FISH : a practical approach / edited by Barbara Beatty, Sabine Mai and Jeremy Squire.

Format:

Book

Contributor:

Beatty, Barbara, 1944-

Mai, Sabine.

Squire, Jeremy, 1950-

Series:

Practical approach series ; no. 260.

The practical approach series ; no. 260

Language:

English

Subjects (All):

Fluorescence in situ hybridization.

In Situ Hybridization, Fluorescence.

Medical Subjects:

In Situ Hybridization, Fluorescence.

Physical Description:

xvii, 255 pages, 16 unnumbered pages of plates : illustrations (some color) ; 26 cm.

Other Title:

Fluorescence in situ hybridization

Place of Publication:

Oxford ; New York : Oxford University Press, 2002.

Summary:

This convenient laboratory manual covers the theory and application o f all the commonly used FISH procedures for both the research and clinical service laboratory. Readers are provided with the theoretical principles which underlie each procedure, and are then guided through the protocols in a systematic, easy to follow manner. Because FISH procedures are particularly sensitive to background and subtle variations in sample quality, the trouble-shooting sections are invaluable to the beginner interested in developing FISH capabilities in their laboratory. Areas covered include probe production and labeling; FISH gene mapping; murine, fiber, and RNA FISH; CGH and microchips; 3D FISH; SKY and M-FISH; FISH and microarrays; and FISH in clinical cytogenetics.

Contents:

2 FISH probes and labelling techniques / Patricia Bray-Ward 5

2 Fluorescence principles 5

Fluors and haptens 6

Commonly used fluors 7

Choice of filter sets 7

3 Nucleic acid probes 8

Types of probes 8

Preparation of cloned probes 9

Enzymatically amplified probes 15

Synthetic oligonucleotide probes 16

4 Coupling of fluors/haptens to nucleotides 17

5 Labelling of probes 19

Nick translation 20

Random primer labelling 21

RNA transcription labelling 22

PCR labelling 24

6 Post-labelling DNA processing and purification 25

DNase treatment (for FISH and other hybridization protocols) 25

Removal of unincorporated nucleotides and BSA from the reaction mix prior to probe precipitation 25

7 Other labelling systems 26

Coupling of fluors or haptens to amine-modified nucleic acids 26

Other chemical coupling systems 26

Direct chemical coupling of fluors or haptens to proteins 26

3 Human chromosome mapping of single copy genes / Barbara G. Beatty, Stephen W. Scherer 29

2 DNA probes for FISH mapping 30

Identification of FISH probes from WWW sites 30

Preparation of probes for FISH mapping 32

3 Target DNA preparation 34

Metaphase chromosomes 34

Mapping with interphase nuclei 36

Hypotonic treatment and fixation 38

4 Slide preparation 39

Target slide pre-treatment 41

5 Denaturation and hybridization of probe and target DNA 42

6 Post-hybridization washes 44

7 Immunodetection 45

8 Chromosome counterstaining and banding 47

9 Microscopy and image analysis 48

10 FISH mapping points to consider 49

FISH mapping of single probes to metaphase chromosomes 49

Relational mapping with multiple probes 51

4 Murine chromosome preparation / Sabine Mai, Francis Wiener 55

1 Murine chromosome preparation for banding and in situ hybridization procedures 55

2 Giemsa-trypsin banding of mouse chromosomes 63

3 Molecular cytogenetic approaches for murine chromosomes 66

5 High resolution FISH mapping using chromatin and DNA fibre / Henry H. Q. Heng 77

2 Practical considerations for fibre preparation 78

3 General equipment required for fibre FISH 78

4 Chromatin fibre preparation 79

5 DNA fibre preparation 83

6 FISH 85

DNA probe labelling 85

Hybridization 86

Wash 88

Detection and amplification 89

Counterstaining and antifade 90

6 Applications of RNA FISH for visualizing gene expression and nuclear architecture / Rose Tam, Lindsay S. Shopland, Carol V. Johnson, John A. McNeil, Jeanne B. Lawrence 93

2 Cell preparation 96

Detergent-extracted cell preparation 96

Cytogenetic preparations 97

3 Probe preparation 99

4 Hybridization to RNA 100

Basic RNA hybridization procedure 101

Oligonucleotide hybridization 102

5 Hybridization to DNA 103

Detecting heat denatured cellular DNA 104

DNA FISH using NaOH denaturation and RNA hydrolysis 105

6 Multiple label techniques and applications 105

Coupling the detection of RNA with DNA 105

Coupling protein detection with FISH 107

Chromosome paints and RNA FISH 108

Differentiating transcripts with intron and cDNA probes 109

Exon suppression hybridization: an example of the use of specific competition 110

