PCR 3: PCR In Situ Hybridization A Practical Approach

by Herrington, C. Simon; O'Leary, John J.

• ISBN13: 9780199636334
• ISBN10: 0199636338
• Format: Hardcover
• Copyright: 1998-01-08
• Publisher: Oxford University Press

Summary

PCR in situ hybridization allows the detection of specific nucleic acidsequences and their distribution in the cell. It combines two powerfultechniquesin situ hybridization (ISH), which allows cellular localization of DNAand RNA sequences in cells and tissues, and the polymerase chain reaction (PCR),which allows reproducible amplification of rare nucleic acid sequences. Thecombined technique and its variants greatly enhance the sensitivity of in situhybridization and add morphological localization to the sensitivity of PCR. Suchtechniques have enormous potential for research and diagnosis but problems withreproducibility and reliability are often encountered. This book overcomesthese problems by describing the key procedures in step-by-step detail and byproviding the essential advice needed for success. Topics include: DNA in situPCR and DNA PCR in situ hybridization (PCR ISH) for the detection of DNA targetsin cells; reverse trancriptase in situ PCR (RT-PCR) and RT-PCR ISH for thedetection of RNA targets; and PRINS (primed in situ synthesis) for chromosomalanalysis in interphase nuclei and metaphase chromosome spreads. There arefurther chapters on fixation of tissues for PCR, selective ultraviolet radiationfractionation (SURF), application of in situ PCR to human tissues, applicationsand modifications of PCR-ISH, and automation of in situ amplification. PCR InSitu Hybridization is a unique and timely collection of well-tested protocolsfor the amplification of DNA and RNA in cells and tissues, drawing on theaccumulated knowledge and experience of leading exponents of these techniques.For each topic covered, the authors provide detailed guidance on the key stepsin the protocols, numerous hints and tips for success, and advice ontrouble-shooting. PCR In Situ Hybridization will be invaluable to molecularbiologists, pathologists, geneticists, and all those seeking to perform in situanalyses of nucleic acid molecules.

