen reveal the various tricks that make the impossible possible. It just takes a little longer. The first trick is to permeabilize the cells to allow entry of the reagents but little leakage of the reaction products. This is achieved by a brief heat treatment at 105⬚C for 5–120 seconds, which appears to immobilize the tissue on the slide and to alter the cellular components so that the amplicons that are synthesized subsequently are held at the loci within the cells where the target nucleic acids originated. After heat treatment, the tissue is fixed and treated with
proteinase K, which permeabilizes the membranes and makes available the target mRNA or DNA. If the target is an mRNA it is first converted to DNA with reverse transcriptase and, depending on the nature of the PCR primers, there might be an additional DNase treatment required to prevent genomic DNA from providing an alternative target site for amplification. The subsequent rounds of amplification require that the reactions are sealed under the coverslip to prevent evaporation. Bagasra and Hansen describe the various alternatives available for creating these incubation chambers. Some exponents of in situ PCR advocate a hot start to the amplification reactions, which is achieved by adding the polymerase when the reaction is at 60–70⬚C. There is a useful discussion of the pros and cons of a hot start and, if it is to be included, then how best to perform it. The book also describes in situ PCR on chromosome spreads. In this case the naked chromosomes are covered with ‘ghost membranes’ made from the original cell culture from which the spreads were made. This prevents the diffusion of the amplification products and ensures the signals are localized. The heat fixation step is also eliminated and fewer cycles of amplification are used. In situ PCR on plant material requires removal of chitin from the cell walls.
Microbiology – on a plate An Electronic Companion to Beginning Microbiology by J.L. Ingraham, M. Schaechter and F.C. Neidhardt
An Electronic Companion to Microbiology for Majors by M.L. Wheelis Cogito Learning Media, 1997. $125.00 each Since the first complete genome sequence of a free-living microorganism, Haemophilus influenzae, was determined in 1995, more than 10 microbial genomes have now been sequenced and 50 more are expected to be sequenced by the turn of the century. A surge of whole-genome sequencing will have a profound impact on microbiology, greatly
advancing our understanding of the microbial world. At the same time, microbiology is fast becoming a major discipline that is essential for understanding all aspects of life sciences with microbes as model organisms. The Electronic Companions by Cogito Learning Media are CD-ROM collections of key concepts in TIG APRIL 1998 VOL. 14 NO. 4
Copyright © 1998 Elsevier Science Ltd. All rights reserved. 0168-9525/98/$19.00
165
Chitin presents a barrier to diffusion and also binds magnesium, which is essential for the amplification. This problem is overcome by enzyme digestion of the plant tissue releasing protoplasts that can be spread on a glass slide for easy assay. Following amplification there are many copies of the target DNA and this is easily visualized by using DNA or RNA probes. These can be quite short and labelled with digoxigenin-, biotinor fluorescein-tagged nucleotides. Signals are detected directly by fluorescence or by an enzyme-conjugated antibody and a chromogenic substrate. The technique is more powerful than conventional in situ hybridization in that single RNA or DNA molecules can be detected per cell. However, it requires well-designed controls and careful interpretation. These are discussed in detail by the authors. In the right hands and by using an automated scanning microscope, the authors claim that a single viral infected cell can be detected in 100 million negative cells. This has application in screening for malignant tumour cells in bone marrow and is a powerful tool in designing better cancer therapies. Althought the book does falter in places (one or two figures have the wrong legend and the some of the colour photographs of presumably the authors’ clearest results are not very clear), in general the book is authoritative and convincingly written.
Trevor Jowett [email protected] Dept of Biochemistry and Genetics, University of Newcastle on Tyne Medical School, Newcastle on Tyne, UK NE2 4HH.
