Protocols for Nucleic Acid Analysis by Nonradioactive Probes

1. Front Matter

   Pages i-xii

   PDF

2. About Nonradioactive Nucleic Acid Detection

   • Peter G. Isaac

   Pages 1-7

3. Isolation of DNA from Plants

   • Justin Stacey, Peter G. Isaac

   Pages 9-15

4. Isolation of High-Molecular-Weight DNA from Animal Cells

   • Ian Garner

   Pages 17-23

5. Restriction Enzyme Digestion, Gel Electrophoresis, and Vacuum Blotting of DNA to Nylon Membranes

   • Justin Stacey, Peter G. Isaac

   Pages 25-36

6. Isolation of Plant RNA

   • Rachel Hodge

   Pages 37-40

7. Isolation of Total and Poly A+ RNA from Animal Cells

   • Ian Garner

   Pages 41-47

8. Preparation of RNA Gel Blots

   • Rachel Hodge

   Pages 49-54

9. Preparation of RNA Dot Blots

   • Rachel Hodge

   Pages 55-57

10. Isolation of Plasmids for the Preparation of Probes

    • Peter G. Isaac, Justin Stacey

    Pages 59-66

11. Production of Hybridization Probes by the PCR Utilizing Digoxigenin-Modified Nucleotides

    • Tom McCreery, Tim Helentjaris

    Pages 67-71

12. Production of DNA Hybridization Probes with Digoxigenin-Modified Nucleotides by Random Hexanucleotide Priming

    • Tom McCreery, Tim Helentjaris

    Pages 73-76

13. Digoxigenin Labeling of RNA Transcripts from Multi- and Single-Locus DNA Minisatellite Probes

    • Esther N. Signer

    Pages 77-81

14. Labeling of Double-Stranded DNA Probes with Biotin

    • Angela Karp

    Pages 83-87

15. Preparation of Horseradish Peroxidase-Labeled Probes

    • Ian Durrant, Timothy Stone

    Pages 89-92

16. Random Prime Labeling of DNA Probes with Fluorescein-11-dUTP

    • Bronwen M. Harvey, Claire B. Wheeler, Martin W. Cunningham

    Pages 93-100

17. Labeling of Oligonucleotides with Fluorescein

    • Patricia M. E. Chadwick, Ian Durrant

    Pages 101-105

18. Hybridization of Digoxigenin-Labeled Probes to Southern Blots and Detection by Chemiluminescence

    • Tom McCreery, Tim Helentjaris

    Pages 107-112

19. Southern Blot Hybridization of Digoxigenin-Labeled RNA Minisatellite Probes and Color Detection

    • Esther N. Signer

    Pages 113-120

20. Hybridization and Detection of Digoxigenin Probes on RNA Blots

    • Elizabeth Davies, Rachel Hodge, Peter G. Isaac

    Pages 121-125

In assembling this book, Protocols for Nucleic Acid Analysis by Nonradioactive Probes, I have endeavored to select protocols that have wide applicability. My aim in doing so is to allow nucleic acid analysis to move from the confines of specialized research labora- ries and into general purpose and teaching laboratories. In particular, disciplines such as population biology and clinical diagnostics (where the application of the technology is important, not the technology itself) will find the newer techniques easier to perform with the qu- tity of samples that are normally required in these fields. Die-hard molecular biologists with hot fingers will find, by trying some of the protocols in this book, that nonradioactive protocols are normally faster than their radioactive brethren, and give more reliable results. An expanded description of the subject areas covered by this book is given in Chapter 1. The technology herein has very broad application; for instance, in conventional molecular biology research, population biology, plant and animal breeding, genetic mapping, paternity testing, forensics, prenatal diagnosis, and clinical and food microbiology. I have tried to include comprehensive protocols for both basic and more complex analyses. There are chapters dealing with the fundamentals of nucleic acid extraction from plants and a- mals, and with the procedures necessary to immobilize nucleic acids on solid supports. A range of labeling procedures is included, f- lowed by a selection of hybridization procedures.

...this book provides a collection of good protocols which are in general clearly written. They can be easily employed on the bench without being a specialist in a particular fields. - FEBS Letters

...Covers the basics of a variety of probe-labelling systems very well...a very interesting read. It could save you money and trouble from the radiation safety officer.-Biotechnology and Applied Biochemistry

• Nickerson BIOCEM Ltd., Cambridge, UK

  Peter G. Isaac

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