Table of Contents

List of Contributors	xv	(2)
Abbreviations	xvii
1. Fixation of tissues for the polymerase chain reaction	1	(10)
David Hopwood
1. Introduction	1	(1)
2. Reaction of fixatives with nucleic acids	2	(1)
3. Fixation	3	(5)
Fixatives	3	(1)
Diffusion	4	(1)
Kinetics of fixation	5	(1)
Temperature	6	(1)
Concentration of fixatives	6	(1)
Duration of fixation	6	(1)
Hydrogen ion concentration and buffers	6	(1)
Microwave fixation/stabilization	7	(1)
4. Artefacts	8	(1)
5. Hazards	9	(1)
References	9	(2)
2. In situ genetic analysis with selective ultraviolet radiation fractionation (SURF)	11	(16)
Adrian Ireland
Darryl Shibata
1. Introduction	11	(2)
Principles of SURF	11	(1)
Advantages of SURF	12	(1)
2. SURF	13	(10)
Suitable tissues and PCR targets	13	(1)
Staining of sections	13	(3)
Dotting of selected cells	16	(3)
Ultraviolet irradiation	19	(2)
Isolation of DNA	21	(1)
PCR	22	(1)
Data presentation and analysis	22	(1)
3. Applications	23	(3)
Tumour evolution: out-of-Africa, out-of-adenoma	25	(1)
Acknowledgements	26	(1)
References	26	(1)
3. In situ hybridization	27	(26)
Shirley A. Southern
C. Simon Herrington
1. Introduction	27	(1)
2. Probes	28	(1)
3. Choice of label	29	(1)
4. Probe labelling	30	(1)
Nick translation	30	(1)
Random primer extension	30	(1)
Oligonucleotide tailing	30	(1)
Probe labelling check using nylon membranes	30	(1)
5. Controls	31	(3)
6. Specimen collection and fixation	34	(1)
7. Preparation of RNase/DNase-free solutions, glassware, and plastics	34	(1)
8. Slide adhesive	35	(1)
9. Section cutting	36	(1)
10. Pre-hybridization	36	(2)
Permeabilization	36	(1)
Blocking of non-specific probe interactions	37	(1)
11. Denaturation	38	(1)
12. Hybridization parameters	38	(2)
T(m)	38	(1)
Stringency	39	(1)
Hybridization buffer	39	(1)
Hybridization time	40	(1)
13. Post-hybridization washes	40	(1)
14. Detection systems	41	(5)
15. Substrates	46	(4)
16. Troubleshooting	50	(1)
References	50	(3)
4. PCR in situ hybridization (PCR ISH)	53	(34)
John J. O'Leary
1. Introduction	53	(1)
2. Definitions and terminology	53	(1)
3. Principles of the techniques	54	(1)
4. Materials and equipment	54	(1)
5. Tissue fixation and preparation	55	(1)
Cell and tissue fixation	55	(1)
Cell and tissue adhesion	55	(1)
Exposure of nucleic acid template	55	(1)
6. Amplification: reagents and conditions	56	(1)
7. Post-amplification washing and fixation	56	(1)
8. Visualization of PCR product	57	(1)
9. Reaction, tissue, and detection controls	57	(1)
10. Problems associated with the techniques	58	(1)
Sequestration of reagents	59	(1)
Diffusion of amplicons	59	(1)
Patchy amplification	59	(1)
11. Current applications	59	(1)
12. PCR ISH methodology	60	(25)
Preparation of slides, cells, and tissue specimens	60	(2)
Tissue and cell pre-treatment	62	(3)
Amplification	65	(4)
In situ hybridization	69	(8)
Post-hybridization washing	77	(1)
Detection of hybridization signal	78	(7)
References	85	(2)
5. Reverse transcriptase in situ PCR for RNA detection	87	(16)
M. Jim Embleton
1. Introduction	87	(1)
2. Preparation of cells for RT-PCR	88	(2)
3. Reverse transcriptase reaction (first-strand cDNA synthesis)	90	(2)
4. PCR with fluorescent primers	92	(8)
5. In situ PCR with biotinylated primers	100	(2)
References	102	(1)
6. Primed in situ DNA synthesis (PRINS)	103	(14)
John R. Gosden
Ernst J. M. Speel
Yoshiro Shibasaki
1. Introduction	103	(1)
2. Specimen preparation	104	(1)
3. PRINS	104	(11)
Denaturation	105	(1)
The basic PRINS procedure	106	(1)
Detection methods	107	(3)
Variations on the basic method	110	(5)
References	115	(2)
7. Application of in situ PCR techniques to human tissues	117	(22)
Omar Bagasra
Lisa E. Bobroski
Muhammed Amjad
Roger J. Pomerantz
John Hansen
1. Introduction	117	(1)
2. Preparation of glass slides and tissues	118	(3)
Glass slides with Teflon-edged wells	118	(1)
Preparation of cells and tissues	119	(2)
3. In situ amplification: DNA and RNA targets	121	(10)
Basic preparation, all protocols	121	(3)
Reverse transcriptase variation: in situ RNA amplification	124	(7)
4. Special application of in situ amplification	131	(1)
In situ amplification and immunohistochemistry	131	(1)
Multiple signals, multiple labels in individual cells	131	(1)
5. Hybridization	131	(5)
Summary of the basic concepts of in situ hybridization	131	(5)
6. Validation and controls	136	(1)
References	137	(2)
8. Applications and modifications of PCR in situ hybridization	139	(24)
Bruce K. Patterson
1. Introduction	139	(3)
General	139	(1)
Technical	140	(2)
2. Single parameter analysis	142	(8)
Cells	142	(4)
Tissue	146	(4)
3. Multiparameter analysis	150	(2)
4. Fluorescence in situ 5'-nuclease assay and fluorescence detection	152	(9)
References	161	(2)
9. Automation of in situ amplification	163	(32)
1. Technologies available for in situ amplification	163	(1)
John J. O'Leary
2. The GeneAmp(R) In Situ PCR System 1000	164	(10)
Steve Picton
David Howells
Introduction	164	(1)
PCR amplification in situ: problems and pitfalls	165	(1)
In situ PCR: the case for dedicated instrumentation and sample containment systems	166	(1)
A dedicated high-performance system for PCR amplification on glass slides	167	(5)
Dedicated and qualified reagent system for in situ PCR amplification	172	(2)
Summary	174	(1)
3. Automation and the Omnislide System	174	(19)
Jacqueline A. Starling
Introduction	174	(1)
Factors and problems to be addressed in order to semi-automate PCR ISH	175	(2)
System design: theoretical and practical considerations	177	(7)
Hybaid's In Situ Systems	184	(3)
Results achieved	187	(6)
Summary	193	(1)
Acknowledgements	193	(1)
References	193	(2)
A1. List of suppliers	195	(6)
Index	201