7 Visualizing and analysing results 112

Microscopy 113

Digital imaging 114

7 FISH on three-dimensionally preserved nuclei / I. Solovei, J. Walter, M. Cremer, F. Habermann, L. Schermelleh, T. Cremer 119

2 Preparation and fixation of cells 123

Preparation of slides 123

Cultivation and fixation of adherent cells 125

Preparation, attachment, and fixation of cells growing in suspension 126

3 Preparation of cells directly isolated from peripheral blood 127

4 Pre-treatments needed for hybridization 128

5 Hybridization set-up 131

Probe labelling 131

Probe preparation 132

DNA denaturation and hybridization 132

6 Post-hybridization washes, detection, nuclei counterstaining, and slide mounting 134

Post-hybridization washes 134

Detection of hybridized probes 135

Counterstaining of nuclei and mounting cells in antifade medium 137

7 Combined 3D FISH and replication labelling 139

Replication labelling 139

Detection of incorporated halogenated deoxyuridines after FISH 140

8 Combined protein immunodetection and 3D FISH 142

9 Preservation of the chromatin structure during 3D FISH 144

10 Confocal microscopy 144

Selection of the filter configuration 147

Conditions of image acquisition 149

Calibration of the instrument 150

Visualization 150

Quantitative measurements and deconvolution 152

8 Comparative genomic hybridization on metaphase chromosomes and DNA chips / Stefan Joos, Carsten Schwanen, Peter Lichter 159

2 Preparation of metaphase chromosomes 161

3 Isolation of genomic DNA 161

4 Isolation of single cells by micromanipulation 167

5 Amplification of genomic DNA from small cell populations by universal polymerase chain reaction (PCR) 168

6 Probe labelling 172

7 Comparative genomic hybridization 174

Denaturation of metaphase chromosomes 174

Probe mixture 175

In situ hybridization and signal detection 176

8 Image acquisition and evaluation 177

9 Troubleshooting of CGH experiments 178

10 Troubleshooting of CGH experiments in combination with universal PCR 179

11 New developments: matrix-CGH 180

9 FISH in clinical cytogenetics / Jeremy A. Squire, P. Marrano, E. Kolomietz 183

2 Probes commonly used for FISH in the clinical laboratory 184

3 Preparation of clinical samples for FISH analysis 185

Preparation of metaphase chromosomes for FISH 185

Preparation of interphase nuclei derived from clinical specimens for FISH 187

4 Criteria for assessing and reporting FISH results 194

General considerations when selecting cells for FISH microscopy 195

Scoring criteria for interphase FISH signal evaluation and enumeration 196

Special considerations concerning interphase FISH interpretation 197

5 Some of the commonly used FISH probes in clinical cytogenetics 198

FISH analysis of microdeletion syndromes 198

Use of the three-colour fusion (translocation/inversion) probes 199

Use of FISH probes in assessing gene amplification 201

6 Appendix (useful web sites for molecular cytogenetics clinical sources) 202

10 Multicolour FISH and spectral karyotyping / Jane Bayani, Jeremy A. Squire 205

2 Spectral karyotyping (SKY) 206

3 M-FISH 208

4 M-FISH and SKY protocols 208

5 General considerations for image acquisition and analysis 214

Image analysis using SKY 215

Image analysis using M-FISH 216

6 Troubleshooting 216

11 cDNA microarrays for fluorescent hybridization analysis of gene expression / Javed Khan, Lao H. Saal, Michael L. Bittner, Yuan Jiang, Gerald C. Gooden, Arthur A. Glatfelter, Paul S. Meltzer 221

Serial analysis of gene expression 221

Oligonucleotide arrays 222

cDNA microarrays 222

2 Fabrication of cDNA microarrays 223

Culturing cDNA bacterial clones 223

Microarray slide printing 230

3 Target production 232

4 Hybridization 235

5 Image acquisition 237

6 Image analysis and normalization 237

7 Sensitivity and specificity 238

8 Data mining and statistical analysis 238.

Notes:

Includes bibliographical references and index.

ISBN:

0199638837

0199638845

OCLC:

47726691