microbiology that are intended to be used in conjunction with most textbooks to introductory and intermediate college courses. In fact, Beginning Microbiology and Microbiology for Majors are suitable not only for non-biology majors and biology majors, respectively, but also for professionals who wish to use microbial analogy for understanding life sciences. The two CD-ROMs are created by different authors. Beginning Microbiology covers introductory materials in genetics, biochemistry, and molecular and cellular biology, while Microbiology for Majors contains more specialized topics in microbiology. The contents are classified into 20 topics in Beginning Microbiology and 13 topics in Microbiology for Majors. Each topic is further divided into sections and each section into subsections. A subsection sometimes consists of multiple pages, totaling a few hundred
The impossible takes a little longer In situ PCR Techniques by Omar Bagasra and John Hansen Wiley-Liss, 1997. £32.50 pbk (142 pages) ISBN 0 471 15946 8 The technique of in situ PCR is a relatively recent invention and is not routine in many laboratories, unlike the individual techniques of PCR amplification and in situ hybridization. This is partly because the combination of the techniques appears to achieve the impossible. The principle is to perform a PCR amplification of target DNA or an RT–PCR of target RNA in situ on either tissue sections or on cell spreads on modified microscope slides. The amplification products are then visualized by in situ hybridization to the newly synthesized DNA. The sceptics among us say that this cannot possibly work reliably because the amplification products will diffuse away throughout the reaction solution and lead to unlocalized signals after in situ hybridization. Bagasra and Hansen reveal the various tricks that make the impossible possible. It just takes a little longer. The first trick is to permeabilize the cells to allow entry of the reagents but little leakage of the reaction products. This is achieved by a brief heat treatment at 105⬚C for 5–120 seconds, which appears to immobilize the tissue on the slide and to alter the cellular components so that the amplicons that are synthesized subsequently are held at the loci within the cells where the target nucleic acids originated. After heat treatment, the tissue is fixed and treated with
proteinase K, which permeabilizes the membranes and makes available the target mRNA or DNA. If the target is an mRNA it is first converted to DNA with reverse transcriptase and, depending on the nature of the PCR primers, there might be an additional DNase treatment required to prevent genomic DNA from providing an alternative target site for amplification. The subsequent rounds of amplification require that the reactions are sealed under the coverslip to prevent evaporation. Bagasra and Hansen describe the various alternatives available for creating these incubation chambers. Some exponents of in situ PCR advocate a hot start to the amplification reactions, which is achieved by adding the polymerase when the reaction is at 60–70⬚C. There is a useful discussion of the pros and cons of a hot start and, if it is to be included, then how best to perform it. The book also describes in situ PCR on chromosome spreads. In this case the naked chromosomes are covered with ‘ghost membranes’ made from the original cell culture from which the spreads were made. This prevents the diffusion of the amplification products and ensures the signals are localized. The heat fixation step is also eliminated and fewer cycles of amplification are used. In situ PCR on plant material requires removal of chitin from the cell walls.
Microbiology – on a plate An Electronic Companion to Beginning Microbiology by J.L. Ingraham, M. Schaechter and F.C. Neidhardt
An Electronic Companion to Microbiology for Majors by M.L. Wheelis Cogito Learning Media, 1997. $125.00 each Since the first complete genome sequence of a free-living microorganism, Haemophilus influenzae, was determined in 1995, more than 10 microbial genomes have now been sequenced and 50 more are expected to be sequenced by the turn of the century. A surge of whole-genome sequencing will have a profound impact on microbiology, greatly
advancing our understanding of the microbial world. At the same time, microbiology is fast becoming a major discipline that is essential for understanding all aspects of life sciences with microbes as model organisms. The Electronic Companions by Cogito Learning Media are CD-ROM collections of key concepts in TIG APRIL 1998 VOL. 14 NO. 4
Copyright © 1998 Elsevier Science Ltd. All rights reserved. 0168-9525/98/$19.00
165
Chitin presents a barrier to diffusion and also binds magnesium, which is essential for the amplification. This problem is overcome by enzyme digestion of the plant tissue releasing protoplasts that can be spread on a glass slide for easy assay. Following amplification there are many copies of the target DNA and this is easily visualized by using DNA or RNA probes. These can be quite short and labelled with digoxigenin-, biotinor fluorescein-tagged nucleotides. Signals are detected directly by fluorescence or by an enzyme-conjugated antibody and a chromogenic substrate. The technique is more powerful than conventional in situ hybridization in that single RNA or DNA molecules can be detected per cell. However, it requires well-designed controls and careful interpretation. These are discussed in detail by the authors. In the right hands and by using an automated scanning microscope, the authors claim that a single viral infected cell can be detected in 100 million negative cells. This has application in screening for malignant tumour cells in bone marrow and is a powerful tool in designing better cancer therapies. Althought the book does falter in places (one or two figures have the wrong legend and the some of the colour photographs of presumably the authors’ clearest results are not very clear), in general the book is authoritative and convincingly written.
Trevor Jowett [email protected] Dept of Biochemistry and Genetics, University of Newcastle on Tyne Medical School, Newcastle on Tyne, UK NE2 4HH.
microbiology that are intended to be used in conjunction with most textbooks to introductory and intermediate college courses. In fact, Beginning Microbiology and Microbiology for Majors are suitable not only for non-biology majors and biology majors, respectively, but also for professionals who wish to use microbial analogy for understanding life sciences. The two CD-ROMs are created by different authors. Beginning Microbiology covers introductory materials in genetics, biochemistry, and molecular and cellular biology, while Microbiology for Majors contains more specialized topics in microbiology. The contents are classified into 20 topics in Beginning Microbiology and 13 topics in Microbiology for Majors. Each topic is further divided into sections and each section into subsections. A subsection sometimes consists of multiple pages, totaling a few hundred